Systemic Bacteriology

Staphylococcus
1. Characteristics of pathogenic staphylococci

Staphylococci are Gram-positive cocci that occur in grape-like clusters that are non-motile
They are catalase positive unlike streptococci
The three pathogenic strains are S. aureus, S. saprophyticus, and S. epidermidis.
Staphylococci produce two types of diseases: infections and intoxications
i. In the former, the cocci gain access to damaged skin, mucosal or tissue sites, colonize by adhering to
cells, evade host mechanisms, and cause tissue damage
ii. In intoxications, the disease is caused by the bacterial toxins that are produced
iii. Virulence factors include polysaccharide peptidoglycan, teichoic acid, capsular polysaccharide, protein
A, clumping factor, coagulase, lipid hydrolases, Hyaluronidase, nuclease, hemolysins, enterotoxin,
exfoliative toxin, leukocidin, and toxic shock syndrome toxin.
Staph infections are characteristically localized pyogenic lesions.
i. Common pyogenic infections are
1. Skin and soft tissue wound infection, impetigo, cellulitis, folliculitis, abscess
2. Musculoskeletal: osteomyelitis, arthritis, bursitis,
3. Respiratory: tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess,
empyema, pneumonia
4. CNS: meningitis
5. Endovascular: bacteremia, septicemia, pyemia, endocarditis
ii. Toxin mediated staphylococcal diseases:
1. Food poisoning
2. Toxic shock syndrome
3. Scalded skin syndrome

2. Toxic Shock Syndrome

It is a potentially fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, diarrhea,
mucosal hyperemia, and an erythematous rash.
It is associated with infection of mucosal sites by toxic shock syndrome toxin (TSST-1) producing Staph aureus.
TSST-1 antibody is seen in convalescents which is protective.
TSST-1 is a superantigen so it can activate T lymphocytes without a secondary signal. The large scale activation
of T cells and release of cytokines like IL1, IL2, TNF, and IFN cause cellular damage.

3. Coagulase Test

Coagulase test can be used to differentiate S. aureus from S. saprophyticus and S. epidermidis.
Coagulase is an enzyme that brings about clotting of human plasma. It acts with coagulase reacting factor (CRF)
in plasma and binds with prothrombin to convert fibrinogen to fibrin.
There are two ways to the coagulase test: tube or slide
Tube coagulase test
i. Detects free coagulase
ii. About 0.1 ml of a young broth culture is added 0.5 ml of human plasma in a test tube. Heparin can be
used as the anticoagulant for preparing the plasma
iii. Positive and negative controls are set up.
iv. The tubes are incubated in a water bath at 37oC for 3-6 hours. If positive the plasma clots and does not
flow when the tube is tilted.

Slide test
i. Detects bound coagulase and is easier than the tube test
ii. The isolate is emulsified in a drop of saline on a slide. After checking for the absence of
autoagglutination, a drop of human plasma is added to the emulsion and mixed.
iii. If there is clumping its positive. Positive and negative controls should be set up.

4. Coagulase negative staphylococci

Constitute a major component of the normal flora of the human body.

Includes S. epidermidis, S. hemolyticus, and S. saprophyticus
S epidermidis is present on normal human skin.
i. It is non-pathogenic but can cause disease when the host defenses are breached
ii. It is common to grow on implanted foreign bodies like artificial heart valves, shunts, intravascular
catheters, and prosthetic appliances leading to bacteremia.
iii. Biofilm formation is an important factor in the pathogenesis of S. epidermidis. It is an extra cellula
polysaccharide matrix which protects bacteria from antibacterial agents and helps in colonization and
resistance of infections
S. saprophyticus may be present on normal human skin and periurethral area and can cause urinary tract
infection particularly in sexually active young women.

Streptococci
5. Classify streptococci

Streptococci are gram-positive cocci arranged in chains or pairs. They are part of the normal flora although
some are pathogenic.
There are several ways to classify streptococci. Either hemolysis on blood agar or Lancefield groups are used.
Based on hemolysis on blood agar:
i. Based on their ability to lyse blood, streptococci are categorized into three groups
ii. Beta () hemolytic
1. Produce clear, defined colorless zone of hemolysis where RBCs are completely lysed
2. Streptococci pyogenes, S. agalactiae
iii. Alpha () hemolytic
1. Produce a greenish discoloration (due to metabolite of hemoglobin) due to partial hemolysis
2. Viridans streptococci, streptococci pneumoniae (differentiate by optochin test. Pneumoniae
are killed by optochin while viridans are resistant)
iv. Gamma ()/ non-hemolytic streptococci
1. Produce no change in the medium and include Enterococcus
Based on carbohydrate antigen or Lancefield groups
i. Hemolytic streptococci are classified serologically into groups depending on the nature of the
carbohydrate (C) antigen. There are about 20 Lancefield groups
ii. A group: S. pyogenes
iii. B group: S. agalactiae, viridans
iv. D group: Enterococci
Fig. 22.2

6. Give an account of morphology, cultural characteristics, and pathogenicity of streptococci

pyogenes.

