INTRODUCTION

MICROBIOLOGY
-is a science that deals with the study of microorganism and their activities. It is concerned with
their form, structure, reproduction, physiology, metabolism and identification. The science also
includes the studyof their distribution in nature, their relationship to each other as well as to other
livimg things, their beneficial and detrimental effects on men and the physical and chemical
changes they make in their environment.
SCOPE OF THE MICROBIOLOGY:
1. Bacteriology- concentrates on the study of structure functions and activities of bacteria
2. Phycology- is the study of various type of algae
3. Mycology- is the study of fungi
4. Protozoology- is the study of protozoa and their activities.
5.Virology- encompasses in the study of viruses and their effect on living cells.
6. Immunology
Virologist and cell biologist may become genetic engineers who manipulate genetic
materials (DNA) from one cell to another.
Prions and viroids are infectious agents smaller than viruses.
Division of Microbiology
1.GeneralMicrobiology- the study and classification of microorganism and how they function. It
encompasses all areas of microbiology.
2. MedicalMicrobiology- involves the study of pathogens, the disease they cause and the body
defense against disease.
3. VeterinaryMicrobiology- study of the spread and control of infection disease among animals.
4.AgriculturalMicrobiology- studies of both beneficial and harmful of microorganism in soil
formation, fertility and disease in plants.
Food microbiology- concerned with the production. Processing, storage serving of food,
prevention of food spoilage, food poisoning and toxicity.
Dairy microbiology- oversees the grading, pasteurizing and processing of milk and cheeses.

5. SanitaryMicrobiology- includes the processing and disposal of garbage and sewage wastes and
purification of water. Inspect food processing installations and eating establishments.
6.IndustrialMicrobiology- deals with the proper growth and maintenance of certain microbes to
produce beers, wines, alcohol and antibiotics.
7. MicrobialPhysiologyandGenetics- deals with the study and structure of DNA ang genetics.
8. EnvironmentalMicrobiology or microbialecology- encompasses the areas of soil, air, water
sewage, food and dairy.

2. In 1798, Edward jenner demonstrated that inoculation

Material provides human with immunity from small pox.
3. About 1880, Pasteur discovered that avirulent bacteria could be used as a
fowl cholera, he coined the word

vaccine for

4. Modern vaccines are prepared from isolated components of pathogens and by recorbinant
DNA techniques.
Birth of Modern Chemotherapy: Dreams of a magic bullet
1. Chemotherapy is the chemical trertment of a disease.
2. Two types of chemotherapeutic agents are synthetic drugs (chemically prepared in the
laboratory) and antibiotics (substance produced naturally by bacteria and fungi to inhibit the
growth of microorganism.
3. Paul Ehrlich introduced an arsenic containing chemical called salvarsan to treat syphilis.
(1910)
4. Alexander Filerniag observed that the mold penicilliumnotalium inhibited the growth of a
bacterial culture. He be name the active ingredient penicillin.
5. Penicillin has been used clinically as antibiotic since 1940s.
6. In 1939, Rene Dubos discovered two antibiotics produced by bacterium Bacillus.
7. Researches are tackling the problems of drug resistant microbes.
8.Domagle- prohtosil
Development of Microbiology

1. Bacteriology is the study of bacteria, mycology is the study of fungi and parasitology is the
study of parasitic protozoa and worms.
2. The study of AIDS, analysis of the action of interferon and the development of newer vaccines
are among the current research inherent in immunology.
3.New techniques in molecular biology and electron microscopy have provided tools for the
advancement of your knowledge in virology.
4.The development recombinant DNA TECHNOLOGY has helped were all areas of
microbiology.
CLASSIFYINGTHE MICROORGANISM
1.In a nomenclature system of designed by carolusbuinaeus (1935), each living organism is
assigned two names.
2. The two names consist of genus and a specific specie both in which are underlines and
italicized.
THE DIVERSITY OF MICROORGANISM
1. Bacteria are unicellular organism beacausthey have no nucleus prokaryotic cells.
2. The three major basic shapes of bacteria are bacillus coccus and spirili.
3. Meat has pychoglycan cell wall, they devide by binary
4.. cell wall is made up of chitin.
Protozoa-priotine.
1. are unicellular and rytes and are classified according bto their
2.obtain nourishment by absorption or ingestion through specified structures.
Algae- is isolation word for seaweeds
1.unicellular and multicellular eukaryotes that obtain nourishment by photosynthesis.
2. produce oxygenandcarbohydratesthatare used by oraganism.Cellwall is made up of cellulose.

Viruses
1. Non cellular entities that are parasites of the cells, no capacity to synthesized their own food.

2. consist of nucleic acid (DNA or RNA) surrounded by protein coat and envelope may surround
the coat.
Multicellular Animal Parasites
-These are flat forms and the round worms called Helminthes.
USES OF MICROORGANISM
1. Microorganism degrade dead plants and animals and recycle chemical element to be used by
living plants and animals.
2. Bacteria are used to decompose organic matter in sewage.
3.Bioremedation processes used bacteria to clean up toxic wastes.
4. Bacteria that cause disease in insects are being used as biological controls of insect pest.
Biologicalcontrol are specific for thepestiside harm the environment.
5.Using microbes to snake products such as foods and chemicals is called biochenolopy.
6. Using recombinant DNA, bacteria can produced important substances or missing genes into
human cells.
8. Genetically engineered bacteria are used in agriculture protect plants from host and insects
that shelf life of produce.
hAaerc-nodpygtil w.
Cyanobcteri-hdugsl fy.

CHAPTER1
THEGNRALCSIOFB

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(2)
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layers,
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(4)
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ofDNA,celwaypmrsndbi.
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Transtio-mRchegl(f)
thegnomribs.
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CYTOPLASMGIRNUEu(nsiocl)

Moetachmriuvnlgsd fer.
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CMROISPEXANTFB

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ndayurisemtclpohf.
(8)
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(9)
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(egy.Cstalrvio)
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10-3second
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acidfst.

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i.Coverwthucabnlf,smgy5 die.
i.hWwaster (15)
ivo.Dlecnrzadhuytfpkmis.
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vi.Countersa10f-3cdwhmylb
vhi.Wwaster,ndyxmAcfogpa
rednagstiubkloc.

b.nKyoui'smethd(cl)

dProuce:
xi.Fsmearbyht
i.Sntao(hecsry)wkublf

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d.uBmgartenh'sMo
ThdisutoefrnaM.blcmp is
easilyntdbucohfgrwileM.ts
doesnytrakiluph b.

Specialtns-duomhzr faen(16)
norgismaS.pe(dxA)
1.CelWnaSti
a.DyhrModet

2.pCsuahlrModet
a.HhisModet-fnubcpl
bn.Gh'sMiodet-rga
c.hAnoyt'sMdeu-apligrkbcn
dh.W'selcMot-buap\vi
e.Tylr'smthod
f.Muir'sethod-caplb,

gd.Whswoa'rtme
hN.Mealc
i.wLsona
3. Stain for Metachromatic Granules
a. LAMB ( LoefflersAlk. Methylene Blue)- deep blue granules, light blue bodies MB+KOH
b. Neissers
c. Alberts- blu black granules, cytoplasm green
d. Ljubinsky Method- deep purple granules, brown bodies
e. Gohar
4. Spore Stain
a. Acetic Acid Method
b. Dorners Method- 10% nequosine- red spores(carbolfuchsin), colorless background
c. Wirtz and Conklin

- Use Malachite Green such that spores

d. Schaeffer and Fulton -(most commonly used)are green and safranin as counterstain

(17)

5. Flagellar Stain
a. Grays

Used as A Special Mordanting solution

b. Leifson- flagella red

such as Tannic Acid which swells,

c. Fisher and Conn

coats and forms a

d. Casares - Gils

precipitate with

e. Van Ermengens

the flagella.

D. Indirect/ Relief/ Negative Staining a staining process in which the microorganisms appear as
bright colorless objects against a dark background.
1. India Ink (Burris Method)
2. Nigrosin Method

Stains for Spirochetes- Burris India Ina and Fontana

Polychrome- is a mixture of eosin + MB to stain pus + exudates
Stains Commonly Used Results Principle
Primary
Crystat violet
Carbolfuchsi
n
Auramine

Mordant
Decolorizer
Alcohol/aceton
e
Acid alcohol
Acid alcohol

Counterstain

Positive

Negative

Safranin

Purple

Pink

Methylene
blue
Km0

Pink

Blue

Orangefluoresc
e

No fluoresce

Rhodamine
Calcofluor
10% KOH

Bluish
white No fluoresce
with fluoresce

(18)
CHAPTER II
BACTERIAL GROWTH AND CULTIVATION
Bacterial Growth- increase in bacterial numbers nor increase in the size of individual
Growth is the orderly increase of all chemical constituents of the cell. A process that entails the
replication of all cellular structures, organelles and protoplasmic components from the nutrients
present in the surrounding environment. Under favorable conditions almost all bacteria are able
to reproduce rapidly. The average time required for an organism to double its number is referred
to as the generation time or doubling time. ( e.g., E. coli 20 minutes)
Binary Fission divides into 2 equal parts. Steps:
1.
2.
3.
4.

Cell elongation & replication

Cell wall & cell membrane
Cross wall formation
Cell separates as indole cells

GROWTH REQUIREMENT
Nutrient Requirements
A Carbon- is the structural backbone of living matter
Classification:
1. Autotrophs (lithotrophs)- require only water, inorganic salts, and carbon dioxide for
growth; they dont require organic nutrient for growth.
2. Heterotrophs (organotrophs)- require an organic form of carbon for growth from CHON,
CHC
a. Parasites-requires living organism matter for growth.These includes pathogens.
b. Saphrophytes- those which live dead organic matter.
B

Nitrogen

Nitrogen is a major component of proteins and nucleic acids of a typical bacterial cell. The
end product produce it from amines.
Sulfur- used to synthesis S containing a vit.like thiamine
Phosporus- essential for the synthesis of nucleic acid and phospholipids

(19)
C.

Growth Factors

Growth factors are organic compounds necessarily needed by a bacterium in order to grow.
These substances are usually provided in the culture medium.
Classification:
1. Prototrophics- do not require an exogenous source of growth factor since they synthesize
their own.
2. Auxotrophics- require the addition of growth factor to culture media for growth to occur.
Examples of Growth Factors :
1.
2.
3.
4.
5.
6.

B- complex vitamins
Amino acids
Purines
Pyrimidines
Pentoses
Fatty acids

Fastidious- are microorganisms with demanding nutritional requirements.

D. Inorganic Salts
Salt in small amounts stimulates the growth of some organisms. Organisms requiring high salt
concentrations are called halophilic.
Vibrio- req.a higher salt concentration.
E. Oxygen
Classifications:
1. Aerobes- grow in the presence of atmospheric (free) oxygen.
a. Obligate aerobes- grow only in the presence of oxygen
b. Facultative anaerobes- fundamentally aerobes, but can grow in the absence of
oxygen.
c. Microaerophiles- grow best at low or reduced oxygen tensions.
2. Anaerobes- grow in the absence of atmospheric oxygen.
a. Obligate anaerobes- grow only in the absence of oxygen
b. Facultative aerobes- fundamentally anaerobes, but can grow in the presence of
oxygen.
c. Aerotolerant anaerobes- do not grow well but do survive in the presence of oxygen.
Oxygen Toxicity
The toxicity of oxygen results from its reduction by enzymes in the bacterial cell to
hydrogen peroxide (H2O2) and by ferrous ion to the more toxic free radical, superoxide. Aerobes
and aerotolerant anaerobes, by the presence of superoxide dismutase prevents the accumulation
of the superoxide ion.
The hydrogen peroxide formed in the above reaction is rapidly destroyed by catalase, also
present in aerobes and aerotolerantanerobes.

F. Carbon Dioxide
Some organisms such as Neisseria, Brucella, etc. require for growth a higher concentration
(3-10%) of CO2. These organisms are called capnophiles.

G. Moisture

This is indispensable for bacterial growth. It serves as a solvent for food and forms the major
portion of the protoplasm. Organisms requiring an increased moisture content are termed
humidophilic.
Physical Requirements
A. Temperature
Every has an optimal temperature, the temperature at which the organism grows best.
Classification:
1. Psychrophilic (cold-loving)- grows at 10-20C
2. Mesophilic- grows at 20-40C (most pathogens grows at 37C)
3. Thermophilic (heat-loving)- grows at 50-60C
4. Thermodiuric- important in organic composed piles.
Minimum growth temperature- lowest temperature at which the specie will grow
Optimum growth temperature- the temperature at which the specie grow best
Maximum growth temperature-highest temperature at w/c growth is possible
Refrigeration- slow down growth of most spoilage microorganisms and will entirely prevent the
growth of all
Freezing- no significant growth

(21)

B. Hydrogen Ion Concentration

Most pathogenic bacteria have an optimal pH of 7.2-2.6
Classification:
1. Acidophiles- grows at pH 6.5-7.0
2. Neutrophiles- grows at pH 7.5-8.0
3. Alkalophiles- grows at pH 8.4-9.0

C. Osmotic Pressure
Organism requiring high osmotic pressures are called osmophilic.
Plasmolysis-osmotic loss of H2O(shrinking of cells plasma membrane)

THE BACTERIAL CURVE-This characteristic growth curve is obtained by plotting the

logarithm of the number of cells against the time of growth.
Major Phases of the growth curve

A. Lag Phase ( Phase of Rejuvinescence or Phase of Physiologic Youth)

The period of adaptation of the organisms to their new environment characterized by little
or no multiplication. However, the cells in this phase are very active metabolically. Active
synthesis or enzyme and other essential constituents occurs all elongation.
B. Exponential Phase (Logarithmic Phase or Phase of Active and Young )
-Best for culture media
-Culture reproduction of Bacteria
-The period at which the cells re in a state of balanced growth characterized by maximal
rates of cell division and mass increase. It is during this phase that the generation time is
constant.
C. Stationary Phase (Phase of Equilibrium or Plateau Phase)
-The period at which the rate of cell reproduction equals the rate of cell death. Growth ceases due
to:
1. Accumulation of waste products
2. Exhaustion of nutrients
3. Change in pH and other factors.
D. Death Phase (Phase of Decline)

(22)

- Rapid decrease in cell membrane.

-Dead cells exceeds new.
-The period at which complete cessation of multiplication occurs such that the death rate in the
medium increases rapidly.
Fig. 2-1. The Bacterial Growth Curve: a, Lag Phase; b, Exponential Phase; c, stationary phase; d,
Phase of decline.
E.coli- 20 minutes in vitro/ in vevo 12 hrs
M.Tuberculosis 12 hrs in vitro and requires 6 days to produce colonies
T.palledum 30 hrs in rabbit testes
M. leprae 13 days in armadillo

MEASUREMENT IN BACTERIAL GROWTH

A. Determination of Bacterial Mass
1. Getting the dry weight.

2. Measuement of optical density of a broth culture in a spectro-photometer. (most

widely used method).
3. Turbidimetric and nephelometric methods (McFarland Standards- most practical
method).
4. Nitrogen determination.
5. Measurement of cell volume after cetrifugation.
B. Determination of Cell/Bacterial Number
1. Stained slide method
2. Counting chamber (Petroff-Hausser counter)
3. Coulter counter
4. Calibrated wire loop
5. Pipet dilution method
6. Pour plate method
7. Sereae delution
8. Most probable # method

Physics agents that affects the bacterial growth:

1.
2.
3.
4.
5.
6.
7.

Temperature
Dessication
Lights
Electric current
Agesetation
Pressure
Vibration

(23)

BACTERIAL CULTIVATION
THE CULTURE MEDIUM
A culture medium is any material containing the necessary nutritional and environmental
requirements for bacterial growth.
CLASSIFICATION OF CULTURE MEDIA
A. According to Physical State and Consistency:
1. Agar- red algae polymer (derived from a marin algae)
2. Liquid- without any trace of agar or solidifying substances
3. Semisolid- contains 0.5-1.0% of agar (e.g, SIM)
4. Solid- contains 2-3% of agar (e.g, BAP, CAP, TSI)
B. According to Composition
1. Synthetic- exact chemical composition of the ingredients is known (e.g, commercially
prepared culture media).

2. Nonsynthetic- precise composition of some or all of the nutritive substances used is

not known (e.g, meat extract broth).
3. Tissue culture media- contains living tissue (e.g, embryonated egg for viruses).
C. According to How the Medium is Dispensed:
1. Plated- distributed in streile petri dishes- 20ml-25ml
2. Tubed- distributed in sterile test tubes- 5-10ml
Types:
a. Slant
b. Butt
c. Butt/slant
Aerobic Media:
Bacteroids Bile Asculine Agar (BBE) for B. fragiles groups- Black
solonies
Kamamycin Vancomycin laked Blood- Bacteriosides sp:- enhamce piment
production
Cycloserine- Cefalexin Fructose- C. defficile
(24)
CDC Amaeroluc BAP- anaerobes enriched with Hemin cystein
Cooked chopped meat medium- anaerobes
Lowenstein jensen agar- mycobacterium
Middle brook- mycobacterium
Petragnani- mycobacterium

According to Use:
1. Simple or Supportive- contains nutrients that allow most nonfastidious organisms to grow
at their natural rates, without providing a growth advantage to any particular organism ;
utilize only for routine cultivation of microorganisms ( e.g., nutrient agar, brain heart
infusion agar)
2. Enrichment- designed to enhance the growth of particular organisms( usually pathogens
and suppress the growth of normal flora in a specimen (e.g.,
Selenite, GN, and Tetrathionate Broths)
Sheep Blood Agar preferred blood because it will give a good hemolytic creation.
3. Differential- distinguishes organisms growing together by their differences in cultural
characteristics (e.g., Blood agar)
CAP- fastidious isolates hemo
BAP- most isolation protocol
4. Selective- contains one or more agents inhibitory to all organisms except the organism
being sought (e.g., bismuth sulphite agar, phenylethyl alcohol and Salmonella-shigella
agar)
Some of the inhibitory agents added to media:

a.
b.
c.
d.
e.
f.
g.

Gentian violet-inhibit growth of G(+) organisms

Sodium deoxycholate and other bile salts
Basic Fuchsin
Alcohol- prevent swarming of proteus
Chloral hydrate
Potassium tellurite- inhibit growth of G(-) organisms
Sodium azide

5. Selective and Differential- some media are both selective and differential like the media
used in the primary isolation of enteric gram negative organisms (e.g., Endo agar,
MacConkey agar, XLD agar and Eosin methylene blue agar)
6. Special-specially prepared to support the growth of specific microorganisms
Examples:
a. Middlebrook 7II-10 agar M. tuberculosis
(25)
b. Fletcher medium Leptospira
c. W medium Brucella
d. Bordet-Gengou agar Bordetella pertussis
e. Thayer-Martin Neisseria
f. McBride Listeria monocytogenes
g. Dieudonnes medium V. cholera
General Steps in Preparation of Culture Medium (Tubed Medium)
1. Weighing weigh the different ingredients and then place in clean, dry containers
2. Dissolving- add the exact amount of solvent to the ingredients and then dissolve by
heating
3. Titration- adjustment to the right ph (Ph7.2)
4. Distribution- distribute in the test tubes
5. Sterilization
NOTE: PLATED MEDIUM- sterilized first before distribution.
Special Media:

Bordet Gengou Agar- B. perstusis

Kellys medium- Borreliaborgdorferi
Skerrow agar- Gelicobacterpyleri
Thiosulfate citrate file salts (TCBS) Vibric

Inoculation of Media
A. Inoculation of Tubed Media
1. Liquid Medium
a. With the use of a sterile Pasteur pipet
b. Inoculate by shaking a previously heated wire loop or needle.
2. Slant Medium

With the use of a wire loop or needle, transfer the inoculum to the bottom of the slant
and streak in a zig-zagging manner across the entire surface toward the mouth of the
tube.
3. Butt Medium
Just stab the medium with an inoculating needle.
4. Butt/Slant Medium
Inoculate the butt first by stabbing the needle to the bottom of the medium and then
streak the surface in a zig-zagging manner toward the mouth of the tube.
B. Inoculation of Plated Media
1. Streak Plate Technique
a. Purpose: To isolate organisms in pure culture.
(26)
b. Procedure:
I.
With a sterile loop, deposit the inoculum at one edge and streak back and forth
over the area progressing across the agar until one fourth of the plate surface is
covered.
II.
Flame the loop, streak at right angles to the originally inoculated agar, and
cover the second quarter of the plate.
III.
Flame loop again and repeat procedure until third and fourth quadrants are
covered.
IV.
Turn plate upside down (to prevent contamination by water condensation) and
incubate at 35-37 degree Celsius under 5-10 CO2
Note: 5-10% is not recommended for media in which Ph alteration is used to
differentiate colony types (e.g, XLD agar, Hektoen enteric agar).
V.
Examine initially after 18-24 hours of incubation.

2. Pour Plate
a. Purpose:
i. To determine the approximate no. of viable organisms in a liquid such as water, milk , urine, or
broth/culture. Data are expressed as the no. of colony-forming units per milliliter (CFU) of the
substances examined.
ii. To determine the type of hemolysis produced by streptococci.
b. Procedure:
i. prepare serial dilutions of the specimen.
ii. transfer a prescribed volume of diluted specimen into tubes of melted agar.
iii. mix the tubes and pour them into petri dish.

iv. allow to solidify and incubate.

3. Streak-pour plate
a. purpose: for studying hemolysis.
b. procedure:
streak a blood agar plate as described for a streak plate and cover streaked surface with a melted
blood agar.
(27)
Inoculum- growth in culture media

THE CULTURE
Types of culture
1. Pure (axenic) culture- made up of organisms belonging to one specie.
2. Mixed culture- made up of organisms belonging to different species.
3. Stock Culture- is a pure culture of organisms used as a soured of supply for industry,
reseach, and students use
4. Contaminated culture- is culture accidentally contains more than , species of bacteria
Methods of obtaining pure culture:
1.streak plate method
2.pour plate method
3. use of selective media and media containing antibiotics
BACTERIAL COLONY
Colonies are visible macroscopic masses of growth on a solid culture medium as a result
of repeated divisions of one or few microorganisms.
TYPES OF COLONY
A. Mucoid (M) Colony
1. exhibits a water-like, glistening, confluent appearance.
2. Characteristic of organisms that form slimes or well-developed capsules.
3. ex. K. pneumonia, S. pneumonia, H. influenza, etc.
IMPORTANT DESCRIPTION OF CULTURE CHARACTERISTIC
Form: circular , filamentous, pinpoint irregular

Education: flat, convex (dome-shaped), raised, umbilicate (depressed center)

Margin: entire, undulate, filamentous curled
Surface: dull, glistening, moist
Consistency: mucoid, creamy, mucous butyrous
Density: translucent transparent opaque.

(28)

B. Smooth (S) Colony

1. Has uniform texture and homogeneity.
2. Easily emulsified in NSS.
3. Characteristic of freshly isolated wild type organisms (virulent microorganisms)
4. Ex. Salmonella, Shigella, E. coli, Serratia and proteus
C. Rough (R) Colony
1. Granulated and rough in appearance.
2. Hard to emulsify in NSS.
3. Usually produced by mutant strains that lack surface proteins or polysaccharides
indicating loss of virulence.
4. Ex. Rough forms of enteric bacteria
5. Exception: R forms of B. anthracis and human and bovine types of M. tuberculosis
(most virulent).

FUNGI

All fungi are heterotrophs

-Sugar conc. 4%
-3.8 to 5.6 ph acidic environment

PROTOZOA

-PH-6.8
-aerobic, heterotrophs
-amino acids/melanin/CHO

(29)

QUALITY CONTROL: is a system used to monitor the quality of performance and result in the
cli. Lab

1. MONITORING OF LAB. EQUIPMENT:

Temp. of ref, incubator, water
Anaerobic jars should be monitored for anaerobic using methylene blue indicator

2. PROCEDURE MANUAL- through organized current procedure manual that contains the
policy and regulations procedures, location of reagents supplies
3. CULTURE MEDIA AND RGT.: each new shioment of C.M should be tested w/ control
org. by known results stock org. should be clean so both g+ & g- can be observed
4. PERSONNEL- properly educated & attend continuing education courses to human
updated
5. SAFETY POLICIES

CHAPTER III
BIOCHEMICAL TESTS

These are tests designed to demonstrate the physiological and chemical char. of microo.
w/c enables the bacteriologist by elimination.

CARBOHYDRATE FERMNETATION
Fermentation is the anaerobic breakdown of subs. (CHO) resulting often in the production
of large amounts of organic acids.

CARBOHYDRATE FERMNETATION TEST

A.CHO Fermentation medium
The CHO fermentation medium is a liquid or semisolid meat extract brith to w/c
an indicator and a CHO are added.

1. The base medium used for fermentation tests is a sugar-free medium

(a) Peptone water, (b) trypticase or (c) tryotone broth
2. pH indicators commonly used
(a) Bromcresol purple- purple(alk) to yellow(acid) at pH 6.3
(b) Andrades acid fuschin-pale yellow (alk) to reddish pink (acid) at pH 5.5
(c) Phenol red- red (alk) to yellow (acid0 at pH 7.9

3. CHOs commonly used are glu., lactose, sucrose and mannitol.

B.Durham Tube:
This consist of a tests tube w/a small inner tube.
(31)
C.Principle:
Organisms that ferment the CHO in the broth produces acid thus, changing the
color of the medium (depending upon the indicator used) to the acidic color. Gas formation may
accompany this reaction in some micro.

D.Procedure
1. Inoculate broth lightly.
2. Incubate overnight at 37C.
3. Examine daily for 4-5 days

Confusing tests:
1. Schick-C. diptheriae
2. Dick-susceptability to scarlet fever
3. Schuloz Charlton-scarlet fever
4. Alik-confum toxin prod. For C. diptheriae in vitro.
E.Result:
1. Positive-purple to yellow (bromcresol purple)
- pale yellow to reddish pink (Andrade indicator)
-red to yellow (phenol red)
-gas formation in the inverted insert tube
2. Negative-color of medium remains the same (e,g. , red w/ phenol red)

-no gas formation

CHO Fermentation W/:

Triple sugar iron agar (TSI) and Klingers Iron Agar (KIA)
(32)
TSI is a butt slant medium considered to be a modification of KIA.The only diff. bet. The
2 is that sucrose is not included in KIA.

A.CHO Content
1. TSI-glucose (0.1%), Lactose (1%), and sucrose (1%)
2. KIA-glucose (0.1%) and lactose (1%)
B.pH indicator: phenol red
C.H2S Indicator: ferrous ammonium sulfate (black deposit)
D.Principle:
1.Glucose utilization by certain micro. Occurs both aerobically on the slant where oxygen is
available and anaerobically in the butt. 6 hrs of incubation.
2.After depletion of the limited glucose, some org. utilize lactose or sucrose and continue making
acid end product. The slant and butt will remain yellow after 18-24 hrs incubation.
3.Some org. are not able to utilize lactose but instead breakdown the peptone in the medium.
After 18-24 hrs incubation the TSI will thus show a red slant and a retained yellow butt. The
reaction is called Alkaline over acid, signifying that only glu. Was fermented.
4.Glucosenonfermenters may also produce alkaline products from peptone utilization.
5.Some org. have the ability to produce gas from the fermentation sugar while others produce
large amount of hydrogen sulfide gas.
E.Procedure:
1.inoculate medium and incubate overnight.

2.record pH changes only after 18-24 hrs of incubation.

F.Result:
1.yellow in acid pH, red in alkaline pH.
2.blackprecitate (+) H2S production.
3.bubbles or cracks in the medium (-) gas production.

(33)

METHODS OF DETERMINING CHO FERMENTATION

RUSSELS DOUBLE SUGAR (RDS)

Contains glu. And lactose w/ andrades acid fuschin as indicator. The positive result is a
change of colony from pale yellow (alk) to reddish pink (acid).
With the use of the Smith Fermentation Tube

(INDOLE, METHYL RED, VOGES-PROSKAUER, CITRATE)

INDOLE TEST
Principle:
Organisms that produce the enzyme tryptophase are able to degrade tryptophan into
indole, w/c can be detected by its ability to combine w/ certain aldehydes to form a colored
compd.

Methods
1.Kovacs Method-Pink to deep red color (+ result)
a.Medium: tryptone broth or trypticasebroth
b.indoleindicator:Kovacsrgt. (p-methylaminobenzaldehyde)
c.procedure and result:

i.add 0.5 ml of kovacsrgt. To 2-3 ml of a 48 hrs old broth culture.

ii.gently shake the tube and observe for the development of a pink to deep red color w/c is a (+)
result.

2.Erlichs method-Brilliant red ring bet. Solvent and medium

a.Medium:tryptone broth or trypticase broth

(34)

b.IndoleIndicator:Erlichsrgt. (p-methylaminobenzaldehyde)
c.Procedure and result:
i.add 1 ml xylene to a 48 hr old broth culture.
ii.shake the tube well and allow to stand for a few mins. Until the solvent rises to the surface.
iii.gently add 0.5 ml of erlichsrgt. Down the slidesof the tube and observe bet. The solvent and
the medium.
3.SpotIndole Test-Blue or blue green (+ result)
This is a rapid method for the detection of indole.
a.Indole Indicator:1% p-dimethylaminobenzaldehyde
b.Procedure and result:
i.saturate a filter paper in the bottom of a petri dish w/ the rgt.
ii.With a wooden stick or loop, rub a portion of the colony on the filter paper and observe for the
rapid development of a blue or blue green color.(+ result).

METHYL RED TEST

A.Medium: MR-VP medium or clark and lubs dextrose broth medium.

B.pHIndicator:methyl red
C.Principle :

Some organisms produce large amounts of acid from dextrose while others produce
less.
D.Procedure:
Add 5 drops of methyl red to 5 ml of a 48 hr old broth culture and observe for the
results.
E.Result:
1.Positive-red color (pH 4.5 or below)

(35)

2.Negative-yellow color (over 4.5)

VOGES-PROSKAUER TEST
Differentiate
Enterobacter and K. pneumoniae (+) from E. coli (-)
Moraxella (+) from actinobacter (-)
A.Medium:MR-VP medium
B.Rgts.:
1.5% alpha-naphthol in 95% ethyl alcohol.
2.40% KOH in distlled water (provides alkalinity)
3.0.3% creatine in distilled water (prevents false negative results)
C.Principle:
Some bacteria have the abilty to produce acetoin (acetylmethylcarbinol) from
glucose.w/c reacts with guandine compd. Present in the broth to give a red-colored complex.
D.Procedure and result:
1.Inoculate NR-VP broth and incubate at 35c to 37 for 48 hrs.
2.to 1 ml of the culture broth, add 2 drops creatine solution, addition of each rgt.
3.Observe for a pink to cherry red color w/c is the (+) result.

CITRATE TEST- Differentiate Entero and Klebselle (+) from E. coli

A.Medium:Simmons citrate slant

B.pH Indicator: bromthymol blue
C.Principle:

(36)

Some organisms can utilize citrate as their sole source of CO2 producing acetate and
other alkaline carbonate end products in the process. These products change the color of the
indicator from green to blue.
D.Procedure:
Inoculate Simmons citrate slant incubate for 24 hrs or up to 4 days at 35-37C
E.Result:

1.Positive-prussian blue color (alkaline) (Entero, Klebsella, and Salmonella)

2.Negative-no color change (green)

CATALASE TEST- rapid evolution of bubbles of gas

A.Principle:
This test is based on the presence of catalase in some micro. Catalizing the release of
oxygen and water from hydrogen peroxide.
B.Procdure:
Pour 1 ml 0f 3% hydrogen peroxide over a culture grown infusion agar slant or pick a
colony and mix it with 1 drop 3% H2O2 on a clean glass slide.
C.Positive result: rapid evolution of bubble gas.

*NOTE:The test should not be performed with colonies grown on blood agar (red blood cells
contains catalase thus, give a positive test).

OXIDASE TEST

A.Principle:
Some organisms produce cytochrome oxidase which in the presence of air, acts on
certain aromatic amines to produce colored compd.
(37)
B.Methodes:
1.Spot Oxidase Test (Kovacs Method)-blue or black purple
a.rgt.:1% tetramethyl-p-phenylenediaminedihydrochloride
b.Procedure and result:
i.Place a filter p[aper into a petri dish and moisten it wuth several drops of the rgt.
ii.With a platinum wire or wooden stick ruba 24 hr old colony on the moistened filter paper.
iii.Observe for color-change to blue or dark purple w/c is a (+) result.
2.Oxidase Test for Neiserria-Pink to red and to purple black
a.rgt.:1% dimethyl-p-phenylenediamine hydrochloride.
b.Procedure:
Add several drops of the rgt. Onto the colony on a soilidmrdium or add 1 drop
of the reagent to a filter paper and spread the colony onto it .
c.Positive Result:
The colonies become pink then red and finally purple black.
3.IndophenolMethod:Intense blue
a.rgt.:
i.p-aminodimethylaniline hydrochloride (or oxalate)
ii.alpha-naphthol
b.Procedure:
i.To an 18-4 hrs old culture grown on a nutrient agar slant add 2-3 drops of the above rgt.

ii.Tilt the tube to mix the rgts. And flow over the growth.
c.Positive result: intense blue color within 2 mins.

(38)
UREASE TEST-Hydrolyze urea to ammonia by urease enzyme

AMedium: Christensens urea agar or urea broth

B.pH Indicator: phenol red
C. Principle:
This is based upon the ability of some bacteria to hydrolyze urea into ammonia. The
medium becomes alkaline and the indicator turns from yellow to red.
99.9% of the original inoculum is the minimum bactericidal concentration(MBC) or minimum
lethal concentration (MLC).

2. Agar dilution method - used to test a numbers of strains simultaneous

a. Most commonly used medium : Mueller-Hinton agar

b. Inoculum:
The organisms are diluted to slightly greater turbidity than that of a McFarland 0.5
standard.
c. Procedure:
The same as in broth dilution method except that solid agar plates are used instead of
tubes. The agar plates are inoculated with a steer-Foltz replicator, a device equipped are
inoculated with calibrated to pick up and deliver 0.001ml of the bactericidal suspension.
d. Result:
The same as in broth dilution method.(This method does not determine the MBC or

MLC)
e. Uses:
A good research laboratory technique for testing anaerobic bacteria.

3. Microdilution or microbroth dilution method

(39)

The principle of this method is identical to that of the broth dilution metjod.The utilizes
small plastic microtiter trays which contain a final volume of organism-antimicrobial
suspension 0.1 ml

B. Agar Diffusion Test

This test utilizes a filter paper disk impregnated with a specifief amount of antimicrobial
agent which is applied onto an agar surface that has been freshly inoculated with a
microorganism. The use of this test is largely limitef to rapidly growing aerobic and
favultatively anaerobic bacteria.

The Kirby-Bauer Technique - this is a paper disc method of sensitivity that is indicated for those
microorganism whose susceptible cannot be predicted after their identity is known.

1. Procedure:
a. With a sterile wireloop, pick (4-5) pure isolated colonies of organism and make a
subculture using MHB, or any other suitable broth medium.
b.Incubate at 35C for 2-8 hours(up to 18 hours) then compare the turbidity of the
subculture to a McFarland 0.5 barium sulfate standard. If the turbidity is greater than
the 0.5 bacterial suspension) by adding sterile NSS and if lesser than the standard

adjust by incubating the subculture further or by adding some more colonies.

c. Using a sterile cotton swab dipped into the standardized bacterial suspension, streak a
Mueller Hinton agar (4-6 mm in depth) evenly in three different planes.

d. Allow the inoculated plate to dry for 3-5minutes.

e.With an alcohol-flamed, fine pointed forceps or a disk dispens distributed the disk
evenly on the agar not closer than 15mm the edge of the petri dish. For the 90 mm plate not
more than 8 disk should be used and with the 150 mm plate 12-14disks may be used without
crowding.
f. Incubate overnight at 35C.
g. Measure the zone of inhibition of each disk and determine whether susceptible,
moderately susceptible, intermediate or resistant comparing the zone diameter obtained
with the standard zone diameter provided by the manufacturer of each disk.

2. Factors affecting the technique :

a. Depth , pH, cation content, supllements and soirce of the Mueller Hinton agar
b. Agevand turbidityb(amount) of inoculum
c.temperature,atmosphere and duration of incubation
d.method of reading results
e. Diffusibility of the antimicrobial disks, their age and storage condaitions.
f.the way the inoculum is spread on the plate.

C. Disk Elution Method

In this method antimicrobial impreganated filter paper disks are into the broth or agar medim The
antimicrobial agent present in the paper disks diffuses from the disk throughout the broth or agar
medium(elute) to provide a desired concentration of the antimicrobial in the mediumThe

principle of this method is the basis of some rapid, semi automated systems(Autobac MS-2or
Avantage) used for susceptibility testing of aerobic and facultatively anaerobic bacteria. Disk
elution is used also in susceptibility testing of anaerobic bacteria and Mycobacteria.

Interpretation of Results:

A. Susceptible

(41)

This implies that an infe tiondue to an organisms can be appropriate treated with the
recommended dosage of antimicrobial agent for that of infectionand infecting organism, unless
otherwise contraindicated

B. Moderately Susceptible
This implies that the amount of the antimicrobial agent given to patient can be increased
beyond or recommended dosages without serious risk of adverse reaction to obtain higher
levels in the infection site.

C. Intermediate
Also known as the intetmediate category , indicates that the test result is equivocal and that
susceptibility of that particular organisms cannot be predicted. This is generally applied to
antimicrobial agents a narrow toxic to therapeutic ratio in which higher dosages are like be
associated with adverse side effects.

D. Resistant
This implies that an infection due to an organism is unlikely to respond or will not reliably
respond to the antimicrobial agent.

SENSITIVITY TESTING
DISCUSSION:

Antibiotics differ from disinfectants and antiseptic in that they are chemicals that are
biosynthesized. Biosynthesized refers to the production of substances by living organisms.
These antibiotic substances are of no use to the microbe so we call them by products of
microbial metabolism. The genera of microbes that produce the largest numbers of
antibioticsuseful to humans are bacillus, penicillium and streptomyces. Antibiotics also differ
from disinfectants and antiseptics in that they are administered internally and travel in the blood
stream to all parts of the body. This means that the antibiotic must have low toxicity to body cells
while
being
toxic
or
destructive
to
the
parasitic
invader.
(42)
Other antimicrobial chemicals used therapeutically are artificially synthesized in the
laboratory. Because of their artificial synthesis they are often called as chemotherapeutic
agents. More commonly they are reffered to a narrow and or broad spectrum antibiotic.
In vitro is the Latin term for "in glass", as opposed to in vivo meaning "in life" or in a
living animal.
The kirby -Bauer method for determining bacteridical sensitivity to antibiotics is
employed in diagnostic laboratories because it is a standardized and therefore gives more
accurate results. It involves growing pure colonies of the etiological agent in a special broth,
diluting the broth culture to match a comparative standard, and inoculating a specific medium
before the sensitivity disks are added. A certain size zone of inhibition for each different
antibiotic has been empirically determined to be effective therapeutically for each bacterial type
and an organism is not considered to be sensitive to the antibiotic unless its zone of inhibition is
as large as longer than the predetermined zone for that antibiotic.
The kirby Bauer method has the advantage in that it standardizes MOST OF THE
FACTORS THAT INFLUENCE INHIBITION ZONE SIZE EXCEPT bacteticidal susceptibility.
These factors are inoculum size, pH of medium, depth of medium, concentration of agar, rate of
diffusion of drug (a function of the molecular weight of the antibiotic) concentration of antibiotic
impregnated disks, and incubation.
The mutant resistant strains of bacteria often arise because of indiscriminate use of
antibiotics or from undertreatment with the proper antibiotics. Therefore, it is important for
the patient to take all of the correctly prescribed dosage. In the case of antibiotic therapy,
undertreatment can be more harmful than overtreatment. In low dosage it is possible that the
antibiotic can act as a selective agent by killing the normally susceptible bacteria thereby

selecting out the drug resistant mutant cells.

Antibiotics kill bacteria in several ways. A few examples are the following: Penicillin
inhibits the synthesis of bacterial cell walls. Streptomycin competes with PABA as a substrate for
an enzyme reaction. Streptomycin actually enters the reaction in place of PABA, thereby
blocking the synthesis of an essential cellular component. Some broad spectrum antibiotics
interfere with enzyme synthesis. All of the above result in the death of the cell. The death of
bacteria in the antibiotic sensitivity test to be performed is manifested by a zone of inhibition
around the antibiotic impregnated disk.
(43)
SERUM BACTERICIDAL TEST ( SCHLITCHER TEST)
This test determines the antibactericidal activity of serum of patients receiving antomicrobial
drugs, serving as a guideline to aid clinicians in deciding whether a patient is being given
effective therapy for a serious disease. The highest dilution of patient's serum required to kill a
microorganism is known as the serum bactericidal or lethal titer (SBT or SLT) whle the lowest
dilution of patient's serum that kills a standard inoculum of the organism is called the serum
bactericidal level. (SBL).

Factor that affect results of susceptibility

1. Size of inoculum
2. Temperature and incubation time
3. Medium used (typed and concentration of agar)
4. pH and depth of the medium
5. Stability(potency) of antibiotic
6. Rate of growth of microorganism

Millimeter scale or vernier or dial caliper scale- usd to measure the zone of zone of inhibiyion
0.5 McFarland standard (Barium sulfate turbidity standard) - 10^8org/m

The preaparation
* 150mm petridish
* 60ml of MH
* 0.5 ml of 1%BaSO4
* 99.5 ml of 1%H2SO4

7 or 6 disc- in a 9-10 cm plate

15 mm- distance between each disc
6-8hrs incubation intial reading can be done
35 C temprature inval7date the results of methecillin and oxacillin
14hrs is the optimum time
B- lactam producing staph- growth within the ZI should be reported as resistant

Action of Antimicrobial

1. Inhibits cell synthesis

ex. Penicilli , vancomycin, cephalosporins

2. Act as competitive enzymes inhibitors to block the formation of essential metabolites

ex, sulfa drugs +PABA, Trimethoprim, ethambutol

3. Inhibits protein synthesis by acting on 70S ribosomes

ex. Tetracycline, erythromycin, streptomycin,neomycin,chloramphenicol

4.Damage plasma membrane

(44)

ex. Polymuxin B

5. Inhibit nuvleic acid synthesis

ex. Refampicin, nalidixic acid, norfloxacin

6. 2 or more drug can be used simultanrously

ex. Co-trimoxazole- trimethoprim sulfamethoxazole

(45)

Agar Culture

Clean the skin auditory canal with 1% tincture of iodine

Touch the infected with sterile cotton swab
Place the swab in the container

C. Blood Culture
1. Most basic blood culture media contain a nutrient broth and an anticoagulant ( SPS).
Examples of blood culture bottles are:
a. Trypticase soy broth
b. brain heart infusion agar (BHIA)
c. Supplemental peptone
d. thioglycollate broth
2.
e.Columbia broth
a. Brucella broth
b. Castaneda medium ( biphasic medium-agar slant with broth)
3. Penicillinase may be added to blood culture media to specifically inactivate penicillins
and other beta-lactam antibiotics present in the patients blood.
4. The ideal dilution factor is 1:10 ( 1 part blood and 10 parts medium). This combines the
largest feasible amount of blood collected (usually 10 ml) with the smallest amount of
medium that can still encourage growth of bacteria and dilute out the antibacterial
components of the system.
5. The use of 2 different bottles of blood culture media, one vented ( aerobic- TSB) and the
other unvented (anaerobic-thioglycollate) is recommended for optimal recovery of
pathogens.
5. Blood culture should be incubated at 35 oC examined visually daily (with subcultures
done at intervals) for 7 days before reporting as negative or no growth. But in patients
suspected of having endocarditis, brucellosis and fungemia, incubation should be
prolonged for 2-4 weeks
6. Growth is usually indicated by the following:
a. Hemolysis of red cells

b. Gas bubbles in the medium

c. Turbidity
d. Colony formation
7. Newer (faster) methods of blood culture:
a. Lysis - centrifugation (e.g. , Isolator)
b. Self contained subculture (e. g. , septi-Chek and vacutainer agar slant) a
modification of the biphasic blood culture medium.
c. Detection of microorganisms through changes in electrical impedance (e.g. , the
bactometer).
(46)
d. Detection of CO2 end product of metabolism (e. g. , bactec system).
Cerebrospinal Fluid done by physicians ( or medical technologist)
A. CSF collection and handling clear colorless 65
1. Collection of CSF is by aseptically inserting a needle into the subarachnoid space at
the level of the lumbar spine.
2. Three to four tubes are usually filled; tube 3 or 4 is used for differential count while
the other tubes are for microbiological and chemical studies. The specimen for
microbiological examination should be placed in a sterile , screw-capped and
airtight tubes.
Genical kroct exudate from urethrae orefect ourva 3-4 cm small deanela siruf well be inserlca to
the urethra. Do not allowed to dry as imposed in cold temp. do GS.
Women: circular spectman cm35- be collected by the physicians up the aid of speculum a slecrite
coton swaf is inserted to the cercux, rotated and allowed to remain for at least
1. Glucose protein analysis
2.
3.
Differential count
4.
3. GSF should be transported immediately to the laboratory without refrigerator . if a
delay is unavoidable the specimens should be incubated for viral studies should be
refrigerated up to 24 hours or frozen at -70oC if a longer delay is anticipated.