S. pyogenes is gram-positive cocci in chains or pairs. They form chains because of daughter cells which divide in
one plane and failing to separate properly. They are non-motile and non-sporing.
Cultural characteristics:
i. It as an aerobe and a facultative anaerobe. It grows best at 37oC

ii. Pyogenes absolutely requires media containing fermentable carbohydrates or media enriched with
blood or serum
iii. Colonies appear after 24 hours on blood agar. There are clear areas of hemolysis around them because
they are hemolytic.
iv. In liquid colonies, such as glucose broth or serum broth, growth occurs as granular turbidity with a
powdery deposit
v. Streptococci ferment several sugars producing acid. They are catalase negative.
Antigens of pyogenes are its carbohydrate antigen, M protein (inhibits phagocytosis), and fimbria. S. pyogenes
antigens cross-react with antigens of normal human heart proteins of the myocardium and valves.
Toxins of S. pyogenes include hemolysins (streptolysin O (oxygen labile, antigenic), streptolysin S (oxygen
stable, nonantigenic), erythrogenic/pyogenic exotoxin (causes scarlet fever, can possibly be a superantigen and
induce T cells which release cytokines and cause toxic shock syndrome), streptokinase, DNAase, hyaluronidase,
and anti-C5a peptidase.
Pathogenicity
i. Suppurative
1. Respiratory infections:
a. Primary site of invasion is the throat. Sore throat is the most common streptococcal
disease. May include red swollen tonsils and pharyngitis.
b. Pyogenes adheres to the pharyngeal epithelium
c. Tonsillitis is more common in adults while pharyngitis is more common in children
d. From the throat the streptococci may spread to surrounding tissues possibly causing
otitis media, mastoiditis, suppurative adenitis, meningitis etc.
2. Skin and soft tissue infections:
a. S. pyogenes also causes a variety of skin infections like pyoderma, cellulitis (deep
infection of the skin cells, producing red, swollen skin which is hot to touch), etc.
b. Erysipelas
i. Diffuse infection involving the superficial lymphatics. The affected skin is red,
swollen, and indurated and also sharply demarcated from the surrounding
healthy area
c. Impetigo
i. A vesicular, blistered, eruption. Most commonly in children. Becomes crust and
flaky. Found usually around the mouth.
d. Necrotizing fasciitis
i. Certain pyogenes have M proteins that block phagocytosis which allow the
bacteria to move rapidly through the tissue
ii. The streptococci enter through a break in the skin and follow a path along the
fascia which is between the subcutaneous tissue and muscle.
iii. Patient develops swelling, heat, and redness that moves from the site of
infection. The skin changes from red to blue and large blisters form (bullae)
iv. Later the skin dies and muscles may become infected (myositis)
v. Antibiotics and surgical removal of the fascia is necessary. Mortality is >50%
3. Genital infections
a. Pyogenes resides naturally in the female genital tract. It is an important cause of
puerperal fever.
4. S. pyogenes can also cause abscesses in internal organs like the brain, lungs, liver, and kidneys
ii. Non-suppurative, post-streptococcal sequelae
1. Pyogenes can also cause two delayed antibody mediated diseases: rheumatic fever and
glomerulonephritis

2. They appear 1-3 weeks after acute infection so pyogenes is often not present
3. Rheumatic fever
a. It occurs only after throat infection of pyogenes and not skin infections
b. Myocarditis (heart inflammation) is seen. Rheumatic fever is often recurrent and the
recurrent myocarditis eventually leads to permanent heart damage, mostly the mitral
valve and the aortic valve. So rheumatic valvular heart disease develops
c. There is also fever, arthritis, chorea (uncontrolled dance like movements of the
extremities), subcutaneous nodules, and erythema marginatum (red margin spreads
out from its center)
4. Glomerulonephritis
a. Antibody-mediated inflammatory disease of the glomeruli of the kidney
b. Can occur after infection of either the throat or the skin if the strain is nephritogenic
c. Antibody-antigen complexes travel to and are deposited in the glomerular basement
membrane where they activate complement which leads to local glomerular
destruction.
d. Affects children mostly. Child presents with puffy face (caused by urinary retention),
dark urine (tea colored due to hematuria), and hypervolemia
e. Good prognosis

7. ASO (Anti-streptolysin O) Test

Streptolysin is an oxygen labile toxin secreted by streptococci. It is also antigenic

Estimation of the antibody to this antigen is a standard serological procedure to detect S. pyogenes
Due to the complexity of the hemolysis inhibition test, the ASO test is now done by the serological method of
latex agglutination
An ASO titer in excess of 200 units is considered significant.

8. Bile solubility test

It is a biochemical test to differentiate S. pneumoniae

S. pneumoniae is a gram-positive lancet-shaped diplococci
It is bile soluble
If a few drops of 10% sodium deoxycholate solution is added to 1 ml of an overnight broth culture, the culture
clears due to lysis of the cocci.
Bile solubility is a constant property of S. pneumoniae and an important diagnostic tool
The test is based on the presence of an autolytic amidase that cleaves the bond between alanine and muramic
acid in the peptidoglycan in S. pneumoniae. Bile activates the enzyme by acting on surface-active agents. This
results in lysis of the organism

9. Quelling Reaction

A test to differentiate S. pneumoniae

A suspension of S. pneumonia is mixed on a slide with a drop of the type-specific antiserum (serum with
antibodies to the capsular antigens) and a loopful of methylene blue. In the presence of the homologous
antiserum, the capsule becomes apparently swollen and sharply delineated.
The quelling test can be done directly with sputum from acute pneumonia cases.

Neisseria
10. Enumerate the organisms causing meningitis. Give the morphology, cultural characteristics,
pathogenesis, and laboratory diagnosis of Neisseria meningitidis (Meningococcus)

Meningitis is the inflammation of the meninges. When an organism invades the subarachnoid space, there is an
inflammatory response in the meninges.
Classification of organisms causing meningitis.

i. Bacteria
1. Neonates/Infants: E. coli, S. agalactiae, Listeria monocytogenes (These three are most common
before 3 months), S. aureus, H. influenzae, S. pneumoniae, Klebsiella,
2. Children: H. influenzae, N. meningitidis (most common from 6 months to 2 years), S.
penumoniae
3. Adults: N. meningitidis, S. pneumoniae
4. Elderly: S. pneumonia, S. aureus
ii. Viruses: Enteroviruses, Paramyxoviruses, Herpesviruses, Adenoviruses, Arbovirus
iii. Fungi: Cryptococcus neoformans, Candida albicans, Aspergillus, Histoplasma capsulatum, Coccidioides
immitis
iv. Parasites: Entamoeba histolytica, Naegleria, Acanthamoeba, Toxoplasma gondii
Neisseria consists of Gram-negative, aerobic, non-sporulating, non-motile cocci arranged in pairs.
Neisseria diplococci appear somewhat kidney shaped with the concave edges facing each other so they look
like minature doughnuts. Meningococci are capsulated while gonorrhea are not.
Cultural Characteristics
i. Meningococci have specific growth requirements. Growth requires media enriched with blood, serum,
or ascitic fluid which promote growth by neutralizing other inhibiting substances rather than providing
additional nutrition.
ii. They are aerobic but grow better with 5-10% CO2 at 35-36oC at a pH of 7.4-7.6
iii. On solid media, after incubation of 24 hours, the colonies are small (1mm), round, bluish grey. Weak
hemolysis occurs in blood agar. Growth is poor in liquid media.
iv. Blood agar, chocolate agar, and Mueller-Hinton starch casein hydrolysate agar are the most commonly
used for culture.
v. Thayer-Martin VCN is a very good selective medium. It contains:
1. Vancomycin: destroys gram-positive organisms
2. Colistin: Destroys all gram-negative organisms except for Neisseria
3. Nystatin: Destroys all fungi
vi. Meningitidis can ferment maltose and glucose while gonorrhea can only ferment glucose. Gonococci
use most of the same cultures.
Antigens of meningococci are the capsules which are classified into 13 serogroups although groups A, B, C are
the most important.
Pathogenicity
i. N. meningitidis can cause cerebrospinal meningitis and/or meningococcal septicemia
(meningococcemia). They usually inhabit the nasopharynx asymptomatically but in some cases they
disseminate and cause symptoms. Classic clue of meningeal infection is petechial rash.
ii. Meningitis
1. Usually affects infants under the age of 1.
2. Cocci usually spread to the subarachnoid space via the blood stream. Upon reaching the CNS, a
suppurative lesion of the meninges is set up at the base of the brain.
3. Cocci are found in spinal fluid especially inside leukocytes.
4. If untreated it can lead to blindness and deafness.
5. Bulging open anterior fontanelle may indicate meningitis in neonates.
6. Stiff neck is seen in meningitis in older children/adults. There is also positive Kernigs Sign
(while lying supine and hips flexed at 90o, the knee cannot be fully extended) and Brudzinskis
sign (While lying supine, flexing the neck causes automatic flexion of knees and hips)
iii. Meningococcemia
1. Meningococci in the blood presents with acute fever with chills, malaise, prostration, arthralgia
(joint pains), and muscle pains.