B. Processing of CSF

glucose
satts
white cells

1. Specimens are centrifuged and decanted, using the sediments for smears (Gram
stain, India ink) and culture (specially of H. influenza , Neis-seria meningitides, S.
pheumoniae, and Crytococous neoformans). The supernatant can be used to test for
the presence of antigens or for chemistry examinations.
Examples of Serological Tests;

bacterial

fungal

multiple sclerosis

a. Counter current immuno electrophoresis (CIE) for bacterial antigen

(47)
b. Coagglutination test PHADEBACT (staphylococcal protein A coated with
antiserum)
c. Latex agglutination tests.
2. Culture media used in CSF culture:
a. enrichment broth = TSB or thioglycollate (37oC for at least 5 days)
b. CAP = for Gram (-) cocci
c. BAF = for Gram (+) cocci
d. EMB, McConkey = for Gram (-) bacilli
NOTE: plated media should be incubated at 370C in 5-10% CO2 for at least 72 hours.
e. fungal media (e. g., non blood containing media brain heart infusion, mycosel)
should be incubated at 300C for 4 weeks.
f. Tissue culture = for viruses
Inraat and Nasopharyngeal Specimens
A. Throat Swab the tongue should be depressed before swabbing, wet the tonsillow sellar
and heherid the ovula never rouchthe
1. The most abundant normal flora of the throat is alpha streptococcus.
2. The most common pathogen is S. pyogenes or group A beta-hemolytic
streptococcus which should be diagnosed with the following precaution.
a. The posterior pharynx, tonsils and any areas of purulence, inflammation or
ulceration should be cultured.
b. The swab is inoculated to a soybean-casein digest agar containing 5% sheep
blood.
c. A bacitracin disk test is placed directly on to a blood agar plate with a purified
subculture.
d. Plates are incubated at 35oC for 18-24 hours with reincubation of 1day if no
growth, before being discarded as negative.
3. The patients trough should be swabbed from the uvula to the tonsil on both sides of
the throat.
4. Throat swabs are adequate for recovery of adenoviruses and haemophilus species.

B. Nasopharyngeal Swab- A flexible urine nasopareangeal swab should be generally inserted

through the most, into the posterior nasopharyngeal rotation then removed.
1. A nasopharyngeal swab is recommended for the isolation and detection of carries
2. For C. diptheriae - Cultured on Loefflers agar slant and cystinetellurite agar plate
3. For B. pertussis charcoal cephalexin medium or Bordet- Bordet-Gengou agar
(48)
4. For Neisseria meningitides and gonorrhocae- Modified Thayer- Martin or Martin
Lewis agar.
5. Nasopharyngeal swabs are also suited for recovery of respiratory syncytial virus,
parainfluenza virus and viruses causing rhinitis
SPUTUM Patient should gargle with H 2O (not mouth wash) immediately before early morning
specimen are best
A. Collection - rusty tinged
1. Sputum should be collected from a deep cough (saliva is most of the time cultured
mistakenly) and expectorate into a sterile swab mouth contain mycopeasemac
2. Specimens with more than 10 squamous epithelial cells or less than 25 pus cells/hpf
are not suitable for culture.
3. Other means of obtaining sputum:
a. Gasrtric aspiration- for acid fast bacilli
b. Transtracheal aspiration
c. Transthoracic needle biopsy
d. Bronchoscopy with aspiration or biopsy
e. Thoracentesis
f. Open lung biopsy
g. Bronchoalveolar lavage (BAL)
h. Protected cather bronchial brushing and etc.
Quality of sputum evali Bartlet classification No. of neutrophil/LPO less than 10 10.25
greater than 25 mucus # of epithelial cell/LPO 10-25 > total scores of 0.02 lack of presser sole
B. Culture
1. Cultured on McConkey agar, BAP, and CAP
2. Anaerobic cultures are not done on expectorated sputum,specimens obtained by
bronchial washing and lavage,ostomy or endotracheal tube aspirates
3. Only specimens obtained by percutaneous aspiration (e.g., transtracheal aspiration)
and by protected bronchial brush are suitable for anaerobic culture.
4. Grams and acid fast stains are done on direct smears
FECES
A. Collection and Handling stool exam are rectal preffered than rectal swab

1.Walnut-sized specimen in a clean waxed cardboard or plastic container is sufficient for

most procedures.
2.Specimens should be delivered to the laboratory within 1 hour if there is a delay of more
than 2 hours, the specimen should be placed in a transport medium (eg., Cary-Blaire & (49)
Stuart)- decrease availability of the changed of ph overtime temperature is determinate to
shigella
3. If culturing for shigella species, specimen should be inoculated at bedside. However, if
this is inconvenient a glycerol transport medium (with equal parts of glycerol and 0.033
M phosphate buffer) should be used. ( polyricinyl alcohol)
4. Stools for viral culture must be refrigerated if not inoculated to media within 2 hours.
5. Stools for detection of C. Difficile cytotoxin should be refrigerated for a maximum of
48 hours until tested.
6. A rectal swab may be substituted as a specimen for bacterial or viral culture (if stool is
not available). The swab should be placed in Cary-Blair or Stuarts or transport medium.
B. Staining of smear
1. Gram staining is not routinely done since gram (-) bacilli are usually seen in stool.
However, it may help in the detection of certain etiologic agents:
a. many clumps of typical gram + cocci staphylococcal infection
b. many thin, comma-shaped gram (-) bacilli Campylobacter and Vibrio cholerase
indicated cholera
c. Large numbers of large and thin gram (+) bacilli- C. difficle
2.
Acid-fast stained should also be used to detect Cryptosporidium species and
Mycobacteria.
C. Culture
1. Specimen is inoculated to the following culture media:
a. General supportive medium BAP For yeast species, staphylococci, enterococci (-)
bacilli
b. Moderately selective agar

MacConkey, EMB supportive Enterobacteriaceae, Vibrios and othe pathogens

Hektoen enteric (HE), xylose-lysin desoxycholate (XLD), and salmonella and Shigella
species to be detected

c. Highly selective agar

Brilliant green (BG), Bismuth sulfite (BS) for Salmonella Species

Campy-blood agar- Campylobacter species
CIN agar- Y. enterocolitica
Cycloserine cefoxitin egg fructose agar (CCFA)-C. difficle

*NOTE: All of the above media are incubated at 35-75oC in air

and examined at 24 and 48 hours for colonies except:
= Campy-blood agar incubated in a microaerophilic atmosophere at 42o\C and examined at 24
& 48 hours ( C. jejuni & C. coli)
= TCBS incubated in air at 35-37oC for 48 hours
= CIN agar incubate at room temperature for 48 hours
= CCIA-

incubated anaerobically for 48 hours

d. Enrichment broth ( selenite F, Hajna Gram-negative (GN), tetrathionate) for enhanced

recovery of Salmonella, Shigella, and Campylobacter which are incubated in air at 35 oC for 1218 hours
(Hemophilus)
Pyelitis
UTI - Lower UT- pyelonephilipitis glomerulone
Hyper UT- cystitis (UB) inflamation urethritis (uretha)
URINE- Normally sterilize UB
Enterobactereiae- common cause UTI
Like E.coli- 90% cause of UTI in an klebsille, anterobacts and the patient faccalis pseudomonas
A. Collection
1. Clean-caton midstream urine specimen collection midstream urine specimen collection \
2. Suprapubic aspiration- infants young children S. saphropyticus culturing of foley cachelic
lerefectea.

B. Handling
1. Urine should be cultured within 2 hours after collection.

(51)

2. Urine may be stored as long as 24-hours under refrigeration (4oC)

C. Gram Stain of Uncentrifuged Specimen
1. This is the simplest and most rapid method for detecting signification bacteriuria
2. Presence of 1 organism /oil immersion field (examining 20 fields) correlates with
significants bacteriuria ( more than 105 CFU/ml)
D. Culture and Colony Count
1. Culture media used:
a. 5% sheep blood agar plate is preferred because other source like horse rabbit blood may
guide errarealus in hemolysis
b. MacConkey agar plate
c. Columbia colistin-nalidixic acid agar (CAN)
d. Phenylethyl alcohol (PEA)
2. Colony count can be performed by pour plate method or calibrated loop method.
a. Pour plate method

Prepare a 1: 100 dilution of urine with sterile water

Transfer 0.1 ml of the dilution to a petri dish
Add method, cooled agar and mix well
Incubate and examine for at least 48 hours
Count the colonies and multiply by 100
1 um ( 0. 001) is used to deliver 1 um of .001 urine x10 # of colonies or

CPU/ ml of urine
b. Calibrated loop method
A calibration loop is designated to deliver a known
Volume, either 0.01 0r 0.001 ml of urine
insert the calibrated loop vertically onto the urine specimen

place the loop at the center of the plate and spread the inoculum in a line across the
diameter of the plate
\without flaming, draw the loop across the entire plate, crossing the first inoculum streak
numerous times to produced isolated colonies.
(52)
Count the colonies and multiply by 1000 (if using 0.001 ml loop) or count the colonies
and multiply by 100 (if using 0. 01 ml loop)
161- 0.00ml X 1000
0.145 Colonies x 1000
145, or 1.45 x 10 CFU/ml
100000- infection
1000-1000000- contaminated
3. interpretation of culture results:

Colony counts of >105 CFULml (100,000 CFU/ml) for 1 or 2 bacterial species are
clinically significant of infection
Colony conts of > 105 CFU/ml for 3 or more bacterial species most likely represent
contamination
A colony count of >10 3/ml of bacterial specie from a midstream urine of a male is also
considered clinically significant
A colony count of 102 10 3/ml of 1 or 2 bacterial species from females wiyh lower
urinary tract symptoms may also be clinically significant
Colony count of < 50, 000 CFU/ml are seen in contaminated specimens aspiration is
significant.

COLLECTION AND CULTUREOF ANAEROBIC SPECIMENS

Collection
A. Specimen foe anaerobic culture is aspired with a sterile needle and syringe to avoid contact
with normal flora or:
B. Specimens are swabbed and immediately put into an evacuated tube filled with oxygen-free
CO2 or nitrogen (gassedout tube) containing an agar or broth indicator system or other
anaerobic transport device like:

Anaerobic Culturette
Bio-Bag
Anaerobic Pouch
GasoPak Pouch

Specimen to the rejected

(53)

1. Swabs
Voided urine
Sputum
Flees,gastric,iodin,small intestine
C. Never use a dry swab or a regular aerobic transport media
Anaerobic Culture Methods
A.Requirements

Exclusions of oxygen
Low redox potential ( E reducing capacity of certain culture media ;
It becomes more negative as the hydrogen ion concentration decreases)

B. Methods of promoting Anaerobiosis

1.Anaerobic jars
Brewer jar
Oxygen is removed by means of an electrically heated platinized catalyst with
electrical connection outside the jar.
Torbal Jar
Similar to the bewer jar but uses a rubber o ring rather than Plasticine and a catalyst
active at room temperature thus requiring no electrical heating.
GasPak jar- This is the most convenient and widely used anaerobic jar.
a. Components:
Hydrogen and CO2 generator envelope- activated with water
Palladium- coated alumina pellets-catalyst
Methylene blue or resazurin indicator strip- upon exposure to atmosphere 0 2 turns blue
or pink
b. Principle and results:

With water added to the CO2 and H2 generator envelope and oxygen catalyzed with H2 to
water via the pellets, anaerobiosis is achieved
Indications of anaerobiosis:

-Production of heat
-Moisture inside jar
-decolorization of indicator strip (white or colorless)

(54)

Jars utilizing the Evacuation-replacement system

a. Air is removed by drawing a vacuum of 25 inches of mercury
b. The jar is filled with oxygen-free gas such as nitrogen between evacuation of air
c. Anaerobiosis is achieved more quickly with this method but less convenient for routine use
2. Glove box or anaerobic chamber
This is an enclosed system which consists of a large clear plastic, airtight bag or chamber
Filled with oxygen-free gas mixture of nitrogen, hydrogen and CO 2. These chambers allow
materials to enter through an air lock. Anaerobiosis is maintained by palladium catalysts and
hydrogen gas in the chamber. The operator uses gloves or sleeves that form airtight seals
around his or her arms to manipulate items inside the chamber.
3. Roll tube technique
In this anaerobic culture system a pre-reduced anaerobically sterilized (PRAS) agar is
distributed under anaerobic conditions as a thin layer around the inner walls of test tubes.
The tubes are rolled and cooled until the melted agar forms the thin layer referred to. The
tubes are inoculated in two ways:
a. Close method by Hungate
- a syringe and needle are used to inoculate through the rubber seal
b. Open method
- consists of removing the rubber stopper and inserting a cannula that has oxygen-free gas
flowing from the tip

CHAPTER VII
HOST- PARASITE RELATIONSHIP
DEFINITONS
1. Host an animal or plant that harbors or nourishes another organism
2. Parasite an organism which dependent on another organism for food and shelter
3. Infection- the process by which a parasitic organism enters into a prolonged relationship with
the host.
(55)
4. Parasitism a relationship whereby one organism derives benefits at the expense of another.

5. Commensalism a relationship wherein the parasite derives benefits from the host with out
causing injury or harm to the host.
6. Symbiosis- refers to a mutually beneficial relationship between the two species.
7. Pathogens organisms capable of producing disease
8. Vectors are arthropods or invertebrates which serve as a means by which infectious agents
are transmitted from one host to another.
Mechanical Transmission- feel a blies biological bites
9. Carriers are individuals who harbor the infectious agents without manifesting symptoms but
who serve as a means of patnogenicity of a particular microorganism
Comprised host- is one whose resistance is impaired by disease or burns
ABILITIES OF MICROORGANISMS TO CAUSE DISEASE
1. Pathogenicity the ability of the organism to produce disease
2. Invasiveness- the ability of the organism to enter host tissues, multiply and spread further.
3. Toxigenecity the ability of the organismto produce toxins (poisonous substances produced
by some organisms)
DIFFERENCES BETWEEN THE TYPES OF TOXINS

(56)

ENDOTOXINS

EXOTOXINS

1. Found only in gram- negative organisms

1. Usually produced by gram-positive organisms.

2. Part of the cell wall and produced only when 2. Produced extracellularly even though the cells are
the cells are lysed.
intact and viable.
3. Lipopolysacharide in nature.

3. Proteins in nature.

4. Not acted up by enzymes

4. Acted upon by enzymes except the exotoxin of

clostridium botulism

5. Heat stable: can withstand heat over 60 oC for

hours without loss of toxicity
Dipetheria toxins
6. Less potent and antigenic.

Erythrogenic

7. Cannot be converted to toxoids

Bolulinum toxin

8. Usually produce fever in the host by release Vibrio

of interleukin-1 and other mediators.
Staphilococal
9. Synthesis directed by chromosomal genes
10. Specific receptors not found on cells.

5. Heat- labile: toxicity often destroyed rapidly by

heating over 60 oC.
6. Can be converted to toxoids
8. Usually do not produce fever in the host
9. Frequently controlled by extrachromosal genes
10. Usuallt bind to specific receptors on cells.

TOXOIDS (ANATOXINS)\
These are non-poisonous forms of toxins which can be used for vaccination. The following
are ways of converting toxins to toxoids:
a. by aging
b. by exposure to heat
c. by exposure to 50% alcohol, formaldehyde and delute acids

(57)

EPIDEMIOLOGY : PHAGE TYPING AND SEROTYPING

In tracing the source of outbreaks for certain bacterial diseases, bacteriophage or serologic
typing have been to be useful. For example: bacteriophage typing of S. aureus amd serotyping of
Salmonelle.

The development of the diseases

1. incubating prd

2. Pordormal
3. Period of illness
4. Peiod of decline
5. Period of convalescence regain strength

Disease caused by exotoxins

1. Botulism
paircily
2. Gas gangrenes

clostridium botulin

flaccid

clostridium prefigenes

Food poisoning
3. diphtheria

C. diphteria

4. Scalded skin syndrome

Staph. aureus

Food poisoning
5. Cholesa
dehydration
6. scarlet fever

Vibrio cholera
strep. Pyogenes

7. Travelers fever
dehydration

rashes
enterogenes
(58)

E. coli
8. Tetanus
muscle

C. Titani

uncontrolabe
Contraction

Pathogenes the manner in which a dss develops

Ethology_ is the scientific study of the dss
Infection invasion and colonization of P.O in the body
Disease- infection result to change from state of health

Incidence- is the fraction of population period of time

Prevalence_ fraction of a dss of the population having the dss at the specific time

(59)
MIDTERM LECTURE
PERIOD OF INFECTION:

1. Incubation period- the time between the entry of organisms into the body and the onset of
the first sign and symptoms. An average is usually 7-10 days
Ex. Tetanus 4-21 days, rubella is 10-14 days
2. Period of illness - incubation period up to the first appearance of illness or sickness.
3. Period of convalescence extends from the time the symptoms start to abate until the person
returns to a normal state of health. healing stage

FACTORS OF INFECTION :

1. number of organisms
2. virulence
3. resistance of the host
4. portal of entry
5. toxigenicity
A. Endotoxin these are synthesize components of the cell structure that are extremely
poisonous. Ex. Salmonella typhosa , Neisseria gonorrheae and Myobacterium tuberculosis
B .exotoxins comes from toxigenic bacteria that form toxic proteins that diffuse out the from
the cell as waste product. Ex. Corynebacterium diptheriae and Staphylococcus aureus

diphtheria toxin- is an exotoxin that injuries the kidney, nervous tissue and heart
tetanus toxin- injuries certain nerves thus produced spasms of lockjaw. Enterotoxin refers to the
toxin that acts in the intestine.
C. Susceptibility to infection

BODY DEFENSE MECHANISM

1. anatomical defense
2. chemical defense
3. inflammation
4. phagocytosis
5. immune system

MANNERS OF TRANSMISSION

normal microbiota- symbiosis

1. Direct contact person to person. Touching, kissing, sexual intercourse

Parental route- punctures injection bites cute wounds surgery
2. indirect contact fomile non- living thing involved in the spread of infections
3. mother of the child transmission
4. droplet transmission travel in short distance

HOW PATHOGENS ENTERS THE BODY (Portal of entry)

1. inhalation respiratory tract

2. ingestion GIT
3. open wound- skin
4. sexual contact genitourinary system

Portal of entry

(61)

1. mucous membrane
2. skin

Centers for disease control

L- incidence of notifiable diseases morbidity
y- number of death from these disease mortality
morbidity rate- is the number of people affected by a disease in a gioven period of time in
relation to total population

Mortality rate- the number of the death resulting from a disease on a population
Etiology cause
Pathogenesis development
Epidemiology is the study of the transmission, incidence and frequency of the disease

TYPES OF INFECTION BASED ON OCCURRENCE :

1. endemic disease constantly present within a stated geographic area or locality

Ex. Common colds
2. epidemic- disease occurring in excess of normal expectancy
Disease found in a large number of individual in a community within a short of
period of time
Ex. Influenza, measles, cholera
3. sporadic- a particular disease occurs occasionally
4. pandemic- when an epidemic disease become widespread ex. Bubonic plaque
(62)
a. descriptive epidemiology- entails collecting all the data that describe the occurrence of the
disease under study
b. analytical epidemiology analyses a particular disease to determine its probable cause.
c. experimental epidemiology- begins a hypothesis about a particular disease.

ACUTE AND CHRONIC INFECTION

1. Acute infection- are those which develop rapidly and usually result a high fever and severe
sickness. Most severe stage of a disease: usually short duration or period of time.
Ex. Acute appendicitis

2. chronic infection- develops more slowly with milder but long lasting symptoms usually
undetected
Ex. Tuberculosis and leprosy
3. Sub- acute between 1 and 2 case reporting best way to establish the chain of transmission
4. Talent disease- C.A remain inactive classification of infectious disease

LOCAL AND SYSTEMATIC INFECTION

1. local infection- is one in which the causative agent is limited to one locality of the body.
Ex. Boil, diphtheria
2. systemic infection is one in which the infecting agent spreads throughout the body
Ex. Typhoid fever
1. Communicable spread from one host to another either directly
Contagious are disease that easily spread from one person to another
2. Non communicable- do not spread from one person to another
(63)
PRIMARY AND SECONDARY INFECTION

1. primary infection initial infection

2.secondary infection opportunitists who take advantage of the primary infection
3. inapparent infection- an illness in which symptoms are so mild that it goes undetected and
undiagnosed
Pyemia- presence of pyogenic bacteria
Bactremia- refers to the presence of non multiplying bacteria in the blood
Viremia- refers to the presence of non multiplying virus in the blood.
Toxemia- presence of toxins in the blood

FACTORS
THAT
MAY
MICROORGANISMS:

CONTRIBUTE

TO

THE

PATHOGENICITY

OF

1. Capsule for protection and surrounds the cell wall: prevent phygocytosis
Ex. Streptococcus pnumoniae
2. enzymes
a.hemolysins bring about the lysis of the host red blood cells
b. leukocidins- kills the host leukocytes
c. hyaluronidase- reffered to as the spreading factor various pathogenic bacteria
excretes
enzymes capable of breaking down hyaluronic acid, the intracellular material of
connective tissue.
d. streptokinase- causes the dissolution of blood clots and thus allows the spread of the
organisms.
e. coagulase- organisms producing coagulase causes the plasma to clot.
f. collagenase is formed by some clostridia that cause gas gangrene. It causes the
breakdownof collagen
3. endotoxins
4. exotoxins
5. fimbrane for attachment/adherence

-develops from production of specialized lymphocytes & abs

- is the state of being resistant to infection
-ability to overcome infection
-resistance

2 TYPES OF IMMUNITY

(64)

1. Inhale/ natural or non specific immunity- inherent in the body

a. specific immunity- an organisms that is pathogenic to one species but not in another species
that is unable to cause disease to another species.
b. racial immunity _ some race might be susceptible to a certain disease and resistant to another
but in any other race is not.
Ex. Negro race is less susceptible to TB or any other respiratory tract infection but more
resistant to skin diseases than white race
c. individual immunity- individual differences that is acquired since childbirth

2. Acquired or specific immunity _ developed subsequent to birth

a. active immunity long lasting or lifelong immunity to reinfection production of its own
immunity

aa. natural immunity- sickness

ab. Artificial immunity- vaccine
(65)
4.vaginal secretion- slightly acidic that discourage the growth of bacteria
5. transferring in the blood is an iron binding enzyme that reduced the amount of available iron
to be used by bacteria
6. urine by cleansing the urethra
7. vaginal secretions move microorganisms out of the female body.

Second line of defense

1. Phagocytosis which means eat and cell cell eating

-is the engulfment of particulate matters such as bacteria by the phagocytic cells

Phagocytic Cells
a. neutrophils
b. monocytes
c. fixed macrophages found in the tissue
Kupffers cells- liver
Alveolar macrophages lungs
Microglial cell- brain
Wandering macrophages- roam around the tissues and gather around the sites of
infection or inflammation

Mechanisms of Phagocytosis

1. chemotaxis is the chemical attraction of the phagocytes to organisms

2. adherence is the attachment of phagocyte plasma membrane to the surface of another
microorganism
(66)
Opsorization is the coating process of the microorganisms by the Phagocytocytes
3. Ingestion
4. Digestion

2. inflammation is the defensive local response of the body against tissue injury

4 signs of inflammation
1. swelling (RUBOR) Edema due to the movement of blood to tissue
2. redness ( erythema) - due to the increased blood flow to the site of injury
3. pain (dolor) due to the release of lactic acid

4. health (calor) results from the increased of temperature.

Loss of function is the fifth sign of inflammation

3 stages of inflammation

1. Vasodilation and increased penneability pf blood vessels

Chemical mediators with vasodilatation effects
a. histamine released by the damages cells and causes the dilatation of the blood vessels
b. serotonin - causes smooth muscle to contract., acts as neurotransmitter
c. leukotrienes produced by mast cells and increased permeability
Pus- a mixture of dead phagocytic cells and body fluid
Abscess- localized accumulation of pus
2.Phagocytic migration and phagocytosis
a. margination is the sticking process of the phagocytic cells on the walls of the blood
vessels
(67)
b. emigration also called diapedesis is the amosboid movement of the phagocyte from the
blood vessels to the site of the injury
c. phagocytosis
3. Tissue repair- is the final stage of inflammation
- the stroma amd parenchyma produces new cells to replaced damaged cells
Fever- an abnormal high body temperature controlled by the hypothalamus that will released
interleukin 1 in the presence of infection. Interleukin will also cause the release prostaglandins
that will reset the hypothalamus to a higher temperature an overall response of the body against
infection.
Shivering the skin remains cold though body temperature climbing high than normal body
temperature.
Chill is a definite sign that the body temperature is rising

Crisis- the skin becomes warm and the person begins to sweat, indicates that the body
temperature is falling

Increased temperature:
1. intensifies the effect of interferon, an antiviral agent
2. helps set up the production of T lymphocytes
3. speed up body reaction like tissue repair
4. decrease the amount of iron available for the microorganisms

Complication of fever:

1. tachycardiac rapid heart rate which may compromised elderly with cardiopulmonary
2. increased metabolic rate which may produced acidosis
3. dehydration
4.electrolytes imbalance
5. convulsion- seizures in young children
6. delirium
7. coma

Death results if the body temperature rises above 44C-46C

Antimicrobial substance
a. complement these are serum proteins that causes bacterial cell to lyze
b. interferon antiviral agent produced in response to viral infections
3 types of interferon

(68)

a. interferon ab. interferon B

c. interferon gamma

Third line defense

SPECIFIC IMMUNITY- ADAPTIVE OF ACQUIRED IMMUNITY

Types of acquired Immunity

Active Acquired Immunity - immune system produced the antibodies, long lasting immunity.
a. natural active acquired immunity acquired from an infection and recovery
b. artificial active acquired immunity vaccination of the attenuated microorganisms
(69)
vaccination or immunization introduces specially prepared antigens called vaccines into the
thereby stimulating the immune system to produce the specific antibodies

2. Passive acquired Immunity- antibodies is produced by another animal, short time immunity
a. natural passive acquired immunity colostrums, placental transfer of antibodies
b. artificial passive acquired immunity- antiserum
antiserum blood derived fluid containing antibodies produced by other animals or individual.

Types of Immunity

Humeral Immunity or antibody mediated immunity-involves the production of antibodies that

act against the foreign antigen or substance

B-cells are responsible for the production of antibodies

-

In the presence of antigen B cell differentiate into memory B cells and plasma cells

Plasma cells are differentiated B lymphocytes and the one in the production of antibodies

Cell Mediated Immunity- involves specialized lymphocytes called T lymphocytes that act against
the foreign organisms or tissues.
Tcells produced the cylokines these are the chemical messengers with in the immune system
Types of T cells
1. T helper cells released the cytokinase that will active the B cells to undergo
differentiation
2. Cytotoxic T cells destroys target cells in contact
3. Suppressor T cells turns off the production of antibodies if the antigen is already
eliminated
4. Memory T cells
Antigen ( immunogen) any substance that causes antibody formation and reacts only
with each specific antibodies.
Characteristics of antigen
1. Foreign to the host
2. High molecular weight
3. Lipid and nucleic acid contents
2 parts of an Antigen
1. Epitopose or antigenic determinant region of antigen that recognizes the
antibodies
2. Hapten which cannot stimulate antibody production unless attach to a carrier
molecule
Antibody (immunoglobulins) a Y shaped protein produced by the body in response to the
antigen and capable of combining specifically with that antigen.

2 Parts of an Antibody
Antigen binding site fragment binds with the antigenic determinant to crystalline fragment

Of Immunoglobulin

1. IgG Most abundant immunoglobulin about 80% of all antibodies

- Can cross the placenta
- Present in the blood
- Produced during secondary response
2. IgM 5- 10% of the circulating antibodies
- A pentamer hence the largest antibody
3. IgA 10-15% in the circulation
- present in the blood and body secretions
4. IgE- 002% and present in the mast cells and basophils
- active in allergic reaction and lysis parasitic worms
5. IgD - 0.2% function not known
Antibody protect the host by:
-

Phagocytes ingest and kill microbes

Neutralize microbial toxins
(71)
Promote bacterial clumping to facilitate clearing from infection site
Inhibits bacterial motility
Combine with microorganisms to active complement and inflammatory response

Diagnostic Immunology

a. IFA Immunoflourescent Assay

b. RIA Radioimmunoassay
c. ELISA Enzyme link immunosolvent assay
Vaccines these are preparationof killed, inactivated or attenuated microorganisms or
toxoids to induce artificially acquired active immunity
Of Vaccines
a. Live attenuated bacterial cells TB (BCG) Intradermal ID )
Typhoid oral
b. Live attenuated virus measles SQ - 12-15 mos
- Mumps
12-15 mos
- Poliomyelitis ( sabin )- oral
2,4 12 mos, 4-6 yo
- Rubella SQ
12-15mos,4-6 yo, 11-12 yo
- Chicken pox SQ
12 mos
- Influenza inhaled
all people with RTI, and healthy people
over 65

Adenovirus SQ
Vesicella Var SQ

12-15 mos, 11-12 yo

Activated whole agent vaccine

a. Formaldehyde inactivated bacterial exotoxins

- diptheria IM
2,4,6,12-15 mos
- tetanus IM
2,4,6,12-15 mos
- Pertusis IM
2,4,6,12-15 mos
- Botulism IM
B. Inactivated virus rabies IM
- influenza IM

Yearly

(72)

- Japanese encephalitis SQ
- Hepatitis A M

12 hours after birth + parents, at birth to 3 mos

Unit Vaccinea. Recombinant vaccine- hepatitis B (IM)

Conjugated vaccine b. acellular vaccine capsular polysaccharides or proteins
-

Anthrax SQ
Meningococcal SQ
Meningitis (H Influenzae)Hib IM Pneumococcal pneumonia IM or SQ
Pertusis IM

2,4,6,12-15 mos

Nucleic acid vaccine newest and the most promising vaccine but still no commercial vaccine for
human has been introduced

Adjuvant is added to enhance the effectiveness of the vaccine ex. Alum

ORDERS ASSOCIATED WITH IMMUNE SYSTEM

HYPERSENSITIVITY - Is an altered enhanced reaction leading to pathological changes

-

Also called allergy

Negative immune response

TYPES OF HYPERSENSITIVITY
a.
b.
c.
d.

Type 1 Anaphylactic or immediate hypersentisitivity

Type II Cytotoxic hepersensitivity
Type III Immune complex hypersensitivity
Type IV- Cell mediated or delayed hypersensitivity
(73)

Autoimmunity is an action of the immune system in response to self antigen and causes
damage to
ones own organs.
-

Loss of self tolerance

TYPES OF AUTOIMMUNE DISEASE

a. Type I autoimmunity involve antibody that attack self
Ex. Hepatitis c sequence similarities between the virus and self proteins
b. Type II CYTOTOXIC Autoimmune reactions
Ex. Graves disease (goiter) a disfiguring swelling of the thyroid gland and markedly
Due to the production of autoantibodies by the receptors cells attach to the thyroid
glands, thus
Myasthenia gravis- is a diseas that muscles become progressively weaker
The autoantibodies coating the acetylocholine receptors at the junction where nerve
impulses reach the muscles including the muscles of the diapraghm and rib cage.
Respiratory anest and death may result.
c. Type III Immune complex autoimmune reaction
Ex. Systemic lupus erythematosus- is asystematic autoimmune disease that mainly affects
woman. Antigen anibody complexes are deposited in the glomerulus, thereby demaging
the kidney
Rheumatoid arthritis- is a chronic inflammation due to immune complex
Of IgG, IgM and the complement deposited in the joints
d. Type IV Cell mediated autoimmune restions
Ex. Multiple scierosis a neutological disorders in which the T cells and the
macrophages attack the myelin shealth of the nerves
Hashimotos disease distruction of the thyroid glands by the T cells

Insulin Dependant Diabetes Militus immunological destruction of the insulin secreting

cells of the pancreas.
Leukocyte Antigen Antigen responsible for rejection in orga transpaltation
Compatibility typing is done to test if the HLA is compatible with the donor and the
reciepient typing is serological method of determining the HLA typing of a person
IMMUNE DEFICIENCIES the absence of sufficient immune response
2 types of immune deficiencies
1. Congenital immune deficiency defects in, or the absence of a numbered of inherited
genes
Ex. DiGeorge syndrome- do not have thymus gland therefore lack cell mediated
immunity
(74)
2. Acquired immune deficiency cause by variety of drugs, cancer and infectious agents
Ex. Hodgkins disease
- Atype of cancer that lowers cell mediated response
Deficiencies
T Helper cells
B,T cells
B,T cell, decreased Ig
B,T and stem cells
T cells
B,T cells and platelets
B cells

Antigens of hypersensitivity

Reaction
Type1 (anaphylactic)

Time before
Clinical signs
Less than 30 min

(75)

characteristics

EXAMPLES

IgE binds to mast

cells of basophils
causes degranulation
and
released
of
reactive substance like

Drug injection
Insect venom
High fever
Asthma

Type2(cytotoxic)

5-12hours

Type3(immune
complex)

308hours

Type4(delayed)

24-48 hours

histamine
Antigen
causes
formation of IgM and
IgG antibodies that
binds to target cells
when combined with
complement will lyze
target cells
Antigen
and
antibodies
formed
complexes that cause
damaging
inflammation
Antigen active T
cytotoxic cells to kill
the target cells

Transfusion reaction
Rh incompatibility
Hemolytic disease of
Newborn (HDN)

Arthus reaction
Serum sickness
Rheumatoid arthritis

Organ
rejection
contact dermatitis like
poison ivy tuberculin
test

(76)
CHAPTER VIII
GRAM POSITIVE COCCI
STAPHYLOCOCCI
GENERAL CHARACTERISTICS
1. Nonmotile, spherical, gram-positive organisms, usually arranged in irregular grapelike
clusters (may also be seen singly in pairs or in tetrands)
2. Nonencapsulated except for a few strains that produce a capsule or slim layer
3. Aerobic or facultatively anaerobic.
4. Catalase positive (this test separates staphylococcus from streptococcus
5. Often present in solid, water and skin of humans
6. Differences between stapgylococcus and micrococcus genera

Genus

Resistant to
Lysostaphin
(200 ug/ml)

modified oxidase
and benzidin

Resistant to
bactericin
(0.04 U)

Micrococcus

Staphylococcus

Staphylococcus aureus
-

The most pathogenic among the staphylococcus, produce exotoxin, grows in the
presence of a high concentration

A . Cultural Characteristics

1.
2.
3.
4.

Grow well in most routin laboratory media like nutrient agar or trypticase soy agar
For primary isolation from clinical specimens, sheep blood agar is recommended
Colonies on solid media are round, smooth, opaque and butyrous
Most strains of S. aureus produce gray to deep golden yellow colonies about 2-3mm in
decimeter entire edge
5. On blood agar a zone of beta hemolysis surrounds the colonies
(77)
*NOTE: Pigmentation and hemolysis are variable characteristics of the staphylococcus
genera
B . Antigenic structure

1. Teichoic acid s. aureus contains ribitol teichoic acid in their cell wall.
2. Peptidoglycan together with teichoic acid it protects the organisms from lysis a d
probably aids in adherence. Elecits the hemoral cellular response
3. Protein A this is a group specific antigen unique to s aureus strains; it prevents
antibody-mediated phagocytosis by PMNS
4. Clumping factor a component on the cell wall of S. aureus responsible for the clumping
of the while staphylococci in the presence of plasma
5. Capsular polysaccharide protects the organism from phagocytosis
C. Determinants of pathogenecity

1. Surface antigens
a. Polysaccharids
b. Protein receptors (e.g., fibronectin, fibrinogen, IgG
2. Extracellular enzymes
a. Coagulase the single most reliable chac of the ID of S. aureus
b. Lipase
c. Hyaluronidase- facilitates the spread of infection
d. Staphylocokinase ( Fibrinolysin)
e. Phosphatase (DIS, RDS)
f. Thermostable deoxyribonuclease
g. Gelatinase
h. Protease
3. Toxins

A. Cytolytic toxins
i. Alpha toxins (alpha-hemolysin) lethal and dermonecrotic
ii. beta toxin (staphylococcal sphingomyelinase) lethal and dermonecrotic ; produces
hot cold
(78)
iii. delta toxin
iv. gamma toxin
v. leukocidin ( Panton- valentine) cause cell lysis

B. Enterotoxins exotoxins implicated in food poisoning enterotoxin A.) and antibiotic

induced membranous colitis
C. Toxic shock syndrome toxin 1 (TSS 1)- for disinfection
D. Exfoliative toxins cause staphylococcal scalded skin syndrome
Pyogenic accumulation of neutrophil, bacterial cells in infection site
D . CLINICAL INFECTION
1. Cutanous infections
a. Folliculitis _ infection of the hair follicle pimples
b. Furuncle or boil a focal suppurative lesion which has sulted from an infection
(folliculitis that has extended into the subcutaneous tissue
c. Carbuncle similar to furuncle but has multiple foci on extends into the deep layers
of fibrous tissue
d. Impetigo characterized by the formation of the encrusted pus when crust are
removed, a red weeping denudes surface is exposed ; common in newborns and
young children
e. Scalded skin syndrome comprised of the following entity
I . generalize exfoliative dermatitis (Ritters disease)
ii . bullous impetigo
iii . staphylococcal scarlet fever
2. Pneumonia
3. Osteomyelitis
4. Pyoarthrosis
5. Bacteremia and endocarditis
6. Metastatic absecesses
7. Food poisoning characterized by the occurrence of vomiting diarrhea between 1 and 6
hours following ingestion of food with enterotoxins
Catalase breakdown H2O2 to water and O2 + bubbles

8. Toxic shock syndrome a multisystematic disease which primarily occurs in young

menstruating whos age is 12-52 yo
E. Laboratory Diagnosis
Specimens: pus, purulent fluids, sputum urine, and blood

(79)

1. Gram stained smears- typical gram-positive cocci in irregular clusters

2. Culture media Columbia colistin nalidixic acid (CAN)
3. Coagulase test - most useful single criterion for the recognition of S. aureus
Principle : based on the presence of coagulase , which binds plasma fibrinogen,
causing the organisms to agglutinate or plasma to clot
Media used : fresh human plasma or rabbit plasma with EDTA OR citrate
Methods :
A. Slide method a rapid screening test
Demonstrates the presence of cell bound coagulase (clumping
factor)
Procedure:
I.
Emulsify a loopful of organisms to a drop of coagulase plasma on a slide
II.
Observe for a clumping within 30 seconds
B. Test tube method use to confirm all slide test negative results on
clinically significant isolates, demonstrates the presence of extracellular
coagulase (free coagulase)
Procedure :
I.
To a 0.5 ml of diluted plasma, emulsify growth from isolated colonies and
incubate for 1-4 hours at 35-37oC
II.
Observe for the presence of a gel or clot
III.
If no clot forms after 4 hours, reincubate at room temperature overnight.
*NOTE: Results should be read within 4 hours to prevent a false (-) reaction
because most strains of S. aureus will produce a clot within this time and
some strains produce a fibrinolysin which lyses the clot formed within 4 hours
Coagulase plasma with citrate is not suitable for this test because organisms
such as pseudomonas and enterococci use the citrate and release calcium,
thus, forming a clot in the absence of coagulase (false (+) result)
4. Growth on tellurite lysine agar
a. S. aureus can reduce tellurite producing jet- black colonies
b. Other staphylococci inhibited if growth occurs gray colonies are
seen
5. Mannitol fermentation test
a. S. aureus can ferment manitol and can tolerate high salt concentration (7.5
10% NaCL)
b. Medium used: mannitol salt agar (MSA)
c. Ph indicators: phenol red
d. (+) result : colonies surrounded by a yellow halo

6. Lysotaphin sensitivity test

a. S. aureus is sensitive to lysostaphin while the micrococci are resistant
b. Procedure and result:
I . A 1:10 dilution of culture is exposed to 2 ug/ml of lysostaphin at 37 oC for 30
minutes
ii. (+) result : reduction of 90% or more in the number of organisms
7. Polymixin B sensitivity test
a. S. aureus resistant in bacitracin
b. Other staphylococci susceptible
8. Thermostable deoxyribonuclease test (+) in S. aureus
9. Particle agglutination test
a. Staphylatex
b. Staphylochrome
c. Sero-STAT
d. Bacto staph latex
e. Staphaurex
f. Accu-staph use latex particles
g. Hemostaph
h. Staphyloslide use sintesized sheep erythrocyte
10. Bacteriophage typing for epidemiologic purposes
F. Treatment
1. Cutaneous infections oral cloxacillin or dicloxacillin; if allergic,
erythromycin may be substituted
2. Systematic staphylococcal disease parenteral nafcillin or oxacillin; if allergic
vancomycin or cephalosphorins may be used
3. Methicillin resistant staphylococci - vancomycin alone or in combination
with rifampicin
Coagulase negative staphylococcus
Staphylococcus epidermidis
-

Glycerol techoic acid , bacterial flora coagulase negative

1. Resistant flora of the human skin and mucus membranes
2. Microscopic morphology is very similar to s. aureus
3. Colonies are circular, smooth and usually pale translucent white
4. With relatively low virulence and more likely to cause infection in
compromised host.
(81)
5. Has a distinct predilection for foreign bodies (e. g . , artificial heart valves
indwelling intravascular catheters, CNS shunt hip prostheses
6. Can cause stitch abscess, UTI especially in elderly hospitalized men,
endocarditis and bacteremia
7. Coagulase(-) and mannitol fermentation (-)

8. Produce extracellular slime matrix that enhance bacteria adhesions in foreign

bodies

Staphylococcus Saprophyticus
1. Present on the normal skin and in theperiurethral and urethral flora.
2. Common cause of the urinary tract infections in young sexually active women.
3. Closely resembles S. Epidermidis.
4. Distinguished from S. Epidermidis by virtue of its resistance to novo-biocin and nalidixicacid
and absence of phosphatase production.
5. Coagulase (-) and mannitol fermentation (+).
6. Common cause of UTI
7. Second most common cause of cystitis after E. coli
8. Only Staphylococcus resistant to novobiocin.

Streptococci

General characteristics
1. Spherical to avoid, catalase (-) , gram (+) organisms that tend to occurs in pairs or chains
when grown in liquid media.
2. Nonsporeformer and generallynonmotileexcept
streptococci.

for rare motile strains of group D

3. Generally nonencapsulatedexcept for some strains of groups A, B , C , & D.Facultatively

anaerobic but some strains required added CO2 for their initial isolation ( microaerophilic
strains viridans streptococci )
(82)
4. Colonies on agar surface are translucent to milky, circular, and small or pinpoint with a shiny
surface.
5. Vancomycin sensitive
Classification of streptococci

There have been taxonomic changes recently. Streptococci designated as S. Milleri, S .