2. Petechial rash is common due to endotoxin release which causes vascular necrosis,
inflammatory reaction, and hemorrhage into the surrounding skin. The cocci can be biopsied
from the rashes.
3. Few people develop fulminant meningococcemia (Waterhouse-Friderichsen syndrome) which
is septic shock and usually fatal. Bilateral hemorrhage of adrenal glands causes adrenal
insufficiency leading to hypotension and tachycardia, DIC, and multisystem organ failure.
Lab Diagnosis
i. For proper treatment of meningitis, the exact cause and organism should be identified.
ii. Specimens: CSF, blood (early stages only), nasopharyngeal swab, and skin scrapings from petechial
lesions can be collected.
iii. Examination of CSF:
1. The fluid will be under pressure and turbid. There will also be a large number of pus cells.
2. To examine it properly, the CSF is split into three portions:
a. One portion is centrifuged and Gram stained to find meningococci. Supernatant will
contain antigens which may be demonstrated by latex agglutination.
b. The second portion is inoculated on blood agar or chocolate agar plates and colonies
appear after 18-24 hours.
c. The third portion is incubated over night after adding glucose broth and then
subcultured on chocolate agar.
iv. Blood culture: In early cases, blood culture is often positive. Cultures should be incubated for 4-7 days.
v. Nasopharyngeal swab: Useful for detection of carriers.
vi. Petechial lesions: Meningococci can be demonstrated by microscopy and culture
vii. Serology: Detection of antibodies to the polysaccharide antigen can be used retrospectively.
Treat with i.v. penicillin G although resistance is growing. Third-generation cephalosporins (ceftriaxone,
ceftazidime) can also be used. Close contacts of patient are treated with rifampin or ciprofloxacin
prophylactically.

11. Lab diagnosis of N. gonorrhea

Specimen
i. Discharge, urethral swab, or endocervical swab
1. A sample of the discharge is collected with a loop for culture or placed on a slide for a smear.
2. In women, cervical swabs should also be collected in addition to discharge. This should be done
using a speculum.
ii. It may also be possible to demonstrate gonococci in the centrifuged deposits of urine in cases where
no urethral discharge is available
Microscopy
i. In acute gonorrhea, urethral discharge contains gonococci in large numbers. Gram-negative diplococci
can indicate gonorrhea
Culture
i. Specimens should be inoculated immediately. If not possible, they should be sent in Stuarts transport
medium.
ii. Cultures can be readily obtained on chocolate agar or Mueller-Hinton agar incubated at 35-36oC under
5-10% CO2.
Serology
i. Tests like the complement fixation test can be used with varying degrees of success. However, no
serological test has been found useful for routine diagnostic purposes.
Molecular methods
i. It may not be possible to obtain gonococci from some chronic cases so PCR molecular methods have
improved the sensitivity of the assay

12. Non-specific urethritis

There has been an increase of cases of chronic urethritis where gonococci cannot be demonstrated
This is called non-gonococcal or non-specific urethritis
Urethritis forms part of a syndrome consisting of conjunctivitis and arthritis (Reiters syndrome)
Some of these cases may be due to gonococcal infection but most are from the result of infections of diverse
origin.
The management of this syndrome is difficult

Corynebacterium
13. Give the morphology, cultural characteristics, pathogenesis, and lab diagnosis of Corynebacterium
diphtheria

Corynebacteria are Gram-positive, non-acid fast, non-motile rods with irregularly stained segments.
C. diphtheria is the most important being the causative agent of diphtheria
Morphology
i. They show club-shaped swellings so they are named Corynebacterium
ii. Diphtheria bacillus is a slender rod with a tendency to club at one or both ends. They are pleomorphic.
iii. They are non-sporulating and non-capsulated.
iv. Metachromatic granules called polar bodies are often situated at the poles. They are also called volutin
or Babes-Ernst granules.
v. They often appear as Chinese letter like or cuneiform arrangement due to incomplete separation of the
daughter cell after binary fission
Cultural characteristics
i. Growth is scanty on ordinary media. Enrichment with blood, serum, or egg is necessary. It is an aerobe
and a facultative anaerobe
ii. The usual media used are Loefflers serum and Tellurite blood agar.
iii. Loefflers serum slope
1. Diphtheria grows very rapidly, colonies can be seen in 6-8 hours.
2. Colonies are at first small, circular white opaque discs but enlarge later.
3. They may acquire a distinct yellow tint
iv. Tellurite blood agar
1. Several modifications are used such as McLeods and Hoyles media
2. Tellurite inhibits the growth of most other bacteria, acting as a selective agent.
3. Diphtheria reduce tellurite to metallic tellurium which is incorporated in the colonies making
them a grey or black color.
4. Colonies may take up to two days to appear
v. Diphtheria have been classified into three types: gravis, intermedius, and mitis
Pathogenesis
i. The incubation period in diphtheria is 3-4 days
ii. The site of infection may be facial, nasal, conjunctival, laryngeal, otitic, genital, or cutaneous
iii. Cutaneous infections are usually secondary to pre-existing skin lesions.
iv. Facial diphtheria is the most common
v. Diphtheria commonly colonize the pharynx and form a grayish pseudomembrane consisting of fibrin,
leukocytes, and necrotic epithelial cells. They the produce a powerful exotoxin which damages heart
and neural cells.
vi. Diphtheria toxin contains two subunits. The B subunit binds to target cells and the A subunit enters the
cell. The A subunit blocks protein synthesis by inactivating elongation factor (EF2) which is an important
part of translation

vii. Diphtheria need to be lysogenized by a bacteriophage to code the toxin similar to how Group A
streptococci need to be lysogenized to get erythrogenic toxin
viii. According to clinical severity, diphtheria can be classified into:
1. Malignant or hypertoxic where there is severe toxemia with adenitis. (bull neck) Death is due
to circulatory failure. There is high incidence of paralytic sequelae
2. Septic: Leads to ulceration, cellulitis, and even gangrene around the pseudomembrane
3. Hemorrhagic: Characterized by bleeding from the edge of the membrane, epistaxis,
conjunctival hemorrhage, purpura
ix. Common complications are:
1. Asphyxia due to mechanical obstruction of the respiratory tract by the pseudomembrane.
2. Acute circulatory failure
3. Post-diphtheric paralysis which occurs in the third or fourth week of disease. There is
spontaneous recovery
4. Septic such as pneumonia and otitis media
5. Heart problems like myocarditis causing AV conduction block and dysrhythmia
6. Neural problems like peripheral nerve palsies, Guillain Barre-like syndrome, palatal paralysis,
and cranial neuropathies
Lab Diagnosis
i. Needed for epidemiological studies but if there is suspicion of diphtheria treatment should be started
immediately without waiting for tests.
ii. Specimen is a swab from the lesions using a tongue depressor
iii. Microscopy
1. Smear examination: Smears stained with methylene blue will show the bacilli with
metachromatic granules. Alberts stain will show delicate green bacilli with purple/blue
granules. Bacilli may not always be demonstrable so smear examination alone is not sufficient
to diagnose diphtheria
iv. Culture isolation
1. Swabs are inoculated on Loefflers serum slope, tellurite blood agar, and ordinary blood agar
(used to differentiate strep/staph pharyngitis which may simulate diphtheria).
v. Demonstration of toxicity (Virulence testing)
1. Any isolate of the diphtheria bacillus should be tested for toxigenicity for the bacteriological
diagnosis to be complete.
2. In vivo tests
a. Subcutaneous
i. The growth from Loefflers serum is emulsified in broth and is injected
subcutaneously into two guinea pigs on of which has the antitoxin. If the bacilli
are toxigenic the unprotected one will die. Not used anymore
b. Intracutaneous test
i. Broth emulsion is inoculated into two guinea pigs. One is given 500 U of
antitoxin while the other is given 50 U of antitoxin to prevent death. After
several hours, the second guinea pig should have an inflammatory reaction if
the bacilli were toxigenic.
3. In vitro tests
a. Eleks gel precipitation test:
i. A rectangular strip of filter paper impregnated with diphtheria antitoxin is
placed on the surface of a 20% normal horse serum agar in a petri dish while
the medium is still fluid

ii. Once the agar has set, the surface is dried and narrow streaks of the strains are
made at right angles to the filter paper strip
iii. Positive and negative controls should be set up
iv. The plate is incubated at 37oC for 24-48 hours. Toxins produced by the
bacterial growth will diffuse in the agar and where it meets the antitoxin will
produce a line of precipitation
v. The presence of such arrowhead lines indicate that the strain is toxigenic.
vi. Fig. 25.2
b. Tissue culture test
i. Strains of diphtheria are overlaid cell culture monolayers in agar. The toxin
diffuses into the cells below and kills them