Intermedius , S.Constellatus and minute or small colony forming beta-hemolytic streptococci
of Lancefield groups A , C,F and D are now designated as S. Anginosus. The enterococcal
group D. Streptococci , including S. Faecalis , S. Fecium , and S. Durans now belong to the
aenus Enterococcus.

A. Academic or Bergeys Classification

( Based on physiologic properties )

Pyogenic group
-grows at neither 10C nor at 45CExample: S. Pyogenes

Viridans group
-grow at 45C butnot at 10C
Example: S. mutan( cause of dental plague )S. sanguis , S. mitis , S. salivarius and S.
anginosus
- resists classification by Lancefield precipitation test.

Enterococcus
grows at both 10C and 45C ; can stands temperature above 60C.
( Enterococcus0faecalis ( part of the normal faecal flora )

Example:

S.
(83)

Lactic group
grow at 10C but not at 45C
Example: S. Lactis

B. Smith and Brown`s classification

( Based on hemolytic reactions on BAP )

1. Beta-hemolytic Streptococci Produce a clear colorless zone around the colony indicating a
complete hemolysis of the red blood cell (e.g. , S. pyogenes, S. aga-Lactiae
2. Alpha-hemolytic Streptococci Produce a zone of partial hemo-lysis with a greenish or
brownish discoloration (e.g. ,viridans species, S. pneumoniae)
Gamma-hemolytic or non-hemolytic or indifferent streptococci-colonies do not produce any
hemolysis (e.g. , S. faecalis)

*NOTE:Hemolysis is enhanced by stabbing the inoculating loop

into the agar several times.
: Plates are always examined for hemolysis by holding them
infrent of a light source.

C.Lancefield Classification
(Based on the antigenic characteristics of group-specific C substance, which is a cell-wall
polysaccharide.
1. Group A Streptococci: Streptococcus pyogenes.

A.Diseases Produced
Sore throat, pharyngitis, tonsillitis, skin infection- pyoderma, impetigo, cellulitis and scarlet
fever.
(84)

B.Cultural characteristics
Colonies are transparent to translucent, convex or domed entire, c

ircular, shiny and surrounded by a wide zone of beta-hemolysis.

C. Cellular components and extracellular products

Lipotechoic acid
present in the cell wall; responsible for the adherence of the bacteria to respiratory epithelial
cells.
Protein
major virulence factor of group A streptococci which renders the orgarnisms resistant to
phagocytosis.
Hyaluronic acid capsule
assists the organisms in avoiding phagocytosis.
Hemalysins
Streptolysin O (SLO): Oxygen labile,
antigenic toxin
: involve in ASO titer test
: responsible for subsuraface
hemolysis.
Streptolysin S (SLS) : Oxygen
stable, non antigenic.
: responsible for surface hemolysis
Erythrogenic (pyrogenic) toxins-responsible for the characteristics rash seen in scarlet fever
Hyaluronidase
spreading factor
Streptokinase
enzyme that dissolve cloths.

(85)

Streptodornese or DNase, NADase, proteinases and other enzymes.

Identification

1. Bacitracin disk test (taxo A)

A presumptive test which differentiates group A beta haemolytic streptococci (positive)
from other beta hemolytic streptococci (negative)
Based on the inhibition of the growth of group A streptococci by a power disc container
0.02-0.04 units of bacitracin
Any zone of inhibition regardless of the diameter is a (+) reaction.

2. PYR test
Based on the presence of the L pyroglutamylaminopeptidase enzyme which hydrolyzes
an amide substrate to form the free beta-naphthylamire, which when reacted with a
cinnamaldehyde reagent (PYR reagent) forms a brightened end product (+)
Aside from S. pyogenes, the enterococcus species also posses the aminopeptodase
enzyme enzyme.

3. Confirmatory Test
Lancefield Precipitin Test
Direct FA Test
Coagglutination (Phadebact)

Group B Streptococci: Streptococcus agalactiae

Cultural characteristics

(86)

Colonies are large, mucoid, more translucent to opaque, whitish gray, soft , smooth
colonies surrounded by a smaller zone of beta-homolysis

Identification

i. Sodium hippurate hydrolysis positive (purplecolor)

II. CAMP (Christie, Atkins, and Much-Peterson) test
Presumptive test for group B streptococci
The CAMP factor is produce by group B beta-hemolytic streptococcus which has the
ability to act synergistically with the beta hemolysisin produce by S. aureus to produce even
more potent hmolysis.

(+) result is an arrowhead-shaped zone of enhanced hemolysis at the juncture between the
(+) streptococci and the staphylococcus.

S. aureus streak

(+) arrowhead-shapedhemolysis

Group strep streak

Group C . Streptococci

(87)

All species of group C (S.equi, S. equisimilis , S. zoo epidermicus ) are B-hemolytic

except for S. dysgalactiae, which may be alpha-hemolytic or nonhemolytic. They can be
differentiated by carbohydrate fermentation.

Group D Streptococci
Traditionally divided into two groups, the enterococcal species (E. faecalis , E. faecium,
E. durans) and the nonenterococci (S. quinus& S. bovis). They both grow in the presence of 40%
bile and hydrolyzeesculin. The enterococci are differentiated from the nonenterrococci by their
ability to grow in the presence of 6.5% sodium chloride.

Veridans Streptococcus sub acute endocarditis (Bacterial)

Non-enterococcal Enterococcus 40% NaCl, 6.5%

Group G Streptococci
This group does not have species designations. Colonies are large with a wide zone of
beta-hemolysis resembling S. pyogenes.

DISEASES PRODUCE AND TREATMENT

Group A Beta-Hemolytic Streptococci- S. pyogenes

Mode of Transmission

Primarily by aerosols and respiratory secretions (droplets)

Food and milk-borne in cases of epidemics.

Diseases produced (predominantly human pathogens)

Most common cause of acute pharyngitis

(88)

Impetigo
Cellulitis
Erysipelas
Wound infections
Puerperal sepsis
Scarlet fever

i. Dick`s test- susceptibility test for scarlet fever

TEST Arm

Control Arm

With 0.1 ml Dick`s toxin

with 0.1 ml Dick`s toxoid

Read reaction after 24 hours

(+)

(-)

(+) reaction: area of irritation or redness

Interpretation: susceptible to scarlet fever.

ii. Schultz-charlton Reaction (Blanching Phenomenon)

Based on the neutralization of erythrogenic toxins when an anti toxin is injected into the skin of a
patient with scarlet fever skin rasher fade or blanch (+)
Used to diagnose whether the skin rashes are due to scarlet fever or not.

Sequelae of Acute Streptococcal Infection

Acute Rheumatic Fever (ARF)- occurs only after pharyngitis
Acute Glormerulonephritis (AGN)- seen after pharyngeal or cutaneous infection

(89)

Treatment
Intramuscular benzathine penicillin as single dose
Oral penicillin V for 10 days
For penicillin-allergic patients- erythromycin, clindamycin and cephalexin.

Differentiation of Gram (+) Cocci

BAP
Staphylococcus
Pneumococcus

Streptococcus

Colonies
Large, round, white,
yellow/gold.
Small, round, surface
colonies,
lancetshaped in polar plate
Small, round surface
colonies, round in
pour plate

Hemolysis
Beta type

Morphology
Clusters

Alpha type

In pair or single lancet

shape

Alpha, Beta and Delta

In pairs, single short

chain, round

Group B Beta-Hemolytic Streptococci- S. agalactaie

Normal flora: of the pharynx, gastrointestinal andogenitourinary tracts and vagina

Diseases Produce
Important etiologic agent of bovine mastitis
Skin infection
Endocarditis
Puerperal infection
Most common etiologic agent of neonatal sepsis and meningitis

Urinary tract infection

Osteomyelitis etc.

Treatment
Penicillin G- antibiotic of choice
Erythromycin ekloramphenicol, cephalosporins, vancomycin, Imipenem and clindamycin

Group C Streptococci

Disease Produced
The members of this group are primarily animal pathogens but they may infect humans as well:

A. S. equi
Causes disease in horses
S. equisimilis
May cause pharyngitis, puerperal sepsis, endocartisis, bacteremia, osteomyelitis, brain
abscess, post-operative wound infection and pneumonia in humans.
Source of streptokinase used in thrombolytic therapy in humans.

Treatment
Similar to group A streptococci

Group D Streptococci (including Enterococci)

(91)

Normal flora
Skin
Upper respiratory
Gastrointestinal
Genitourinary tracts.

Diseases Produced
Frequent cause of bacterial endocarditis in the elderly
Urinary and biliary tract infections
Septicemia
Wound infection and intra-abdominal abscesses

Treatment
Enterococcal strains
Generally resistant to penicillin G, ampicillin and penicillinase-resistant penicillins
Penicillin in combination with an aminoglycoside (gentamicin or streptomycin)
For penicillin allergic patients- vancomycin and erythromycin
Nonenterococcal strains- penicillin G.
(92)
Thioglycollate
Staphylococcus
Pneumococcus
Streptococcus
Viridans Streptococci

Normal flora

Growth
Abundant
Inhibited
Abundant

Optochin Test

Bile Solubility

(+) Sensitive
(-) Resistant

(+) Soluble
(-) Not Soluble

Nasopharynx
Mouth
Gingival crevices, gastrointestinal tract and female genital tract. They are opportunistic
pathogens thought to be of low virulence.

Diseases Produced
Major etiologic agent of bacterial endocarditis especially S. sanguis
Major etiologic agent of dental caries (plague)- S. mutans
Cellulitis and wound infection
Meningitis
Sinusitis
Biliary or intra-abdominal infections

Treatment
Aqueous penicillin G or with the additional of gentamicin
For penicillin allergic patients- vancomycin
(93)
LABORATORY DIAGNOSIS
Specimens: threat swabs, vesicular or pustular fluids, skin lessions, aspirated tissue fluids, blood,
etc.

A. Stained smears
B. Culture
Sheep blood agar is preferred for primarily isolation because it is inhibitory to the
growth of Haemophilushaemolyticus( a normal throat commensal whose colonies may be
mistaken for beta haemolytic streptococci).

Other culture media used are chocolate agar, Columbia agar with colistin and nalidixic
acid (CAN), phenylethyl alcohol agar (PEA) and Todd-hewitt broth.

C. Bacitracin disk test, PYR test, hippurate hydrolysis, CAMP test, bile-esculin test, growth in
6.5% NaCI

Serological Tests
a. Lance field precipitin test
confirmation test wherein the cell wall antigens are extracted either physically be heating
or chemical or enzymatic extraction of a cell suspension grown overnight in Todd-hewitt broth
b. Direct flurescent antibody test
c. Coagglutination (Phadebact)
d. Enzyme-linked immunosorbent assay (ELLISA)
e. Latex agglutination test
f. ASO titer for group A streptococcal infection

STREPTOCOCCUS PNEUMONIAE (Diplococcuspneumaniae/pneumococcus)

(94)

Morphology and Phsiology

1. Encapsulated gram-positive coccus, characteristically lancet-shaped which occurs singly , in
pairs and short chains.
2. Facultative anaerobe requiring and increased CO2 tension (using a candle jar).
3. Has an absolute rutritional requirement for choline.

Cultural Characteristics

1. On blood agar plates young colonies are circular , glistening, dome-shaped while some are
larger and more mucoid. As the colonies becomeolder, autolytic changes result in a collapse of
the center of the colony (with raised margins and depressed centers), giving it an umbilicate or
doughnut appearance ( checker or nailhead colonies).
2. Colonies incubated aerobically produce a zone of alpha hemolysis. Colonies incubated
anaerobically produce a zone of beta hemolysiswhich is due to the oxygen-labile pneumolysin 0.

Determinants of Pathogenicity
1. Polysaccharide capsule
2. Adherence
3. Enzymes
Neuramidase- enzyme that degrades surfaces surface structures of host tissue.
Proteases-enzymes that facilitate bacterial colonization on mucosal surfaces by
eliminating immunoglobulins such as IgA, IgG and IgM.
4. Pneumolysin 0- an oxygen-sensitive toxin that is cytolytic for cells.
5. Autolysin facilitates the release of pneumolysin 0 and other toxic proteins or inflammatory
substances from cells.
6. C-subtance- a component of the cell wall of pneumococci which is a teichoic acid that reacts
with C-reactive proteins (CRP) resulting in the activation of some nonspecific host immune
responses.
(95)

Diseases Produced
Most common cause of bacterial pneumonia (lobar)
Second most common cause o bacterial meningitisis
Otitis media, purulent sinusitis and occasionally peritonitis

Laboratory Diagnosis
Specimens: Sputum, swabs, pus and blood

1. Gram- stained smears

2. Culture
3. Optochinsansativity Test
Most widely used presumptive test for differentiating pneunomococci from other alphahemolytic streptococci (e.g. , the viridans streptococci)
The optochin disk or Taxo P (ethylhydrocupreine hydrochloride) is placed on a blood
agar plate heavily inoculated with suspected organisms and incubated overnight:
(+) reaction is a 14-16 mm zone of inhibition depending on the size of optochin disk (6 &
10 mm )

Bile solubility test

Based on the presence in pneumococci or an autholyticamidase which when activated by
bile results in the analysis of the organisms.
Differentiation of B. streptococci
Disc (Bacitracin)
(+) Sensitive
(-) Resistant
(96)

Facultative Anaerobic Streptococcus

Streptococcus
Aerococcus
Leuconostoc
Pediococcus
Gemella
Enterococcus
Lactococcus

Strictly Anaerobic Streptococcus

Peptococcus
Peptostreptococcus
Ruminococcus
Coprococcus
Sarcoma
Cultural Characteristics
1. On blood agar plated young colonies are circular, glistening, dome- shaped while some
are larger and more mucoid. As the colonies become older, autolytic changes result in a
collapse of the center of the colony (with raised margins and depressed centers), giving it
an umbilicate or doughnut appearance (checker or nail head colonies).
2. Colonies incubated aerobically produce a zone of alpha hemolysis.
Colonies incubated anaerobically produce a zone of beta hemolysis which is due to the
oxygen-labile pneumolysin O.
C. Determinants of Pathogenicity
1.
2.
3.
a.
b.

Polysaccharide capsule
Adherence
Enzymes
neuraminidase enzymes that degrades surface structures of host tissue
proteases enzymes that facilitates bacterial colonization on mucosal surface by
eliminating immunoglobins such as IgA, IgG and IgM.
4. Pneumolysin O an oxygen-sensitive toxin that is cytolytic for cells
5. Autolysin facilitates the release of pneumolysin O and other toxic proteins or
inflammatory substances from cells
6. C-substances a component of the cell wall of pneumococci which is a teichoic acid that
reacts with C-reactive protein (CRP) resulting in the activation of some nonspecific host
immune responses
D. Diseases Produced
1. Most common cause of bacterial pneumonia (lobar) (rusty sputum)
2. Second most common cause of bacterial meningitis
3. Otitis media, purulent sinusitis and occasionally peritonitis, bacteremia
E. Laboratory Diagnosis
Specimens: sputum, swabs, pus and blood

(97)

1. Gram-stained smears
2. Culture
3. Optochin sensitivity test Taxo P

4.

5.
a.
b.
c.
6.
a.
b.
c.
7.

a. Most widely used presumptive test for differentiating pneumococci from other alphahemolytic streptococci (e.g. , the viridans streptococci)
b. The optochin disk or Taxo P (ethylhydrocupreine hydrochloride) is placed on a blood
agar plate heavily inoculated with suspected organisms and incubated overnight
c. (+) reaction is a 14-16 mm zone of inhibition depending on the size of optochin disk
(6&10 mm)
Bile solubility test
- Based on the presence in pneumococci of an autolytic amidase which when activated
by bile results in the lysis of the organisms.
Mouse virulence test
The mouse is particularly sensitive to even a small inoculum of pneumococci
Sputum containing pneumococci is injected intraperitoneally to a mouse which
eventually dies within 16-48 hours
The heart blood of the dead mouse harbors a pure culture of pneumococci
Neufeld/ quelling or Capsular precipitation reaction
The most useful, specific and rapid method for the identification of S. pneumoniae
Test is performed by mixing on a slide of a loopful of emulsified sputum with a loopful
of antipneumococcal serum and methylene blue
(+) reaction capsule appears swollen due to a change in refractive index which in turn is
due to a serological reaction
Other serologic test like pneumoslide

F. Treatment
1. Penicillin drug of choice
2. Cephalosporins or erythromycin (for pneumonia) and chloramphenicol ( for meningitis)
for patients allergic to penicillin
Table 8-1 Tests Differentiating Pneumococci and Streptococci
TESTS
Bile Solubility Test
Inulin Fermentation Test
Capsular Swelling Test
Quinidine Test
Optochin Test
Mouse Virulence Test

PNEUMOCOCCI
Bile soluble
Fermenter
Swelling of capsule
Susceptible
Susceptible
Mouse dies within
hours

STREPTOCOCCI
insoluble
Non- fermenter
No swelling
Resistant
Resistant
16-48 Wont die

Francis Test skin test for determining the presence of antibodies against pneumococci

ANAEROBIC GRAM-POSITIVE COCCI

(98)

This group includes Peptococcus, Streptococcus (S. intermedius, S. constellatus).

Gemellamorbillorum and Peptosstreptococcus. They generally occur in singles, pairs, tetrads,
chains and irregular clumps. Many of these have similar colony appearance that are minute to
small, convex, gray to white and translucent to opaque with entire edges and strippled or pocked-

marked surface. These species are normal flora of the mouth, urogenital tract and gastrointestinal
tract but may cause pleuropulmonary disease, brain abscess and gynecologic infections.
a) Peptococcus commonly known as the anaerobic staphylococcus, catalase + and in
clusters
b) Peptostreptococcus commonly known as the anaerobic streptococcus
c) Gaffykatetragena arranged in packets of 4 or tetrads
d) Sarcinalutea occurs in cubical packets of 8s, 16s, 32s or more. Also known as the
saphrophyticinterococci. Produce a bright yellow pigment. Contaminants in the lab.
(99)

CHAPTER IX
GRAM NEGATIVE COCCI
NEISSERRIA
There are two species of this genus that are major of medical importance; they are N.
meningitidis (causative agent of meningococcal meningitis) and N. gonorrhea (causative agent of
gonorrhea). Humans are the only known reservoir of these species.
General Characteristics
1. Gram (-) kidney or coffee bean shaped with adjacent sides flattened.
2. Most are encapsulated; nonmotile and nonsporeformer.
3. Aerobic or facultatively anaerobic organisns requiring an increased CO2 tension for
growth (3-10% CO2).
4. Highly susceptible to drying, chilling and exposure to unfavorable pH or to sunlight.
5. Most species can grow on blood agar except for N. gonorrhea which requires the added
nutrients of chocolate agar or special enrichments.
6. Catalase and oxidase (+)
Cultural Characteristics
1. Fastidious organisms requiring complex nutritional growth and requirements.
2. They appear as translucent, grayish, convex, shiny colonies with entire margins that are
nonhemolytic and nonpigmented.
Culture Media
1. For normally sterile specimens (blood, CSF, synovial fluid, etc.)
- Blood agar and chocolate agar
2. For contaminated specimens (discharges and swabs)
a. Thayer-Martin medium (Specific, Selective)
- Enriched chocolate agar with supplement B or Isovitale X and antibiotics:
I.
Vancomycin inhibits gram (+) organisms
II.
Colistin inhibits gram (-) rods
III.
Nystatin inhibits yeast (fungi)
b. Modified Thayer-Martin medium
- With the addition of hemoglobin solution and trimethoprim lactate which inhibits the
spreading of proteus
c. Transgrow medium
- A Thayer-Martin medium with glucose, 2% agar, trimethoprim lactate and CO2
incorporated in bottle
d. Martin-Lewis medium
- A modified Thayer-Martin medium with the substitution of anisomycin, an antifungal
agent with a longer half-life for nystatin
e. New York City medium
- Also a modified Thayer-Martin medium with the substitution of amphotericin B for
nystatin.

Neisseria gonorrhea
Gonorrhea is the most common sexually transmitted disease (STD) seen in adults, children and
infants.
Clinical Manifestation

1. In men, it usually presents as acute urethritis with dysuria and purulent urethral discharge,
although up to 25% have only minimal discharge and some have no symptoms.
2. In women, between 20% to 80% are asymptomatic, though there may be increased
vaginal discharge, dysuria menstrual abnormalities, and lower abdominal pain and
physical examination may show a friable erythrematous cervix with mucopurulent
discharge.
3. Pharyngeal gonorrhea occurs in both sexes and is usually asymptomatic.
4. Gonococcal arthritis-dermatitis syndrome is the most common manifestation of
disseminated gonococcal disease which is heralded by fever, chills, malaise, intermittent
bacteremia, polyarticular arthritis or tenosynovitis and development of typical skin
lesions.
5. Gonorrhea may be seen in sexually abused children but in infants it usually results from
contamination during passage through an infected birth canal. The infant develops
gonococcal opthalmianeonatrum, an eye infection which cause blindness. This is
prevented by Credes prophylaxis (1-2 drops of 1% silver nitrate), erythromycin or
penicillin drops instilled in the eyes of all newborn babies. Children may also acquire the
disease from fromites.
Laboratory Diagnosis
Specimens: discharges from urethra, cervix and anal canal; specimens from the oropharynx,
skin lesions, inflamed joints, prostate and blood; swabs of eye discharge
1. Stained smear
- Typical gram (-) diplococci, intracellular (within PMNs) and/or extracellular (outside
PMNs); intracellular existence is indicative of active infection
2. Culture
Four colony types:
a. T1 and T2 produced on 1 culture; small and dome-shaped colonies; piliated thus
virulent (pili or fimbriae principal virulence factor)
b. T3 and T4 produced on subsequent cultures; larger and flatter colonies; piliated thus
avirulent
3. Presumptive Identification Test
4. Confirmatory Identification Test
a. Sugar degradation
i.
CTA (cystinetrypticase agar)
ii.
Gonobio Test a rapid 4 hour micro method
iii.
Gonochek II
*N. gonorrheae ferment only glucose producing acid without gas
b. Direct fluorescent antibody technique
5. Antibody-based particle agglutination
a. GonoGen
b. Phadebact
c. Meritec GC
6. ELISA (Gonozyme) for direct detection of gonococcus in specimens
7. Limulus test rapid screening test for N. gonorrhea
8. Superexol test (+) production of gas bubbles from 30% H2O2
Treatment
1. Amoxicillin, ampicillin and aqueous procaine penicillin G
(101)
2. Ceftrimoxazole most commonly recommended for uncomplicated cases due to
increasing incidence of penicillinase-producing N. gonorrhea (PPNG) or chromosomally
mediated resistant N. gonorrhea (CMRNG)
3. Other alternatives are spectionomycin, trimethoprim-sulfamethoxazole, ciprofloxacin and
cefuroxime

Neisseria meningitidis
Disease Produced: Epidemic meningococcal meningitis/ cerebrospinal fever/ spotted fever (due
to the presence of petechial lesions)
Clinical Manifestation:
1. Meningococci enter the body thru the upper respiratory tract including the conjunctiva
and establish on the mucous membrane of the nasopharynx. Then dissemination occurs
via the bloodstream
2. Signs and symptoms include mild fever, meningitis, pharyngitis, prostration,
erythematous macular rash usually superseded by a petechial eruption. This vasculitic
purpura is the hallmark of meningococcal disease.
3. Hemorrhage into the adrenal tissue with resultant hypoadrenergic state is known as the
Waterhouse-Friedrichsen syndrome ( a highly fatal fulminating meningococcemia).
4. Sequelae include eight nerve deafness, CNS damage, skin or tissue necrosis due to
vascular thrombosis.
Laboratory Diagnosis:
Specimens: blood, CSF, synovial fluid, pleural fluid, aspirates of petechiae, and nasopharyngeal
or throat swabs (for carrier)
1.
2.
3.
4.
5.
a.
b.
6.

Stained smears
Culture
Presumptive oxidase test
Confirmatory test: CHO fermentation test (CTA)
- N. meningitidis ferments glucose and maltose
Rapid detection of capsular polysaccharide antigen in urine, serum, CSF and synovial
fluid by:
Countercurrent immunoelectrophoresis (CIE)
Particle agglutination techniques such as latex and coagulatination
Limulus test for lipopolysaccharide endotoxin

Treatment
1. Penicillin drug of choice
2. Chloramphenicol and ceftriaxone for penicillin sensitive patients
Prophylaxis for carriers
1. Rifampicin and minocycline primarily
2. Ciprofloxacin

Neisseria cinerea
A pathogen associated with bacteremia, conjunctivitis, nosocomial pneumonia and proctitis. It
has a biochemical resemblance to N. gonorrhea by its ability to grow on Mueller-Hinton agar (N.
gonorrhea unable to grow on MH) and inability to grow on Thayer-Martin medium.
(102)
Branhamella (subgenus of Moraxella)
Branhamellacatarrhalis
-

Morphologically and biochemically resembles the Neisseria species

Separated from Neisseria due to to differences in DNA base composition
Normal flora of the upper respiratory tract but has been isolated in cases of
septicemia, laryngitis, pneumonia and bronchitis
Does not ferment any sugar

Veilonella (V. parvula)

-

Tiny, strictly anaerobic gram (-) cocci that occur in masses or as diplococci which are part
of the normal flora of the mouth.
Produce small, convex, translucent to transparent colonies with entire edge which may
show red fluorescence under long-wave ultraviolet light
Fatty acid end products are propionic and acetic acids
Other anaerobic gram-negative cocci commonly found in human clinical
infections are Megasphaera and Acidaminococcus.

(103)
CHAPTER X
AEROBIC, NONSPOREFORMING GRAM POSITIVE BACILLI
MYCOBACTERIA
General Characteristics
1. Gram positive, acid fast, strictly aerobic, nonmotile, nonencapsulated, slender, slightly
curved or straight rods

2. Often have a beaded appearance due to the presence of much granules which are non-acid
fast, gram (+) granules.
3. May have palisades or X, Y, V & L (snapping) formation.
4. Stain with difficulty, but once stained, they are resistant to decolorization.
A. Mycobacterium tuberculosis
1. Morphology same as above
2. Cultural characteristics
a. able to grow on simple synthetic medium but for primary isolation, a more
complex medium containing either an egg-pota
b. to base or serum-agar based is required
c. the very slow growth of the organisms requires an increased CO2 tension (5-10%
CO2) at an optimal temperature of 37c; it takes 10-20 days before growth can be
visualized
d. growth first appear as small (1-3mm), dry, friable colonies that are rough, warty,
granular and buff-colored which then become typical colonies having a flat
irregular margin and a cauliflower center after several weeks
e. the growth is luxuriant enhanced by glycerol thus termed eugenic; other
mycobacteria are dysgonic (with smooth scanty growth)
3. Viability and Pathogenicity
a. Tubercle bacilli can resist adverse environmental conditions such as drying, heat
and chemical agents; this is due to the lipids present in the cell wall which are
responsible for the hydrophobic nature of the organism
b. In the body, the organisms are protected due to their intracellular existence (since
they are phagocytosed by alveolar macrophages)
c. Does not produce any toxin but have the following:
i. Cord factor (trehalose-6, 6-dimycolate) glycolipid present in virulent
strains of M. tuberculosis responsible for the formation of tight serpentine
cords in smears and in cord media
ii. Sulfatides glycolipids responsible for the neutral red reacting associated
with virulent strains of M. tuberculosis

Laboratory Diagnosis

Specimens: sputum (series of 3-5 single early morning samples),

urine, CSF, gastric lavage material, synovial fluid,
pleural fluid, pus, tissue biopsy specimens.

Decontamination and concentration techniques

i. N-acetyl-L-cysteine (mucolytic agent) with IN (4%) NaOH
(decontaminant) recommended technique
ii. 4% NaOH traditional decontamination & conc. Soln.
iii. 4% H2SO4
iv. zephiran ( benzalkonium chloride ) trisodiumphospate
v. 6% oxalic acid
vi. sputolysin (dithiothreitol) oxalic acid

(104)

vii. 20% chlorox

viii. 1% cetyl-pyridium chloride + 2% NaCl
ix.

sputolysin

Used for
specimen
cholorox

For complete liquefaction of mucous in the specimen

Procedure:

a.

effective inhibition
with
low
toxicity
can
also

2%

specimen

of
to

NaOH

contaminants presents in the

acid
fast
bacilli
(however
kill
m.
tuberculosis)

digestant
mix and stand at room
temperature
for several minutes centrifuge
decant sediments for smears and cultures

stained smears with: (see p.7)

i. Ziehl-Neelsen
- scan at least 100 fields before reporting smear as
negative
or

sputum
positive

specimen
for

are
reported
as
acid
fast

acid
fast
bacilli,

bacilli
seen
semiquantitation
recommended:

* 1-2/smear report number seen and suggest new specimen

* 3-9/slide rare (+)
* 10 or more/slide few (++)
* 1 or more/oil immersion field numerous (+++)
ii. kinyoun

iii. Auramine-rhodamineflourochrome stain(truant)

- advantages: ease, speed (examination on LPO, better
Contrast, minimal eye strain.
-

decolorizer
permanganate

(105)
-

acid

fluorescent yellow organism on black background

acid fast stain may be done on same smear after

Fluorescent staining to confirm all positive results

b. culture and culture media

Agar-based

alcohol;

counter-stain

potassium

i.

Dubos oleic acid albumin

ii.

Middlebrook 7H10} good for antimicrobial susceptibi iii.Middlebrook 7H11} lity test but 7H10 is much preferred
iv. Mitchisons selective 7H11
Egg-based
i.Petragnani more inhibitory, reserved
ii. Lowenstein-Jansen good for niacin test
iii. Dorset egg medium
iv. American Thoracic Society (ATS) medium less inhibitory
- all of the above contains eggs, potato flour, glycerol
with malachite green to inhibit the growth of contaminants.
Liquid
i.Bactec 12B medium
ii.Middlebrook 7H9 Broth for aseptically collected specimens from normaly sterile sites
Note:

Cultures are examined weekly for growth and reported and discarded as negative (no growth) after
8 weeks of incubation.

c. virulence tests for M. tuberculosis

i.

serpentine cord formation

- virulent strains tend to orient themselves in parallel
chains resembling serpentine cords
- best observed in smears from the condensation water or
by direct observation of colonies on a cord medium
ii.

neutral red dye test

- sulfolipidspresent in virulent M. tuberculosis bind
the red dye.

inc. 37C

procedure: organism + 50% methyl alcohol ----------

1 hour

decant organism + fresh barbital

buffer and neutral red dye solution (yellow)
inc. 37C
--------------
1 hour

d.

biochemical tests and characteristic M. tuberculosis

i. niacin positive (yellow color)

ii.
nitatre positive
iii.
tween 80 hydrolyzed in 10-20 days (pink color)
iv.
catalase at 68C : negative
v.
no growth on McConkey medium
vi.
Arylsulfatase negative
vii.Pyrazinamidase (+)
7. Treatment

(106)

a.

first-line antituberculosis drugs isoniazid (INII), rifampicin

pyrazinamide (PZA), Streptomycin, and ethambutol.
b. second-line agents cycloserine, ethionamide p-aminosalicylic acid, capreomycin
c. for newly diagnostic patients INH & rifampicin for 9 months
(appropriate for both pulmonary
and extrapulmonarytb) ; pyra zinamide may be added for the
first 2 months , in which case
the duration of treatment can
be shortened to 6 months
d. in cases of drug resistance for INH and rifamcin the
addition of ethambutol or streptomycin or both is necessary
8. Prevention
a.

BCG (bacillus of calmette and Guerin) vaccination the

vaccine is prepared from an attenuated mutant strain of
M. bovis

b.

INH prophylaxis

9. Other Species Causing Tuberculosis in Man

a. Mycobactererium tuberculosis in cattle
i.

Mode of transmission
-

ii.

transmitted to human via ingestion of raw milk

cultural characteristic
-

slow growers which appear as tiny, translucent, smooth

pyramidal colonies when grown at 35c

glycerol selectively inhibits gworth thus should not

Be included in media

iii.

Biochemical characteristic
-

forms serpentine cords in smears

niacin negative ( useful single test for differentia
ting M. bovis from M. tuberculosis)
niacin negative
susceptible to thiopene-2-carboxylic acid hydrazide
(TCH) useful test for differentiation fron other
mycobacteria

b. Mycobacteriumafricanum
- rarely isolated in the US
- inactive biochemically but urease (+) ; variably (+) growth
in TCH.

Mycobacterium leprae (Hansens bacillus)

(107)

1.Morphology
a.

2.

acid fast rods predominatly in modified mononuclear or epitheloid structure known as lepra cells
b.

found in parallel or palisade arrangement which appear as cigar

packets.

c.

contains phenolase obtained from lepromatous skin nodules

which differentiates it form other mycobacteria

Cultivation
a.

non-culturable on artificial medium

b.

can be grown experimentally only in animals:

i. footpad of mine success is in the low temperature

30C of footpads
ii. Armadillos - animals with low nody temperature
3.

Clinical Infection: Leprosy or Hansens Disease

a.

mode of transmission

i. humans are known to be only natural host

ii. infection is acquired by contact with patient with
lepromatous leprosy who shed numerous organism in their
nasal secretion and ulcer exudates
iii. Major portal of entry is probably the respiratory tract;
biting insects and breast milk mayalsobe potential
b.

pathogenesis and clinical manifestation

M. leprae is an obligate intracellular parasite that

multiplies very slowy within mononuclear phagocytes and has
a strong predilection for nerves. There are 5 forms of leprosy :
tuberculoid (TT), borderline tuberculoid (BT), borderline (BB),
borderline lepromatous (BL) and Lepromatous (LL), but only
two (TT & LL) are stable. (BL,BT,BB) are unstable.
i. tuberculoid or nodular type (TT)
- non-prgressive and benign course
- fewhypopigmented macules
- few bacilli in the lesions
- (+) lepromine test
- progressine and malignant

(108)

4. Lepromin Test
A

skin

test

consisting

of

heat-killed

suspension

of

m.

leprae

24-48

hours

Types of reaction
a.
early/
Fernandez
reaction

induration
appears
b. late/ Mitsuda reaction indurated nodule develops after 3-4 weeks.

in

5. Laboratory Diagnosis
- conventional acid fast stains or via wade-fite technique of
Skin lesion, nasal scrapings, ear lobes, tissue secretions,
lymph node, bone marrow and breast milk.
6.

treatment

a. combined regimen of dapsone or DDS (4,4-diaminodiphenyl sulfone), rifampicin and clofazime for lepromatous leprosy
b. combineddapsone and rifampicin for tuberculoid leprosy
7.

Prevention
a. BCG vaccination

b. chemoprophylaxis with DDS

Mycobacterium ulcerans
- associated with skin lesions in tropical areas( buruli ulcers in Africa)
- slow-growing, requiring 6-12 weeks of incubation
- produces rough domed, lemon-yellow colonies
- biochemical unreactive
- treatment is by wide surgical excision and skin grafting
Mycobacteria Other Than Tuberculosis
Often reffered to as atypical mycobacteria classified on the
Basis of their rate of growth and pigment production (Runyon scheme).
Definitive diagnosis is based on a battery of biochemical tests.

1. Biochemical Tests
a.

niacin
principle: accumulation of niacin in the medium due of lack

test

of an enzyme that converts niacin to another metabolite is characteristic for

M. tuberculosis and

a few other species

i. add 1 ml of sterile distilled water to growth on egg
based medium and stand for 15-30 minutes
ii. extract 0.6 ml distilled water by puncturing medium
with pipet
iii. transfer 1-2 drops of extract to a white spot plate
and add 2 drops 4% aniline in 95% alchocol and 2 drops

(109)

10% aqueous cyanogen bromide (CNBr)

Results:
i. positive yellow color
ii. Negative clear

b. nitrate reduction test

principle :
- presence of nitroreduetaste in M. tuberculosis and other
species of mycobacteria produce a colored end product
from substrates that combine with nitrite
procedure
i. inoculate 4-week-old culture into nitrate broth and incubate in a 36C water bath for 2 hours
ii. acidify with one drop 1:2 dilution of HCl, followed by
2 drops of sulphanilamide solution and 2 drops of coupling reagent.
Results:
i. positive pink to bright red color; for paper strips blue color
ii. Negative no color change
c. catalase test
- most species of mycobacteria produce catalase, which splits
hydrogen peroxide to water and oxygen; it is measured in two ways:
i. semi quantitative by the relative activity of catalase, as determined by the height
of a column of bubbles of oxygen
formed by the action of untreated
enzyme produced by the organism.
ii. heat-stable by the ability of catalase to remain
active after heating at 68C for 20 minutes
d. Tween 80 hydrolysis test

i. certain species of mycobacteria are capable of hyrolyzing tween 80, a derivative of sorbitanmonoleate,
with the production of oleic acid
ii. In the presence of the pH indicator neutral red or phenol red
the oleic acid is indicated by a change from amber to pink (positive result)
iii. Runyons groups II and III are positive in 5 days
iv. M. tuberculosis nis positive in 10-20 days

e. sodium chloride (NaCl) tolerance test

i. useful for the general separation of rapid growers (+)

from slow growers (-)
ii. It also aids in the identification of M. triviale (+)
and M. flavescens (sometimes positive)
iii. 5% NaCl medium is inoculated and observed for the growth
at weekly intervals for 4 weeks.

f.

arylsulfatase test

principle
- based on the ability of arylsulfatase to breakdown
phenolphthalein disulfate into phenolphthalein
Procedure and result
i. inoculate test medium and incubate at 35C for 3 days
ii. Add sodium carbonate and observe for color change to
pink or red (+)
g. tellurite reduction black ppt.
Principle
- telluritereductase reduces potassium tellurite to metallic
tellurite, visualized as a black precipitate; M. avium and
rapid growers posses this fast-acting enzyme

Procedure and result

i. inoculate 7H9 broth and incubate for 7 days at 37C
ii. Add 2-3 drops of tellurite, vortex and reincubate for
3 more days at 37C
iii. Black precipitate (+); no color change (-)
h. thiopene-2-carboxylic acid hydrazide (TCH) susceptibility
- used to distinguish M. bovis (inhivited) from M. tuberculosis (not inhibited)
i. growth on mcConkey agar
- used to differentiate M. fortuitum and M. chelonei, which
grow on the medium in 5 days, from other group IV rapid
growers, which are inhibited
2. Runyons Classification
a. slow growers requiring 7 days or more to yield visible
colonies.

(111)

i. photochromogens (group I) producing yellow to orange

pigmented colonies only on
exposure to light.
M. kansasii strongly catalase (+), nitrate (+)
niacin (-)
M. marinum causative agent of swimming pool granuloma
- weakly catalase (+), nitrate (-)
M. simiae - niacin (+), nitrate (-), hydrolyzes tween 80 slowly
M. asiaticum

ii. Scotochromogens (group II) - producing yellow to orange

pigmented colonies even
withoutlight exposure
( pigmented in light and darkness)

M. scrofulaceun (M. marianum) tween 80 hydrolysis (-)

urease (+)
M. szulgai nitrate (+)
M. xenopi
M. gordonae tap water bacillus, tween 80 hydrolysis (+)
M. flavescens nitrate (+), tween 80 hydrolysis (+)

iii. nonphotochromogenic (group III) - producing nonpigmented

colonies both in light
and darkness
M. avium M. intracellulare ( battey bacillus) complexopportunistic pathogens in patients with
AIDS and other immune dysfunctions
Niacin (-), nitrate (-), small amount of catalase, between 80
hydrolysis (-), reduce tellurite within 3 days.
M. gastri (J bacillus)-hydrolyze tween 80 rapidly,
catalase (-) at 68oC
nitrate (-)
M. malmoense- niacin (-), nitrate (-) hydrolyzes tween 80, produces heat labile catalase.
M. haemophilum
M. terrae (radish bacillus) hydrolyzed tween 80,
nitrate (+), strongly catalase (+)
M. trivial (v bacillus)
M. shimoidei

(112)

b. rapid growers (group IV) produce visible growth within less than 7 days.
M. fortuitum- nonchromogenic, strongly arylsufutase (+) grows on MacConkey agar, niacin (-)
nitrate (+)

M. chelonae ( formerly M. borstelense) strongly arylsulfatase (+)

M. smegmatis
M. phlei ( hay bacillus)- produces large amount of co2 utilizd to stimulate 1o growth of
M. tuberculosis
-produces the growth factor mycobactin required by some M. avium
complex species.
M.vaccae
Unclassified Mycobacterium
-associated with Crohns disease
-colonies are brilliant white and mucoid
-weakly niacnin (+) (+) 14-day arylsulfatase, thermostable catalase (+)
CORYNEBACTERIA
General characteristics
gram (+) aerobic rod shaped organisms that are non acid fast, none motile, non sporeformers.
they are pleomorphic showing variation on size and shape.
posses irregular swellings at one end of the cell as preferential site for synthesis of cell wall
components that gives a club shaped appearance.
CorynebacteriumDiphtheriae (kelbs-loeffler bacillus)
1. Morphology
a. slender, gram (+), rod shaped non acid fast, non motile, nonsporeforming organism.
b.highly pleomorphic, characteristically appear in palasides (picket fence), X, Y, V, and L
formations; these Chinese letter character like formation caused by snapping during binary
fission.

c. with club shaped swellings and beaded and barred forms (meta-chromatic granules or babes
Ernst bodies- responsible for the beaded appearance and when situated at both ends are called
polar bodies)
Cultivation
a. on loefflers coagulated serum media, they appear as minute, grayish white glistening colonies
b. on modified tinsdale, colonies are black with dark brown halos.
(113)
c. on cystinetellurite media, colonies are gray or black with garlic like odor and are differentiated
into 3 major colonial types.

Major types

colonies on CT-BAP

i. gravis

large, flat, gray or black

with irregular edges and
radial striations
(daisy head colonies)
medium sized colonies
that are blacker, glossy or
moist and more convex
(coolie hat colonies)

ii. Mitis

hemolysis

hydrolysis of starch granules

(-)

(+)

(+)

(-)

(-)

(-)

iii. Intermedius

very small either, smooth or

rough flat, gray or black
colonies.