Treatment
i. Treatment should be started immediately.
ii. There is a 3-step method
iii. Antitoxin: Diphtheria antitoxin only inactivates circulating toxin which has not yet reached its target
tissue
iv. Penicillin or erythromycin: Either antibiotic will kill the bacteria preventing further toxin production
and renders the patient non-contagious
v. DPT vaccine: prophylaxis against future infection

14. DPT vaccine

Diphtheria can be controlled by immunization. The objective of immunization is to increase protective levels of
antitoxin in circulation
Three methods of immunization are available: active, passive, and combined but only active immunization can
lead to eradication of the disease. Passive and combined can only provide emergency protection to susceptible
individuals to risk
Active immunization is given using a killed vaccine. (toxin is incubated with formalin)
Diphtheria toxoid is given in children as a trivalent preparation along with tetanus toxoid and pertussis vaccine
as DPT.
For younger children diphtheria toxoid is given in a dose of 10-25 Lf
DPT is given at 2, 4, 6, and 18 months. Followed by booster at school entry then boosters every 10 years.

Bacillus
15. Bacillus anthracis and malignant pustule

Bacillus anthrax is a Gram-positive sporulating, rod-shaped bacteria that is aerobic. It is non-motile with a
unique capsule that is made of protein.
The bacilli appear somewhat like a bamboo stick in culture. They are arranged end to end in long chains
Anthracis can be grown on agar plates. PLET medium is selective for anthracis
Pathogenicity
i. Anthrax is normally a disease of cattle and sheep. (zoonosis) Humans acquire the spores of anthracis
from infected animals, the soil, or handling infected animal products like wool. The spores then get
activated in the human body and produce toxins
ii. Anthracis has two virulence factors:
1. Capsule which prevents phagocytosis. It is encoded by a pXO2 plasmid.
2. Anthrax toxin which is encoded by the pXO1 plasmid. The exotoxin has three different parts:
a. Edema factor (EF): It is a calmodulin-dependent adenylate cyclase. It increases cAMP
which impairs neutrophil function and causes massive edema (impairs water and
electrolyte homeostasis)
b. Protective antigen (PA): promotes entry of EF into phagocytic cells

c. Lethal factor (LF): it is a zinc metalloprotease that inactivates protein kinase. It

stimulates macrophages to release tumor necrosis factor and IL-1 which contribute
to death.
iii. Anthrax can take the form of cutaneous, pulmonary, or intestinal depending on the form of ingestion
of spores but cutaneous is most common:
iv. Cutaneous anthrax:
1. Infection through skin. Face, neck, hands, arms, and back are the usual sites. Lesion starts as a
papule 1-3 days after infection and becomes vesicular containing fluid which may be clear or
blood stained.
2. The whole area becomes congested and edematous. Several satellite lesions filled with yellow
fluid are arranged around a central necrotic lesion which is covered by a black eschar.
3. The lesion is called a malignant pustule
4. Revolves spontaneously usually. In 10-20% of untreated patients it may develop into fatal
septicemia or meningitis.
v. Pulmonary anthrax
1. Called wool sorters disease as it usually because inhalation of dust from infected wool
2. There is hemorrhagic pneumonia with a high fatality rate
3. Hemorrhagic meningitis may occur as a complication
vi. Intestinal anthrax
1. Very rare as people dont eat contaminated meat
2. Violent enteritis with bloody diarrhea occurs with high case fatality
Treatment and prophylaxis
i. Rapid identification and prompt use of ciprofloxacin or doxycycline are critical in preventing the high
mortality associated with systemic infection
ii. Individuals taking part in high risk activities: spending time with cows or sheep and military personnel
are given a vaccine composed of the protective antigen (PA)

Clostridium
16. Clostridium perfringens

Clostridium consists of Gram-positive anaerobic, spore forming, motile bacilli. Clostridium are responsible for
gas gangrene, food poisoning, and tetanus. However, C. perfringens and C. tetani are not motile. C. perfringens
is capsulated unlike the other bacteria
Clostridium are anaerobic so a useful medium is Robertsons cooked meat broth.
Clostridium perfringens is the most important clostridia causing gas gangrene. It also produces food poisoning
and necrotic enteritis. It is a normal inhabitant of the large intestine.
Toxins
i. C. perfringens is one the most prolific toxin-producing bacteria. There are four major toxins called
alpha, beta, epsilon, and iota which are predominantly responsible for pathogenesis.
ii. The alpha () toxin is the major toxin responsible for toxemia of gas gangrene. It is lethal,
dermonecrotic, and hemolytic. It is lecithinase C which in the presence of Ca and Mg splits lecithin into
phosphorylcholine and diglyceride.
iii. Beta (), epsilon (), and iota () toxins have lethal and necrotizing properties.
iv. C. perfringens also has dangerous enzymes
1. Enzymes that destroy the blood group substance
2. Histamine
3. Fibrinolysin
4. Hemolysin
5. Hemagglutinin against RBCs