Clinical Infection: Diphtheria

a. mode of transmission droplet nuclei from the respiratory tract or by contact from cutaneous
foci of infection.
b. pathogenesis and virulence
i. the powerful exotoxin is produced only by strains of C.diphtheriae infected with a
bacteriophages (bacterial virus) carrying the TOX gene for toxing production of C. diphtheria
exhibits lysogenecity (potebtiality of a bacterium to produce phage) producing an exotoxin
which inhibits protein synthesis and is more poisonous than a cobra venom.
ii. Initial lesions usually occurs on the tonsils and pharynx which spreads to the
nasopharynx, larynx and trachea.
iii. The organism and their exotoxin produce a serum exudates and celllar infiltrate of the
mucous membrane in the pharynx, leading to formation of a very tough adherent grayish to black
pseudomembrane.
C. clinical manifestation
i. low grade fever, malaise and mld sore throat
ii. Edematous and tender cervical lymph nodes (bull neck appearance)

iii. mechanical obstruction of the airway may ensue due to the membrane and accompanying
edema of the larynx and trachea.
iv. Skin ulcers
v. complications involve the cardiovascular and nervous systems
Schicks test
a. susceptibility test for diphtheria
b. test arm is injected intracutanously with 0.1 ml of diphtheria toxin and on the opposite arm
(control arm) 0.1 ml of diphtheria toxoid.
c. results are read after 24-48 hours an after 6 days.
d. the test is positive if redness, induration and necrosis appear at the site of injection (test arm)
which persist up to the 6th day and fade gradually leaving a brownish pigmented area, and no
reaction in the control arm.
(114)
e. Interpretaion of results:
i. (+) = t.a. (+) and c.a. (-) patient is susceptible to diphtheria
ii. (-) = t.a. (-) and c.a (-) indication of immunity
Laboratory diagnosis
specimen: nasopharyngeal and throat swabs
a. stained smears
b. culture
i. BAP
ii. Tinsdale
iii.Pais coagulated egg medium

iv. Loefflers coagulated serum slant

c. biochemical characteristics
i. nitrate reduction positive except for c. diptheriae var. mitis (+/-)
ii. Urease negative
iii. Catalase positive
iv. Ferments glucose and maltose but not sucrose
v. pyrasinamidase negative
d. virulence tests (for toxin production)
i. in vivo toxigenixity test: animal inoculation
-inject intracutanously 0.2 ml of culture into a guinen pig followed 5 hours after with 500 units
of antitoxin injected intraperitoneally.
-after 30 minutes a second 0.2 ml culture is injected into the control area opposite the test site.
-the test site after 48-71 hours and a pinkish nodule on the control site (there is prevention of
necrosis due to prior administration of antitoxin.

ii. in vitro toxigenicity test: Elek plate/gel diffusion test

-soak a filter paper strip in an antitoxin (100u/ml) and embed or lay on the surface of a serum
agar medium.
-streak the unknown organism, positive and negative controls perpendicular to the filter paper
-incubate at 35oc for 1-3 days
-if the unknown organism is toxigenic, awjite line of toxin-antitoxin precipitate apperars at a 45
degree angle between the filter paper and the streak.
-(+) control
- unknown
- (-) control

Treatment

(115)

a. antitoxin
b. penicillin G. drug of choice; erythromycin for penicillin allergic patients.
Prevention
a. active immunization with DPT (below 7 y/o) and Td (7y/o and above)
b. prophylaxis for case contacts
i. boosters dose of toxoid- for previously immunized persons within 5 years.
ii. Completion of immunization and antibiotics- unimmunized or inadequately immunized
persons.
iii. Passive immunization with antitoxin- may be employed for nonimmunized persons with
heavy exposure.
otherCorynebacteria( Diphtheriod)

these are opportunistic invaders infecting immunocompromised cysts which are oftenly confused
with C. diptheriae.
C. minutissimum
a. causative agent of erythrasma, a skin disease occurring in the intertriginous areas especially
the axillae, genitocrural crease, and webs between the fourth and fifth toes.
b. skin lesions show a corak re fluorescence when exposed to woods light due to the presence of
prophyrin.
c. ulcerans- is now considered a variant of C. diphtheria
c. pseudotuberculosis- also produce an exotoxin and causes subacute relapsing lymphadenitis
c.xerosis- inhabits skin and pharynx especially the conjunctiva.
c. pseudodiphthericum-may cause endocarditis.
othercorynebacteria species are c. bovis, areanobacteriumhaemolyticum (c. haemolyticum),
actinomycespyogenes (c. pyogenes), rhodococcusequi (c.qui) c.jeikeium (group jk)
CDC d2 A4 and G2, c matruchotii. Etc.

LISERIA ( L.monocytogenes)
Morphology
a. small gram (+) , aerobic to microaerophiliccoccobacilli that are nonacid-fast and
nonsporeformers
b. they occur in pairs and short chains (resembling streptococci) or in typical
diphtheriodpalasides arrangement; they are also confused with haemophilus species on over
decolorized
preparations.
o
o
c. with tumbling motility at room temperature (20 -25 c) due to the presence of 4 peritrichous
flagella; less or nonmotile at 35o-37oc
Cultivation
a. Optimum temperature for growth is at 37 oc but it has the ability to grow at low temperature
(4oc) being the basis for the cold enrichment technique.
b. on BAP smooth, translucent gray colonies with narrow zone of beta hemolysis.
c. on clear tryticase agar or nutrient agar smooth, translucent blue-green colonies.
Disease produced: Listeriosis
a. may present from sepsis and meningitis in neonates and immunocompromised patients to a
nonspecific febrile illness in pregnant women.
(116)
b. transplacental infection is also common.
c. in adults, meningitis is the most common presentation of listeriosis.
Laboratory Diagnosis
Specimen: CSF, blood, amniotic fluid and genital tract secretion.
a. stained smears
b. motility test refers to p.23
-umbrella-shaped motility pattern in semisolid agar
c. culture
i. sheeps blood agar
ii. Tryptose agar
iii.Mcbride agar
iv. PEA agar- for contaminated specimens (inhibits gram negative organism)

v. nalidixic acid selective medium.

vi. Cold enrichment technique where in suspect cultures and tissues are held at 4c for
several weeks and replated after storage for 6 weeks and 3 months to enhance recovery of
the organism.
d. biochemical characteristics.
i. catalase (+)
ii. Ferments glucose, trehalose + salicin
e. virulence test:Anton;s test
-development of purulent conjunctivitis in rabbits eye inoculated with L. monocytogenes
suspensions
f. FAT- unreliable because L. monocytogenes cross reacts with other gram (+) organism
TREATMENT
a. Penicillin G or ampicillin in combination with amioglycosides
b.trimethoprim- sulfamethoxazole- for penicillin allergic patients
c. other alternatives- erythromycin and tetracyclines.

ERYSIPELOTHERIX ( E. rhusiopathiae)
Morphology
a. nonsporogeneus, non motile, nonencapsulated, microaerophilic gram positive rods.
b.in acute cases of Erysipelothrix disease, colones are smooth with short slender, straight or
slightly curved organism while in chronic cases, colonies are rough with long-filamentous
organisms.
Cultivation
a.
on
BAPalpha-hemolytic,
small
round
b. on gelatin medius- lamp brush or test
c. tellurite medium- black colonies.

and
tube

grayish
white
colonies
brush type of growth.

Clinical Infection: Erysopeloid

a. mode of transmission
(117)
-transmitted to man from animals by means of skin wounds produced with contaminated
object or
in contact with blood, flesh, viscera or feces of infected animals, fishermans,
butchers and rose
gardeners.
b.clinical manifestation.
i. it presents as a local cutaneous infection manifested by pain, itch swelling and a
cutaneous
eruption that is slowly progressive, slightly elevated, non suppurative purplish
erythematous
zone at the site of inoculation.
ii. Lymphangitis may also occur
iii. Rarely, dissemination may occur causing infective endocarditis and septic arthritis.
Laboratory diagnosis
Specimen: aspirated material and biopsy specimens; blood in cases of endocarditis.

a. stained smears
b.culture
i. heart infusion agar with 1% flucose and incubated in 5% co2
ii. BAP etc.
c. Biochemical characteristics.
i. oxidase and catalase negative
ii. Produce H2S in TSI
iii.nitrate (-)
iv. Ferments glucose and lactose
Treatment
a. penicillin- drug of choice
b. erythromycin- for penicillin allergic patients.
c. other alternatives tetracyclines, cephalosporins, clindamycin and chloroampenicol.

NOCARDIA
Morphology
a. aerobic, gram-positive, partially acid fast bacilli that are delicate, multiply branched with
beaded filaments
b. partially acid-fast when stained according to kinyouns method; with 1-2% h2so4 as
decolorizers, most strains of nocardia strain acid fast.
c. the organisms can also be stained with Gomorimethenamine silver stain.
Cultivation
a. grow on the most laboratory media like Lowenstein-jensen, brain-heart infusion, sabouraud
dextrose agar etc.
b. colonies are heaped, irregular, waxy shiny, bumpy or velvety.
c. utilized parafiin as a source of carbon and energy.
Clinical infection: nocardiosis
a. Habitat: soil and water
b. species causing nocardiosis: mainly N. asteroids and rarely N. brasiliensis and N.
otitidiscaviarum. (N. cavaie)
c. mode of transmission
i. inhalation of organism
ii. Inoculation through breaks and the skin.
(118)
d. epidemiology and manifestations
i. infection is chronic but occasionally fulminant and usually see in immunocompromised
patients, particularly following renal or cardiac transplantation.
ii. Begins as chronic lobar pneumonia which may disseminate hematogenously to any
organ but with preference to the central nervous system resulting in single or multiple brain
abscesses.
Laboratory diagnosis
Specimens: sputum, skin lesions, tissue biopics or surgical material
a. stained smears
b. culture
c. biochemical characteristics: urease (+) lysozymes resistant
d. gas liquid chromatography (GLC) for cell wall analysis.

Treatment
a.sulfonamides
b.trimethoprin
c.amakacin and imipenem
OTHER AEROBIC NONSPOREFORMING GRAM-POSITIVE BACILLI
Other aerobic actinomycetes- noncadiopsis, actinomadura (causative agent of
actinomycetoma),dermatiphilus, streptomyces, thermoactinomyces, sacharomonospora and
Micropolyspora
Rothia, kurthia, Oorskovia and lactobacillus.

(119)
CHAPTER XI
AEROBIC SPORE-FORMING GRAM POSITIVE BACILLI

BACILLUS
A. Bacillus anthracis
1. Morphology
a. Facultative, large, square ended, boxcar-shaped, encapsulated, nonmotile, gram
positive rod with an ellipsoidal to oval centrally located spore; sporangium is not
swollen (Bamboo fishing rod)
b. They occur in long chains, giving a bamboo pole appearance.

2. Cultivation
a. Optimal growth temperature is at 35C
b. On BAP, colonies are nonhemolytic, large, raised, opaque, grayish white with
irregular, fringe-like margin and cut glass appearance
c. Comma-shaped outgrowths or tangled masses of long hairlikecurlsare common
giving rise to Medusa or lion head colonies
d. The colonies are tenacious thus when pushed and lifted with an inoculating needle
they stand up like beaten egg whites
e. In aelatine, growth is described as inverted fir or pine tree
3. Determination of Pathogenecity
a. Capsule
b. Anthrax toxin consists 3 components: protective antigens, lethal factor, and
edema factor; responsible for the signs and symptoms.
4. Clinical Infection : Anthrax
a. Anthrax infection is primary a disease of lower animals (sheeps and cattles) which
rarely infects humans
b. Three forms of anthrax:
i.

Cutaneous anthrax (malignant pustule)

-

ii.

Pulmonary anthrax (Woolsorters disease)

-

iii.

Most common form

Infection initiated by entrance of bacilli through an abrasion of the
skin
Pustule appears on hands or forearms which develops into a vesicle
filled with dark bluish black fluid.
Rupture of the vesicle reveals a black eschar

Contracted by inhalation of spores during shearing, sorting, and

handling of animal hair

Gastrointestinal anthrax (violent enteritis)

-

Most severe and rarest form

Contacted by ingestion of bacilli or spores from infected meat
resulting to invasion and ulceration of the gastrointestinal mucosa

c. Septicaemia can occur in all three forms and may lead to fatal meningitis
5. Laboratory Diagnosis
Specimens: swabs of vesicles and eschar, sputum, stool, blood and CSF(cerebrospinal
fluid)
a.
b.
c.
d.

Stained smears gram stain and fluorescent-antibody stain

Culture
Animal inoculation tests or virulence tests
String of pearls test
i.
ii.
iii.

This test reflects the susceptibility of a stain to penicillin

Organism is streaked on Mueller-Hinton agar with a 10 unit penicillin disk
placed over the streak and incubated for 3-6 hours at 37C
The cells become large and spherical occurring in chains as seen on the
surface of agar, resembling a string of pearls (+ result)

e. Ascoli test
i.
ii.

Diagnostic precipitin test for B. anthracis

Extracts of infected tissues show a ring of precipitate when layered over
immune serum

f. Use of gamma bacteriophage

g. Indirect hemagglutination
h. ELISA (Enzyme-linked Immunosorbent Assay)
6. Treatment
a. Penicillin drug of choice
b. Other alternatives tetracycline, erythromycin, & chloramphenicol
7. Prevention: active immunization

B. Bacillus cereus
1. Morphologically similar to B. anthracis but usually motile
2. Cultivation
Colonies are beta-hemolytic, small, shiny, and compact or large, feathery and
spreading.
3. Differential Points Between B. cereus and B. anthracis
(121)
a.
b.
c.
d.
e.
f.
g.

Motility
Capsule
Hemolysis
Growth at 45C
Salicin fermentation
Penicillin sensitivity
Gamma phage

B. cereus

B. anthracis

motile
nonencapsulated
beta-hemolytic
(+)
(+)
resistant
resistant

nonmotile
encapsulated
nonhemolytic
(-)
(-)
sensitive
sensitive

4. Clinical Infection
a. An important cause of food poisoning which are of two types :
i. short incubated type associated with fried rice
ii. long incubated type associated with meat or vegetable dishes
b. Seen in serious infections associated with immunocompromised hosts
5. Treatment
- Susceptible to chloramphenicol, aminoglycosides, clindamycin, erythromycin, and
vancomycin
Bacillus subtilis (Hay bacillus)

- A very common laboratory contaminant

- colonies are usually large, flat and dull with a ground glass appearance
- morphologically similar to B. cereus

(122)
CHAPTER XII
ANAEROBIC GRAM-POSITIVE BACILLI

CLOSTRIDIUM
General Characteristics
1. Most are obligate anaerobes but some are aerotolerant like C. histolyticum and C. tertium
2. All are motile with peritrichous flagella except C. perfringens, C. ramosum, and C.
innocuum.
3. All can readily sporulate in vitro except C. perfringens, C. ramosum, and C. clostridioforme.
4. All have swollen sporangia except C. perfringens and C. bifermentans.
5. All are encapsulated except C. perfringens.
6. Colonies are large with irregular with irregular edges or swarming growth (which occurs only
in anaerobic plate) but some may form small, convex, nonhemolytic colonies with an entire
edge (e.g., C. ramosum, C. clostridioforme.)
7. All are usually catalase negative
8. All are Nagler-negative except C. perfringens, C. baratti, C. bifermentans and C. sordellii.
9. They are widely distributed in soil, dust and water and are common onhabitants of the
intestinal and genital tract.

A. HistotoxicClotridium (Gas Gangrene)

This group causes a severe infection of muscle known as
Clostridialmyonecrosis(gas gangrene/clostridial myositis). It includes the following
species (the first three are the most important):
1. C. perfringens
2. C. novyi
3. C. septicum

4. histolyticum
5. C. sordellii
6. C. fallax

ClostridiumPerfringens (C. welchii or Bacillus aerogenescapsulatus or gas gangrene

bacillus)- produces 12 toxins & spores

1. Morphology
a. Short, plump, boxcar-shaped, gram-positive rod which is encapsulated and
nonmotile
b. Does not produce spores in ordinary media
c. Subterminal spores but impossible to demonstrate
2. Cultural and Biochemical Characteristics
a. On BAP circular and smooth colonies with a characteristic target or double zone
hemolysis resulting from a narrow zone of complete hemolysis due to
theta-toxin and a much wider and a much wider zone of incomplete (123)
hemolysis due to alpha-toxin.
b. In chopped-meat glucose media abundant growth with gas formation
c. In milk media stormy fermentation due to the excessive gas production
d. Lecithinase test some Clostridia species like C. perfringens possess lecithinase
which splits lecithin (a normal component of egg yolk) to
insoluble diglycerides, resulting in an opaque halo surrounding
a colony growing on egg yolk agar.
e. Nagler or Lecithovitellinrection
Procedure
i. Swab one-half of an egg yolk agar plate with C. perfringens type A
antitoxin and allow to dry
ii. Inoculate test organism on both halves of plate
iii. Inoculate anaerobically 24-48 hours at 37C
Result and Interpretation
i. Disappearance or reduction of the opacity on the antitoxin half of the plate
which denotes neutralization of the type a lecithinase (+Nagler test)
ii. The reaction is caused by alpha-toxin (a lecithinase C) which is
specifically inhibited by C. perfringens antitoxin.
f. Ferments glucose, lactose, maltose and fructose but not xylose
3. Clinical Infection
a. Wound and tissue infection caused by Type A strains of C. perfringens ;
symptoms are attributed to alpha-toxin
i. Simple wound contamination

ii.
iii.
iv.
v.

Anaerobic cellulitis
Clostridialmyonecrosis
Uterine infection
Clostridialsepticemina

b. Food poisioning caused by type A strain that produce heat resistant spores
and minimal amounts theta toxin; mode of transmission is
by ingestion of enterotoxin (108 - 103 viable bacteria).
Third most common cause of food poisoning in US.
c. Enteritis necroticans (necrotizing jejunitis or necrotic enteritis)
- caused by type C strains ingested from inadequately
cooked pork; also called pig-bel and Darmbrand :
symptoms are due to beta-toxin.
d. Post abortion sepsis
4. Laboratory Diagnosis
Specimens: wound tissues, aspirates, deep swabs, feces and samples of ingested food.
a. Direct and Gram stained smears
b. Culture and biochemical tests
i.
ii.
iii.
iv.
v.

(124)

Double zone of hemolysis theta toxin

Lecithinase (+)
Ferments glucose, maltose & fructose
Nagler plate
Reverse CAMP test

c. ELISA
5. Treatment
a. Surgical removal or debridement of necrotic tissues
b. Intensive antibiotic (Penicillin) and antitoxin therapy

B. Clostridium tetani (Pack head bacillus)

1. Morphology
a. Spore is terminally located
b. Sporangia are swollen giving a characteristic drumstick or lollipop or tennis
racket appearance
c. Possess numerous peritrichous flagella
2. Cultural Characteristics
a. On BAP swarming is observed ; colonies consist of a flat network with a
delicate edge of projecting filaments; faint beta-hemolysis is also seen
b. Does not ferment any carbohydrates
c. MOT puncture wound, gunshot, burn and animal bite
3. Tetanus Toxin
a. All symptoms of tetanus are attributed to an extremely toxic neurotoxin,
tetanospasmin (released upon death and lysis of C. tetani)
b. It becomes bound to gangliosides within the CNS and suppresses the central
inhibitory balancing influences on motor neuron activity, thus leading to

intensified reflex response to afferent stimuli and to spasticity and convulsions

(this is proven by Wasserman-Takaki Phenomenon)
4. Clinical Manifestation
a. Trismus or lockjaw spasm of the masseter muscles
b. Risussardonicus grinning expression produced by spasms of facial muscles
c. Opisthotonus arching of the back
5. Laboratory Diagnosis
a.
b.
c.
d.

Diagnosis is made on clinical grounds (signs and symptoms)

History of punctured wound
Material from wound site may be stained and cultured
Gelatinose (+), indole (+), lecithinase (-), lipase (-), unable to ferment lactose
(125)
6. Treatment
a.
b.
c.
d.
e.

Antitoxin
Debridement of wound and removal of foreign bodies
Penicillin, tetracycline or metronidazole for penicillin-allergic patients
Barbiturates and diazepam for control of spasms
Careful control of environment

7. Prevention active and passive immunization. DPT 2,4,6 months and booster 12-15
months 1-6 years
C. Clostridium botulinum (Bacillus botulinus or Canned-good bacillus)
Produces 7 types of botulinum toxins. The most common are A,B,C and F.
1. Morphological and Cultural Characteristics
a.
b.
c.
d.

Straight and slightly curved gram-positive rod with rounded ends

Produces heat resistant spores that are oval and subterminal
Motile with peritrichous flagella
On BAP beta-hemolytic

2. Botulinum Toxin
a. One of the most potent exotoxin known
b. A neurotoxin which attaches to individual motor nerve terminals, blocking the
release of acetylcholine at the nerve endings
3. Types of Botulism
a. Food-borne botulism lethal food poisoning which results from ingestion of
preformed toxin in contaminated food
b. Infant botulism related to ingestion of spores, multiplication of organisms
withinthe gastrointestinal tract and subsequent absorption of
toxin.
c. Wound botulism- the least common which occurs with the introduction of spores
or bacteria into wound.
d. Unclassified botulism- occurs in patients over one year of age who have clinical
symptoms of botulism but with no, identifiable vehicle of
transmission.
4. Laboratory diagnosis

Specimens: blood, stool, gastric washings, and food specimen

a. Toxin identification by:
i. Mouse toxicity
ii. Neutralization
iii. ELISA
b. Isolation of organism from clinical from clinical specimens
(126)
c. Resistance to cycloserine, sulfamethoxazole and trimethoprim in an egg yolk agar
medium
d. Lipase (+), lecithinase and indole (-)
e. Ferments glucose but not lactose
5. Treatment
a. Antitoxin
b. Saline enemas
c. Guanidine hydrochloride
D. Clostridium difficile(isolated in 1975)
1. Cultural characteristics
a. On BAP fluoresce yellow-green colonies with a horse stable odor
b. On cycloserine-cefoxitin egg yolk fructose agar yellow ground glass colonies
2. Toxins Produced
a. Toxin B or cytotoxin which is cytopathic for most tissue culture cell lines
b. Toxin A which is a potent enterotoxin causing severe damage to the
intestinalmucosa and an excess fluid response
3. Antibiotic-Associated pseudomembranous colitis
a. A severe, potentially lethal disease of the GIT caused by an over growth of C.
difficile as a side effect of antibiotic like ampicillin, clindamycin and
cephalosporin
b. When normal flora are reduced by these antibiotics, C. difficile multiplies and
produces its toxins
4. Laboratory Diagnosis
a.
b.
c.
d.
e.
f.

Demonstration of cytotoxin in stool specimens by microtiter cytotoxicity assay

ELISA test for toxin A
History and clinical picture
Culture of school specimens
Lecithinase, lipase, indole, gelatinose (-)
Ferment glucose and fructose

5. Treatment
a.
b.
c.
d.

Discontinue implicated antibiotics

Maintain fluid and electrolyte balance
Acid anti-motility drugs
Administer vancomycin; metronidazole and bacitracin are also effective

Nonpathogenic Clostridia C. sporogenes, C. bifermentans, C. tertium, C. innocuum,

C.ramosum, etc.

(127)

ACTINOMYCES
1. Normal flora of the mouth, GIT, and female genital tract.
2. Morphological and Cultural Characteristics
a. Branching and beaded with diptheroid and coccobacillary forms that are nonsporogenous
and nonacid-fast
b. Gram stain of sulfur granules shows tangled mass of long filamentous forms
c. Most are facultative anaerobes, growing best in the presence of CO 2; A. israelii and A.
bovis are anaerobes
d. Generally Actinomyces produce colonies that are smooth, flat to convex, gray-white, and
translucent with entire margins; some may show beta-hemolysis like A. pyogenes and A.
odontolyticus
e. A. israelii with molar tooth colony on brain-heart infusion agar described as white,
heaped, rough and lobate; may also produce breadcrumb-like, raspberry or smooth
colony; tight aggregates (balls) in thioglycollate broth
3. Clinical Forms of Actinomycosis
Actinomycosis is a chronic suppurative and granulomatous infection characterized by
pyogenic lesion with interconnecting sinus tracts that contain granules (also called grains or
sulfur granules). These granules are composed of tissue elements and the organisms.
a. Cervicofacial actinomycosis
- Caused by a trauma to the oral mucosa permitting endogenous bacteria to breach the
normal defenses of the mucosa (e.g., following dental caries and gingival disease)
b. Thoracic actinomycosis
- Acquired by aspiration of the bacteria or as extension from the cervicofacial form
leading to pulmonary infection
c. Abdominal actinomycosis
- Initiated by a traumatic perforation of the intestinal mucosa (e.g., ruptured appendix
and ulcer)
d. Genital actinomycosis
- Seen in women using intrauterine devices (IUD)
4. Laboratory Diagnosis
Specimens: sputum, pus, tissue-sections and cervical exudates
a. Examine specimens for presence of granules
- Yellowish color is due to abundance of macrophages containing lipid vacuoles
b. Stained smears
i.
Gram stain
ii.
Fluorescent antibody stain

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-85c. Culture media

i.
metronidazole-cadmium sulfate-containing medium
ii.
metronidazole disk on PEA plate
d. Biochemical characteristics
i.

Most are nitrate positive

ii.

Indole negative

iii.

All are urease negative except A. naeslundii

iv.

All are catalase negative except A. viscosus

5. Treatment
i. Penicillin G drug of choice
ii. Alternative tetracycline, clindamycin and erythromycin and sulfonamide
BIFIDOBACTERIUM
1. Normal flora of the GIT and oral cavity.
2. Morphology
-

Various shapes, with clubbing or bifurcated ends and may show diphtheroidal and
branching forms

3. Culture
-

B. dentium (B. eriksonii) produces white, convex, shiny colony with irregular edge and
has a diffuse growth in broth; they are acidophilic and grow best on low pH agar (e.g.,
tomato juice agar)

4. Biochemical Characteristics
a. Produces large amounts of acetic and lactic acids in peptone yeast glucose broth, with
acetic acid in much greater quantity than lactic acid
b. Generally indole-, nitrate-, and catalase-negative with rare catalase-positive strain
5. Very rarely causes Actinomycosis
EUBACTERIUM
1. Normal Flora of the GIT and mouth.
2. Morphology
a. Generally pleomorphic rods to coccobacilli occurring in pairs or short chains but also as
straight uniform or curved rods
b. E. alactolyticum sea-gull-wing shape forms
c. E. nodatum beading, filamentous and branching similar to Actinomyces
d. E. lentum (most common Eubacteria) small, straight rod with rounded ends
3. Culture
a. Colonies are raised to convex and transparent to translucent
b. E. nodatum colonies are similar to A. israelii
4. Identification
a. Usually identified by exclusion
b. They are obligate anaerobes, nonmotile and catalase negative

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-865. Clinical Infection

a. Isolated from blood, abscesses, dental infections (periodontitis) and wounds with other
anaerobes
b. E. nodatum has been isolated from actinomycosis of the jaw and IUD-associated
infections
LACTOBACILLUS
1. Normal flora of the GIT, mouth, and vagina.
2. Morphology and General Characteristics
a. cells are straight and uniform with rounded ends and may form chains
b. some are short and coccobacillary in form
c. organisms are facultative or strict anaerobes, usually preferring
microaerophilically
d. on sheep blood and chocolate agar colonies are small with greening

to

grow

3. Identification
a. produce large amounts of lactic acid with or without smaller amounts of acetic acid
b. nonmotile, indole-, catalase-, and nitrate-negative
4. Clinical Infection
a. pleuropulmonary infections caused by L. catenaforme
b. dental caries, UTI, bacteremia, endocarditis, local suppurative infections, and
chorioamnionitis
5. Treatment: penicillin
PROPIONIBACTERIUM
1. Normal flora of the skin, GIT, upper respiratory tract (ant. Nares), and urogenital tract
2. Morphology and General Characteristics
a. irregular, pleomorphic, and club-shaped with one end round and the other tapered
(anaerobic diphtheroids); but sometimes short branching and beading resembling
Actinomyces
b. colonies of P. acnes are small and white to gray-white becoming yellow as they get
older and are sometimes beta-hemolytic
c. species are aerotolerant but grow better anaerobically
d. grow well in broth especially with Tween 80
3. Identification
a. produce large amount of propionic and acetic acids with lesser amount of lactic,
succinic and isovaleric acids in peptone yeast glucose broth
b. catalase positive except for P. propionicus (Arachnia propionica) and a few other
strains
4. Clinical Infection
a. frequently contaminants of blood culture and other sterile body fluids
b. P. acnes

i. Implicated in infection related to heart valves and prosthetic devices

ii. Plays a role in acne
c. P. propionicus (A. propionica) also a cause of actinomycosis
5. Treatment: penicillin
-87CHAPTER XIII

ENTEROBACTERIACEAE
GENERAL CHARACTERISTICS
1. Small, straight sided, facultative anaerobic, gram negative, non-sporeforming rods
2. All motile members possess peritrichous flagella except Tatumella ptyseos which has polar
flagella. Shigella and Klebsiella are non-motile. Some species of Escherichia, Salmonella and
Yersinia are non-motile too.
3. All members grow luxuriantly on BAP as moist, smooth, gray, shiny, entire, covex and
opaque colonies. Smooth to rough variations can occur. Beta-hemolysis is seen in some
strains.
4. All are catalase positive except for one group of Shigella species and a species of
Xernorhabdus.
5. All members ferment glucose but do not liquify alginate and are oxidase negative.
6. Almost all are able to reduce nitrate to nitrites.
7. Predominant facultative flora in the human bowel.
8. Most species are not intestinal pathogens but opportunistic organisms which are responsible
for the majority of nosocomial infections.
CULTURE MEDIA
1. For fecal specimens (including rectal swabs)
a. Differential media
i.
MacConkey agar divides Enterobacteriaceae into:
- Lactose fermenters pink or reddish colonies or bile lactose media and
are usually non-pathogenic
- nonlactose fermenters colorless, translucent colonies and are usually
pathogenic
ii.
Eosin methylene blue (EMB) also divides Enterobacteriaceae into lactose
and nonlactose fermenters; aside from
lactose it also contains sucrose
iii.
Desoxycholate agar
b. Selective media
i.
Hektoen enteric agar (HE) contains lactose, sucrose and salicin; able to
detect H2S production
ii.
Xylose lysine deoxycholate (XDL) contains lactose, sucrose and xylose;
able to detect H2S production
iii.
Salmonella-Shigella agar
iv. Desoxycholate citrate agar
2. For other specimens like material from wounds, respiratory tract secretions, aspiration from
abscesses, sterile body fluids, urines, tissue, and blood cultures
a. BAP or chocolate agar (supportive medium to allow growth of all enterobacteriaceae) and
b. McConkey agar (differential medium for gram negative bacilli)
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Enrichment broths
a. selenite broth
b. G-N broth
c. tetrathionate broth
CHEMICAL TEST (refer to Chapter III)

Lysine iron agar (LIA)

a.
b.
c.

detects H2S production, lysine decarboxylation and phenylalanine deamination

pH indicator: bromcresol purple
H2S indicator : ferric ammonium citrate
principle and results:
i.
thiosulfate is reduced by some bacteria to hydrogen sulfide which reacts with the iron
(ferric ammonium citrate) giving a black precipitate
ii.
organisms that are able to decarboxylate lysine (a reaction which takes place
anaerobically in the butt) produce alkaline end products resulting in a purple color;
bacteria that do not decarboxylase lysine ferment glucose, yielding acid by-products
and causing the pH indicator to turn the butt yellow
iii.
deamination of phenylalanine is detected by a change in the color of the slant from
purple to reddish

ONPG (o-Nitrophenyl-beta-D-Galactopyranoside) test for beta-Galactosidase

- rapid test to detect slow lactose fermenters
a. principle
- beta-galactosidase acts on substrate ONPG in the same way as the enzyme hydrolyzes
lactose to galactose and glucose, although the end product of ONPG hydrolysis is a
yellow product, orthonitrophenol
b. procedure
i.
inoculate ONPG broth with a heavy suspension of organism
ii.
incubate overnight at 37 C
c. results
i.
positive yellow
ii.
negative no color change
Gelatin Hydrolysis test
a. principle
- Serratin, Proteus, Xernorhabdus and some Yersinia produce proteinase like gelatinase
which liquifies gelatin
b. procedure and result
i.
inoculate two gelatin broth tubes and incubate one at 37 C and the other at room
temperature
ii.
an uninoculated tube is also incubated at 37 C as control
iii.
after sufficient growth (up to 30 days) tubes are placed in the refrigerator for 30
minutes or until the control tube resolidifies
iv. if gelatinase is present, either one or both of the inoculated tubes will remain liquid,
indicating breakdown of gelatin
DNase test
a. principle
extracellular nucleases are produced by the same species that produce the protease
gelatinase
b. procedure and result
i.
streak DNase test medium (with methyl green indicator) with organism
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ii.
incubate overnight
iii.
after growth has been obtained, flood plate with 1N HCl
iv. DNase-positive cultures show a distinct clear zone around the streak
Escherichia
This genus includes several other species aside from E. coli, but are rarely isolated from human
disease.
E. coli (Colon bacillus)
1. Major facultative inhabitant of the large intestine

2. Determinants of Pathogenecity
a.
b.
c.
d.
e.

polysialic acid capsule called Kl

S type fimbriae or pili
adhesins
enterotoxins heat stable, nonantigenic (ST) and heat labile antigenic (LT)
verotoxins
i.
human-derived or porcine-derived cytotoxin which cause an irreversible cytotoxic
affect on Vero tissue culture cells (a cell line developed from African green
monkey kidney cells)
ii.
similar to Shiga toxin thus reffered to as Shigalike toxins (inhibits protein synthesis
like Shiga toxin)
f. invasiveness
g. hemolysin
3. Disease Produced
a.
b.
c.
d.
e.
f.

urinary tract infection

pneumonia
neonatal meningitis
wound infection
septicemia
diarrheal disease

Strains of E. coli that cause diarrhea in man

i.
enteropathogenic E. coli (EPEC)
- does not produce enterotoxin
- associated with infatile diarrhea
ii.
enterotoxigenic E.coli (ETEC)
- produce enterotoxins
- major cause of of travelers diarrhea; may resemble cholera
iii.
enteroinvasive E. coli (EIEC)
- causes bacillary dysentery in all age group similar to shigellosis
- Sereny Test : a classic test for invasiveness in which a suspension of the organism is
instilled within the conjunctival sac of a guinea pig or rabbit causing a
purulent and exudative conjunctivitis
iv. enteroadherent E. coli (EAEC)
- produce nonfimbrial adhesions that attach the organisms to their target cells (also
produced by EPEC and VTEC)
v. enterotoxin-producing E. coli (VTEC)
- associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome
4. Laboratory Diagnosis

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a. stained smears not diagnostic

b. culture
i.
MacConkey dry pink colonies due to lactose fermentation
ii.
Eosin methylene blue colonies with greenish metallic sheen
iii.
XLD dry yellow colonies
c. biochemical characteristics
i.
TSI A/A + gas
ii.
IMVIC - ++-iii.
Lysine decarboxylase (+)
iv. utilizes acetate as a carbon source
d. MacConkey/sorbitol/agar medium that substitutes sorbitol for lactose in MacConkey
agar and used as a screening agar for VTEC detecting the most common serotype
0157:H7
e. Serotyping
5. Treatment

a. management of fluid and electrolyte imbalance

b. antibiotics

Edwardsiella
E.tarda
1. Type specie of this genus
2. Isolated from humans and associated with diarrhea, wound infections and sepsis.
3. Biochemical Characteristics
a. MacConkey nonlactose fermenter
b. TSI- K/A + H2S +gas
c. IMVIC - ++-d. Lysine decarboxylase (+)
e. Ornithine decarboxylase (+)
Shigella
1. General Characteristics
a. Slender, aerobic, nonmotile, nonencapsulated, gram negative rods
b. Generally nonlactose fermenters and do not produce H2S
c. Do not produce gas from glucose (anaerogenic)
d. All species can cause bacillary dysentery and are the major cause of it.
2. Classification
a. According to mannitol fermentation
Nonmannitol fermenters
S. dysenteriae (Dysentery of Shiga Bacillus) type specie of the genera
Mannitol fermenters
i.

Nonlactose fermenters
S. flexneri (Strongs Bacillus)
S.boydii (Boyd Bacillus or S. ambigua)

ii.

Late lactose fermenter

S. sonnei (Sonne Duval Bacillus)

b.

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According to serogrouping
i. Group A S. dysenteriae
ii. Group B - S. flexneri
iii.
Group C S. boydii
iv. Group D sonnei
3. Factors contributing to Virulence
a. Smooth lipopolsaccharide (LPS) structure may be the one responsible for the
organisms ability to resist gastric acidity
b. Invasiveness
c. Shiga toxin interferes with protein synthesis and is neurotoxin, cytotoxic and
enterotoxic
4. Clinical infection (Bacillary dysentery or Shigellosis)
a. S. sonnei is the most common isolate worldwide followed by S. flexneri and to a
lesser extent S. boydii and S. dysenteriae, however in developing countries S.
dysenteriae and S. boydii are more frequently isolated, followed by S. flexneri
then S. sonnei.
b. Mode of transmission: fecal-oral route
c. Clinical manifestations

i.

Acute toxigenic gastroenteritis: diarrhea with profuse, watery stool and

fever of a self- limited course: or
ii.
Acute tissue- invasive gastroenteritis: diarrhea with tenesmus, bloody or
blood-streaked and mucoid stools, vomiting, fever, abdominal
pain/cramps, and abdominal tenderness.
5. Laboratory Diagnosis
Specimen rectal swab of ulcer talem by sigmoidoscopy best specimen
- Feces usual specimen
- Specimen must be placed quickly on isolation media or transport medium
a. Stool examination
i. If toxigenic, no abnormal findings
ii. If invasive: pus cells, red blood cells, and macrophages are seen
b. Stained smears- not diagnostic
c. Culture
i. Colorless, transparent, colonies on EMB, MacConkey& SSA
ii. Reddish colonies in XLD
iii.
Green to blue green colonies in HEA
d. Biochemical characteristics
i. TSI K/A
ii. IMVIC V+-- : S. ambigua + S. flexneri are indole (+)
iii.
Lysine decarboxylase (-)
iv. Do not utilize acetate
e. Serotyping with polyvalent and specific antisera
6. Treatment
a. Amoxicillin or amoxicillin drug of choice
b. Cotrimoxazole alternative
c. Management of fluids and electrolyte imbalance
7. Control
a. Infected person should be isolated until culture is negative
b. Carriers should be treated and not allowed to handle food
c. Proper sewage disposal and chlorination of water

Salmonella
1. General characteristics
a. Nonencapsulated, nonsporeforming, gram (-) rods
b. All members are nonlacyose fermenters
c. All are motile except S.pullorum and S. gallinarum
d. All produce gas from glucose except S. typhi and S. gallinarum
e. Most strains produce H2S from thiosulfate
2. Classifications
a. According to Ewing and coworkers (based on O & H antigens known as the
Kauffmann-White antigenic scheme) there were only three species of Salmonella:
S. typhi (S.typhosa or Eberths bacillus) : all other species or serotypes were
identified as serotypes \s. enteritidis like:
i. S. paratyphi A
ii. S. paratyphi B or S. schottmuelleri
iii.
S. paratyphi C or S. hirschfeldii
iv. S. typhimurium
v. S. sendai
vi. S. pullorum
vii. S. gallinarum
viii.
S. derby
b. According to DNA hybridilization studies all Salmonella and organisms formerly
referred to as genus Arisona belong to the same species, S. enterica and now
classified as:

i.
ii.
iii.
iv.
v.
vi.
vii.

Salmonella subgroup 1, subspecie designation enterica

2,

salamae

3a,

arizonae

3b,

diarizonae

4,

houtenae

5,

bongori

6,

indica
Majority of human isolates belong to subgroup L.
3. Antigens produced
a. O antigen somatic antigen
b. H antigen flagellar antigen which exist in 2 phases:
i. Phase 1 (specific phase) shared by a few organisms and
react with homologous antisera
ii. Phase 2 (nonspecific phase) - shared by many organisms
and react with heterologous antisera
c. Vi (Virulence) antigen a type of capsular (K) antigen found in serotype typhi
which may prevent the intracellular destruction of the organism.
4. Factors Contributing to Virulence
a. Surface antigen
i. O antigen - somatic
ii. Vi antigen virulence
b. Invasiveness
c. Endotoxin
d. Enterotoxin similar to both the heat-labile (LT) and heat-stable enterotoxin (ST)
of E.coli
e. Cytotoxin
5. Clinical infection: Salmonellosis
a. Mode of transmission
i. Typhoid fever is transmitted by ingesting food or water contaminated with
the feces of a carrier, often a chronic carrier (person who has recovered
from typhoid fever ut harbors the organism asymptomatically in the
gallbladder for long periods of time) : silent carriers (asymptomatic
carriers without ever suffering the disease) also transmit the disease and
contribute to continued episodes of infections: humans carrier is the sole
source of the organism (S. typhi)
ii. Nontyphoidal salmonellosis is transmitted by ingestion of contaminated
food (usually eggs, poultry and beef products) and water; fecal-oral
routes : aside from the human source, animals, and animal products are the
major sources.
*NOTE: the mean infective dose to produce infection is 105-103 organisms
b. Three distinct clinical entities
i. Gastroenteritis
- most commonly causedby S. serotype typhimurium
- characterized by diarrhea, fever and abdominal pain
- self-limiting, last from 2-5 days
ii. Typhoid fever and other enteric fevers
- typhoid fever is the most severe enteric fever which is caused by
serotype typhi\
- other Salmonella, particularly serotypes paratyphi A and B, can also
cause enteric fevers with milder symptoms
- signs and symptoms lethargy, fever, malaise, constipation and body
aches and pain during the first week followed by sustained fever 104F,
tender abdomen with rose-colored spots and diarrhea during the 2nd and
3rd week
- complications intestinal perforation, severe bleeding, thrombophlebitis,
cholecystitis, pneumonia, and abscess formation

iii.

Septicemia with focal lesions

- frequently caused by serotype cholerasuis
- characterized by fever, chills, anorexia and anemia
- focal lesions may develop and produce 2 osteomyelitis, pneumonia,
pulmonary abscess, meningitis or endocarditis
6. Laboratory Diagnosis
Specimen: Blood during the first week
Urine during the first two weeks
Stool throughout the course of illness but more positive isolation during the
rd
3 week
a. stained smears- not diagnostic
b. culture:
i. EMS, MacConkey, Salmonella-Shigella (SS) agar- colorless, transparent
colonies
ii. XLD, Bismuth Sulfite Agar black colonies *BSA best medium for
isolation of S.typhi
iii.
Brillliant Green Agar slightly pink to white opaque colonies surrounded
by brilliant red medium. It is highly recommended for isolation of
Salmonella except S.typhi
iv. Hektoen enteric (HE) green to blue green colonies with black center
v. Enrichment media- selenite-F, gram negative (GN) broth
vi. Chromatic culture medium- (CAROM AGAR)
c. Biochemical characteristics
i. S. typhi
TSI- K/A + small amount H2S
IMVIC --+-Lysine decarboxylase (+)
ii. Other Salmonella subgroups 2-5
TSI K/A + gas + H2S
IMVIC - - + - +
Lysine decarboxylase (+)
d. Widal test fibrile agglutination test
i.
Done on the 8th or 10th day and repeated by the 4th week
ii.
(+) result; fourfold rise by the 4th week
e. FAT
f. ELiSA
7. Treatment
a. For gastroenteritis supportive therapy and maintaining fluid and electrolyte
balance; antibioyic treatment only prolongs the carrier state
b. For enteric fever or septicemia ampicillin or chloramphenicol is the drug of
choice: cotrimixazole(alternative)
c. For chronic carriers of typhi serotype - ampicillin (drug of choice) ;
cholecystectomy

8. Control
a. Proper cooking and storage of food
b. detection and treatment of carriers
c. oral vaccine of attenuated typhoid bacillus
Arizona (Salmonella enterica subgroup 3a-arizonae)
1.
2.
3.
4.

A. hinshawii type specie of genera

Associated with reptiles and birds
Can rarely cause disease in humans similar to salmonella infections.
Distinguished from other Salmonella species by their late lactose fermentation and
growth in sodium malonate medium (green-blue)
5. Biochemical Characteristics
a. TSI A/A + gas + H2S or K/A + gas + H2S
b. IMVIC - + - +

c. Lysine decarboxylase (+)

6. Treatment: ampicillin or chloramphenicol
Citrobacter
1. Has 3 species: C. freundii (type specie) C. diversus& C. amalonaticus
2. Morphologically similar to E.coli
3. Biochemical Characteristics (C.freundii)
a. TSI A/A + gas + H2S or K/A due to delayed lactose fermentation
b. IMVC = - + - +
c. Lysine decarboxylase (-)
d. Grows in KCN medium
e. Malonate (+)
f. Urease (+)

Table 13-1. Reactions of Citrobacter

Test
*Indole
*H2S
*Growth in KCN
*Malonate

C. amalonaticus
+
+
-

C. freundii
+
+
+

C.diversus
+
+

4. Disease Produced (Opportunistic Infection)

a. UTI caused by C. freundii and C. diversus
b. Neonatal meningitis and brain abscess C. diversus
c. Diarrhea enterotoxigenic C. freundii
5. Treatment: aminoglycosides, tetracycline, and chloramphenicol

Klebsiella
General Characteristics
- genus consist of 5 species: K.pneumoniae, K.oxytoca, K.planticola, K. terrigena, and K.group
47: two former species, K.ozanae and K. rhinoscleromatis, are biochemically inactive strains of
K.pneumoniae.
- all are encapsulated and nonmotile
- all are lactose fermenter except K. ozanae and K.rhinoscleromatis
- colonies are large, moist and mucoid
- they possess O and K antigen
- urease (+) and ornithine decarboxylase (-)
1. K.pneumoniae (Friedlanders bacillus or baciluusmucosuscapsulatus)
a. Type specie of the genera
b. Disease produced
i.
Community-acquired and nosocomial pneumonia
ii.
Can also cause UTI, wound infections, bacteremia, and meningitis
iii.
Tropical sprue caused by an enterotoxigenic strain
c. Laboratory diagnosis
i.
Stained smears

ii.
iii.

I.

Culture large, oist, mucoid colonies on EMB, MacConkey and XL, that
have a tendency to coalesce( (+) to strings test)
Biochemical characteristics
TSI = A/A + gas
IMVIC = - - + +
Urease (+)
Grows in KCN medium
Quellang reaction for presence of capsule
Serotyping

iv.
v.
2. K. oxytoca
- Resembles K.pneumoniae in its disease spectrum
- IMVIC = + - + +
3. K.ozanae
- May contribute to the condition called ozaena which is a chronic atrophic
rhinitis characterized by a fetid odor
4. K.rhinoscleromatis
- Causative agent of rhinoscleroma, a chronic, granulomatous infection of the
nasal passages, pharynx and larynx
5. K.planticola UTI, has been implicated in human urinary tract and wound infections
6. K.terrigena isolated only from the environment
7. K.group 47 isolated from resoiratory tract and blood
Enterobacter (formerly Aerobacter)
1. General Characteristic
a. Inhabits soils and water and , to a lesser extent , the large , the large bowels of man and
animals
b. Oftenly confused with klebsiella
c. Most species are rapid lactose fermenters and urease (+)
d. All are motile and with the exception of E. agglomerans ,
e. All are ornithine decarboxylase (+)
2.

Other Important Biochemical Reactions

a. TSI = A/A + gas

b. IMVIC = - - + +
c. KCN (+)
3. Diseases Produced (usually nosocomial)
Most of the infections are caused by E. cloacae followed by E. aerogenes and E.
agglomerans.
a.
b.
c.
d.
e.

UTI
Bacteremia
Tropical sprue caused by enterotoxigenic E. cloacae
Meningitis and brain abscesses
Wound infections

4. Members ( associated with human disease)

E. cloacae , E. aerogenes , E. agglomerans , E. gergoviae ,
E. sakazakki , E taylorae , E. asburiae , E. hormaechii

5. Treatment 2nd and 3rd generation cephalosporins

Serratia

1. General characteristic
a. Motile gram- negative rods which are nonlactose fermenters but sucrose fermenters
b. Found in soil and water and are associated with plants and animals
c. Majority of S. rubidaea and some strains of S. marcescens produce a non water soluble
red to pink pigment (Prodigiosin)
d. S. orodifera produces a very musty , pungent odor
2. Biochemical Reactions
a. TSI = K/A or A/A
b. IMVIC = - - + +
c. With the exception of S. fonticola , all are extracellular Dnase , lipase and
gelatinase positive and resistant to colistin and cephalotin
3.

Associated with nosocomial outbreaks of urinary tract and wound infections ,

pneumonia and septicemia , most of which are caused by S. marcescens.
4. Treatment : gentimicin ,amikacin , chloramphenicol , ciprofloxacin , cotrimoxazole are
effective against S. marcescens

Proteus
1. General Characteristic
a. Motile , pleomorphic , gram- negative rods which are nonlactose fermenters
b. All are urease and phenylalanine deaminase (+)
c. All produce a bluish gray confluent surface growth or translucent sheet of growth on
moist blood agar that gives off a burnt gun powder odor
2. Other Biochemical Reactions
a.TSI = K/A + GAS + H2S
b. IMVIC
i
P. vulgaris = + + - V (indole (+) member)
ii.
P. mirabilis = -+ V V
Ornithine decarboxylase
i.
P. vulgaris negative
ii.
P. mirabilis positive
3. Dienes Phenomenon
Different strains of proteus when inoculated separately on a culture media swarm
towards each other but they do not merge and are separated by a narrow demarcation
line between them.
4. Antigenic Structure
a. All posses O, H and K antigens
b. P. vulgaris have the same antigenic structure as rickettsia such that O antigens (OX19 , OX-K , OX-2) of some strains are used to detect the rickettsial antibodies in the
the Weil-Felix test.
5. Clinical Infections
a. Majority of human infections are caused by P. mirabilis.
b. It is the second leading causes of community- acquired UTI and is a major cause of
nosocomial infections.
6. Treatment : sensitive to ampicillin and cephalosporins

K.

Providencia
1. Biochemically related to proteus.
2. Consists of four species :

a.

P. alcalifaciens (type specie )

b.

P. stuartii

c.

P. rettgeri (Proteus rettgeri)

d.

p. rustigianii

3. All possess O , H , and K antigens

4. Biochemical Reactions

a. TSI = K/A
b.

IMVIC = + + - +

c. All produce phenylalanine deaminase

d.

P. rettgeri and some strains of P. stuartti are urease (+)

5 . Causes nosocomial infections and are resistant to multiple antibiotics.

L.

Morganella
1. M. morganii (Proteus morganii) is the only species.
2. Biochemical Reactions
a. TSI = K/A + gas
b. IMVIC = + + - c. Phenylalanine deaminase (+)
d. Urease (+)

M.

Yersenia ( Pandemic plague)

Only 3 species are pathogenic for humans : Y. pestis , Y. enterocolitica , and Y.

pseudotuberculosis. They are facultatively intracellular or primarily animal pathogens. Humans
are only accidental hosts. This genus was previously classified under pasteurella.
Y. pestis
1. Morphology
a. facultative anaerobic , nonmotile , gram- negative coccobacilli
b.shows marked bipolar staining with Waysons stain , staining the polar bodies blue and
giving the cells a safety pin appearance.
c. grows slowly on nutrient agar and rapidly on BAP producing non hemolytic
d. gram negative
e. microaerophilic organism (stalactite streamers)

2.

Cultivation

a. Optimal temperature for growth is 28 C

b. Can grow on ordinary laboratory media
c. On nutrient agar small mucoid colonies
d. On deoxycholate agar very small red colonies
e.

In old broth cultures surface growth pellicle with stalactite streamers

f. Anaerogenic and usually non lactose fermenters

3. Virulence- Associated Factors

a. Ca2+ dependency when deprived on Ca2+ the bacteria are stimulated to produce V
& W antigens and several outer membrane proteins (Yops)
b. V & W antigens render the organism less susceptible to intracellular killing
c. Outer membrane proteins
d. F-1 envelope antigen may resist phagocytosis
e. Pigment binding and iron-regulated surface proteins
f. Pesticin I (a bacteriocin) , coagulase and plasminogen activator promotes
invasiveness
g. Murine toxin , endotoxin , etc.

4. Disease Produced : Plague

-

Y.pestis is primarily pathogenic to rodents and humans are accidental host only

a. Mode of transmission (to humans)

i. via bites of infected rat fleas (Xenopsylla cheopis)
ii . by handling the carcass of an infected animal
iii. via infected aerosol droplets from person to person

b. Clinical forms

i.

bubonic plague
- characterized by infection and swollen lymph nodes (called buboes) which occur
most commonly in the groin and less frequently in the axillary and cervical nodes

ii.

The bubo may be preceded by prodromata of fever, chills, body malaise,

confusion, nausea and pains in the back and limbs

septicemic plague
-

Mortality rate (97-99%)

Prominent finding is disseminated intravascular coagulation (DIC) with

generalized Shwartzman phenomenon (presence of black areas of hemorrhages all
over the body)

Highly fatal

iii.

Pneumonic plague (the Black Death; Pandemic plague)

Mortality rate (85-89%)

Usually arises from septic embolization to the lungs

Transmitted by inhalation and is highly contagious

5. Laboratory Diagnosis

Specimens: aspirates from buboes , pus from flea bites area , sputum , throat swabs or
blood; these specimens should be placed in Cary- Blair transport medium

a. Stained smears by Wayson or Giemsa

b. Culture
c. Biochemical reactions

i. TSI = K/A
ii. IMVIC = - + - iii. urease (-)

d. Animal inoculation test

e. FAT
f. Serologic diagnosis

6. Treatment : plague vaccine

Y. pseudotuberculosis and Y. enterocolitica

1. Morphology & Biochemical reactions

a. Unlike Y. pestis , they are motile gram negative coccobacilli

b. TSI = A/A
c. Urease (+)
d. Y. enterocolitica ornithine decarboxylase = Voges-Pros-kaurer (+);
Y. pseudotuberculosis OD and VP (-)
Produce Hydrogen Sulfide

2. Disease Produced :

a. Yersiniosis a term denoting infection with Yersenia species other than Y. pestis ;
enteritis that mimics acute appendicitis
b. Mode of transmission : accidentally acquired via fecal-oral route
c. Clinical manifestation :

i.
involvement

Gastrotestinal symptoms including diarrhea and mesenteric lymphatic

ii.

headache , malaise and fever with convulsions

iii.

severe abdominal pain

iv. complications : septicemia and hepatic abscesses

3. Laboratory Diagnosis

Specimens : mesenteric lymp nodes , feces , blood , effusions , from serous cavities and
organ specimens

a. Culture
i. noncontaminated samples blood or nutrient agar
ii. for selective enrichment and holding specimens are placed in isotonic saline with
or without potassium tellurite and promptly refrigerated
iii. CIN agar for Y. enterocoloitica (cefsulodin-irgasan-novobiocin)

b. Biochemical test
c. Widal type agglutination test most specific serologic test wherein a titer of 1:160 or
greater is indincative of yersiniosis

4. Treatment
i. aminoglycosides and cotrimoxazole
ii. correction of fluid and electrolyte imbalance
iii. supportive theraphy

CHAPTER XIV
NONFERMENTATIVE GRAM-NEGATIVE BACILLI & COCCOBACILLI

Basis of classification
1. This group of organisms is differentiated from the Enterobacteriaceae in the
way they utilize carbohydrates. They do not ferment sugars (in the absence of
air ) but utilize them oxidatively (in the presence of air ) producing tiny
amounts of acid.

2. The Hugh & Leifsons Oxidative-Fermentative Medium

a. evolution of the medium

i. Hugh & Leifson realized that organism utilizing carbohydrates very

minimally produce tiny amounts of acid. The conventional fermentation
medium contains a high content of peptone (1%) which these organisms
utilize the small amount of acid produced, thereby no pH reaction is seen.

ii.
To detect acid production in these organism Hugh and Leifson
developed a low-peptone medium (0.2%) , the oxidative-fermentative
mediem (O-F medium)

b. Procedure and result

i.

colonies are inoculated by stabbing (4x at approx. 5mm deep)

into two tubes of 1% O-F glucose

ii. one of the media is overlayed with sterile melted petroleum or vaspar
(paraffin with petroleum jelly); this is the closed O-F medium and the
other one is the open O-F medium
iii. tubes are incubated at 35C for 4 days and examined daily for acid
production, that is a change in the bromthymol blue (pH indicator) from
green to yellow(positive reaction)

c. interpretation of results

i. fermenters organism that are able to ferment glucose in the closed

tubes and oxidize it in the open tubes

ii. oxidizers- organism that give a yellow reaction only in the open tubes
indicating oxidative utilization of glucose
iii.
nonoxidizers- do not utilize glucose , either fermentatively or
oxidatively.

PSEUDOMONAS

A. Pseudomonas aeruginosa

1. Morphology

a. Aerobic , motile with a single polar flagellum , gram negative rods that
occur singly , in pairs and occasionally in short chains
b. Produces an extracellular slime layer, similar to a capsule usually seen
in mucoid strains isolated from patients with cystic fibrosis
c. Frequently posses pili that promote attachment to host cell surfaces

2. Cultural Characteristics

a. Can grow on media for isolation of enterobacteria and sometimes on vibrio

isolation media due to its ability to tolerate alkaline conditions.
b. Grows well at 37-42C , its growth 42C helps differentiate at from other
Pseudomonas species
c. On BAP- large , flat ,beta hemolytic colonies with a feathered edge and ground
glass appearance
d. Colonies tend to spread to give off a characteristic odor resembling that of
overripe grapes (or corn tortillas) which is due to substance known as 2aminocetophenone
e. Most strains produce pyocyanin , a blue water soluble and chloroform-extractable
pigment , seen on uncolored media like :

i. Muller-Hinton agar
ii. Pseodomonas P. Agar
iii. Tech agar

*NOTE : This characteristic is unique to P. aeruginosa only , thus it is called the

agent of the blue pus

f.

some strains produce other pigments :

i. pyoverdin (fluorescein) yellow to green water soluble pigment

ii. pyorubin- red pigment
iii. pyomelanin- brown to black

3. Virulence Factor
a. Motility
b. Presence of glycocalyx (slime layer)
c. Pili of fimbriae
d. Endotoxin
e. Extracellular enzymes including elastases, proteases , and 2 hemolysin; a heat
labile phospholipase C and a heat-stable glycolipid
f. Exotoxin A- causes tissue necrosis
g. Exoenzymes S- play a role in necrotic injury
h. Cytotoxin and enterotoxin

4. Diseases Produced

P. aeruginosa inhibits soil and water and causes disease in human with impaired host
defenses.

a. Burn wound infections; traumatic and operative wound infections

b. Nosocomial infections like pneumonia (especially in cystic fibrosis patients) , UTI
;endocarditis , osteomyelitis , etc.
c. Eye infections seen in contact lens wearer
d. Dermatologic infections

5. Laboratoty Diagnosis

a. Flagella media
b. Culture (isolation of organism)
i. selective media
= Pseudosel agar- containing cetrimide (cetrimethyl ammonium bromide)

=irgasan agar
=30 ug/ml C-390
ii. media enhancing fluorescent pigment production
=Pseudomonas F agar
=GNF agar
=Flo agar
c. biochemical reactions
i. TSI-K/K with a metallic sheen on slant surface
ii. open OF medium (+) & closed OF medium (-) utilizing glucose oxidatively
iii.oxidase and catalase (+)
iv. unable to oxidize lactose, sucrose and maltose
v. unable to decarboxylate lysine or ornithine
vi. able to dihydrolyze arginine
d. serotyping of O antigen, bacteriophage typing and pyocin typing
- for epidemiologic purposes
6. Treatment
a. aminoglycosides (amikacin, gentamicin,& tobramycin)
b. extendedspectrum penicillins (azlocillin, carbenicillin)
c. 3rd generation cephalosporins (ceftazidime and cefoperazone)
d. quinolones and carbapenems

B.Pseudomonas cepacia
-most commonly isolated from cystic fibrosis patents and has been associated with
endocarditis,
septicemia and wound and urinary tract infections in immunocompromised patients
-colonies are yellow-yellow green, rough with serrated edges which do not fluoresce
-strains are motile and able to oxidize glucose, lactose, maltose and mannitol; lysine
decarboxylase
(+)
-resistant to polymyxin B and sensitive to chloramphenicol, cotrimoxazole and ceftazidime

C. Pseudomonas mallei (Glanders bacillus)

-causes

glanders, a disease of horses occasionally transmitted to humans by direct

contact
through skin abrasions and inhalation of organism
-they are small, nonmotile, aerobic, pleomorphic, cocoid to rod shaped organisms which are
oxidase negative
-colonies on heart-infusion agar are grayish white and translucent later becoming
yellowish and
opaque

-Straus test: test use to diagnose glanders, wherein male guinea pigs are inoculated
intraperitoneally with suspected material; within 2-3 days the animals develop
orchitis (tender swollen scrotum), with tumefaction and purulent inflammation of
testicles
-glanders is treated effectively with tetracycline plus an aminoglycoside

D.Pseudomonaspseudomallei(Whitmores bacillus)

-causes melioidosis, a glanderslike disease in humans transmitted via inhalation,

ingestion or
through skin abrasions
-the disease occurs in four forms: acute, subacute, chronic and latent, which may
abscesses
and septicemia; both the chronic and latent forms may be reactivated to a symptomatic form
after many years,thus nicknamed Vietnamese timebomb
-they are small, motile with polar tufts of flagella, aerobic, gram negative rods
-colonies are wrinkled at first becoming umbonate exhibiting varying degrees of
hemolysis with a characteristic putrid odor followed by an aromatic, pungent odor
-treatment of choice is cotrimoxazole; other derivatives are tetracycline, chloramphenicol
and sulfadiazine

E. Xanthomonasmaltophilia (Pseudomonas maltophilia)

-important causes of hospital-acquired infections in immunocompromised patients

-they are motile with multitrichous polar flagella
-on BAP; colonies are rough, lavender-green or gray which may be slightly alphahemolytic and have an ammonia-like odor; unable to grow on centrimide
-they are oxidase (-), arginine dihydrolase (-), ornithine decarboxylase (-) but lysine
decarboxylase (+)
-susceptible to trimethoprim-sulfamethoxazole

F. Other Pseudomonas
-represent contaminants or colonizers but may cause opportunistic infections
ALCALIGENES
1. Opportunistic pathogens isolated from blood , respiratory secretions, infected wounds, urine
and other sources

2. Consist of Four Species

a. A. faecalis (A. odorans)-produce a sweet odor, reminiscent of fresh apple cider

b. A. xylosoxidans subsp. xylosoxidans (Achromobacterxylosoxidans)
c. A. xylosoxidans subsp. dniterificans
d. A. piechaudii

3. General Characteristics

a. possess peritrichous flagella

b. on blood agar, colonies are flat, spreading and rough with a feathery edge
c. oxidase and catalase (+)
d. open OF medium (-) & closed OF medium (-)
e. asaccharolytic and utilizes acetate as sole carbon source

ACINETOBACTER

1. Commonly found in soil and water but occasionally found on skin and mucous membranes of
healthy persons.

2. Clinically important species: A. calcoaceticus

2 biovars:
a. A. calcoaceticus var. anitratus (Herelleavaginicola) - oxidizer
b. A. calcoaceticus var. Iwoffi- ( Mimapolymorpha) nonoxidizer

3. General Characteristics

a. aerobic, nonmotile, plump, diplococcoid rods resembling Neisseriae on smears

b. colonies on BAP are convex, gray to white and variably hemolytic
c. oxidase negative and catalase positive
d. do not reduce nitrates

4. Occasionally causes nosocomial infection and may be involved in wound infections, UTI,
sepsis and other infections

5. Treatment: gentamicin, amikacin or tobramycin and cephalosporins

MORAXELLA

General Characteristics

1. Nonmotile, nonpigmented, gram-negativecoccobacillithat do not utilize carbohydrates and

are oxidase positive.
2. All are normal flora of the mucous membranes of humans and other animals.
3. Isolated as etiologic agents of nosocomial and wound infections, pneumonia and other
opportunistic infections.
4. All are susceptible to penicillin.

A. Moraxella lacunata (MoraxAxenfeld bacillus or Haemophilus duplex)

1.Isolated from patients with blepharoconjunctivitis.

2. Fails to grow on McConkey agar but may produce pitting of blood agar.

B. Moraxellaosloensis

1. Oftenly mistaken for Neisseria gonorrheae.

2. Differentiated by the cysteine trypticase agar (CTA) glucose medium:

a. N. gonorrheae ferments the medium

b. M. osloensis does not ferment the medium

C. Moraxella urethralis( named Oligellaurethralis)

1. Small coccoid bacilli that are asaccharolytic, oxidase and catalase positive.

D. Moraxella phenylpyruvica

1. Urease and phenylalanine deaminase (+)

EIKENELLA (E. corrodens)

1. Normal flora of mouth and gastrointestinal tract.

2. Morphology: small, even, straight-sided, capnophilic, facultatively anaerobic gram-negative

bacillus

3.Cultivation

a. blood is required for growth and chocolate agar is the most supportive medium.
b. colonies are tiny with pitting (ability of organism to corrode the agar), yellowish, opaque and
consist of three distinct zone of growth:

i. moist, shiny central zone

ii. refractile circle of growth with a pearly sheen, resembling a mercury drop
iii. outer flat, rougher spreading zone

c. colonies emit a bleach like odor

d. in broth form discrete granules that are often adherent to the sides of the tube

4. Biochemical Reactions

a. oxidase (+) but unable to utilize carbohydrates

b. catalase, urease, indole and arginine dihydrolase (-)
c. lysine decarboxylase and nitrate (+)

5. Common etiologic agents of human bite and clenched fist wounds.

6. Susceptibility

a. susceptible to ampicillin and newer penicillin and cephalosporins

b. resistant to clindamycin

FLAVOBACTERIUM

1. The most clinical significant specie is F. meningosepticum, an etiologic agent of neonatal

meningitis and sepsis.

2. Morphology

-Long, thin, filamentous, nonmotile gram negative bacilli with occasional swollen ends.

3.Cultivation

a. on BAP yellow pigmented (flavin) colonies usually surrounded by lavender-green

discoloration due to extensive proteolytic enzyme production
b. can grow on McConkey agar

4. Biochemical Reactions: oxidase and catalase (+)

5. resistant to penicillins and polymyxins.

WEEKSELLA(W. zoohelcum)

-nonmotile, gram-negative bacillus that is nonsaccharolytic, unable to grow on McConkey agar

and urease positive
- with sticky colonies on rabbit blood agar
- associated with animal bite wounds and scratches

CHAPTER XV
GRAM-NEGATIVE FACULTATIVELY ANAEROBIC BACILLI

AND AEROBIC COCCOBACILLI

FRANCISELLA (F.tularensis)

1. Morphology

a. small, highly pleomorphic, nonmotile, encapsulated, weakly staining gram-negative rods

b. strictly aerobic and are found intracellularly
c. with bipolar staining on Giemsa stain

2. Cultavation

a. requires cysteine or cysteine for growth

b. culture media

i. glucose-cysteine-blood agar
ii. peptone-cysteine agar
iii. cystine heart agar
iv. chocolate agar
v. rarely on Thayer-Martin agar

c. does not grow on McConkey agar

d. colonies are transparent droplike, mucoid and easily emulsified

3. Virulence Factors

a. being an intracellular parasite it is able to survive in the reticuloendothelial cell

b. invasiveness
c. capsule

Strains of F. tularensis

biovartularensisbiovarpalaearctica

i. or Jellison type A

i. or Jellisons type B

ii. highly virulent

ii. less virulent

iii. oftenassociatd with tick-borne disease

iii. associated with water-borne disease

of rabbits
iv. exhibitcitrullineureidaseactivity
v. ferment glycerol

of rodents
iv.do not exhibit citrullineureidase activity
v. seldom ferment glycerol

vi. seen mostly in North America

vi. seenmostky in eastern US, Europe,

Russia,aand the Far East

5. Disease Produced:

A disease of rodents (particularly rabbits) that is usually seen in hunting men. It is also
one of the most common laboratory acquired infections.

a. mode of transmission

i. contact with infected animals (usually rabbits)

ii. bite of an arthropod vector (deerflies and ticks)
iii. ingestion of infected animals
iv. inhalation

b. clinical forms

i. glandular presents with adenopathy and systemic manifestations

ii. ulceroglandular most common form; with ulceration on the site of inoculation and
regional lymphadenitis; with systematic manifestation such as anorexia,
back pain, headache, chills, and fever, sweating and prostration;
pneumonia may also occur.

iii. oculoglandularsimilar to ulceroglandular with the conjunctival sac as its primary source
of inoculation.

iv. typhoidal characterized by general and severe systematic symptoms;

hepatosplenomegaly is common.

6.Laboratory Diagnosis

Specimens: blood, pharyngeal secretion, gastric aspirates, pleural fluid, tissue secretions,
exudates, etc.

a. generally smears and culture are not contributory and the diagnosis relies on serological
tests.

b. serologic tests

i. direct and indirect fluorescent antibody staining best technique for rapid specific
diagnosis from exudates or tissues impressions

ii. agglutination test (Foshays antiserum test) commonly used serologic method

iii. ELISA
iv. lymphocyte stimulation assay
v. tularemia skin test

c. intraperitoneal inoculation of guinea pigs for demonstration of virulence

d. biochemical reactions

i. weakly catalase positive

ii. oxidase negative

7. Treatment

a. streptomycin drug of choice

b. gentamicin and tetracycline alternatives

8. Prevention

a. avoidance from infected animals, protection from biting insects and provision of clean
watersupplies

b. immunization (by multiple puncture) with live attenuated strain of F. tularensis for all
laboratory personnel handling culture of organisms

BRUCELLA

1. Pathogenic Specie

Preferred

CO2

H2S

Host

Req.

Prod.

Growth on media with

ThionineFuchsin
(1:25,000)

B. abortus

cattle

(1:50,000)

goats, sheep

B. suis

swine

B. canis

dogs

(Bangs bacillus)

B melitensis

2. Normal flora of genital and urinary tracts of animals such as goats, sheeps, pigs, cows and
dogs.

3. Morphology

a. strictly aerobic, small, nonmotile, nonsporeforming, gram-negative coccobacilli

b. capsules can be demonstrated on smooth and mucoid variants

4. Cultural Characteristics

-colonies are small, convex, smooth, translucent and slightly yellow to opalescent which may
become brownish with age.

5. Primary Virulence Factor: ability to survive intracellularly

6. Disease Produced: Brucellosis (Undulant or Malta Fever)

a. mode of transmission

i. contact with infected tissues of animals (common among abattoir workers, farmers and
veterinarians
ii. ingestion of infected milk and milk products
b. pathogenicity

i. B. abortus causes mild disease without suppurative complication and is self-limited;

noncaseating granulomas of the reticuloendothelial system are found
ii. B. canis also causes mild disease
iii. B. suis chronic with suppurative lesions
iv. B. melitensismost invasive and is more acute and severe

c. clinical manifestations

i. gradual onset of nonspecific systemic symptoms including fever, malaise, chills, sweats,

anorexia and fatigue

ii. may be accompanied by associated mental depression and increase nervousness
iii. localized lesion are granulomatous or suppurative and destructive which may occur in
any organ or bone
iv. some may present with lymphadenopathy or splenomegaly
7. Immunity
After natural infection in both humans and animals, an initial IgN antibody response,
followed by an IgG antibody response is observed.

8. Laboratory Diagnosis
Specimens: blood (specimen of choice), bone marrow, material from lymph nodes, other
tissue, CSF, and urine.

a. Culture
i.

ii.

Cultures should be incubated in 5-10% CO2 humidified atmosphere at 3537C and should be retained for 3-4 weeks before being discarded as
negative.
Culture Media
- Castaeda biphasic medium (for blood and other body fluids)
- Trypticase soy agar
- Tryptose agar

b. Serologic Test
i.
particle agglutination test with antismooth Brucella serum (most rapid
presumptive identification test)
ii.
ELISA
iii.
2-mercaptoethanol test
iv.
Complement fixation
v.
Fluorescent antibody staining

c. Skin test (using brucellin and aborsin)

d. Abortus bang ringprobe test (ABR test)
e. Biochemical reaction
i.
catalase and oxidase (+)
ii.
urease and nitrate (+)

9. Treatment
a. Doxycycling plus rifampicin for 30 days- for adults
b. Cotrimoxazole for 3 weeks and gentamicin IM for 5 days- in children less than
8 years old.
c. Doxycycling or oxytetracycline for 3 weeks with gentamicin- more than 8 years
old.

10. Prevention
a. Animal immunization
b.

BORDETELLA
Bordetella pertussis (Bordet-Gengou bacillus)

1. Morphology
a. Slightly aerobic, nonmotile, small, gram-negative coccobacilli that occurs
singly, in pairs and in small clusters.
b. Capsules are produced and demonstrated only by special stains.
c. Bipolar metachromatic granukes can be demonstrated with toluidine blue
stains.

2. Cultural Characteristics
Colonies are pinpoint, smooth, convex, glistening with a pearly luster, resembling
mercury drops and show a diffuse zone
of hemolysis.

3. Virulence Test
a. Portussis toxin - an exotoxin promotes lymphocytosis
-role in adhesion to ciliated epithelial cells.
b.

Filamentous hemagglutinin- mediates adhesion to ciliated epithelial cells.

c.

Adenylate cyclase

d.

dermonecrotic toxin (heat-labile)

e.

Lipopolysaccharide (heat-stable toxin)

f.

Tracheal cytotoxin- inhibits DNA synthesis in ciliated cells.

g.

Pertactin

h.

Capsule of virulent strains.

4. Disease Produced: Pertussis or Whooping Cough

a. Mode of Transmission: Via respiratory route.
b. Stages of disease
i.
Catarrhal (prodromal) stage
- With mild coughing and sneezing
- Patient is infectious but not very ill

ii.

paroxysmal stage

-Cough develops its explosive character (5-20 forcible

hacking coughs/15-20
seconds)
-With the characteristic whoop upon inhalation
-This leads to rapid exhaustion and may be associated with
iii.
iv.

convalescent stage (Cell limiting)

Complication: Rarely and encephalitis

5. Laboratory Diagnosis:
Specimens: nasopharyngeal swab (most commonly
nasopharyngeal aspirates, cough droplets expelled onto a cough plate.

recommended),

a. Culture Media
i.
Bordet-Gengou agar (potato-glycerol-blood agar)
ii.
Jones-Kendrick charcoal agar- for transport and cultivation
iii.
Regan-Lowe (half-strength charcoal agar medium with horse blood and
cephalexin)- as transport and selective enrichment medium
iv.
Buffered-charcoal-yeast-extract
v.
Cold casein hydrolysate medium
vi.
Casamino acid broth
vii.
modified Stainer- Scholte agar with cyclodextrin and cephalexinpreferred for 1 isolation
b. direct fluorescent antibody staining
c. Slide agglutination test
d. ELISA
e. biochemical reactions
i.
oxidase (+)
ii.
urease (-)

6. Treatment
a. erythromycin- drug of choices
b. tetracycline, chloramphenicol or cotrimoxazole- alternative

7. Prevention
-Active immunization with killed phase I B. pertussis combined with diphtheria
and tetanus toxiods (DPT)

B. Bordetella parapertussis
-Nonmotie, urease and oxidase positive
-Can cause mild respiratory disease similar to B.pertussis

C. Bordetella bronchiseptica
-Motile with lateral flagella
-Urease and oxidase negative
-Can cause mild respiratory disease similar to B. pertussis

HAEMOPHILUS
General Characteristics
Small, pleomorphic, nonmotile, aerobic, gram- negative bacilli.
Require the following factors present in the blood adequate growth:

a. X-Factor- a heat stable substance known as hemin associated with hemoglobin

b. V-Factor- a heat labile substance known as nicotinamide adenine dinucleotide (NAD)
or coenzyme I which can be supplied
by yeast, potato extract and several
bacteria such as Staphylococci, pneumococci and neisseria (usually applied by s.
aureus)

H. influenza (Pfeiffers bacillus)

1. Morphology
a. small, pleomorphic, nonmotile gram-negative coccobacilli
b. may exisr in an encapsulated or nonencapsulated type
c. exhibit bipolar staining with Gram stain

2. Cultural Characteristics
a. colonies usually are very small and dewdrop-like, transparent and colorless
with a distinct mousy or bleachlike
odor.
b. exhibits the satellite phenomenon wherein colonies of H. influenza grow
luxuriantly next to the S. aureus streak
(due to the production of V factor)

3. Virulence Factors
a. capsule- associated with H. influenza type b invasiveness
b. outer membrane proteins- responsible for attachment, invasiveness and
resistance to phagocytosis
c. lipooligosaccharide (LOS)- exerts a paralyzing action on the ciliated respiratory
epithelium & promotes
proliferation of the organism
d. adherence
e. IgA proteases- hydrolyze human IgA

4. Clinical Infection
a. Mode of transmission

- inhalation of infected droplets from clinically active cases, comvalescent

patients and asymptomatic
carriers who harbor the organisms in the
nasopharynx.
b. Diseases produced (H. influenza type b most common pathogen)
i. acute bacterial meningitis in infancy and early childhood
ii. acute bacterial epiglotittis
iii.pneumonia
iv.bacteremia without focus
v.cellulitis
vi.pericarditis
vii.otitis media
viii.septic arthritis
5. Immunity ( Hib vaccine= 2mos)
a. passive immunity in neonates from the mother
b. natural immunity is usually acquired by 8 years of age
6. Laboratory Diagnosis
Specimens: CSF, blood, middle ear exudates, joint fluids, respiratory tract
specimens
a. Gram-stained smears and quelling reaction
b. culture
i.
facultative anaerobe that grows best under aerobic condition
ii.
fastidious organism requiring both X and V factors for growth
iii.
culture media
- chocolate agar- contains X & V factors
- BAP of plain agar with commercial X, V and XV strips or disk
- Levinthal and Fildes enriched media
- horseblood- bacitracin by Klein & Blazevic (selective)
c. antigen detection
i.
counter immunoelectrophoresis (CIE)
ii.
latex particle agglutination
iii.
ELISA

d. Porphyrin or ALA (delta-aminolevulinic acid) test for X factor requirementpresence of brick red to orange
fluorescence indicates that porphyrins
were produced and that the organism
does not require X factor.
8. Treatment
a. chloramphenicol alone or in combination with ampicillin- for meningitis and
epiglotittis
b. ampicillin or amoxicillin- drug of choice for otitis media in children;
alternatives are cotrimoxazole, cefaclor or
penicillin with sulfonamide.
c. ampicillin- for sinusitis
d. passive immunotherapy
9. Prevention
a. active immunization with purified capsular polysaccharide
b. passive immunization with intramuscular human hyperimmunoglobulin

B. H. aegyptius (Koch-Weeks bacillus)

- associated with a communicable purulent conjunctivitis (pink eye)

- now part of H. influenza sometimes called H. influenza biotype III

C. H. parainfluenzae
- normal flora of mouth and nasopharynx
- infection after dental disease, dental procedures or other oral trauma
- ampicillin alone or with gentamicin is the recommend treatment

D. H. ducreyi
- sexually transmitted
1. Morphology: pleomorphic gram-negative coccobacilli which may occur extracellularly or
intracellularly.

2. Cultivation
a. requires X-factor for growth
b. media: chocolate agar medium containing vancomycin

3. Disease Produced: Chancroid (soft chancre)

4. Transmission: sexually

5. Clinical Manifestations
a. male ragged ulcer in the genitalia
- with enlarged painful inguinal lymph node
- multiple lesions occur by autoinoculation
b. female- asymptomatic or with mild vaginitis

6. Laboratory Diagnosis
a. Gram or Giemsa-stained smears of ulcer exudates or bubo aspirate showing the classic
microscopic appearance of a school of a
red fish
b. Ducreyis skin test
- a nonspecific test which is positive 1-2 weeks after injection and may remain
positive for years.

7. Treatment
- Peniciliin- drug of choice

FIGURE 15.1
Potential Properties of Haemophilus Species that colonize Humans
Species

V
Factor X
Factor Increased
Req.
Req.
Co2 Req.

Hemolysis

Acid fr. DXylose

Influenzae
Aegyptius

+
+

+
+

+
-

Hemolyticus
Ducreyi

+
-

+
+

Parainfluenzae
Parahaemolyticus

+
+

+
+
+

+
-

Paraphrohaemolyticu
s
Hrophilus
Paraphrophilus
H.Agnis

+
-

+
+

+
-

+
+

+
-

ACTINOBACILLUS ( A. actinomycetemcomitans)
Most members are pathogens and commensal organism in domestic mammals birds. Only
A. actinomycetemcomitans is a human parasite.

Normal flora of human oral mucosa.

1. Morphology
-gram- negative bacilli, often displaying cocoid forms which may be located at the end of
bacilli, giving the overall appearance of dots and dashes of Morse code.

2. Cultivation
a. grows on BAP and CAP but not on McConkey Agar
b. growth is enhanced in a CO2 incubator or candle jar.
c. in blood cultures bottles- microcolonies may be seen as tiny puffballs growing on the
blood cell layer or as tiny puffs on the sides of
the bottle.
d. on BAP- rpugh and sticky colonies surrounded by a light greenish tinge

e. on brain-heart infusion agar- four to six pointed starlike configuration in the center of
the colony.

3. Diseases Produced
a. often found in actinomycosis
b. severe periodontal disease in adolescents
c. endocarditis
d. abscesses, osteomyelitis and other infections.

4. Treatment
a. tetracycline or chloramphenicol usually
b. penicillin G, ampicillin or erythromycin sometimes

PASTEURELLA
Primarily parasites of domestic and wild animals and birds but may also produce disease
in humans. P. multocida is the most frequently associated with human infections.

1. Morphology
a. small, nonmotile, facultatively anaerobic, coccobacillary or rod shaped
organisms that occur singly, in pairs or in short chains and often exhibit
bipolar staining.
b. some strains show pleomorphism
c. virulent organisms produce capsule
2. Cultural Characteristics
a. does not grow in MacConkeys agar
b. on BAP colonies are small and translucent with a brownish discoloration of
the medium and amiting a musty or mushroom-like

3. Virulence Factors
a. capsule- major virulence factor
b. somatic antigens
c. endotoxin
d. neuraminidase and hyaluronidase
e. fimbriae

4. Clinical Infection
a. mode of transmission

i.
majority are through dog or cat bite or scratch
ii.
possibly inhalation
b. diseases produced
i.
wound infection from animal bites that may progress to
pyarthrosis, necrotizing synovitis and osteomyelitis
ii.
infection of lung in patients with preexisting chronic pulmonary
disease (usually lower respiratory tract disease)
iii.
other foci of disease that are secondary to septicemia like
meningitis, cerebellar abscess and infectious endocarditis.

5. Laboratory Diagnosis
Specimens: Sputum, bronchial washing, nasal swabs, purulent exudates from
animal bites, spinal fluid, blood, joint fluid, biopsy specimens from osteomyelitis
patients, etc.
a. stained smears
b. culture- most important
c. biochemical reactions
i.
catalase and oxidase (+)
ii.
ferments glucose, mannitol and sucrose
iii.
nitrate and spot indole (+)

6. Treatment
a.
b.
c.
d.
e.

f.
g.

h.
i.
j.

k.
l.

m.

penicillin- drug of choices

tetracycline, chloramphenicol, ampicillin & cephalotin- alternatives
KINGELLA
1. Contains 3 species which are normal flora of the oropharynx;
i. K. denitrificans
ii. K. indologenes rarely isolated from eye infection
iii. K. kingae etiologic agent of bacteremia, skin lesions and
septic arthritis
2. Laboratory diagnosis
a. stained smears: short, plump gram-negative bacilli with square
off ends that may occur in
chains
b. culture
i. K. kingae
-an increased CO seems to enhance growth
-colonies on BAP are small with a clear zone of hemolysis
-colony morphology may vary; one type pits the agar giving
a fried egg
appearance with a thin, spreading haze of growth
surrounding the central colony.
ii. K . denitrificans
-nonhemolytic colonies on BAP
-grows in Thayer-Martin medium (may be mistaken for N.
gonorrhea since it is
also oxidase (+) and a glucose fermenter; but most K.
denitrificans are nitrate (+)
while N. gonorrhea is nitrate (-)
c. biochemical reactions

n.

i.

all are oxidase (+) and non motile and able to ferment

ii.

Most are catalase negative

glucose

o.
p.
q.
r.
s.
t.

u.
v.

iii. Only K. indologenes is indole (+)

iv. None produce acid in the butt of TSI
3. Treatment
a. penicillin drug of choice
b. gentamicin and chloramphenicol alternatives
CAPNOCYTOPHAGA
1. Normal flora of the oral cavity
2. Morphology
a. fastidious, capnophilic, flexible, fusiform, shaped, gramnegative bacilli with one
rounded end and one tapered end and occasional
filamentous form.
b. with a gliding motility
3. Cultivation
a. does not grow on MacConkey agar
b. cultures are incubated at 37C in 5-10% CO
c. colonies on BAP are opaque, shiny and nonhemolytic, with a
pale beige or yellowish color
d. gliding motility is observed as outgrowths from the colonies or
as a haze of the agar surface

w.
x. -120y.
z. 4.
Biochemical reactions
aa.
a.
there are 3 species: C. ochracea (formerly bacteriodes), C.
sputigena, and C. gingivalis
b.
they are oxidase, catalase and indole(-)
c.
glucose and sucrose fermenters
d.
urease, OD and LD(-)
ab. 5. Virulence Factors
ac.
a. proteases
b. heat-stable factor that alters polymorphonuclear neutrophil
chemotactic activity
ad. 6.
Diseases Produced
ae.
a.
periodontal disease; periodontal lesions in juvenile diabetes
patience
b.
sepsis in leukemia and granulocytopenic patients
c.
osteomyelitis, septic arthritis, endocarditis and soft tissue
infections
af. 7.
Treatment
ag.
a.
susceptible to penicillin, carbenicillin, clindamycin, and
erythromycin
b.
resistant to aminoglycosides
ah. CARDIOBACTERIUM (C. hominis)
ai. 1.
Normal flora of the upper respiratory tract, mouth and maybe
other mucous membranes
aj. 2.
Morphology
ak.
-pleomorphic, microaerophilic, non motile, gram-negative
bacillus that tends to form rosette clusters
or a serpentine pattern
al. 3.
Cultivation
am.
a. grows well on BAP, CAP, trypticase soy agar or heart
infusion agar
b. does not grow in McConkey agar
c. cultures are incubated at 37C in 5% CO
d. colonies are slightly alpha-hemolytic, smooth, round,
glistening and opaque
an. 4.
Biochemical Reactions

ao.

ap.
aq.
ar.
as.

a. oxidase (+) indole (+)

b. utilizes carbohydrates
c. urease, catalase and nitrate (-)
5.
Diseases Produced
a. bacteremia in compromised individuals
b. endocarditis
6.
Treatment
a. penicillin drug of choice
b. also susceptible to ampicillin, cephalotin,
chloramphenicol, aminoglycosides and tetracycline

at.
au. -121-

av. CHAPTER XVI

aw. ANAEROBIC GRAM NEGATIVE BACILLI
ax.
ay. A. BACTEROIDES (B. fragilis)
az.
The B. fragilis group contains 5 species namely: B. fragilis,
B. thetaiotaomicron, B. distasonis,
B. vulagatus. The most common isolate from infections is B.
fragilis, but it is also present in lower
concentrations in the normal fecal flora.
ba.
1. Morphology
bb.
- pleomorphic, encapsulated, nonmotile, gram-negative bacilli
with vacuoles and swellings
bc.
2. Cultural Characteristics
bd.
a. colonies are low convex, white to gray, semiopaque an d
glistening
b. some strains may be hemolytic
c. growth is stimulated by bile
be.
3. Virulence factor
bf.
a. capsule
b. enterotoxin
c. enzymes such as neuraminidase, DNase, hyaluronidase,
gelatinase, fibrinolysin, superoxide
dismutase and heparinase
bg.
4. Disease Produced
bh.
a. intra-abdominal infections
b. septicemia
c. genital tract infections
bi.
5. Laboratory diagnosis
bj.
a. stained smears
b. demonstration of capsule with:
bk.
i. Ruthenium red staining
ii. India ink
iii. Electron microscopy
bl.
c. culture see p. 42
d. biochemical reactions:
bm.
i. superoxide dismutase (+)
ii. Catalase (+)
bn.
e. serologic test
bo.
6. Treatment
bp.
a. susceptible to metronidazole, chloramphenicol, broad
spectrum penicillins, cefotoxin and imipenem
b. resistant to penicillin, cephalosporins and tetracycline
bq.
br. -122bs.
bt.
bu. PIGMENTING GRAM-NEGATIVE BACILLI (formerly B.
melaninogenicus)
bv.
This group includes two genera: Porphyromonas and Prevotella
however the latter genus also contains some nonpigmented species.
bw.1. Natural Habitat and Diseases Produces
bx.
a. Prevotella melaninogenica, P. denticola, P. loescheii, P.
intermedia, Porphyromonas endodontalis and
P. gingivalis are indigenous in the gingival crevice and most can
cause dental infections like periodontitis
and can occur in infections involving the head, neck and
respiratory tract.

by.

b. Porphyromonas asaccharolytica and Prevotella corporis are

normally found in the lower genital tract and
are seen in nonoral sites of infection.
bz. 2. Morphology
ca.
- small, nonmotile, straight long rods with rounded ends that may
also appear pleomorphic
cb. 3. Cultural Characteristics
cc.
a. colonies on BAP are usually convex, smooth, circular, sometimes
beta-hemolytic and pigmented tan to
black
b. vitamin K and hemin are required for growth
c. all are sensitive to bile
cd. 4. Virulence Factors
ce.
a. collagenase
b. capsule
cf. 5. Laboratory Diagnosis
cg.
a. stained smears
b. culture
c. biochemical test
ch.
- P. malaninogenica (B. melaninogenicus)
ci.
i. does not ferment glucose
ii. Fluoresces brick red under UV light at 366 nm
cj.
d. serologic test
ck. 6. Treatment
cl.
a. susceptible to motronidazole, chloramphenicol, clindamycin,
carbonicillin and cefoxitin
b. resistant to penicillins and cephalosporins
cm.C. FUSOBACTERIUM
cn.
The most commonly encounterd isolate in human infections is F.
nucleatum followed by F. necrophorum.
co.
1. Natural Habitat and Disease Produced
cp.
a. F. nucleatum
cq.
- normal flora of the mouth and occasionally found in the
urogenital tract; associated with oral
infections, lung abscesses other pleuropulmonary infections
and amniotic fluid infections.
cr.
cs. -123ct.
cu.
b. F. necrophorum
cv.
-important animal pathogen found in human infections
particularly abdominal infections and
liver abscesses
cw.
2. Morphology
cx.
a. F. nucleatum
cy.
-nonmotile, thin with pointed or taperd ends and may
resemble scattered wheat straw or appeared
very long, thin filaments
cz.
b. F. necrophorum
da.
-nonmotile, bread with rounded ends and may be short, long
or filamentous, often with bulbous
swellings and rounded bodies
db.
3. Cultural Characteristics
dc.
a. F. nucleatum
dd.
- colonies are alpha-hemolytic, convex and translucent, with
internal flecking or mottling; or more
umbonate, heaped, dull and opaque; they are also described
as white bread crumb-like colonies

de.
df.
dg.
dh.

b. F. necropharum
-umbonate colonies that may be alpha- or beta-hemolytic
4. Identification Characteristics
a. both produce large amounts of butyric acid
b. both are resistant to vancomycin and susceptible to
kanamycin and colistin
c. both are indole positive
d. F. necropherum is lipase (+)

5. Treatment
- susceptible to penicillin G, cephalosporins, metronidazole and
chloramphenicol

CHAPTER VII
VIBRIONACEAE
GENERAL CHARACTERISTICS
1. Gram-negative facultative organisms that are oxidase positive (except V. metschnikovii)
2. Most are motile with a polar flagella.
3. Naturally found in seawater (more commonly, Vibrio) and fresh water (more frequently,
aeromonas and
plesiomonas)
4. Causes sporadic cases or diarrhea and soft tissue infections.