6. Bursting factor which has a specific action on muscle tissue

7. Circulating factor which can cause an increase in the epinephrine sensitivity of the capillary bed
and also inhibit phagocytosis
Pathogenicity
i. Gas gangrene
1. C. perfringens Type A is the predominant agent causing gas gangrene
2. Not all clostridial wound infections lead to gas gangrene. Usually only wound contamination or
anaerobic cellulitis. Only when muscle tissues are invaded does gas gangrene occur
ii. Food poisoning
1. Usually caused by cold or warmed up meat dish
2. When contaminated meat is cooked, the spores may still survive which germinate later and
multiply.
3. When ingested they produce the enterotoxin in the intestines leading to abdominal pain,
diarrhea, and vomiting
4. Illness is self-limiting and recovery occurs in 24-48 hours
iii. Gangrenous appendicitis: Antitoxin is given to patients.
iv. Necrotizing enteritis: Severe and often fatal enteritis with spores germinating in the intestine
producing beta toxin causing mucosal necrosis.
v. Biliary tract infection: Rarely causes acute emphysematous cholecystitis and postcholecystectomy
septicemia
vi. Endogenous gas gangrene: Gas gangrene of the abdominal wall has been reported as complication of
abdominal surgery. The infection is endogenous. The organism is derived from the gut and
contaminates the abdominal wall during surgery.
vii. Thoracic and urogenital infections may occur as well

17. Naglers reaction

When C. perfringens is grown on a medium containing 6% agar, 5% Fildes peptic digest of sheep blood, and
20% human serum with antitoxin spread on one half of the plate, colonies on the other half will be surrounded
by a zone of opacity.
There is no opacity around the colonies that grow on the side of the plate with the antitoxin because of the
neutralization of the alpha toxin. This is the lecithinase effect/Nagler reaction. It is a useful test for the
detection of C. perfringens
Incorporation of neomycin makes it more selective as it inhibits coliforms and aerobic spore bearers.

18. What is gas gangrene? What are the organisms causing it? Give the pathogenesis and lab diagnosis
of gas gangrene.

Gas gangrene is a rapidly spreading edematous myonecrosis occurring usually with severe wounds of extensive
muscle masses contaminated with pathogenic clostridia
Gas gangrene is most common in soldiers in war. In civilian life, it generally follows road accidents or other
injuries where there is crushing of large muscle mass.
Organisms causing gas gangrene are varied. Several species of clostridia are found like C. perfringens, C. novyi,
C. septicum, C. histolyticum, C. sporogenes, C. fallax, C. bifermentans, C. sordellii, C. aerofoetidum, and C.
tertium. Other bacteria include anaerobic streptococci and facultative anaerobes like E. coli, proteus, and
staphylococci.
Clinical presentation
i. The disease develops with increasing pain, tenderness, and edema of the affected part along with the
systemic signs of toxemia.
ii. There is thin, watery discharge from the wound which later becomes profuse and serosanguinous
(serum leaves the body)

iii. Accumulation of gas makes the tissues crepitant (a crackling sound).

iv. In untreated cases, the disease process extends rapidly and profound toxemia and prostration
(physical/mental collapse) develop and death occurs due to circulatory failure
Lab diagnosis
i. Diagnosis of gas gangrene is taken mostly from clinical presentations and laboratory only provides
confirmation.
ii. Specimens:
1. Films from the muscles at the edge of the affected area, from tissue in the necrotic area, and
from exudate in the deeper parts of the wound
2. Exudates from the parts where infection appears to be most active
3. Necrotic tissues and muscle fragments
iii. Microscopic examination
1. Gram-stained films can help identify the species of clostridia.
2. Gram-positive bacilli without spores indicate C. perfringens.
3. Citron bodies and boat/leaf-shaped pleomorphic bacilli with irregular staining suggest C.
septicum.
4. Large bacilli with oval, subterminal spores indicate C. novyi.
5. Slender bacilli with round, terminal spores may be C. tetani or C. tetanomorphum
iv. Culture
1. Aerobic and anaerobic cultures are made on fresh and heated blood agar.
2. Nagler reaction is done
3. Robertsons cooked meat broth is used to differentiate organisms
Treatment
i. Surgery is the most important prophylactic and therapeutic measure in gas gangrene. All damaged
tissue should be removed properly and wounds should be clean to avoid infection.