VIBRIO
The two most important members of this genus are V. cholera and V. parahaemolyticus.
A. Vibrio cholera (V. comma or spirillum cholera asiaticae)
1.

Serogroups or V. cholera (Based on their somatic O antigen)

a. 01 - causes classes epidemic cholera primarily by means of a specific enterotoxin and

biochemically
divided into cholerae or el tor biotypes
b. atypical or nontoxigenic 01 - biochemiocally similar to 01 organisms and type with
the 01 antisera
but does not produce the classic cholera enterotoxin
c. non-01 does not agglutinate in 01 antisera but similar to 01 organisms
biovhemically and
genetically; associated with large outbreaks
2.

Biotypes
TESTS

a.
b.
c.
d.
e.

soluble hemolysin
Voges-Preskeur
chicken red cell agglutination
polymycin B sensitivity
bacteriophage IV sensitivity

Biotype cholerae Biotype

or classic
el tor
+
+

+
+
+
-

*el tor biovars display the typical beta-hemolysis (several years ago) however,
recent epidemics
have been caused by nonhemolytic strains
3.

Serotypes (Subdivide from both biotypes classic and el tor)

a. ogawa or variant P - common in India
b. inaba or original J common in the Philippines
c. hikojima or middle or intermediate common in Japan

4.

Morphology

a. small, facultative, gram-negative rods that are slightly curved or comma shaped
b. shows a characteristic darting motility by means of a single thick, sheathed polar
flagellum
5. Cultural Characteristics
a. requires an alkaline medium for growth
b. growth on TOBS agar shows medium-sized, smooth, opaque, thin-edged yellow
colonies which on
prolonged incubation turns green especially if the organism is the el tor biotype
6. Virulence Factors

a. enterotoxin (cholera toxin or choleragen)

- causes a rapid secretion of electrolytes into the small intestinal lumen resulting to
diarrhea and
profound dehydration
b. adherence
7.

Diseases Produced: (Cholera or Epidemic Asiatic Cholera)

a. mode of transmission
i. usually acquired during foreign travel
ii. ingestion of infected shellfish
iii. Contact with sea water
iv. Ingestion of water and food contaminated with carriers feces
b. clinical manifestations

i. abrupt onset of diarrhea (rice water stools) and vomiting

ii. dehydration wherein eyes and cheeks appear sunken with diminished skin
turgor and
a washerwomans hand appearance
8.

Laboratory Diagnosis
Specimens: stool and rectal swabs
-rice water stools
a. Gram stained smears
b. culture
i. transport media
- Amies
- Cary-Blair modification of Stuarts medium
ii. selective media
- alkaline peptone water (enrichment broth)
- tellurite taurocholate gelatin agar (TTGA)
- thiosulfate citrate bile sucrose agar (TCBS)
- Gohar, Dieudonnes, Monsur and Aronson media
c.

cholera red test (nitrose-indole reaction)

i. 24 hour culture of organism grown in alkaline peptone

water with tryptophan and NO

ii. add concentrated H2SO4

iii. (+) result : red color due to the presence of nitroso-indole
iv.

not specific since it also gives a positive result to indole (+) and nitrate (+) organisms
(
Enterobacteriaciae )

d. bacteriolysis ( Pfeifferss Phenomenon)

- lysis of V. cholerae organism when inoculated into an immuned guinea pig

e. direct fluorescent antibody technique

f. slide agglutination test
g. darkfield or phase contrast microscopy for the characteristics motility.
h. immobilization test
i. Griegs test
- done to demonstrate the soluble hemolysin cl tor
i. mixsheepssrbc with organism
ii. incubate at 370C for 30 minutes
iii. ( + ) result : lysis of red cells.
j. biochemical reactions
i. oxidase ( + ), ornithine and lysine decarboxylase ( + )
ii. sucrose feter, ONPG ( + )
iii. susceptible to 0129 ( 2, 4 diamine- 6-7- diisopropylpteridine ) it also inhibits the
growth of other vibrios.
9. Treatment
a. Fluid and electrolyte replacement
b. tetracycline or furazolidone shortens the course of disease. \
B. Vibrio parahemolyticus
1. Morphology: resembles other Vibrio species
2. Cultivation
a. requires an alkaline environment thus the same selective media as in V. cholerae
b. members are halophilic ( salt loving ) and requires 3% NaCl for growth
c. colonies on TCBS are large, green and smooth which do not ferment sucrose
3. Diseases Produced
a. mode of transmission
i. ingestion of raw or improperly handled seafood.
ii. wounds or tissues contaminated with seawater
b. clinical infections
i. seafood gastroenteretitis
- explosive, watery diarrhea without blood or mucus blood or mucus
- headache, abdominal cramps, nausea, vomiting snd fever

ii. extraintestinal infections

-

Localized infections seen in boat workers, seafood cooks and swimmers

4. Laboratory Diagnosis
Specimens: feces and rectal swabs
a. Staining smears
b. Culture ( similar to V. cholera )
c. Kanagawa phenomenon
- Wherein pathogenic strain of V. parahemolyticus produce hemolyis on a
special high salt medium ( Wagatsuma Blood Agar) distinguishing them from
non pathogenic strains.
d. Biochemical reactions
i. oxidase ( + ), ornithine and lysine decarboxylase ( + )
ii. ONPG ( - ) and sensitive to 0/129
iii. urease ( + )
5. Treatment

a. gastroenteritis produced is selof limiting

b. fluid and electrolyte replacement
c. susceptible to cholramphenicol, kanamycin, tetracyclineand cephalosporins.

C. Other Vibrios
1. V. vulnificus
a. halophilic vibrio from seawater
b. cause intense skin lesions and occasionally enteritis, bacteremia and death in erderly
or debilitated persons.
2. V. mimicus causes diarrhea after ingestion of raw oysters.
3. V. holicas and V. fluvialis cause diarrhea
4. V. alginolyticus - causes eye, ear or wounf infection after seawater exposure.
5. V. damsel causes wound infections.

AEROMONAS

1. Morphology
a. straight sided, medium sized, nonpleomorphic, gram negative bacilli.
b. motile species possess a single polar flagellum
2. Cultivation
a. grow on common laboratory media like BAP and McConkey agar
b. many are beta- haemolytic

3. Virulence Factors
a. heat labile enterotoxin similar to LT of E. Coli and V. cholera enterotoxin
b. heat stable cytotoxic enterotoxin similar to that of shigella dydenteriae
c. extracellular enzymes like protease, amylase, lipase, nucleases, etc.
d. hemolysins
e. adherence
4. Clinical Infection
Aeromonas species are generally considered freshwater organism that are pathogenic to
cold blooded animals ( frogs and snakes ). Human infections are usually caused by motile
species ( A. hydrophilia, A. caviae, A. sobria, A. schubertii and A. veronii )

a. diseases produced
i. gastroenteritis
ii. wound infections after exposure to water or soil.
iii. opportunistic infections of blood or other body sites
b. risk factors for gastroenteritis acquisition
i. derinking untreated water
ii. current gastrointestinal or liver disease
iii. reduced stomach acidity
iv. recent antibiotic administration

5. Laboratory Diagnosis
Specimens: feces, sterile body fluids, tissues and oxidates from wounds
a. Gram stained smears
b. Culture media
i. trypticase soy agar with 5 % sheep blood and 10 or 30 ug/ml ampicillin.
ii. combination of ampicillin blood agar and CIN agar
c. Biochemical reactions
i. all are oxidase ( + ) and resistant to 0/129
ii. they ferment glucose
6. Treatment
a. susceptible to aminoglycosides, tetracycline, cofamandole and trimethoprim
sulfamethoxazole
b. resistant to penicillin, ampicillin, cephalothin and carbonicillin

PLESIOMONAS ( P. Shigelloides )

P. shigelloides is an aquatic orgaism similar phenotypically with Vibrio and Aeromonas. It

probably realted to the Protease the family Enterobacteriaceae.
1. General Characteristics
a. oxidase ( + ) fermentative, gram-negative bacilli
b. may be seen as long filamentous forms on gram stained colonies
c. with lophotrichous flagella
d. ferements inositol
e. lysine and ornithine decarboxylase ( + )
f. arginine dihydrolase ( + )
2. Virulence Factors
a. enterotoxin
b. possible invasiness
c. adherence
3. Cinical Infection
a. mode of transmission
i. eating raw shellfish
ii. foreign travel
b. Diseases produced
i. gastroenterititis
ii. extraintestinal septicaemia, endothalpmitis, septic arthritis , meningitis,
cholecystitis and cellulitis.
4. Laboratory Diagnosis
- isolation from fecal samples and identification by biochemical tests.
5. Treatment
a. susceptible to aminoglycosides, cepaholosporins, imipenom , ciprofloxacin ,
chlorampenicol, trimethoprim-sulfamethoxazole and tetracycline.
b. resistant to penicillin, ampicillin and carbonicillin.

CHAPTER XVIII
CAMPYLOBACTER, HELICOBACTER AND SPIRILLUM
CAMPYLOBACTER
General Characteristics
1. Organisms are small, slender, helicallycurved, microaerophilic, gram negative rods
that may the following morphologic forms: spirals, S shape, commas, seagull winged
and coccoid shapes.
2. Most have a singleunipolar or bipolar flagellum exhibiting a distinctive corkscrewdarting motility on phase contrast or darkfield microscopy.

3. They require a low oxygen tension ( 5% O 2) and an increase3d CO2 level ( 10% CO2)
for growth.
4. C. jejuni, C. coli, and C. laridis are thermophilic with an optimum tempaerature of
420C.
5. 5. Most of the pathogenic species are oxidase and catalase positive.
6. They do notr ferment or oxidize sugars.
7. 7. Most are susceptible to cephalosporins.
A. Campylobacter jejeuni
1. Morphology
- same as with other Campylobacter species,moves with a single polar flagellum.
2. Cultivation
a. plates are incubated at 420C in an atmosphere containing 5% O2, 10 CO2, and 85% N2
b. culture media
i. Butzlers medium
ii. Skirrows medium
-contains blood agar base, 5% lysed horse blood, vancomycin, polymixin, B and
Trimethoprim, cephalothin and ampothericin B.
iii. campy thio medium
-

A thioglycollate broth base with 0. 16% agar and vancomycin, polymixin, B

and Trimethoprim, cephalothin and ampothericin B.

iv. campyn BAP medium

-

Consists of Brucella agar base and 5% sheep erythrocytes as that of campy

thio medium.

c. colonies are gray topinkish or yellowish gray, slightly mucoid looking and
spreading or round and convex ; some may exhibit a tailing effect along the
streakline.
3. Virulence Factors
a. invasiveness
b. cytotoxin
c. enterotoxin
4. Clinical Infections
C. jejuni have birds, dogs, cats , sheep and cattle as its reservoir.
a. mode of transmission
i. ingestion of contaminated milk, water and food
ii. person to person spread via fecal-oral route.
b. diseases produced
i. mainly enteritis
ii. occasionally with systemic invasion leading to bacteremia

iii. meningitis, cholecystitis and UTI (rare )

c. Clinical manifestations of acute enteritis
i.
fever, headache and myalgia
ii.
loose stools to massive watery or bloody stools with inflammatory cells
iii.
crampy abdominal pain
iv.
tenesmus
5. L:aboratory Diagnosis
Specimens: mainly stool: blood and sterile body fluids in extraintestinal infections.
a.
b.
c.
d.
-

Gram stained smears

Darkfield or phase contrast microscopy
Culture
Serotyping with:
i.
Panner Method
An indirect hemagglutination technique for soluble heat-stable antigens.
ii.
Lior method
A slide agglutination technique for heat-labile antigens

e. cephalothin resistant and nalidixic acid sensitive

6. Treatment (most infections are self limited )
a. sensitive to erythromycin, aminoglycosides, tetracycline, and chloramphenicol
b. resistant to cephalosporins
B. Campylobacter fetus
- an opportunistic pathogen that causes systemic infections in immune-compromised patients;
occasionally causes diarrhea
- it is isolated from sheep and cattle
- grows well at 250C; some strains do not grow at 420C
c. Other Campylobacter species
Table17-1. Properties of clinically Important Campylobacter Species
Growth at
250C

Nitrate

420C

Sensitivity to

Reduction

Cephalothin

Nalidixic

a. C. coli

b. C. fenelliae

c. C. fetus

-/+

+/-

e. C. jejuni

f. C. laridis

subsp. fetus
d. C. hyointestinalis

Table 17-2. Reservoir and Disease Produced

Reservoir

Human Disease

a. C. oli

pigs

diarrhea

b. C. laridis

seagulls

diarrhea

c. C. cinaedi, C.

enteritis and proctitis in

hyointestinalis

homosexual men

and C.finelliae
HELICOBACTER ( H. Pylori, formerly Campylobacter pylori )
1. Morphology
a. curved, spiral shaped or bizarre U-shaped microaerophilic gram negative rod
b. with sheathed tuft or polar flagella exhibiting a corkscrew motility
2. Cultivation
a. plates are incubated at 35-370C for 1 week, however, growth is also seen at 420C
b. culture media
i. nonselective agar media like chocolate agar and brucella agar with 5% shhep blood
ii. selective agar media like Skirrows agar
iii. modified Thayer-Martin medium
d. Colonies are small, circular, translucent and nonpigmented.
3. Disease Produced
H. pylori is associated with antral gastritis and of antral gastritis with gastric and
duodenal ulcers.
4. Laboratory
Specimens: antral biopsy specimens ( H. Pylori is seen on the surface of gastric antral
epithelium)
a. Stained smears of tissue ( histologically )
i. Gram stain
ii. Giemsa
iii. Special liver stains
b. Culture
c. Biochemical reactions
i.
Oxidase and catalase ( + )
ii.
Rapid urease ( + ) can be detected by placing biopsy specimens directly into
urea broth
iii.
Cephalothin sensitive and nalidixic acid resistant
iv.
No sugars are metabolized
5. Treatment
a. bismuth compounds alone or with antibiotics

b. susceptible to erythromycin, tetracycline, penicillin, cephalosporins, and metronidazole.

Spirillum ( S. minor)
1. Morphology
a. Short, thick, helical gram-negative organism with tapering endsand two or three
spirals that resembles Campylobactermore than spirochetes.
b. Wit polytrichous polar flagella.
2. Cultivation : non culturable on artificial media
3. Disease Produced: Rat- bite fever ( Soduku fever)
The disease is an acute bacteremic infection caused by S. minus and streptobacillus
moniliformis, both of which are present in the normal oropharingeal flora of rodents. The
Disease is primarily seen in wild rats.
a. Mode of transmission : bite of an infected animal ( usually rats )
b. Clinical manifestations
i.
Local lesions chancre like indurated ulcerwith black crust.
ii.
Regional gland swelling
iii.
Skin rashes- purplish maculopapular eruption
iv.
Relapsing type of fever
4. Laboratory Diagnosis
Specimens: blood, exudates from initial lesion, serum from exanthematous patches,
lymph node aspirates or ground up tissue from lesions.
a. Stained smears
i.
Gram stain
ii.
Giemsa or wrights stains- for blood smears
iii.
Silver imp[regnation methods like Fontana-Tribondeau ( flagella stain )
b. Darkfield or phase contrast microscopy
c. Isolation by animal inoculation, either mice or guinea pigs.
5. Treatment
a. Penicillin drug of choice
b. Streptomycin - alternative
CHAPTER XIX
SPIROCHETES AND CURVED RODS

SPIROCHETES
General Characteristics
1.
2.
3.
4.

Unicellular organisms with flexous structures (helical coils).

All are motile due to axial fibrils long flagella- like unicellular organelles.
Slender, gram negative , facultative anaerobes.
Multiply by transverse fission.

TREPONEMA
Treponema palidum
1. Morphology
a. Contains numerous tight, rigid coils which are more or less regular.
b. Motility is sluggish, with a drifting motion and flexous movements.
c. Obligate anaerobes.
d. Very hard to stain but best observed by darkfield microscopy.
2. Cultivation

- Non culturable in artificial medium but can be maintained in the laboratory in

testicular chancres of rabbits.
3. Clinical Infection
- Syphilis, also known as Lues venerea is a disease of blood vessels and of the
perivascular areas. It was initially called Italian disease, French disease and the great
pox, as distinguished from small pox.
a. Mode of transmission
i.
Sexual contact.
ii.
Direct contact with primary lesions.
iii.
Contact with body fluids or secretions of patients in infections (primary and
secondary stages).
iv.
Prenatal intrauterine infection.
b. Clinical manifestations
STAGES OF SYPHILIS
i.

Primary stage
- typically a single dry lesion, nontender and firm, with a clean surface, raised
border and reddish color.
- lesions known as hard chancre or Hunterian chancre appear on the
genitalia or within the anal canal.
- systemic signs or symptoms are absent but lymph nodes are frequently
enlarged and tender.
-lymphadenopathy

ii.

Secondary stage
- fever, sore throat, generalized lymphadenopathy, headache and rash.
- lesions can be found on other parts of the body; may appear as white mucous
patches on mucous membranes.
- secondary lesions are called condylomas(condylomata acuminata) which
occur around moist areas like the vagina and anus.
- all secondary lesions are highly infectious.

iii.

Latent stage
- patients have no signs and symptoms of
seroactive.

iv.

v.

vi.

active syphilis but remain

Tertiary stage
- involvement of the deep organs of the body.
EXAMPLES:
GUMMAS non progressive localized lesions of the dermal elements or
supporting structures of the body; also known as tertiary lesions
(benign tertiary syphilis)
NEUROSYPHILIS involvement of the CNS
CARDIOVASCULAR SYPHILIS commonly involved organs are the
great vessels of the heart.
Congenital syphilis results from transplacental infection of the developing
fetus and is often very severe mutilating form of the
disease.

Bejel or Endemic syphilis caused by a variant of T. pallidum (T. pallidum

subspecies endemicum).
- non- venereal form of syphilis that is transported by
direct contact.
4. Laboratory Diagnosis
Specimens: tissue fluid from early surface lesions and blood.
Methods:

a. Direct visualization of the organism by darkfield microscopy, fluorescent antibody

technique, or by special stains of infected tissue, like Levaditi Silver Impregnation
Technique and Fontana Tibondeau.
b. Animal inoculation test
c. Serologic tests:
i.
Non specific or non treponemal antibody test
- organism is not used as antigen
- a cardiolipin lecithin antigen is used to detect antibody like substance called
reagin
- cardiolipin lecithin antigen from deep heart muscle; nonspecific
- reagin nonspecific; can be produced in tuberculosis, leprosy and malaria.
* nonspecific flocculation test
VDRL
Eagle
Kahn
Mazzini
Kline
RPR Rapid Plasma Reagin
Hinton
USR Untreated Serum Reagin
* nonspecific complement fixation test
Kolmer
Wasserman
ii.

Specific or treponemal antibody test

- organisms are used as antigen
- used to detect specific treponemal antibodies
ii.a) Treponema Pallidum Immobilization (TPI)
- immobilization of live, motile, T. pallidum organisms by specific
antibodies in serum from infected individuals.
ii.b) Treponema Pallidum Agglutination (TPA)
ii.c) T. Pallidum Methylene Blue (TPMB)
ii.d) T. Pallidum Hemagglutination (TPHA)
ii.e) T. Pallidum Immune Adherence (TPIA)
ii.f) Reiter - Protein Complement Fixation
- antigen for this test is an extract from a nonvirulent treponeme;
frequently nonreactive in late stages of syphilis.
ii.g) Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
- use to confirm the validation of a positive reaginic test to diagnose
congenital syphilis and to diagnose late stages of syphilis.

Application of Methods
a. Primary syphilis dark field identification provides the most definite and earliest means
of diagnosis.
b. Secondary syphilis ( STS) serologic tests or treponemal tests for syphilis are almost
always (+).
c. Tertiary syphilis fluorescent antibody test is the most specific test for syphilis.
5. Treatment
a. Penicillin drug of choice
b. Erythromycin, tetracycline and cephaloridine alternative drugs
Treponema pertenue
1. Morphology
- closely related to T. pallidum; serologically and morphologically indistinguishable and
are differentiated by the type of lesions produced in experimental animals.
2. Clinical Infection
- Yaws (Frambesia) is a spirochetal disease of the tropics caused by T. pertenue
- endemic, particularly among children, in many humid, hot tropical
countries

a. Mode of transmission
- direct contact (person to person) other than sexual contact in children under
age 15
b. clinical manifestations
i.

Primary lesion mother yaw or Framboise

- painless erythematous papule that heals during subsequent 1
or 2 months.

ii.

Secondary lesion daughter yaw

- resembles the primary lesion; occurs 6 weeks to 3 months
later.

iii.

Tertiary lesion gangosa; gummatous ulcerations involving the skin and

bones.

3. Laboratory diagnosis
- no serologic test distinguishes human yaws from syphilis

4. Treatment: Penicillin drug of choice

Treponema carateum

1. Morphology: similar to both T. pallidum and T. pertenue

2. Clinical infection: Pinta is a disease of tropical areas of Central and South America

a. Mode of transmission

i.

Person to person contact

ii.

Rarely by sexual intercourse

b. Clinical manifestations

i.

Primary and secondary lesions flat, erythematous & non-ulcerating

- the healing lesion first becomes
hyperpigmented and later, as scarring
occurs, will be depigmented.

- lesions most commonly occur on the

hands, feet & scalp.
ii.

Tertiary lesions uncommon in pinta

3. Laboratory Diagnosis

- serologically similar to both T. pallidum and T. pertenue and is distinguished by failure

to produce cutaneous lesions in rabbits, hamsters or guinea pigs.

4. Treatment: penicillin

BORRELIA

A. Relapsing Fever
Spirochetes of the genus Borrelia cause the disease in humans known as
relapsing fever. An acute infection characterized by febrile episodes that
subside spontaneously but tend to recur over a period of weeks.

1. Morphology

a. Helical organisms which are loosely coiled with coarser and irregular
spirals
b. The only spirochete demonstrated by direct stain (Wright Giemsa) in the
peripheral blood (blood spirochetes)
c. Microaerophilic and actively motile with a corkscrew-like motion have
11-30 periplasmic flagella per cell end

2. Clinical infection: Borreliosis or relapsing fever (tick fever, famine fever or

yellow plague)

a. Mode of transmission
i.

Transmitted primarily by ticks or human body or head lice

ii.

Transplacental transmission

iii.

Infected blood

b. Types of diseases

i.

Tickborne or endemic or American relapsing fever caused by:

- B. duttoni

B. anserina

- B.turicatae

B. hermsii

- B. parkeri

ii.

Louseborne or epidemic or European relapsing fever caused by:

B. recurrentis

c. Clinical manifestations
i.

Relapsing fever

ii.

Splenomegaly and hepatomegaly

iii.

Jaundice and rashes

iv.

Respiratory symptoms

v.

CNS involvement

3. Laboratory diagnosis

a. Giemsa and Wright stained blood smears

b. Animal inoculation test
c. Serologic tests
d. Culture using
i.

Kellys medium

ii.

Chick embryo

iii.

Barbour Stoenner Kelly medium

4. Treatment
a. Tetracycline drug of choice
b. Erythromycin and penicillin alternatives

B. Lyme Disease
An illness associated with a characteristic skin rash, erythema chronicum migrans
(ECM) caused by Borrelia burgdorferi.

1. Morphology

a. Resembles other members of the family spirochaetaceae

b. Microaerophilic
c. Has 7 to 11 periplasmic flagella per cell end

2. Clinical Infection : Lyme disease (erythema chronicum migrans, Lyme arthritis,

& Bannwarths syndrome)

a. Mode of transmission : Tick bites

b. Vectors : ( ticks)
i.

Ixodes pacificus West Coast

ii.

Ixodes dammini East Coast and Midwest

iii.

Ixodes ricinus Europe

iv.

Ambyloma americanum also a vector of tularemia and Rocky Mt.

spotted fever.

c. Clinical manifestations
i.

First stage highly characteristic expanding lesion showing a papule

at the site of the bite with sharply demarcated borders accompanied by
constitutional symptoms like: malaise, fever, headache, stiff neck

ii.

Second stage onset of neurologic and cardiac involvement

- headache, Bells palsy, radiculoneuropathy,
myocarditis, arrythmias

iii.

Third stage migrating episodes of arthritis with associated fever.

3. Laboratory Diagnosis

Specimens : skin biopsy from lesions, synovial tissue, blood, CSF

a. Stained smears ( Dieterles silver stain)

b. Culture
c. Serologic tests
i.

ELISA

ii.

Indirect immunofluorescence assay

4. Treatment
a. Phenoxymethylpenicillin or tetracycline drug of choice
b. Penicillin and amoxicillin alternatives

LEPTOSPIRAE

Leptospirae are tightly wound spirochetes about the size of treponemes, often shaped like a
shepherds crook. They are saprophytes and animal pathogens, only accidentally infecting man.

1. Morphology

a. Helicoidal, tightly coiled, thin, flexible spirochetes with very fine spirals in which one
or both ends is often bent forming a hook
b. With active rotational motion (corkscrew like)
c. Obligate aerobes, oxidase (+), catalase (-) or peroxidase positive or both
d. The diaminopimelic acid content of leptospira serves to differentiate these organisms
from Treponema and Borrelia, which instead contain ornithine.

2. Characterization of species

a. L. interrogans representing all the parasitic strains or pathogenic organisms

b. L. biflexa includes all the saprophytic strains or water leptospira.

3. Clinical Infection: Leptospirosis ( Swineherds disease, Fort Bragg Fever, Pretibial Fever,
Weils disease, Canicola fever, Autumnal fever)

a. Mode of transmission

i.

Direct and indirect contact with infected urine of an animal (the proximal
convoluted renal tubules of infected animals harbor the leptospira organisms
which are then passed in the urine)

ii.

Ingestion of contaminated food and water

iii.

Infected soil, food and water enter the body through mucus membrane or
breaks in the skin

b. Clinical manifestations

i.

Infecting serogroups
- severe icteric disease L. icterohemorrhagiae (most common cause of
disease)
- less severe & anicteric disease L. australis, L. pyrogenes
- mild L. canicola, L.ballum, L. Pomona

ii.

Stages of leptospirosis (illness is biphasic)

- 1st stage variable febrile onset with jaundice, hemorrhage and nitrogen
retention.

- 2nd stage aseptic meningitis with intense headache,stiff neck, and

pleocytosis in CSF
- nephritis and hepatitis
- skin, muscle and eye lesions
- myocarditis

4. Laboratory Diagnosis

Specimens : blood, CSF, urine

a. Dark field microscopy

b. Animal inoculation test
c. Serologic test
d. Culture
i.

Fletchers medium

ii.

Noguchis medium

iii.

Stewarts medium

iv.

Ellinghausen, McCullough, Johnson & Harris (EMJH)medium

5. Treatment

a. Penicillin drug of choice

b. Streptomycin, tetracycline, doxycycline, macrolide antibiotics alternatives

CHAPTER XX
CHLAMIDIAS, MYCOPLASMA AND RICKETSIAE

CHLAMYDIAE
A. General Characteristics
1. They are obligate intracellular parasites closely related to gram negative bacteria with
a tropism columnar epithelial cells lining the mucous membranes.
2. They cannot generate high-energy phosphate bonds (ATPs) and are totally dependent
on eukaryotic cells for energy thus restricting them to an intracellular existence.
3. All species exhibit two morphologically distinct forms:
a. Elementary body (EB) Small, dense spherical body which is the infectious form
b. Reticulate bod (RB) intracellular, metabolically active form that divides by
binary fission
4. All are non-motile and non-piliated but possesses unusual cylindric surface
projections arranged in a hexagonal array.
5. All share a common genus-specificor group antigen and multiply in the cytoplasm of
their host cells by a distinctive developmental cycle.
6. All require a living cells for growth.
7. They are rapidly inactivated by heat and lose their infectivity completely after 10
minutes at 60C.
B. Developmental Cycle
1. Phase I. Attachment and penetration of the EB
a. EB attaches to the surface of a susceptible host cell;

2.
3.
4.

5.

b. Then induces endocytosis and is enclosed within a cytoplasmic vesicle, a

pHagosome
Phase II. Recognization of the EB to a more metabolically active form, the RB.
Phase III.Growth and division by binary fission of the RB.
Phase IV. Maturation of the RBs into infectious EBs
a. Newly formed reticulate and elementary bodies are still enclosed within the
vesicles, which is visible microscopically as an inclusion
b. Inclusions of C.trachomatis ( Halberstadter Prowazek bodies) are rigid, compact
and consist primarily of glycogen, hence can be stained brown with Lugols
iodine; dark purple with Giemsas stain
c. Inclusion of C.osittaci (Levinthal-Cole-Lillie bodies) are diffuse, irregular in
shape and contain no glycogen; he mocrocolonies stain with the Giemsa and
Machiavella stains.
Phase V. Release of the EBs from the host cell.

Figure 20.1 Schematic Diagram of the Developmental Cycle

C. Differential Characteristics From Viruses

1. Possess both RNA and DNA.
2. Multiply by binary fission.
3. Possess a cell envelope similar to that of gram-negative bacteria.
4. Possess ribosomes and synthsize their own proteins, nucleic acids and lipids.
5. Susceptible to a wide range of antibioics, specially tetracycline and erythromycin
D. Antigen Produced
1. Genus-specific or group antigens
- These are heat-stable lipopolysaccharide (LPS) shared by all chlamydia
2. Species-specific antigens
- These are mainly outer membrane proteins shared by all members of a chlamydia
species.
Table 20.1 Differentiation Among Chlamydia Species
Characteristics
C.pneumoniae

C.trachomatis

1. Host range

Humans, mice

2. Inclusion
Morphology

Oval, vacuolar

C.psittaci
Birds, humans
Lower animals
Variable, dense

Humans
Oval, dense

3. Elementary body
Morphology
4. Glycogen in
inclisions
5. Number of
Serovars
6. Folate bioSynthesis
7. Susceptiblility
To sulfonamides
& D-cycloserine
8. Plasmid DNA

Round
(+)

Round
(-)

Pear-shaped
(-)

15

NA

(+)

(-)

(-)

(+)

(-)

(-)

(+)

(-)

(+)

Trachomatis
A.Biovars and Serovars
a. Biovar LGV- consists of 3 serovars (LGV1-LGV3)
b. Biovar trachoma- have 12 serovars
i.
Serovars A, B, Ba, & C- isolated primarily from eyes of trachoma patients in
endemic
Area
ii.
Serovars D to K- isolated most often from genital tracts and eyes of trachoma
patients in
Non-endemic areas
B. Diseases Produced
a. Endemic trachoma
i.
A chronic keratoconjuctivitis that begins with acute inflammatory changes in the
conjunctiva and cornea progressing to scarring and blindness
ii.
Caused by serotypes A, B, Ba, or C
iii.
Seen in poverty-stricken families (overcrowding)
iv.
Transmitted by droplets, hands and contaminated clothing from eye to another
b. Inclusion conjunctivitis
i.
Caused by serotypes D through K; historically referred to s the TRIC (trachomona
inclusion conjunctivitis) agent
ii.
Infants acquire the infection from passage through an infected birth canal; does
not lead to blindness
iii.
Neonatal pneumonia may also be seen
iv.
Adults acquired the infection via self-inoculation of genital secretions or
following contamination of un-chlorinated swimming pools
c. Sexually transmitted chlamydial disease
i.
Caused by oculogenital serotypes D through k
ii.
Includes nongonococcal urethritis, cervicitis, bartholinitis, epididymitis, proctitis,
salpingitis, etc.
iii.
Associated with Reithers syndrome
d. Lymphogranuloma venereum (LGV)
i.
Other names are lymphogranuloma inguinale, climatic bubo, tropical bubo and
esthiomene
ii.
Sexually transmitted disease caused by serotypes LGV1, LGV2, LGV3
iii.
Presents a painless, small, inconspicuous and vesicular primary lesion with painful,
firm and enlarged matted inguinal ad femoral lymph nodes
iv.
Elephantiasis of the vulva (called esthiomene) may occur 2 to lymphatic obstruction
C. Laboratory Diagnosis

a. Specimens: scrapings of epithelial cells from urethra, cervic, vagina, conjunctiva, rectum
and nasopharynx; throat specimens; salpinx and epididymis aspiration biopsies; pus and
buboes
1. Cytological experimentation of cell scraping fror the presence of inclusion bodies
which are stained with iodine, fluorescent antibody and Giemsa stains
2. Rapid antigen detection methods
i.
Direct fluorescent antibody staining (e.g., Micro Trak DFA)
ii.
ELISA (e.g., Chlamydiazyme assay)
iii.
RNA-directed DNA probe conjugated to a chemiluminescent marker (e.g.,
PACE, Gen-Probe)
b. Isolation
i.
Inoculation into embrayonated eggs (chick embrayo)
ii.
Isolation of experimanetal animals (usually mice, inoculated intracerebrally)
iii.
Selected tissue culture cell lines like McCoy, Hela 229, BHK-21, L 929 and
buffalo green monkey which are inoculated by centrifugation;
iii.a. this is the most sensitive ad specifi diagnostic method
iii.b. cells are pretreated with cycloheximide, cytochalasin B or idoxuridine to
enhance
chlamydia rplication and allow easier recognization of inclusions
iii.c. inclusions are visualized using the above mentioned stains,
immunofluorescence
being the most sensitive
iii.d. cycloheximide-treated McCoy cells have been shown to produce higher
inclusion
counts than other cell lines
c. Serologic testing
i.
Compliment fixation (detects antibody to a genus-specific antigen)- used
commonly for LGV
ii.
Microimmunofluorescence (micro-IF) technique (detects type specific antibodies)
- used mainly in ocular and genital infections and neonatal pheumonia
- may also be used inLGV
iii.
Frei test
- An intradermal skin test used in the diagnosis of LGV which detects a delayed
hypersensitivity response to chlamydia antigen
D. Treatment
- Susceptible to tetracyclines, sulfonamides and erythromycin

C.psittaci
1. Disease produced: Psittacosis or Ornithosis
Primarily a disease of birds especially the psittacine birds (parrots, parakeets,
cockatoos, etc.), which is occasionally transmissible to humans.
a. Mode of transmission
i.
Inhalation of organisms from infected birds (aerosols) and their droppings
ii.
Person to person transmission (rare)
b. Clinical manifestation
i.
Pneumonia
ii.
Severe headache and changes in mentation
iii.
Hepatosplenomegaly
2. Laboratory Dignosis
Specimens: Blood, sputum and biopsy tissue specimens
a. Isolation
i.
Yolk sacs of embrayonated eggs
ii.
Mice, inoculated intracerebrally or subcutaneously
iii.
Grows best in L 929 cells
b. FA test using monoclonal antibody

- Used primarily as research too

c. Serologic testing
i.
Compliment fixation- most commonly used
ii.
Micro-IF technique
3. Treatment: sensitive to tetracyclines and erythromycin

C.pneumoniae
1. History
C.pneumoniae was initially considered to be a psittacosis strain since its inclusion bodies
produced in cell culture resemble that of C.psittaci. the strain was named TWAR, an acronym
reflecting the history of the first two isolates, Taian and acute respiratory.
2. Diseases Produced
a. Associated with pneumonia, bronchitis, pharyngitis, sinusitis and a fluke illness
b. Pneumonia is transmitted from human to human and has been seen in grouped young
adults such as military camps and college campus.
3. Laboratory Diagnosis
Specimen:swabs of the posterior oropharynx
a. Ahdlk
i. kdh
ii. HeLa 229 cells and heteroploid cell
b. FA test using monoclonal antibody
- Used as research tool
c. Serologic testing
i. Complement fixation
ii. micri-IF test- more reliable
4. Treatment: sensitive o tetracycline and erythromycin

MYCOPLASMA(Pleuropneumonia-lke organisms or PPLO)

General Characteristics
1. Mycoplasma are members of the class Mollicutes an occur as free-living saprophytes or
as parasites in many animal and plant species.
2. They are the smallest prokaryotes that are able o survive extracellularly.
3. They are highly pleomorphic due to the absence of a rigid cell wall and instead are
bounded by a trilaminar membrane (cells exhibits plasticity).
4. Since they lack a cell wall, this renders them completely resistant to beta-lactam and othe
cell wall-active drugs such as penicillins and cephalosporins.
5. Morphologically, they range from spherical to pear-shaped structures to filamentous cells
with branching or with terminal structures.
6. Some mycoplasmas exhibit a gliding motility on liquid-covered surfaces.
7. Most mycoplasmas requires sterols for growth.
8. Cultured in vitro in a complex media such as beef-infusion broth supplemented with
horse serum or human ascetic fluid, yeast extract and nucleic acids.
9. On solid media colonies are round, dome-shaped, with a granular surface and dark center
typically buried in the agar which when viewed from above, resembles a friend egg in
appearance.
10. Most are facultative anaerobes but growth is better in an aerobic condition.
11. Resistant to penicillin

A. M.pneumoniae (Eaton agent)

There are 10 accepted human species of Mycoplasma and only a few produced disease,
M.pneumoniae is one of them. It is not a part of a normal flora as are most of the human isolates.
1. Morphlogy
a. Small coccoidal t shor, branched filamentous cells
b. bulbous enlargement with a differentiated tip structure
- most distinctive feature seen in a young filamentous cells
- grown in broth cultures
c. has the ability to attach to glass surfaces, red cells and respiratory epithelial cells
d. when so attached, it exhibits a gliding motility.
2. Cultural Characteristics
- colonies show a mulberry-colony appearance and a typical fried egg appearance after
epeated transfer to artificial media.
3.Determirance of Pathogenecity
a. ability of the organism to attach to the respiratory epithelial cells with a predilection for
the
lower tract.
b. attachment is mediated by a specific adhesion, designated protein Pl
c. it is believe that the gliding motility of M.pneumoniae facilitates penetration and its
minute size and plastic nature enables it to adapt its shape to conform to contours of the
host cells surface(?)
4.Diseased Produced: Primary Atypical Pneumonia (Walking Pneumonia)
a.Mode of transmission
- person to person via aerosol droplets
b. clinical manifestation
i. tracheobronvhitis (most common syndrome)
ii. pneumonia
iii. pharyngitis and rhinitis
iv.ear infection including bullous myringitis
v.meningitis and encephalitis
vi. myocarditis and pericarditis
5. Laboratory Diagnosis (usually serologically)
Specimens: sputum (best), nasopharyngeal and throat swabs and washings, tracheal aspirates and
lung biopsy specimens
a. phase contract and darkfield microscopy
b. isolation

A selective biphasic medium containing broth over agar is preferred with the
addition of enicillin and thallium to inhibit overgrowth of other organisms.
i. SP-4 Mycoplasma medium- for primary isolation
ii. Edward-Hayflick agar
- inoculated with subculture coming from SP-4 medium
- colonies are spherical, grainy, yellowish and embedded in the agar with
thin
Outer layer
- The growth is confirm by a small zone of beta-hemolysis around the
colonies
After a thin layer of agar containing 5% sheep or guinea pig erythrocytes
was
Overlaid on the medium and subsequently re-incubated for 24 hours.
c. Serologic tests
i. cold agglutination reaction
ii. complement fixation
iii. growth inhibition test
iv. immunofluorescence
v. passive hemagglutination
vi.RNA-dorected DNa probe test
vii. ELISA
viii. hemdsorption
ix.radioimmunoprecipitation tests
d. Dienes staining method for PPLO colonies
i.
uses
azure
II
and
methylene
ii. colony stain light blue at the periphery and bright deep blue at the center

blue

6. Treatment: susceptible to tetracyclines and erythromycin

Genital Mycoplasmas
1. Pathogenic Species
a. Ureaplasmaurealyticum
i.
A cause of nongonococcalurethritic in mases
ii.
May possibly contribute to low birth weight of newborns and perinatal
mortality
iii.
originally referred to as T. strain mycoplasmas (T for tiny)
b. M. hominis
- Associated with postpartum fever, postabortal fever, pelvic inflammatory
disease, and pyclonephritis
c. M. genitalium
i.
associated with nongonococcal urethritis
ii.
shares extensive serologic cross-reactivity with M. pneumonia
iii.
possesses an attachment mechanism and surface protein

2. laboratory disease
Specimens: urethral or genital discharges collected on swabs and inflammatory exudates.
a. Microscopic examination: useless
b. Culture
i.
Use of transport media such as modified Stuarts, 2-SP or trypticase soy broth
with 0.5% albumin and 400 U/ml penicillin
ii.
Shepards A7-B agar
iii.
Ureaplasma agar plate and broth
c. Serologic tests
i.
ELISA
ii.
RNA-directed DNA probes (PACE system, Gen Probe)
iii.
Indirect hemagglutination
iv.
Growth inhibition
v.
Nucleic acid dot-blot hybridization methods for M. genitalium
3. Treatment: susceptible to tetracyclines and erythromycin

RICKETSIAE
The family Ricketsiaceae is composed of three tribes, Ricketsiae, Erlichieae and
Wolbachieae. The tribe Ricketsieae has 3 genera that affect man, Ricketsia, Rochalimaea and
Coxiella while genus Ehrlichia has two species, E. sennetsu and E. canis
Morphology and Physiology
1. Small, nonmotil, peromorphic, gram-negative coccobacilli that are obligate intracellular
parasites.
2. They occur singly, in pairs, in short chains or in filaments.
3. They stain poorly with Grams stain but appear blue with Giemsas stain and red with
Machiavellos stain.
Cultivation
1. All require living cells for growth, except Rochalimaea Quintana which can be cultivated
in cell free media
2. All multiply by binary fission intracellularly in the cytoplasm after escaping from
phagosomes, except for C. burnetii which multiplies within vacuoles; occasionally R.
ricketsii (spotted fever group) multiplies also within the nucleus
3. Growth is enhanced in the presence of sulfonamides
Resistance
1. All are quickly destroyed by heat, drying and chemical agents except for C. burnetii
2. C. burnetiis resistance to heat and drying may be due to the formation of endospore-like
structures
Pathogenesis
1. Ricketsiae (except C. burnetii) multiply in endothelial cells of small blood vessels
causing endothelial proliferation and perivascular infiltration leading to leakage and
thrombosis
2. They have the ability to invade and damage vascular smooth muscle cells and the
endothelium of larger blood vessels
3. Centripetal spread from capillaries to arterioles and veins may occur resulting in
widespread infectious vasculitis with rash and organ dysfunction
4. A ricketsial toxin is currently suggested (?)

5. Coxiella is inhaled into the alveoli, picked up by macrophages and carried to lymph
nodes, from which it disseminates into the blood stream; granulomatous inflammation of
the liver (hepatits) and heart (endocarditis) may occur
Clinical infection
Rickettsial infections (except Q fever, in which no rash appears) are characterized by
fever, chills, headache, malaise, myalgia and skin rash

Spotted Fever Group

a. Etiologic agents
i.
Rocky mountain/American spotted fever R. ricketsii
ii.
North Asian tick typhus R. sibirica
iii.
Queensland tick typhus R. australis
iv.
Japanese spotted fever R. japonicum
v.
Boutonneuse fever/South African tick bite fever/Kenya tick typhus/ Indian
Tick Typhus/Mediterranean spotted fever R. conorii
vi.
Ricketsial pox R. okari
b. Vectors
i.
Ixodid (hard) ticks
- Rocky mountain wood tick (Dermacentorandersoni)
- American dog tick (D. variabilis)
- Louse star tick (Amblyomaamericanum)
- Rabbit tick (Haemaphysalisleporispalustris)
ii.
Mite vector (Allodermanyssussanguineus)
c. Transmission
i.
Humans are oonly accidentally infected
ii.
All are transmitted by tick bites except Ricketsial pox which infect humans
via mite bites
d. Clinical manifestations
i.
The spread of the rash is from the extremities to the trunk (centripetally) and
commonly involves the palms and the soles
ii.
In addition to the basic symptoms RMSF have the following:
Gastrointestinal complaints, arthralgias, confunctivities, stiff neck, periorbital
edema, splenomegaly, DIC, andvasculitis including the brain, heart, liver and
kindneys.
iii.
Ricketsial pox is characterized by a local eschar, papulovesicular rash with
regional lymphadenopathy and a mild clinical course
iv.
Other tickborne diseases may cause local eschars and regional
lymphadenopathy and produce a milder disease than RMSF
1. Typhus group
a. Etiologic agents
i.
Epidemic/Louse-typhus R. prowazekii
ii.
Brill-Zinsser disease R. prowazekii (relapse of epidemic typhus)
iii.
Murine/Endemic/Flea-borne/Rat typhus R. typhi
iv.
RicketsiaCanada
- Isolated from rabbit ticks, belonging antigenically to the typhus group
which grows both in the cytoplasm and nuclei of the infected cells
- Has been associated with a human febrile illness

b. Vectors and transmission

i.

Human body louse (Pediculushumanuscorporis) most important and head

louse ( Pediculoushumanuscapitis) during each blood meal the louse
defacates leading to scratching, which produces minor excoriations that in
turn become portals of entry for ricketsiae in the louse feces
ii.
Oriental rat flea (Xenopsyllacheopsis) most important and the rat louse
(polyplaxspinulosus) transmission is the same with that if epidemin typhus
c. Clinical manifestation
i.
Rash appears first on the trunk and later spreads to the extremeties
ii.
In epidemic typhus, systemic infection and prostration are severe; the disease
is more often fatal
iii.
In endemic typhus the manifestations are common with that of epidemic
typhus but the disease is milder and rarely fatal
2. Scrub/Chigger-bone Typhus/Tsutsugamushi Disease
a. Etiologic agent: R. tsutsugamushi (R. orientalis)
b. Vector: larva of trambiculid mites
c. Transmission:
i.
Humans are only accidentally infected
ii.
Transmitted by bites of infected Chigger (larva)
d. Clinical manifestations
i.
Resembles epidemic typhus clinically
ii.
Skin rash is heralded by eschar that may evolve from a small indurated papule
or vesicle in a punched-out ulcer covered with a blackened scab
iii.
Generalized lymphadenopathy and lymphocytosis are common
3. Trench/Shinbone fever
a. Etiologic agent: R. Quintana
b. Transmission: transmitted by the human body louse
c. Clinical manifestations
- Headache, exhaustion, bone pain (especially in the tibial region),
sweating, coldness of extremities and fever associated with a roseolar rash
4. Q fever (Q for query)
a. Etiologic agent: Coxiellaburnetii
b. Transmission:
i.
Primarily by inhalation of infectious aerosols
ii.
Consumption of infected milk
iii.
Handling of contaminated wools or hides
iv.
Soil contaminated with infected animal species
v.
Conjunctival contact with infectious materials

Clinical manifestations
i.
ii.
iii.

Resembles influenza, nonbacterial pneumonia, hepatitis or encephalopathy

rather than typhus
No rash or local lesion involved
Aside from the basic symptoms the following may be seen:
GIT symptoms, sore throat,chest pain, nonproductive cough, painful eyes,
hepatosplenomegaly, granulomatous hepatic lesions and endocarditis

Laboratory Diagnosis
Specimens: Primarily blood
Sputum and urine for Q fever
Vesicular fluid for Ricketsial pox
1. Stained Smears using:
a. Giemsa stain
b. Machiavello stain
c. Gimenez stain the best
2. Isolation using:
a. Embryonated hens eggs

b. Laboratory animals (guinea pigs and mice)

c. Tissue culture
3. Serologic tests
a. Neutralization test
b. Weil Felix test
i.
Based on the fact that Ricketsiae and proteus share certain antigens
ii.
However it lacks specificity and sensitivity
iii.
Neither Q fever nor ricketsial pox induces Weil Felix antibody
c. ELISA
d. Microimmunoflourescent antibody test most widely used
e. Complement fixation
f. Indirect hemagglutination
g. Latex agglutination
h. Microagglutination
4. Others
a. Polymerase chain reaction technique
b. Direct staining of punch biopsis of skin with fluorescent conjugates of antiricketsial
serum for early stages of RMSF but lacks sensitivity
c. Xenodiagnoses (in louse tissue) and primary isolation on enriched blood agar media
for R. Quintana (can also be grown in yolk sacs if embryonated eggs)
Treatment: tetracycline and chloramphenicol

Ehrlichia
E. sennetsu
1. Disease Produced
a. Sennetsuricketsiosis
b. other names: infectious mononucleosis, glandular fever, hyuganetsu and
kagaminetsu
2. Transmission: unknown (probably by ticks)
3. Clinical findings:
a. fever
b. lymphadenopathy
c. atypical lymphocytosis
4. Laboratory Diagnosis
Specimens: blood, lymph nodes and bone marrow
a. Intraperitoneal mice inoculation
b. Specific immunoserologic technique
5. Treatment: Tetracycline
E. canis
1. Disease Produced: Canine ehlichiosis/Tropical canine pancytopenia
2. Transmission: tick bites
3. Clinical Findings:
a. resembles RMSF but only a few cases have rashes
b. leukopenia and thrombocytopenia
4. Laboratory Diagnosis

a. demonstration of intracytoplasmic inclusions in leukocytes

5. Treatment: tetracyclines

20-3. REACTIONS IN WEIL-FELIX TEST

DISEASE

OX-19

OX-2

OX-K

_______

______

______

______

Epidemic typhus

++

+-

Nurine typhus

++

Scrub typhus

RMSF

Boutonneuse fever

Queenland tick thypus

North Asian tickthypus

Rickettsial pox

Q fever

Trench fever

Ehrlichiosis

CHAPTER XXI
MISCELLANEOUS PATHOGENIC BACERIA

BARTONELLA (B. bacilliformis)

1. Morphology

a. Small, highly pleomorphic, motile, aerobic gram-negative coccobacilli that occur in

chains and clusters.
b. Shape varies from small coccoid& ring-shaped structures to long angular forms
2. Cultivation
a. They grow best at room temperature
b. Grow in cell free semisolid media containing agar (BHIA), fresh rabbit, horse or
human blood
c. Colonies are described as white puffs measuring 1-5mm
d. Can also be grown in the yolk sac &chorioallantoic fluid of an embryonated hens egg
& in tissue culture systems
e. In tissue culture the organisms grow in the cytoplasm &extracellularly.
3. Disease Produced: Bartonellosis or Carrions disease
a. Mode of transmission
- bite of sandfly (Plebotumus
b. Pathogenesis
i.
They grow within and on the surface of erythrocytes & occasionally in
vascular endothelium
ii.
Infected red blood cells are hemolyzed by unknown mechanisms
c. Two syndrome
i.
Oroya fever severe febrile hemolytic anemia characterized by fever, diffuse
bone & muscle pain and anemia with
hepatosplenomegaly;
often fatal
ii.
Verrugaperuana cutaneous disease characterized by localized or generalized
angiomatouswats; chronic nonfatal illness
4. Laboratory Diagnosis
5.
Specimens: blood, aspirated materials from lymp nodes and cutaneous lesions
a. Stained smears of blood
i.
Gram stain with carbolfuchsin as counterstain
ii.
Wrights or Giemsa stain (bright red to violet)
b. Culture
c. Serologic tests (e.g. complement fixation)
6. Treatment : penicillin, streptomycin, tetracycline & chloramphenicol
CHROMOBACTERIUM
There are only 2 species recognized , C. violaceum&C.fluviatile but only the former is
associated with human infection.

1. Morphology
a. Medium to long, facultatively anaerobic, motilie, gram-negative bacilli with rounded
ends which are occasionally slightly curved.
b. Possesses a single polar flagellum and lateral flagella.
2. Cultivation
a. Grows best at 25C but can also grow at 37C
b. Colonies are violet on media containing tryptophan (5% sheep BA) due to the
pigment violacein.
c. Cultures may have smell of ammonium cyanide, since the organism produces HCN
d. They also grow on McConkeys agar.

3. Clinical Infection
a. Mode of transmission
a. The organism is on inhabitant of soil and water
i.
ii.
iii.

Punctured wounds
Wounds in contact with soil or contaminated water
Ingestion of large amounts of water in near-drowning cases

b. Disease produced and manifestations

i.
Causes a severe pyrogenic or septicemic infection
ii.
Characterized by fever, skin lesions, liver abscess & pulmonary involvement
4. Laboratory Diagnosis
a. Stained smears
b. Culture
c. Biochemical tests
i.
Resistant to penicillin and vibriostaticagentn 0/129
ii.
Oxidase (+)
iii.
Catalase -, VP-, esculin- and indole-negative
5. Treatment
a. Susceptible to chloramphenicol, amoniglycosides, tetracycline, erythromycin,
trimethroprim-sulfamethoxazole
b. Resistant to ampicillin, cephalothin, carbenicillin, &penicillins

GARDNERELLA (G. vaginalis formerly Corynebacteriumvaginale&Haemophilusvaginalis)

1. Morphology
a. Small, pleomorphic, nonmotile, nonencapsulated, facultatively anaerobic, fastidious
gram-variable or gram-negative coccobacilli and short rods
b. Club forms and metachromatic granules are oftenly present.
2. Cultivation
a. Plates are incubated in 5-10% CO2 or in a candle jar for 48 hours
b. The best selective and differential medium is the HBT (human blood tween) agar;
colonies are convex, opaque and gray, surrounded by a diffuse zone of beta-hemolysis
(on sheep agar they are nonhemolytic and barely visible)
c. Other media are V(vaginalis) agar, Casman agar and proteose peptone starch dextrose
(PSD) agar.
d. They also grow well on Columbia CNA (colistin, nalidixic acid) agar. But do not on
McConkey; colonies are nonhemolytic
e. Growth is inhibited by sodium polyethanolsulfonate (SPS)
f. Do not require X or V factor for growth
3. Disease Produced
a. May play a role in bacterial vaginosis (formerly non specific vaginitis) which is
probably a sexually transmitted disease of women characterized by copious,
malodorous discharge unaccompanied by pain or itching
b. Associated with septic abortion, puerperal fever with bacteremia and
neonatalbacteremia.
c. Urinary tract infections in both sexes
4. Laboratory Diagnosis
Specimens: Swabs of cervical & vaginal discharges, urethral swabs
a. Gram-stained smears
b. Culture

c. Biochemical test
i.
Oxidase (-) and catalase (-)
ii.
Hydrolyzes sodium hippurate
iii.
Fements starch and raffinose
d. Sniff test
- Fishy odor of discharge material after addition of 10% KOH
e. Direct wet mount of vaginal discharge
- Shows masses of bacilli located on the surface of squamous epithelial cells,
giving the cell a stippled appearance called clue cells
f. pH determination of vaginal secretions
- over pH 4.5 (normal pH is less than 4.5)
5. Treatment
a. metronidazole drug of choice
b. susceptible to ampicillin, carbenicillin, oxacillin, penicillin and vancomycin
STREPTOBACILLUS (S. moniliformis)
1. Morphology
a. Extremely pleomorhic, nonencapsulated, nonmotile, facultatively anaerobic, gramnegative rods that occur in long chains and filaments
b. Elongated bulbous swellings may be produced giving the appearance of a string of
beads or necklace-like arrangement
c. The most distinguishing characteristic is the spontaneous development of L forms
(without cell walls)
d. L form colonies when stained with Dienes or acridine orange stain yield
coccobacillary or bipolar staining coccoid forms
e. Highly fastidious
2. Cultivation
a. Requires the presence of blood, ascitic fluid or serum for growth
b. Plates are incubated in a humid environment with 5-10% CO2 at 37C
c. On brain-heart infusion agar with 20% horse serum, colonies are small, smoothn
grayish or colorless, glistening (butyrous) with irregular edge
d. In thioglycollate medium enriched with ascitic fluid, growth is described as fluff
balls, cotton balls, puff balls, or bread crumbs near the bottom of the tube.
e. L-phase colonies are smaller and exhibit at a fried-egg appearance with a dense or
dark center that extends into the agar and a flattened lacy edge
f. Growth is inhibited by SPS
3. Clinical Infection
a. Mode of transmission
i.
Bite of wild or laboratory rats wherein S. moniliformis is part of the normal
flora of their nasopharynx
ii.
Animal bite from mouse, cat and dog
iii.
Less commonly, ingestion of contaminated milk, food or water
b. Disease Produced
i.
Rat-bite fever
- a systemic febrile disease acquired by direct contact with rats or other
small rodents (another causative agent is Spirillum minor)
ii.
Haverhill fever
- acquired by consumption of contaminated milk
c. Clinical Manifestations
i.
Acute onset of chills, fever, vomiting, headache and severe joint pains
ii.
With a characteristic rash with a morbilliform, maculopapular or petechial
involving the palms, soles and extremities
iii.
Complications include endocarditis, septic arthritis & pneumonia

4. Laboratory Diagnosis
Specimens:

blood, joint fluids, lymph node aspirates, pus from lesions

a. Stained smears
i.
Dienes&acridine orange stains for L-forms
ii.
Grams stain with carbolfuchsin as a counterstain &Giemsa stain
b. Culture
c. Serologic test
5. Treatment
a. Penicillin- drug of choice
b. Susceptible to tetracycline, streptomycin, chloramphenicol & erythromycin
c. Resistant to sulfonamides
CALYMMATOBACTERIUM (C.granulomatis)
1. Morphology
a. Pleomorphic, nonmotile, heavily encapsulated, gram-negative rod which exhibits a
single or bipolar condensation of chromatin giving rise to a safety pin appearance
b. Because of its capsular polysaccharide C. granulomatis is antigenically similar to
klebsiella species\
2. Cultivation
a. Initial isolation is very difficult
b. The organism can be cultured on fresh egg yolk media or in the yolk sac of
embryonated chicken eggs
3. Clinical Infection:Donovanosis/Granulosainguinale
a. Mode of transmission
i.
Sexually, however, infectivity is low
ii.
It is an inhabitant of the intestinal tract & skin disease may be contracted
through poor hygiene & abrasions of the skin in the anal and perineal areas
b. Clinical Manifestations
i.
Initial lesions occur in pubic areas and begin as a painless papules that
develop into spreading erythematous, granulomatous ulcerating lesions which
may bleed and become secondarily infected (sometimes mistaken for
neoplasms)
ii.
Lesions spread by direct extension or by contact of skin area with another
(between scrotum and thigh)
iii.
Inguinal lymphadenopathy is common
iv.
If untreated, genital elephantiasis may ensue.
4. Laboratory Diagnosis
a. Demonstration of Donovan bodies (numerous encapsulated bacilli within
mononuclear endothelial cells) by direct examination of biopsy material stained with
i.
ii.

Wright or
Giemsa stain

blue rod with safety pin appearance,

surrounded by a large pink capsule

5. Treatment
a. Tetracycline or ampicillin drugs of choice
b. Susceptible to gentamicin, chloramphenicol, trimethroprim-sulfamethoxazole and
erythromycin
LEGIONELLA (L.pneumophila type species)
1. Morphology

Fastidious, aerobic, small, motile, piliated, gram-negative bacilli

2. Cultivation
a. Culture media
Buffered charcoal yeast extract agar (BCYE) the best
i.
- Charcoal serves t to detoxify the medium , remove carbon dioxide &
modify the surface tension to allow the organism to proliferate more easily
-buffered with N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)
- with growth supplements cysteine (required by Legionella), yeast extract,
alpha-ketoglutarate and iron
- can add antibiotics for selectively (polymyxin B, anisomycin and
cefamandole) and dyes for differentiation (bromthymol blue and bromcresol
purple)
- colonies are gray-white to iridescent pink or blue-green glistening, convex
or flat, circular and may exhibit a cut-glass sort of internal granular speckling
ii.

b.
c.
d.
e.

Feeley-Gorman (FG) medium

- an iron cystein agar in which colonies produce a characteristic brown
pigment (usually seen in media containing tyrosine
iii.
Mueller-Hinton supplemented with hemoglobin (hemin) and isoVitale X or
supplement B
iv.
Special blood culture broth without SPS
v.
Selective agar containing vancomycin
vi.
No growth on blood and , TSA or MacConkey agars
Plates are incubated in air or in a candle jar (5% CO2 , but not over
- may inhibit growth) at 35-37C in a humid atmosphere
can survive up to 65C but do not grow in temperature above 42C
growth is slow
plates should be held for 2 weeks and blood cultures in biphasic media for 1 month
before discarding

3. Clinical Infection
a. Epidemiology
i.
Legionella was discovered in the summer of 1976 as a cause of a fulminant
pneumonia among persons at attending an American Legion convention (thus,
Legionnaires disease)
ii.
Has been isolated from natural ( lakes, streams) and tap waters, air
conditioning cooling towers or evaporative condensers and water supplies of
hospitals & hotels (hot shower or whirlpools), often in association with bluegreen algae and free-living protozoa.
iii.
Risk factors
- smokers, chronic pulmonary disease, high alcohol consumption.
Immunosuppressed patients (patients on corticosteroids, renal transplant
patients, CA patients, patients on dialysis, etc)
iv.
Transmission
- airborne exposure (breathing in aerosols)
- person to person spread has not been documented
b. Virulence factors
i.
Extracellular enzymes phosphatase, lipase & nucleases
ii.
Extracellular toxin - may contribute to the intracellular survival
iii.
Ability to survive within phagocytic host cells primary virulence mechanism
c. Clinical Manifestations
i.
Legionnaires diseases ( Broad street pneumonia)
- a multisystem disease manifested primarily as severe, consolidated
pneumonia, which has a significant mortality
- may produce CNS symptoms, diarrhea and renal problems
ii.
Pontiac fever (also caused by L.feeleii)
- a multisystem disease with respiratory symptoms, fever, myalgia &
headache but without pneumonia

- it is a mild self-limited disease occurring primarily among healthy

individuals
4. Laboratory Diagnosis
Specimens: Respiratory tract secretions (like sputum, bronchial washings,
pleural fluid), blood, lung biopsy specimens,
a. Direct demonstration in clinical specimens ( difficult to stain)
i.
Dieterles silver impregnation test
ii.
Direct immunofluorescent staining (DFA) fluorescent rod with velvety
texture
iii.
Grams stain with carbolfuchsin as counterstain
iv.
Gimenez stain
v.
Giemsa stain
b. Isolation thru culture
- isolation is enhanced when specimens are first washed with an acid solution (acidwash treatment)
c. Antigen detection
i.
ELISA and
ii.
Radioimmunoassay methods for detecting antigen in urine
d. serologic tests- primary method of diagnosis
i. indirect immunofluorescence assay (IFA)
ii. ELISA
e. nucleic acid probe technique
f. biochemical tests
i. weakly catalase and oxidase positive
ii. hydrolyzes starch, gelatin and hippurate
iii. L. pnuemophila fluoresce pale yellow-green like L. micdadei, L. jordanis,
L. wadsworthii, L. oakridgensis & L. longbeachae when exposed to long-wave UV light
(others fluoresce blue-white like L. gormanii, L. dumoffii, L. morrissii
5. Treatment
a. erythromycin- drug of choice
b. rifampicin in combination with tetracycline- alternative
c. rifampicin in combination with erythromycin or doxycycline- another alternative
d. resistant to penicillins, cephalosporins and aminoglycosides
6. Control
a. hot water tanks must be heated to more than 70C for 72 hours
b. hyperchlorination or use of disinfectants
7. Other Common Legionella Species
a. L. micdadei (TATLOCK agent/HEBA agent)
i. second to L. pnuemophila as the leading cause of legionellosis
ii. pneumonia (Pittsburgh pneumonia) developed is similar to L. pneumophila

iii. does not hydrolyze hippurate

iv. does not produce gelatinase nor beta-lactamase
v. does not produce a brown pigment on tyrosine-containing media
vi. treated with erythromycin
b. L. bozemanii (WIGA Agent)
i. third leading cause of legionellosis
ii. oxidase and beta-lactamase variable
iii. produce a brown pigment on tyrosine-containing media
iv. treated with erythromycin
c. Other species mostly cause pneumonia like L. gormanii, L. dumoffii, L. longbeache,
L.wadsworthii, L. jordanis, L. feelei and L. oakridgensis.
CHAPTER XXII
GENERAL CHARACTERISTICS OF FUNGI

Fungi are eukaryotic, achlorophyllous (thus, lacking the property of photosynthesis)

organism which are mostly aerobic & inhabit the water, soil and decaying organic debris. They
are members of the plant kingdom that lack roots and stems & are referred to as Thallophytes.
The study of fungi is known as mycology.
Differences between Fungi and Bacteria
Fungi
1.Nucleus
2.Mitochondria
3.Cell wall

4.Cell membrane
5.Antibiotic susceptibility

6.Dimorphism
7.Chromosome
8.Sedimentation coefficient
9.Cultivation
10.Temperature

-Defined nuclear membrane

-Present
-composed of glucose &
mannose polymers (chitin); no
teichoic or muramic acids
-contain sterols; does not hane
respiratory enzymes
-resistant
to
penicillins,
tetracycline
&
chloramphenicol but sensitive
to
griseofulvin
&
amphotericin B
-can exist in yeast & mycelial
forms
-more than 1 & CHON
associated
-8OS
-less than pH 6
-23C except for pathogenic
fungi 37C at refrigerator

Bacteria
-no membrane
-absent
-no glucose & mannose
polymers with muramic &
teichoic acid
-with respiratory enzymes
-vice versa of fungi

-None
-only one but not CHON
associated
-7OS
-pH 7.2-7.6

Two Basic Morphologic Forms of Fungi

1. Yeast
a. Unicellular organism which are usually spherical to ellipsoidal in shape
b. A few divide by binary fission but most reproduce asexually by budding (also
referred to as the formation of blastoconidia)
c. Colonies are moist, creamy, opaque & pasty on culture media
d. Most yeast specie have similar microscopic & colonial morphology and requirement
physiologic tests for their speciation.
2. Molds
a. Multicellular, filamentous organism
b. Colonies on culture media are fluffy, cottony, woolly or powdery
Structure of Molds
1. Hyphae (singular,hypha)
-branching cylindrical tubules which are the structural units of molds
Kinds of Hypae
a. Aseptate (coenocytic) multinucleate structure without divitions (e.g.,Zygomycetes)
b. Septate hyphae divided into cells by crosswalls
c. Spirals (coiled hyphae) seen in a number of dermatophytes (e.eg., T.
mentagrophytes)
d. Nodular bodies knot of twisted hyphae (e.g., M. canis & T. mentagrophytes)
e. Racquet hyphae- cells entarged at one end or club-shaped hyphe with the smaller end
attached to the large end of an adjacent club-shaped hyphal strand
f. Pectinate body broken comb appearance (e.g., M. audouinii)
g. Favic chandeliers or antler hyphae curved, freely branching & antler-like in
appearance (e.g., T. schoeleini and T. violaceum)
2. Mycelium (plural, mycelia)
-mass or group of countless intertwined hyphae that accumulates during active growth in
the mold form
Types of Mycelia
a. Vegetative or substrate mycelium
- Portion of the growth that penetrates the substrate or laboratory media and
absorbs nutrients
b. Aerial mycelium
- Portion of the growth that projects above the surface of the substrate and produce
asexual spores
Dimorphism
Most fungus species grow only as yeasts or molds, but some pathogenic fungi exhibit
thermal dimorphism. They grow as yeast at 37C and molds at 25C or 30c. other dimorphic fungi
develop under different growth conditions aside from the one mentioned.
Types of Spores for reproduction
A. Sexual Spores
- Formed as a result of nuclear fusion; fungi that have a sexual reproductive stage
are called perfect fungi
1. Ascospores spore contained in a sac-like structure called ascus (asci)
2. Basidiospores spore formed at the end of club-shaped structures called basidium
3. Zygospores formed by conjugation between two morphologically identical cells

4. Oospores formed by heterogenous fertilization or fusion between unlike or dissimilar

cells
B. Asexual Spores
- Formed without nuclear fusion; found in fungi imperfecti
1. Thallospores
- Derived from cells of the thallus or body of the fungus
a. Arthrospores (arthroconidia)
i. formed directly from hyphae by fragmentation at points septation
ii. square, rectangular, or barrel-shaped , thick-walled cells (e.g., Coccidiodes,
Geotrichum)
b. Blastospores (blastoconidia)
i. produced by budding with the daughter cells being pinched off from portions of the
mother cell
ii. most often found in yeasts
iii. in some species, blasconidia may elongate & remain attached and form structures
called psuedohyphae (e.g., Candida)

c. Chlamydospores (chlamydoconidia)
i. formed by enlargement of a hyphal cell in which there is a concentration of protoplasm
and nutrient material
ii. large, round, thick-walled resistant spores (e.g., C. albicans)
iii. types
iii.a. intercalary (within hypha)
iii.b. sessile (to side of hypha)
iii.c. terminal (at end of hypha)
2. Conidia
- Asexual spores produced singly or in groups by specialized vegetative hyphal
strands called conidiophores
a. Microconidia
i. borne directly on the hyphal strand or at the end of a long or short conidiophores
ii. small, unicellular, round, elliptical or pyriform in shape
b. Macroconidia (fuseaux)
i. usually borne on a short to long conidiophores
ii. large usually multiseptate, and club-or spindle-shaped that may be smooth-or roughwalled
3. Other Complex Methods of Sporulation
a. Some conidiophores terminate in a swollen vesicle from which short phialides (flaskshaped projections) form and in turn produce radiating chains of conidia
(phialoconidia)

b. Some conidiophores branch into a penicillus, a structure where each branch

terminates in secondary branches (metulae) and phialides from which chains of
conidia are borne (Ex: Penicillium and Paecilomyces)
c. Zygomycetes produce spores (sporangiospores) in large sac-like structure termed a
sporangium, which is borne on the tip of a supporting structure (sporangiophore);
spores are released by the rupture of the sporangial wall

Classification of Fungi
1. Ascomycotina (the ascomyces)
Sexual fusion result in a sc, or ascus, containing the meiotic products as four or eight
spores (ascospores). Asexual spores (conidia) are borne externally at the tips of hyphae.
Ex. Trichophyton (Arthroderma), Microsporum (Nannizzia), Blastomyces (Ajellomyces).
2. Basidiomycotina (the basidiomyces)
Sexual fusion results in formation of a club-shaped organ called a basidium, on the
surface of which are borne the four meiotic products (basidiospores). Asexual spores
(conidia) are borne externally at the tips of hyphae. Ex. Cryptococus neoformans
Filobasidiella neoformans.)
3. Deuteromycotina (the imperfect fungi)
This is not a true phylogenic group but rather an artificial class into which are
temporarily placed all forms in which the sexual process has not yet been observed. Most
of the members resemble ascomyces morphologically. Ex. Epidermophyton, Sporothrix,
Candida species.
4. Zygomycotina (the phycomyces)
Mycelium usually non-septate; asexual spores produced in indefinite numbers
within a structure called a sporangium. Sexual fusion results in formation of a resting,
thick-walled cell termed a zygospore. Ex. Rhizopus nigrans (opportunistic pathogens
only.)

CHAPTER XXIII
LABORATORY IDENTIFICATION OF FUNGI

Specimen collection and Culturing

A. Respiratory Secretions (Sputum, bronchial washings & tracheal aspirates)
1. These are the most commonly submitted specimens for cultures since most mycoses
(fungal infections) have a primary focus in the lungs.
2. About 0.5 ml or specimen must be inoculated to each medium
3. antibacterial antibiotics should be added in every media used to ensure optimal recovery of
Fungi and prevent overgrowth by contaminants.
4. Cycloheximide, an antifungal agent which prevent rapid overgrowth of contaminants,
Should be added to at least one culture medium.
B. CSF
1. CSF for culture should be filtered through a 0.45 um pore size membrane filter attached to
to a sterile syringe. The filter is then removed and place into a culture medium. The culture
are examined daily with the filter moved to another location on an every-other-day basis.
2. If specimen submitted is less than 2ml, it should be centrifuged and a drop of the
Sediments is placed onto several areas on the agar surface.
3. Media should not contain any antibacterial or antifungal agents.
4. Specimen should be processed immediately but if this is not possible they should be kept at
Room temperature or placed in an incubator at 30C.
C. Blood
1. This is the specimen collected for disseminated fungal infections.
2. Fungal blood culture systems commonly used:
a. biphasic brain-heart infusion agar-broth
b. lysis-centrifugation system (Du Pont Isolator tube)

3. most fungi are detected within the first 4 days of incubation, however, for Histoplasma
Capsulatum it may take 10-14 days for recovery.
D. Hair, Skin and Nail Scrapings
1. Usually submitted for dermatophyte culture.
2. Lesions from skin and nails are scalped with a scalpel blade or microscope slide while
Infected hairs are plucked with forceps.
3. Specimen should be placed in a sterile petri dish or paper envelope prior to culturing
and should not be refrigerated.
4. Specimens are either placed on Mycobiotic or Mycosel agar which contain
chloramphenicol and cycloheximide.
E. Urine
1. Samples are centrifuged and the sediment cultured using loop to adequate isolation of
colonies.
2. Antibacterial agents should be added to media since urine is often contaminated with
gram negative bacteria.
F. Tissue, Bone Marrow and Sterile Body Fluids
1. All tissues are processed by mincing or grinding or placement in a Stomacher prior to
Culturing at least 1 ml of specimen is inoculated.
2. Bone marrow is directly plated to media.
3. Sterile body fluids are concentrated first by centrifugation before culturing and at least
I ml is inoculated.
NOTE: All specimens should be cultured as soon as possible.

Cultivation
A. Requirements
1. A battery of media must be used with the following recommendations:
a. media with and without blood enrichment (since certain fungi have a requirement for
blood 5% sheep blood)
b. media with and without cycloheximide (since some pathogenic fungi such as
C. neoformans, Candida krusei and other species of Candida, Trichosporon biegelii,
Pseudallescheria boydii and species of Asperigillus are partially or completely inhibited
by this compound)
c. all media should contain antibacterial agents

2. Cultures should be incubated at 30C at a 40%-50% relative humidity for 30 days before
Discarding as negative.
B. Fungal Culture Media
1. Primary Recovery Media
a. Brain-heart infusion agar
i. primary recovery of saprobic and pathogenic fungi
ii. isolates and maintains yeast phase of systemic fungi at 27C
b. Brain-heart infusion agar with antibiotics
i. primary recovery of pathogenic fungi exclusive of dermatophytes
c. Brain-heart infusion biphasic blood culture bottles
i. recovery of fungi from blood
ii. isolation and maintenance of yeast phase of systemic fungi at 35C
d. Dermatophyte test medium (DTM)
i. primary recovery of dermatophytes, recommended as screening medium only
ii. most dermatophytes produce a red color on this medium due to a phenol red indicator
e. Inhibitory mold agar
i. primary recovery of pathogenic fungi exclusive of dermatophytes
f. Mycosel or Mycobiotic agar
i. primary recovery of dermatophytes
ii. with chloramphenicol to inhibit bacterial growth and cycloheximide to inhibit growth
of saprophytic fungi
g. SABHI agar (Sabouraud dextrose & brain-heart infusion)
i. primary recovery of saprobic & pathogenic fungi like Blastomyces and Histoplama.

H. Sabouraud dextrose agar (SDA or SAS)

i. Primary isolation and maintenance of fungal cultures
ii. Addition of thiamine
(e.g.,Trichophytonverrrucosum)

enhances growth of dermatophyton

i. Yeast-extract phosphate agar

i. Primary isolation of pathogenic fungi like Histoplasma, Blastomyces and Coccidiodes
excluding dermatophytes
2. Special Purpose Media
a. Ascospore agar
i. Detection of ascospores in ascosporogenous yeasts like Saccharomyees sp.

b. Cornmeal agar
i. Stimulates chlamydospore production for C. albicans
ii. 1% glucose can be added to differentiate T. rubrum from T. mentagrophytes by amount
of pigment formation
iii. Stimulates fungal sporulation- used for slide culture
c. Cornmeal agar with Tween 80 and try pan blue
i. Identification of C. albicans by chlamydospore production; identification of Candida by
microscopic morphology
ii. Tween 80 reduces surface tension of medium and promotes optimal production of hyphae
and blastospores
iii. Trypan blue aids visual observation
d. Cottonseed conversion agar
mold-yeast
i. Conversion of dimorphic fungus B. dermatitis from mold to yeast form
e. Czapek's agar
i. Isolation and identification of Aspergillus sp.
f. Niger seed agar (Birdseed agar or Staib's medium)
i. Identification of C. neoformans which produces a brown pigment indicating the presence
of phenol oxidase
ii. Thisle (Guizotia abyssinica) seeds are used
g. Nitrate reduction medium
i. Detection of nitrate reduction in confirmation of Cryptococcus sp.
h. Potato dextrose agar
i. Demonstrates pigment production by T. rubrum
ii. Preparation of microslide cultures
i. Rice medium
i. Identification of M. audounii differentiating it (negative) from M. canis (positive)
j. Trichophyton agars 1-7
i. Identification of members of Trichophyton genus
k. Urea agar
i. Detection of Cryptococcus sp.
ii. Differentiates T.
rubrum (delayed or no utilization)
iii. Detection of Trichosporon sp.
l. Yeast fermentation broth

mentagrophytes (rapid utilization) from T.

i. Identification of yeasts by determining fermentation

<3<3<3 170 <3<3<3
m. Yeast nitrogen base agar
i. Identification of yeasts by determining carbohydrate assimilation
METHODS FOR DIRECT MICROSCOPIC EXAMINATION OF CLINICAL SPECIMENS
A. Temporary Mounts
1. Potassium hydroxide (KOH), 10-20%
rapid, simple and lack of contrast
KOH - easy to identify fungi
Dimethylsulfoxide - for hard nail or skin
10 gm KOH + 80 mL distilled H20 + 20 mL glycerol = it prevents crystallization
a. Traditionally the recommended method before the existence of the calcofluor white stain
b. Fluid or skin or nail scrapings are placed on a slide & a drop of 10% KOH is added
c. The KOH preparation is left at room temperature for 20-30 minutes to allow clearing of
epidermal cells
dissolve epidermal cells
d. Gentle heat increases the rate of clearing, but it is unnecessary and may be harmful to the
specimen
2. Lactophenol cotton blue
Phenol - killing agent
Aniline blue - stain
It provides blue color the cell wall
For tissue & slide culture
a. Contains lactic acid which preserves the fungal structures, phenol as a killing agent and
aniline blue (cotton blue) which stains the structures for better visualization
b. Can be added to 10% KOH preparation or used alone for wet mount preparations of
fungi
3. India ink or Nigrosin (see Appendix A)
Not diagnostic
Should be mistaken for yeast
Capsule - is not penetrated (clear, hallow)
a. For detection of C. neoformans in CSF through negative staining to demonstrate capsule
C. neoformans - are opportunistic fungi
4. Calcofluor white stain
Cellular

a. Currently preferred method

b. For sharp delineation of fungal elements in clinical specimens by fluorescence
microscopy
Chitin of fungal cell wall
c. Organisms fluoresce blue-white or apple green depending upon the light source
B. Permanent Mounts
1. Periodic acid - Schiff (PAS) stain
For tissues
a. Stains the fungal elements well; hyphae of molds and yeast can be readily distinguished
b. Carbohydrates present in fungus wall stain purplish red
Oxidizes the hydroxyl in the cell wall to aldehyde
c. Hematoxylin and eosin (H &E) slides may be restained by this method if fungus wall is visible
Fast green
2. Gram stain (Hucker modification)
Color violet
C. neoformans - pale lavender with blue inclusion
a. All fungi are gram positive (2-3 wider than gram negative)
b. This method employs crystal violet and ammonium oxalate
3. Acid- fast stain (Kinyoun modification)
Also called Cold Method
Red against green background
a. For detection of Nocardia and B. dermatitis
4. Giemsa and Wright stains
a. For detection of H. capsulatum from blood or bone marrow
H. capsulatum - small, oval, and its yeast cell is light dark blue in color
;O);O);O) 171 ;O);O);O)
5. Papanicolaou stain
Also called PAP smear
a. Cytotechnologists can detect fungal elements with this stain
Vaginal smear
6. Gomori methenamine silver stain
For tissues
Outline in black against a pale background
a. For detection of fungi in histologic sections

7. Acridine orange
a. For tinea versicolor
An-an
Superficial mycosis
Skin
b. Green fluorescent fungal elements and orange epithelial cells
TECHNIQUES FOR MICROSCOPIC OBSERVATION OF FUNGI
A. Wet Mount
1. A small portion of an isolated colony from a point intermediate between the cents and the
periphery is removed containing a small amount of the supporting agar with a wire bent at a 90
angle.
2. This portion is placed on a slide and a drop of Lactophenol cotton or aniline blue is added.
3. A coverslip is applied with a gentle pressure using a pencil eraser or other object to disperse
the growth and agar.
4. The preservation is examined microscopically.
B. Scotch or Cellophane Tape Preparation
1. Touch the adhesive side of a small length of transparent tape to the surface of the colony.
2. Adhere the length of tape to the microscope slide surface to,which has been added a drop of
Lactophenol cotton blue or aniline blue.
3. Observe under the microscope for the characteristic shape and arrangement of the spores.
C. Microslide Culture
Original state
1. This technique allows one to observe microscopically the fungus growing directly underneath
the coverslip.
2. Slide cultures should not be made on slowly growing organisms suspected of being dimorphic
pathogens like H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis or S. schenckii.
SPECIAL TESTS
A. Hair Perforation or Baiting Test
1. Place a filter paper disk into the bottom of a sterile Petri dish.
2. Cover the surface of paper disk with sterile distilled water.
3. Add a small portion of sterilized prepubertal hair into the water.
4. Inoculate the portion of the colony to be studied directly onto the hair.
5. Incubate at 25C for 10-14 days.
6. Observe hairs at regular intervals by placing them into a drop of water on a glass slide.
Position a coverslip and examine microscopically for the presence of conical perforations of the
hair shaft.

T. mentagrophytes = ( + )
T. rubrum = ( - )
:-*^3^:-* 172 :-*<3:-*
B. Exoantigen Test
A serological test
1. Principle
Antibodies developed against particular mycelial antigens will react specifically in a gel
immunodiffusion precipitin test. The mold forms can be identified definitively by an antigenantibody reaction, hence the conversion the yeast phase is not needed.
2. Procedure
l l - positive
/\ - negative
a. A concentrated merthiolate extract of mature fungus culture used in the microdiffusion
tests
b. The extract is placed into wells punched into a plate of buffered phenolized agar adjacent
to the control antigen well and is tested against positive control antiserum
c. The test is read after 24 hours for the presence of precipitin hands of identity after
incubation at 25
C. Rapid Urease Test
1. Principle
Ability of some bacteria to hydrolyze urea into ammonia, water and CO2 by means of an
enzyme urease.
a. The same as urease test.
2. Procedure
Pink color = (+)
Purple color = (-)
a. Use either a UREA-R broth (Difco) or Urea Base (BBL)
b. Transfer a heavy inoculum of a yeast colony (excluding pink yeasts) to a well of a
microdilution plate containing the urea broth
c. Include positive C. neoformans and negative C. albicans controls
C. albicans - yellow after 4 hours
d. Seal the wells with plastic tape and incubate 4 hours at 37C
e. Observe for a pink to purple color as positive result
D. Rapid Nitrate Reductase Test
1. Principle

Similar to test on page 23 except that benzalkonium chloride is added to test medium
to disassociate the cell wall of yeasts to allow the release of nitrate Reductase (Reductase
enzyme).
2. Procedure
Red color = (+)
a. Sweep the tip of an applicator impregnated with the nitrate reduction test reagents across 2 or
3 colonies of yeast
b. Swirl the applicator containing the yeast against the bottom of an empty test tube to get
the yeast cells in contact with the contact with the cotton fibers
c. Incubate the tube and swab at 45C for 10 minutes
d. Remove the swab and add two drops each of N-naphthylenediamine and sulfanilic acid
reagents to the tube and replace the applicator; a change in color to red indicator a positive test; if
no color appears after 10 minutes, add a pinch of zinc dust and observe for a red color indicating
the presence of previously unreacted nitrate and a negative test
>.<>.<>.< 173 >.<>.<>.<
E. Levodopa-Ferric Citrate Test
1. Principle
Formation of melanin
C. neoformans is known to produce the enzyme phenol oxidase which when made to
react with dihydroxyphenylalanine in the presence of ferric nitrate forms a dark pigmented
compound, melanin (for rapid identification of C. neoformans)
2. Procedure
a. The test is available commercially:
i. G/N - screen (Flow Laboratories)
ii. L-DOPA Ferric citrate reagent disks (Difco)
b. Smear test organism onto the surface of reagent disks which were moistened beforehand
with 2-3 drops of distilled water in a Petri dish
c. Incubate disks at 37C and examine at 30 minutes interval for up to 6 hours.
F. Germ Tube Test
1. Specifically used to identify C. albicans.
2. Procedure
a. Suspend a very small inoculum from an isolated yeast colony in 0.5 mL of serum
b. Incubate tubes at 35-37C for 3 hours
c. Place a drop of the suspension on a glass slide and examine under low-power
magnification for the presence of germ tubes (appendages half the width and 3-4 times the length
of the yeast cell from which they arise)
G. Carbohydrate Utilization Tests
1. Yeasts and yeast-like fungi are inoculated onto a carbohydrate-free medium

2. Carbohydrate-containing filter paper disks are added and utilization is determined by a

color change or presence of growth around the disk.
H. Commercial Yeast Identification Systems
1. API-20C yeast system
2. API Yeast-Ident system
3. Uni-Yeast Tek system
4. AutoMicrobic system
5. Abbott Quantum II system
CHAPTER XXXIV
THE MEDICALLY IMPORTANT MYCOSES
SUPERFICIAL MYCOSES
These are caused by fungi affecting the superficial layers of the skin such as the stratum
corneum and hair. They are asymptomatic but aesthetically displeasing.
A. Tinea versicolor (Pityriasis versicolor
1. Clinical Features
Forms spaghetti & meatballs
No cellular response
Fluoresce golden yellow or brownish
a. It is a chronic and nonirritating superficial infection of the stratum corneum occurring on
the chest, upper back, arms, abdomen and rarely on the face
b. Lesions are described as superficial brownish scaly areas on light-skinned persons and
lighter areas on the dark-skinned persons
Hypopigmentation & Hyperpigmentation (brownish scalp)
2. Causative Agent: Malassezia furfur (Pityrosporum orbiculare - yeast phase)
3. Predisposing Factors:
i. Hot and humid climate
ii. Excessive perspiration
iii. Corticosteroid use
iv. Malnutrition
v. Heredity
4.Other Diseases Caused by M. furfur
a. Disseminated infection in infants, young children and adults receiving lipid replacement
therapy
b. Folliculitis
c. Possibly seborrheic dermatitis or dandruff

5. Laboratory Diagnosis
Specimen: skin scrapings
a. Direct microscopic examination
i. 10% KOH
ii. Calcofluor white stain
appearance: blunt-ended short hyphae and clusters of spherical spores that form
spaghetti and meatballs pattern
b. Wood's light (ultraviolet radiation of lesions in a darkened room)
- lesions Fluoresce golden yellow or brownish
c. Culture
i. not required to establish a diagnosis
ii. M. furfur grows on Sabouraud's medium overlaid with olive oil a related species M.
pachydermatis does not require added lipid for growth
:-o:-o:-o 175 :-o:-o:-o
6. Treatment
a. Topical application of 2.5% selenium sulfide for 10 minutes daily for 7 days
b. Topical miconazole and ketoconazole
c. Folliculitis - treated with oral ketoconazole
B. Tinea nigra (Tinea nigra Palmaris)
Blackish-brown palms, soles or elsewhere
1. Clinical Features
a. Blackish-brown macular patches resembling faded silver nitrate stain occurring most
frequently on the palms but also on the soles or elsewhere
b. Lesions are not elevated or scaly
2. Causative Agent: Phaeoannellomyces werneckii (Cladosporium or Exophiala werneckii)
A dimatiacous fungi hydrolysis casein
3. Laboratory Diagnosis
Specimens: skin scrapings
a. Direct KOH or calcofluor white examination
i. Olive or brown-pigmented, branched, septate hyphae and budding yeast cells that are
one-or two-celled
ii. Older colonies exhibit one-or two-celled conidia produced by annelides, that bear
successive rings (annelations)
Safety pins
b. Culture (Sabouraud's medium with and without antibiotics)

i. Initial colonies are glossy, black and yeast like in appearance

ii. With age, colonies become filamentous with velvety-gray aerial hyphae
4. Treatment
a. Topical keratolytic solutions to sulfur, salicylic acid or tinctura of iodine
b. Simple shaving of superficial epidermis with scalpel blade
C. Piedra (Trichosporosis)
Hair
A fungal infection of the hair characterized by hard nodules distributed irregularly along
the length of the shaft which are easily felt palpation.
1. Black Piedra(Piedra hortai)
a. Caused by Piedra hortai
b. Occurs in tropical areas
c. Crushed nodules in KOH preparation shows numerous oval asci, containing 2-8 aseptate
ascospores; ascospores are spindle shaped (fusiform) and have a filament at each pole
d. Any fungal culture medium lacking cycloheximide may be used; colonies are black and
composed of hyphae and chlamydospores
e. Treatment is by cutting the hair

2.

White Piedra

a. caused by Trichosporon beigelii (T. cutaneum) Endotrix soft portion

b. occurs commonly in temperate regions
c. crushed nodules in KOH preparation show hyaline hyphae and arthrospores
d. d. culture shows cream-colored, soft colonies composed of blastospores and septate
hyphae which fragment into arthrospores
e. T. beigelii does not ferment carbohydrates but aerobically utilizes certain substrates
f. Treatment is the same
Manum hands
Pedis athletes foot
Capifis head
Ongium nails
MOT: direct contact

CUTANEOUS MYCOSES (DERMATOMYCOSES)

These are fungal infections involving the superficial keratinized tissue of the body such
as the skin, hair, and nails. They are caused by a group of fungi commonly called dermatophytes
which breakdown and utilize keratin as a source of nitrogen but are incapable of penetrating the
subcutaneous tissue. The three genera associated with dermatomycoses include:

1. Epidermophyton affecting skin and nails Macroconidia

2. Microsporum affecting skin and hair Macroconidia
3. Trichophyton affecting skin, hair and nails - Microconidia

A. Morphology and Identification of Common Dermatophytes

Table 24-1 Characteristics of commonly Isolated Dermatophytes

Dermatophytes

Colonial Morphology

Growth Rate

Micromorphology

E. Floccosum

Center of colony tends

1 week

Macroconidia large,

to be folded and is

thin smooth-walled,

khaki green; periphery is

mutiseptate, cla-

yellow; reverse

vate and borne sing-

yellowish brown with

ly or in clusters of

observable folds

two or three; microconidia are absent;

numerous chlamydospores

M. audouinii

Downy white to salmon

2 weeks

Sterile hyphae: termi-

(epidermiecopitis

pink colony; reverse

nal chlamydospores,

ectorix; apple

tan to salmon pink

favic chandeliers,

green in woodlight;

and pectinate bodies;

10-21 days)

macroconidia rarely
seen- bizarre

shaped
microconidia
absent

is

1 week

seen;
rare or

M. canis

Colony usually mem-

Thick-walled, spin-

(dog and cat ring-

branous with feathery

dle shaped, multisep-

worm; zoophilic;

periphery; center of

tate (terminal knob),

ectotrix)

colony white to buff

rough-walled, macro-

over orange-yellow;

conidia some with a

lemon-yellow or

curved-tip; micro-

yellow-orange apron

conidia rarely seen.

reverse. 8-15 cells

8 septa; grow after

4-5 days

177
M. gypseum

Cinnamon-colored

1 week

(geophilic-soil;

powdery-colony;

elliptical multi-

ectotrix; wood-

reverse light tan

septate macroconidia;

light (-)

Thick-walled rough,

microconidia few or
absent

T. mentagrophytes

Different colonial

7-10 days

(scanty pigment;

types; white to pink-

bose microconidia

spiral hyphae;

ish, granular and

most commonly borne

urease +; nair bai-

fluffy varieties occa-

in grapelike clusters

ting test +; sensi-

sional light yellow

or laterally along the

tivity test; Endotrix) periphery in younger

Many round to glo-

hyphae in 30% of iso-

cultures; reverse buff

lates; macroconidia

to reddish brown

are thin-walled,
smooth, club-shaped
& multiseptate; numerous or rare depending upon strain

T. rubrum

Colonial types very

2 weeks

Microconidia, usually

(anthrophilic;

from white downy to

teardrop, most comm-

Endo & Exo;

pink granular; rugal

only borne along side

woodlight (-);

folds are common;

of the hyphae; macro-

hair baiting test (-);

reverse yellow when

conidia usually absent

urease test (-)

colony is young; how

but when present are

ever, deep wine red color

smooth, thin-walled,

commonly develops with age

and pencil-shaped

T. tonsurans

White, tan to yellow

7-14 days

Microconidia are tear-

(2nd toens capitis;

or rust, suede-like to

drop or club-shaped

black dot ring-

powdery; wrinkled

with flat bottoms;

worm; alopecia)

with heaped or sunken

vary in size but usua-

center; reverse yellow

lly larger than other

to tan to rust red

dermatophytes;
balloon forms-aged
pleomorphic microconidia macroconidia
usually rare

T. schoenleinii

Irregularly heaped,

2-3 weeks

(slow grower;

smooth white to

rile; may antler-type

Mousy odor;

cream colony with

hyphae seen (favic

permanent alopecia; radiating grooves

Hyphae usually ste-

chandeliers)

scarring taeni; fluoresence type of capitis)

T. violaceum

Port wine to deep

(no fluorescence;

violet colony, may

2-3 weeks

Branched, tortuous
hyphae that are sterile

block-dot ringworm be heaped or flat

chlamydospores

appearance)

with waxy-glabrous

commonly aligned in

surface; pigment may

chains

be lost on subculture

T. verrucosum

Glabrous to velvety

(swollen hyphae;

white colonies; rare

2-3 weeks

Microconidia rare;
large and teardrop

concentrate rings

stains produce yellow

when seen; macro-

of lesions Kerion)

brown color; rugal

conidia extremely

folds with tendency

rare, but form

to sink into agar

characteristics

ratsurface

tail types when seen;

many chlamydospores
seen in chains, particu
larly when colony is
incubated at 37 C

1.

Epidermophyton floccosum

(see table 24-1)

(causes epidemic tinea pedis in sumner sample)

2.

Microsporum (for colonial and microscopical morphologies see table 24-1)

a. M. audouinii Gray patch ectotherix

i. infected hair shafts fluoresce yellow-green using a Woods lamp
ii. addition of yeast extract may stimulate growth and production of
macroconidia
iii. grows poorly on sterile rice grains (rice medium)

b. M. canis
i. infected hairs fluoresce bright yellow-green using a Woods lamp
ii. grows well on sterile rice grains

c. M. gypseum
i.

3.

infected hairs generally do not fluoresce using Woods lamp

Trichophyton (infected hair do not fluoresce under a Woods lamp)

The most common species recovered in the clinical laboratory are T. rubrum and T.
mentagrophytes hence, they should be differentiated (for colonial and microscopical
morphologies see table 24-1)

a. T. rubrum

i. no specific nutritional requirement

ii. does not perforate hair in vitro (hair baiting test)
iii. does not produce urease
iv. abundant red colonies when after aged
v. Tinea unguium beneath the nail plates

b. T. mentagrophytes
i. produces urease
ii. perforates hair in vitro (hair baiting test)
iii. teardrop/pencil-shaped
iv. Kerion common cause of tinea pedis
v. Permanent. Alopecia

c. T. tonsurans
i. growth enhanced by thiamine
ii. Black dots Endotrix
iii. club-shaped or flat bottoms, batton-form

d. T. verrucosum
i.

growth enhanced at 53-37 degree C

ii. growth requires thiamine and inositol

iii. grows in media with 4% casein and 0.5% yeast extract
iv. early hydrolysis of casein
v. Rat tail Glaborous and velvety white colonies

e. T. schoenleinii (see table 24-1)

i.

radiating grooves

ii. scutula extensive alopecia, hair breaks off above the scalp
(favic antler type) last for lifetime

f. T. violaceum
i.

growth enhanced with thiamine

ii. diffuse alopecia

iii. hair breaks off at follicle

iv. branched tortuous hyphae
v. port wine to deep violet colonies

B. Clinical Infections

1. Natural Habitat of Common Dermatophytes

Anthropophilic

Zoophilic

Geophilic

(humans)

(animals)

(soil)

E. floccosum

M. canis (dogs, cats)

M. audouinii

T. mentagrophytes var

T. mentagrophytes var
interdigitale
T. rubrum
T. schoenleinii

M. gypseum

mentagrophytes
(rodents)
T. verrucosum
(cattle)

T. tonsurans
T. violaceum

Anthropophilic species produce relatively mild and chronic infections in humans,

whereas zoophilic dermatophytes cause more inflammatory and acute infections that respond
better to treatment and usually do not recur.

2. Transmission

a. contact with dermatophytes in soil or in animals

b. anthropophilic species are transmitted by direct contact or fomites (ex. infected haurs
on hats, caps, combs or barber clippers)

3. Predisposing Factors

a. hot, humid climates

b. crowded living conditions
c. increased perspirations
d. heavy exposure
e. young individuals
f. genetic predisposition

4. Clinical Forms of Dermatophytes

Cutaneous mycoses are usually refered to as tines (ringworm) because of the

raised circular lesion which has an outer ring of an active progressing infection with central
healing within the ring.

Tinea capitis

a. site of lesion

i. scalp
ii. endothrix:

presence of arthroconidia (from fragmentation of hyphae) within

hair shaft but without destruction of cuticle

iii. ectothrix:

presence of arthroconidia (from fragmentation of hyphae) around

or on the surface of hair shaft with destruction of cuticle

b. most commonly isolated organisms

i. epidemic tinea capitis:

T. tonsurans, M. ausouinii (most important),

T. violaceum

ii. nonepidemic tinea capitis:

M. canis, T. verrucosum, M. gypseum

c. manifestations

i.

T. tonsurans and T. violaceum:

endothrax type of invasion; circular, scaly

patches of alopecia; stub of hair remain in
the epidermis of the scalp after the brittle

hair have broken off and give the typical

black dot ringworm appearance
ii. T. verrucosum:

lesions are deep, pustular, and

inflammatory; endothrix and ectothrix type
of invasion; may induce a severe combined
inflammatory and hypersensitivity reaction
called a kerion.

iii. Microsporum species:

ectothrix type of invasion

Tinea favosa (favus)

a. site of lesions

i. scalp
ii. torso

b. causative agent: T. schoenleinii

c. manifestations

i.

formation of yellowish cup-shaped crusts or scutulae

ii. extensive alopecia and scarring of the scalp

iii. mousy odor to scalp

Tinea barbae (barbers itch)

a. site of lesions

i. bearded areas of face and neck

b. commonly isolated organisms

i. T. rubrum
ii. T. verrucosum

c. manifestations

i.

mild superficial edematous, erythematous lesions or;

ii. severe deep pusticular folliculitis

Tinea corporis

a. site of lesions

i.

arms

ii. legs
iii. torso

b. commonly isolated fungi

i. T. rubrum
ii. M. canis
iii. T. mentagrophytes
c. manifestations

i.

circular patches with advancing red, vesiculated border and central scaling

ii. pruritic

Tinea cruris (jock itch)

a. site of lesions: genitocrural folds

b. commonly isolated fungi

i. T. rubrum and T. mentagrophytes

ii. E. floccosum

c. manifestations

i. erythematous scaling lesion in intertriginous area

ii. pruritic

Tinea pedis (athletes foot)

a. site of lesions: feet particularly the toes, webs and soles

b. commonly isolated organisms

i. T. rubrum and T. mentagrophytes

ii. E. floccosum

c. manifestations

i. itching, red vesicle lesions

ii. itching, scaling and tissures

Tinea tonsurans

a. site of lesions: intertriginous area

b. commonly isolated fungi: same as in tinea pedis

c. manifestations

i. usually associated with tinea pedis

ii. hyperkeratotic (white flakes or vesicular or erythematous)

Tinea unguim

a. site of lesions: nails

b. commonly isolated dermatophytes

i. T. rubrum and T. mentagrophytes

ii. E. floccosum

c. manifestations

i. nails thickened or crumbling distally

ii. white patches on surface or invasive infection beneath nail plates
iii. usually associated with tinea pedis
Dermatophytid (id reaction)

An allergic response to fungal antigens wherein a dermatophyte infection in one area

(e.g., tinea pedis) elicits an allergic reaction elsewhere on the body (e.g., the ahnds)

5.

Laboratory Diagnosis

Specimens: skin and nail scrapings, hairs

a. examination of infected hairs under Woodslight

b. microscopic examamination using 10-20% KOH (or calcofluor white)

i. Microsporum species:

form dense sheaths of spores in

A mosaic pattern around the hair (ectothrix)

ii. Trichophyton species:

form parallel rows of spore outside (ectothrix)

or inside (endothrix) the hair shaft

iii. T. schoenleinii:

favic hairs present characteristic air spaces in the

hair which are readily filled with fluid in KOH
preparations

c. culture

i. Mycosel

ii. DTM
iii. cornmeal agar for production of macroconidia
iv. Inhibitory mold agar
v. Sabourauds agar with antibiotics

d. id reaction is diagnosed by a negative microscopic and cultural examination of the

reaction site and finding of dermatophytosis elsewhere on the body

6.

Treatment

a. topical antibiotics such as cream preparation of tolnaftate, miconazole nitrate,

haloprogin, clotrimazole, econazole or ciclopirox
b. oral griseofulvin for long periods most effective especially for scalp and nail
infections
c. oral ketoconazole

SUBCUTANEOUS MYCOSES

These are infections that involve the skin and subcutaneous tissue which may rarely
become systemic or disseminated.

A.

Sporotrichosis (rose gardeners disease)

1. causative agent:

thermally dimorphic Sporothrix schenkii whose natural habitat is

living on dead vegetation

2. Morphologic and Cultural Characteristics

a. Mycelial phase

i. Colonies are usually small, moist, and white to cream colored which on
further incubation become membrabous and coarsely matted, wrinkled, with
the color becoming irregularly dark brown or black and the colony becoming
leathery

ii.

microscopically hyphae are delicate, septate, exhibit branching and bear

one-celled conidia which are borne bouquet-like, in clusters from the tips of
single conidiospores. Flowerettes daisy-like

b. Yeast phase

i. colonies are pasty and grayish

ii. microscopically they are usually spherical, oval, singly or multiply budding
cells but some are fusiform, elongated or cigar-shaped; asteroids may also
seen
iii. mycelia to yeast conversion at 37 degree C in 12-48 hours

3. Transmission

a. through traumatic inoculation with contaminated material

(e.g., thorn or splinter, bushing against a tree bark)

b. inhalation of infectous particles (in pulmonary sporotrichosis)

4. Clinical Manifestation

a. lymphocutaneous sporotrichosis (75% of cases)

i. lesions appear first in the cutaneous and subcutaneous tissues and

progressively involve the draining lymphatics
ii. initially with a small, movable, nontender, subcutaneous nodule develops
but a small ulcer may also be seen
iii. the subcutaneous nodule becomes discolored and the skin darken from red
to black which subsequently erupts to form an ulcer or sporotrichotic chancre
iv. after a few weeks, the primary lesion heals and new ones develop.

b. chronic sporotrichosis

i. After the primary lesion, multiple subcutaneous nodules develop along the
lymphatic channels and become hard and cordlike

ii. the course is the same and if untreated becomes chronic, but may heal
spontaneously

c. fixed sporotrichosis

i. presence of only one lesion without involvement of the lymphatics

ii. lesion may be ulcerative or appear a plaque or rash

d. pulmonary sporotrichosis (rare)

5. Laboratory Diagnosis

Specimens:

exudates from unopened subcutaneous nodules or from open

draining lesions; biopsy material

a. KOH or calcofluor white preparations

b. special stains

a. PAS
ii. methenamine silver stain
iii. fluorescent antibody stain

c. culture
i.

Inhibitory mold agar or

ii.

Sabourauds agar with antibiotics

iii.

others

d. serology

6.

i.

yeast cell agglutination test

ii.

sporotrichin

Treatment

a. oral solution of saturated potassium iodide (SSKI) treatment of choice for cutaneous
sporotrichosis (can be administered topically also)
b. amphotericin B, ketoconazole, dihydroxystilbamidine, griseofulvin and flucytosine

B.

Chromomycosis (Chromoblastomycosis)

This is a chronic fungal infection acquired thru traumatic inoculation of spores, primarily
in the lower extremities. It is characterized by the development of a papule at the site of the
injury that spreads to form warty or tumor like lesions (cauliflower-like). Secondary infection
and ulceration may ensue. Rarely, brain abscess may occur. Elephantiasis and Lymph stasis can
result to secondary infection.

1.

Causative agents

Chromomycosis is caused by dematiaceous fungi which are imperfect fungi that

produce varying amount of melanin-like pigments found in the conidia or hyphae or both.

a. Cladosporium carrionii
b. Phialophora verrucosa
c. Fonsecae pedrosoi and F. compacta

2.

Cultural Characteristics

a. the dematiaceous fungi have similar characteristics

b. all are slow-growing and produce heaped-up and slightly folded darkly pigmented
colonies with a grayish-velvety appearance; the reverse side of the colonies is jet
black
c. yeast species are usually pleomorphic and produce a dark moist colony

3. Microscopic Apperance

a. Cladosporium species:

produce long chains of elliptical conidia (blastoconidia)

borne from erect, tall, branching conidiophores

b. Phialophora species:

produce short, flask-shaped to tubular phialides, usually

with a well developed collarette: clusters of conidia are
produced by the phialides through an apical pore

c. Fonsecae species:

may exhibit Phialophora-type or Cladosporium-type of

Sporulation and also produce lateral or terminal conidia
From a lengthening conidiogenous cell resembling a
series of bent knees (conidia are produced sympodially)
Achothica: series of bent knees

i. F. pedrosoi produces loose conidial heads

ii. F. compacta produces more compact conidial heads

4. Laboratory Diagnosis

Specimens: Scrapings or biopsy from lesions

a. Microscopic examnination

i.

scrapings from crusted areas added to 1-% KOH show presence of muriform
or sclerotic bodies, which appear rounded brown resembling copper
pennies that exhibit transverse septations

b. culture: Sabourauds with or without antibiotics

c. extraction and detection of exoantigens

5. Treatment

a. flucytosine the best

b. amphotericin B and the azoles especially itraconazole

C.

Phaeohyphomycosis

This term refers to infections characterized by presence of darkly pigmented, septate

hyphae in tissues. Clinical forms vary from solitary encapsulated cysts in the subcutaneous
tissue to brain abscess.

1. Causative Agents

a. Exophiala jeanselmei

i.

grows early as a black yeast with dark budding yeast forms seen on
microscopy of culture

ii.

develops long slender conidiophores with tapered tips and conidia arising from
growth rings (annelids)

b. Phialophora (see above)

c. Wangiella dermatitidis

i.

early form same as that of Exophiala

ii.

produces conidia phialides that lack collarettes

d. Xylohypha bantiana (Cladosporium bantianum or Cladosporium trichoides)

i.

Cladosporium type of sporulation

ii.

infection usually recognized only at autopsy

e. other normally saprophytic dematiaceous fungi

2. Laboratory Diagnosis

a. microscopic examination (KOH preparation) irregular dark, hyphae, as well as

yeast and pseudohyphae are seen in tissue
b. culture

3. Treatment: same as in Chromomycosis

D.

Mycetoma (Madura foot or Maduromycosis) Maduromycetoma

lume faction, draining sinuses and granules

This is a chronic granulomatous infection usually involving the lower extremities. It is

characterized by swelling, purplish discoloration, tumor-like deformities of the subcutaneous
tissue and multiple sinus tracts that drain pus containing yellow, red, white or black granules.
There are two types of Mycetoma:

Actinomycotic mycetoma:

caused by Nocardia, Actinomadura, Streptomyces and

aerobic actinomycetes. Granules filamentous 0.5-1.0 um in
diameter

Eumycotic mycetoma:

caused by a true fungi, most commonly Pseudoallescheria

boydii which is found in soil and sewage; transmitted via
traumatic inoculation. Large hypha Granule color

1. Cultural and Microscopic Apperance

a. P. boydii is a hyaline mold (sexual stage) that grows rapidly as white fluffy colony
that changes to a brownish-gray (mousey) mycelium; the reverse of the colony is
black
b. the asexual stage is called Scedosporium apiospermum and microscopically produces
elliptical (sperm-shaped), single celled conidia borne singly from the tips of long or
short conidiophores (annelophores); clusters of conidiophores with conidia produced
at the ends are called coremia
c. P. boydii, microscopically exhibit cleistothecia, which are saclike structures that
contain asci and ascospores; the ascospores are oval and delicately pointed at each
end resembling the condia of the asexual form. SDA + Cycloheximide

2. Laboratory Diagnosis

a. macroscopic examination of granules from lesions are white to yellow

b. microscopically the granules consist of loosely arranged hyaline or pigmented,
intertwined hyphae

3. Treatment

a. topical nystatin and potassium iodide

b. ampotericin B and miconazole

E. Rhinosporidiosis

A chronic infection characterized by the development of polypoid masses of the nasal

mucosa caused by Rhinosporidium seeberi. (cannot be cultured)

1. Laboratory Diagnosis

a. histologic examination of infected tissue

i.

reveals epithelial hyperplasia and cellular infiltrate of neutrophils,

lymphocytes, plasma cells and giant cells

ii.

large thick-walled sporangia; the cell wall of the spherule is mutilayered and
stains with mucicarmine. Mature spores 7-9 um appear lobulated by globular
bodies

b. serologic test for rhinosporidial antigen

2. Treatment

a. topically, surgically and by local injection of ethylstilbamidine

F.

Lobomycosis Keloidal blastomycosis/ Lobes disease

A chronic subcutaneous infection of human and dolphins caused by Loboa loboi

(Paracoccidiodes loboi) Localized and manifested as keloid verrucoid to nodular lesion, well
defined, smooth, painless and easily removed.

1. Laboratory Diagnosis

Specimens: skin scrapings, biopsies or wet preparations of exudative lesions

a. direct microscopic examination

i.

large, spherical or oval yeasts that exhibit multiple budding and form short
chains of three to six or more yeast cells which are multinucleated and thick
walled

b. histologic examination

i.

granulomatous nodules and occasional asteroid bodies be seen inside

macrophages. Grow ns globose cells connected to each other by marrow
nodules in chain in KOH

2. Treatment

a. Sulfa drugs
b. Surgical excision

G. Thinoentomophthoromycosis (Entomophthoromycosis conidiobolae)

A rare infection of the nasal mucosa caused by Entomoohthora coronate (Conidiobolus

coronatus)

SYSTEMIC OR DEEP MYCOSES (Dimorphic; yeast form/mold form) (exoantigen test)

These are fungal infections that may involve any of the internal organs of the body as
well as lymph nodes, bone, subcutaneous tissue and skin. They are caused by inhalation of the
thermally dimorphic fungi which exist in two phases of growth:

1. Yeast phase, parasitic, invasive or tissue form

-

takes place at 35-37 degree C

2. Mycelila phase, mold or saprobic form

-

takes place at room temperature (25-30 degree C)

Traditionally, the definitive diagnosis of a dimorphic fungus has been made by observing
both the mold and yeast form. But now most laboratories consider the exoantigen test as the most
conclusive method.

OPPORTUNISTIC MYCOSES

These are caused by fungi that usually do not induce disease and occur almost
exclusively in immunocompromised patients who are often on treatment with corticosteroids,
cytotoxic drugs, or other immuno-suppressive agents

TABLE 24-2 CHARACTERISTIC FEATURES OF FUNGI CAUSING SYSTEMIC MYCOSES

Infection

Coccidioidomycosis
(valley fever or

Etiologic Agent

Habitat

Clinical Implication

Coccidioides

soil

pulmonary infection, skin

immitis

Growth Rate (days)

2-21

infection, osteomyelitis,

San Joaquin Valley

meningitis, arthritis,

fever, desert fever

disseminated infection

desert rheumatism)

Histoplasmosis

Histoplasma

soil enriched

pulmonary infection, oro-

(intracellular mycoses

capsulatum

with bird manure

pharyngeal lesions, CNS

or bat guano

infection, skin infection,

of the reticuloendothelial system)

(rare), uveitis, perito-

- mimic

nitis, endocarditis
brain abscess, disseminated
infection

5-45

Blastomycosis

Blastomyces

not clearly

(Gilchrists dse.,

dermatitidis

defined

pulmonary infection, skin

5-30

infection, oropharyngeal

Chicago dse., North

ulceration, osteomyelitis,

American blastomycosis)

prostatitis, arthritis,
CNS infection, disseminated
Infection

Paracoccidioidomycosis

Paracoccidioides

not clearly

(south American

brasiliensis

defined

pulmonary infection, skin

21-28

infection, oropharyngeal

blastomycosis)

and nasopharyngeal lesions,

CNS infection and other
disseminated infection

189
Cultural characteristics at 30 degree C

Microscopic Morphological Features

Microscopic Morpho-

Blood-enriched

Blood-containing : Non-Blood Con-

logical Features

Medium

:
:

Medium Without
Blood

Medium

: taining Medium

of Tissue Form

Coccidioidomycosis

Colonies may be

Colonies appear

Chains of alternate, barrel-shaped

Round, nonbudding

white and fluffy

delicate, cobweb-like

arthroconidia are characteristic;

thick-walled sphe-

to greenish on

growth & usually are

some arthroconidia may be elongated;

rules 30-60 um in

blood-enriched

fluffy white nut may

hyphae are small and often arranged

diameter containing

media; some iso-

be pigmented gray,

in ropelike strands and racquet

2-5 um endospores

lates are yeast-

orange. brown, or

forms are seen in young cultures

are characteristic

like, heaped,

yellow; mycelium is

wrinkled, and

adherent to the agar

membranous

surface in some por-

2-5 um, small, oval

tions of the colony

(CAUTION: major biohazard to lab. personnel

Histoplasmosis

Colonies are heap-

Colonies are white,

Hyphae 1-2 um in

Young cultures

ed, moist, wrink-

cream, tan or gray

diameter are

usually have a

to spherical bud-

led, yeastlike,

fluffly to glabrous;

present; some are

predominance

ding cells often

soft, & cream,

some colonies appear

aggregated in

tan or pink in

yeastlike and adhe-

ropelike clusters;

walled macro-

color; tuft of

rent to the agar sur-

sporulation is

conidia that

hyphae often pro-

face; many variations

ject upward from

in colonial morpholo-

colonies

gy occur

rare

of smooth

seen inside of
mononuclear cells

become tuberculate with age;

macroconidia
may be pyriform
or spherical; some
isolates produce
small pyriform
microconidia
in the presence or
absence of macroconidia

190
Cultural characteristics at 30 degree C

Microscopic Morphological Features

Microscopic Morpho-

Blood-enriched

Blood-containing : Non-Blood Con-

logical Features

Medium

:
:

Medium Without
Blood

Medium

: taining Medium

of Tissue Form

Blastomycosis

Colonies are cream

Colonies are white to

Hyphae 1-2 um in

Hyphae 1-2 um in

8-15 um, broad-based

to tan, soft

cream to tan, some

diameter are pre-

diameter are pre-

budding cells with

moist, wrinkled,

with drops of exu-

sent; some are

pyriform conidia

double-contoured

waxy, flat to

date present, fluffy

aggregated in

are produced on

walls are seen;

heaped, and yeast-

to glabrous, and ad-

ropelike clusters

are produced on

cytoplasmic granu-

like; tufts of

herent to the sugar

sporulation is

short to long

lation is often

hyphae often pro-

surface

rare

conidiophores

obvious

ject upward from

(lollipop); some

colonies (prick-

cultures produce

ly state) (BAP)

few conidia

Paracoccidioi-

Colonies are heaped, wrinkled, moist

Hyphae 1-2 um in diameter are present;

10-25 um, multiply

domycosis

and yeastlike; with age, colonies may

some isolates produce conidia similar

budding cells (buds)

become covered with short aerial myce-

to those of B. dermatitidis, chlamydo-

1-2 um resembling

lium and may turn brown; the surface is

spores may be numerous, and multiple

a mariners wheel

often heaped with crater formation (BAP)

budding yeast cells 10-25 um in dia-

may be present;

meter may be present

buds are attached

to the parent cell

by a narrow neck

191
Probable Recovery
Sites

Confirmatory Tests

Serologic Tests

Treatment

for Identification

Coccidioido-

Respiratory secretions

Exoantigen test posi-

Compliment fixation

Amphotericin B,

mycosis

skin, bone, CSF, syno-

tive for III, F, or

Immunodiffusion

miconazole

vial fluid, urine,

TP bands

Latex agglutination

ketoconazole

gastric washings

triazole

fluconazole

Histoplasmosis

Respiratory secretions,

Exoantigen test posi-

Same as above

bone marrow, blood,

tive with H, M, or

treatment;

urine, adrenals, skin,

H and M band

Amphotericin B-DOC

CSF, eye, pleural

most require no

ketoconazole

fluid, liver, spleen,

oropharyngeal lesions,
vagina, gastric washings
larynx

Blastomycosis

Respiratory secretions

1 Broad-based budding

skin, oropharyngeal

cells may be seen

ulcer, bone, prostate

after in vitro conversion on cotton

seed agar
2 Exoantigen test positive with A band 70%

Complement fixation

Amphotericin B and

Immunodiffusion

ketoconazole

exhibit A bands

Paracoccidioidomycosis

Respiratory secretions,
oropharyngeal lesions,
gastric washings, skin,
nose

192

Exoantigen test positive for bands 1, 2, 3

Complement fixation

Ketoconazole-DOC;
Amphotericin B,
ulfa drugs

A.

Candidiasis; A mucocutaneous or deep tissue infection of C. albicans

1. Clinical Classification

a. cutaneous and mucosal candidiasis

- thrush (oral), moniliasis (vaginal), stomatitis, intertrigous candidiasis,

onychomycosis, esophagitis, severe diaper rash, balanitis

b. systemic candidiasis

- esophagitis, intestinitis, infant diarrhea, bronchopulmonary candidiasis,

pyelonephritis, cystitis, endocarditis, myocarditis, endophthalmitis,
meningitis, arthritis, osteomyelitis, peritonitis, macronodular skin lesions

c. chronic mucocutaneous candidiasis

2. Etiologic Agents (C. albicans)

a. Candida albicans and other Candida species which are members of the normal flora
of the skin, mucous membranes and GIT

3. Laboratory Diagnosis (see table 24-3)

Specimens:

swabs and scrapings from surface lesions, sputum, exudates and

material from removed IV catheters

a. Microscopic examination
b. Culture germ tube
c. Serology (immunodiffusion, CIE, latex agglutination)
d. Skin test
e. CHROM agar medium: 48 hours incubation

C. albicans green
C. tropecalis blue gray

f. Oricult N medium: 48/37 degree C, 5 days

C. albicans brown pigmented col.

4. Treatment

a. For cutaneous candidiasis :

topical ketoconazole, nystatin, miconazole or

gentian violet

b. For systemic candidiasis:

amphotericin B and flucocytosine

c. For chronic mucocutaneous candiasis:

flucytosine, amphotericin B, miconazole,

topical chemical solutions (gentian violet)
and transfer factor

B.

Cryptococcosis

(torulosis, European blastomycosis, torula meningitis)

(culture, india ink, serology)

1. Clinical Forms

a. Pulmonary cryptococcosis
b. Disseminated cryptococcosis
- Causing meningitis, may also occur in skin and viscera

2. Etiologic Agent (C. neoformans)

a. Cryptococcus neoformans which is found in the soil and in avian fecal material
(pigeon droppings)
b. Transmitted via inhalation

3. Laboratory Diagnosis (see table 24-3)

Specimens: spinal fluid, aspirates from skin lesions, sputum, urine, serum, tissue

II. Human fetal diploid(HFD)- useful in isolation of varicella-zoster virus & HSV, anderivirus,
picornavirus and RSV & are the only cells in which CMV virus and RSV & are the only cells in
which CMV is recovered.
III. Hep-2 continuous cell line- derived from human epithelial cancer cells; excellent for
recovering adenovirus, HSV and especially RSV.
IV. Hela cells- derived from human cervical cancer cells.
V. African green monkey kidney (AGMK), Verocells, baby hamster kidney (BHK),
Human embryonic kidney (HEK), Human embryonic lung (HEL). Etc.
B. Signs of growth
I. Cytophatic effects (CPES)
-evidence of host cell infection and damage or morphological changes in the cells due
to viral proliferation that can be readily observed in unfixed, unstained cell cultures under
LPO.
II. Plaque formation
-small zones of cell destruction or areas of clearness that can be see with the naked
eye.
III. Production ogHemagglutinins
-detected by hemadsorption/ hemagglitination of guinea pig erythrocytes.
IV. Foci formationdo not destroy cells
- Many tumor viruses do not destroy cells but rather cause them to change
morphology and multiply at a faster rate (called transformed cells)
- Colonies of transformed cells develop into foci that can be visualized with the
naked eye.
G. Serologic tests
-includes the following: complement fixation (CF), neutralization hemagglutation
inhibition (HI), passive hemagglutination (PHA), indireectctfluorescent antibody (FA), Immune
adherence
hemagglutination
(IAHA),
immunoelectronmicscopy
(IEM),
counterimmunoelectrophoresis (CIE), radioimmunoassay (RIA), enzyme-linked immunisorbent
assay (ELISA), Flourescent-focus inhibition (FFI), Anti compliment immunofluorescence
(ACIF), and single radial hemolysis (SRH).
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
-HIV is a retrovirus of the lentovirus group causing acquired Immune Defficiency
Syndrome (AIDS).

A. Introduction and Nomenclature

1. In 1983 and 1984 retrovirus isolates were described by motagnier and coworkers from
hemophiliac patients with lymphadenopathy (called lymphadenopathy-associated virus or LAV1) and from patients with franki AIDS (called ImmunoDefficiency-associated Virus or IDAV
).
2. In 1984 Gallo and Associated isolated HTLV-III (Human T-cells Lymphocytic Virus type
III) From Homosexual and hemophiliac patients with AIDS and AIDS-related while late inh
1984 levy and colleagues isolated AIDS-realted retrovirus or ARV.
3. Subsequent investigations revealed that LAV-1, HTLV-III, IDAV and ARV are all isolated of
the same virus, now called Human immune Defficiecy Virus type 1 (HIV-1)

4. In 1986 Motagnier isolated LAV-2 Which is now a member of a family of human lentivirus
called HIV-2 (this virus can also cause AIDS but limited to west and central Africa and has not
demistrated the virulence of HIV-1)
MODE OF TRANSMISSION
1. Sexual contact
2. Blood transfusion
3. Sharing of contaminated needles between IV Drug abusers
4. Trans placental transmission
5. HIV has been isolated from blood, semen, mothers milk, tears, CSF and saliva; however, to
date, transmission by fluids other than the first 3 has not been reported.
Risk groups
1. Homosexual and bisexual men
2. IV Drug abusers
3. Hatians
4. Recepients of transfused blood and blood products
5. Hemophiliacs who received factor VIII Concentrate
6. Sexual contacts and children of persons with HIV infection.
D. Incubation Period: 5-65 months
E. Pathogenesis

1. HIV has a strong affinity for the CD4 antigen which serves as a receptor for entering and
infecting cells.
2. In AIDS, HIV Selectivity adheres to T-lymphocytes which carry the CD4 antigen on their
surface (formely T Helper cells with T4 surface antigen). This leads to depletion of CD4 T-cells
resulting in severe lymphopenia.
3. Aside from CD4+ T-cells, HIV also infects all CD4+ Cell types which include peripheral
blood monocytes, monocytoid cell lines and tissue macrophages (e.g, follicular dendritic cells or
micrpglial cells in brain).
4. An important characteristics of HIV is that its replication in some cells in vivo appears
restricted, resulting in maintenance of HIV in alatent form.
F. Clinical Manifestation
1. AIDS
a. It is obvious that the primary site of cellular destruction is the host immune system, thus
patients suffer from numerous opportunistics infections, the most common are the following.
Bacterial
i. Mycrobacteriumavium- intracellular are complex
ii. M. Tuberculosis
iii. Other mycobacteria
Fungal
i. Esophangeal&Disseminated candidiasis
ii. Disseminated aspergillosis
iii. Cryptococcosis
Parasitic
i. chronic crptosporidiosa (Enteritis)
ii. Pneumocystis carinii pneumonia
iii. Intestinal and disseminated strongyloidosis
iv. Toxoplasmosis (pneumonia or CNS infection)
Viral

i. Disseminated CMV infection

ii. Chronic or disseminated herpes simplex infection
iii. Progressive multifocal leukoencephalopathy (JC VIRUS)
iv.Condylomataacumita
b. malignation also develop in AIDS patients. The most common of which is Kaposi sarcoma.
Other malignancies include non Hodgkins B cell lymphomas and carcinomas of the rectum and
tongue.
c. Neurologic syndrome recognized recently include encephalitis, progressive dementia, spinal
cord degeneration, acute aseptic meningitis, chronic meningitis and peripheral neuropathy.
2. AIDS- related complex (ARC)
a. This is seen in patients with HIV antibody whose primary evidence of disease of
generalized lymphadenopathy.
b. Patients may also have fever, malaise, fatigue, night, sweats, weight loss, anorexia, diarrhea
or low platelet counts.
c. These features may represent a prod Rome for the development of AIDS.
G. Laboratory Diagnosis
1. Screening Test
a. ELISA
b. Particle agglutination
c. radioimmunoprecipitation (RIPA)
2.Confirmatory test
a. Western blot
b. Indirect FAT
3. Culture
a. specimen: peripheral blood mononuclear cells (PBMS)
b. Culture system:
i.PBMS from HIV-Uninfected donors in coculture with the patients PBMS

ii. These Donor cells should be blast-transformed by exposure to mitogens.

c. Detection of either reverse transcriptase activity or HIV antigen denotes a positive culture
result.
4. Polymerase chain reaction (PCR) method
-for early detection of viral-nucleic acid in infected peripheral blood lymphocytes particularly in
neonates.
H. Treatment
1. Currently there are no available antivirus agents that will eradicate or prevent HIV-1 infection.
2. To date, only 3-Azidothymidine (AZT) or Zidovudine has been approved for use in adults and
children that may prolonged the survival rate among person with AIDS.

HEPATITIS VIRUSES
A. Hepatitis A virus (HAV)
-a picornavirus, classified as enterovirus 72
1.Disease Produced: Hepatitis A or infection hepatitis (short incubation hepatitis)
2. Transmission: Predominantly fecal-oral route
3. Incubation Period: 15-45 days (avg. 25-30 days)
4. Laboratory Diagnosis:
a. demonstration of the virus by electron microscopy
b. detection of anti HAV (antibody to HAV) present at onset of symptoms which persist
throughout lifetime.
c. detection of IgM anti- HAV indicating recent infection with Hepatitis A which is
positive up to 4-6 months after infection.
B.Hepatits B virus (RBV)
-A HEPADNAVIRUS conraining the infection dane particle
1. Disease produced: Hepatitis B or serum Hepatitis (long incubation hepatitis); can end to liver
CA and Cirrhosis.
2. TRANSMISSION
a. Predominantly parenteral route
b. sexual contact
c. transplacental
d. mucous membrane exposure
3. Incubationperiod: 50-180 days (avg., 60-90 days)

4. Serological Tests for HBV

a. HBsAg- indicates active Hepatitis B infection; either acute or chronic.
b.Anti-HBs- indicates protection against reinfection (immunity) which remains for years.

c.Anti-HBc- Indicates active HBV infection cannot be excluded; a recent HBV infection can be
confirmed by examining the sample for high titers of IgM anti HBC.
d.HBeA- indicates active hepatitis infection,
e. Anti-HBe- when present in HBsAg carrier blood is potentially less infectious.
f.HBcAg (Hepatitis B core antigen)- no test available for routine use.

C. Hepatitis D virus (Delta Hepatitis Virus)

1. Unclassified virus which possesses an envelope composed of HBsAg and a very small
viroid-like RNA molecule.
2. HDV is a defective mutant virus that replicates only in HBV infected cells, thus, the delta
agent requires confection with hepatitis B for damage occur.
D. Hepatitis C Virus (HCV)
- related to the toga viruses and flavi viruses which causes the parenterally transmitted Non-B
Hepatitis
1. Disease Produce: most common cause of post transfusion hepatitis
2. Transmission: predominantly
3. Incubation Period: 40-120 days
4. Laboratory Diagnosis: anti- HIV testing as carried out by ELISA or RIA
E. Hepatitis E (HEV)
1. Probably a calci virus which causes the enterically transmitted Non-A, Non-B Hepatitis
(also referred to as water-borne or endemic hepatitis)
2. Transmitted via fecal-oral route.
3. Diagnosed by exclusion.