19. Describe the pathogenesis and lab diagnosis of C. tetani. Give the prophylactic measures.

C. tetani is the causative agent of tetanus. It is widely distributed through soil, intestines of humans, dust, wool,
bandages, wall plaster, clothing etc.
C. tetani is a Gram-positive bacillus that is non-capsulated but motile by peritrichous flagella.
It is an obligate anaerobe that only grows in the absence of oxygen. It grows well with blood and serum but not
glucose. Grows well in Robertsons cooked meat broth.
Toxins
i. C. tetani produces two distinct toxins: a hemolysin (tetanolysin) and a powerful neurotoxin
(tetanospasmin)
ii. Tetanolysin is heat-stable toxin. It is not relevant to the pathogenesis of tetanus.
iii. Tetanospasmin is responsible for tetanus. It is antigenic.
Pathogenicity
i. C. tetani has little invasive power. Germination and pathogenesis only occur if favorable condition
exist. (necrotic tissue)
ii. Toxin produced locally is transported to the CNS intraxonally. The toxin specifically blocks synaptic
inhibition in the spinal cord at the inhibitory terminals that use glycine and GABA as neurotransmitters.
The toxin acts presynaptically. The abolition of spinal inhibition causes uncontrolled spread of impulses
initiated anywhere in the central nervous system.
iii. This causes muscle rigidity and spasms due to simultaneous contraction of agonists and antagonists in
the absence of reciprocal inhibition
iv. Tetanus is characterized by tonic muscular spasms. It starts at the local site but soon spreads to the
whole muscular system.

v. Patient often presents with trismus which is lockjaw. The patient has a grotesque smile. Death is due to
the diaphragm becoming affected and respiratory failure.
vi. The incubation period is commonly 6-12 days.
vii. Fig. 6.3 in MRS
Lab diagnosis
i. Tetanus is diagnosed on clinical grounds and labs only confirm diagnosis
ii. Microscopy
1. Unreliable as the bacilli are difficult to demonstrate and may be confused for other similar
clostridia species.
iii. Culture
1. Is a more dependable method. Isolation is more likely from excised bits of tissue from the
necrotic depths
2. Material is inoculated on one half of a blood agar plate. It is also tested on meat broth.
iv. Toxigenicity testing and in vitro identification can also be used.
Prophylaxis
i. Tetanus is a preventable disease. Wound contamination is, however, unavoidable but it is possible to
build antitoxic immunity to the toxin.
ii. Surgical prophylaxis aims at removing foreign bodies, necrotic tissue, and blood clots to prevent an
anaerobic environment favorable for the tetanus bacillus.
iii. Antibody prophylaxis aims to destroy the tetanus bacilli so the production of toxin is prevented
iv. Immunization
1. Passive immunization is the injection of tetanus antibody as an emergency
2. Active immunization
a. The most effective method of prophylaxis.
b. Formol toxoid is given in DPT
c. Doses are given at 2, 4, 6, and 18 months. Then once again at entrance to school.
Afterwards every 10 years a booster dose is given.
Treatment
i. Neutralize the circulating toxin with human tetanus immunoglobulins.
ii. Give an immunization booster.
iii. Clean the wound and remove any source of C. tetani
iv. Antibiotics to remove the rest of the bacteria
v. Provide supportive therapy until the toxin is cleared. (muscle relaxants and ventilation)

Enterobacterial I: Coliforms
20. Classify enterobacteria

The predominant aerobic bacterial flora of the large intestines are non-sporing, Gram-negative bacilli. Some
are motile and some are non-motile.
They are aerobic and facultatively anaerobic. They ferment glucose into acid and gas.
Oldest method to classify these bacteria is MacConkey agar which can detect lactose fermenters
i. Lactose fermenters: Escherichia, Klebsiella
ii. Lactose non-fermenters: Shigella, Salmonella, Proteus
Classification of bacteria
i. Escherichiae: Genus: Escherichia, Edwardsiella, Citrobacter, Salmonella, Shigella, Yersinia
ii. Klebsielleae: Genus: Klebsiella, Enterobacter, Hafnia, Serratia
iii. Proteeae: Genus: Proteus, Morganella, Providencia
iv. Erwinieae: Genus: Erwinia

21. Enumerate the organisms causing UTI. Describe the pathogenesis and lab diagnosis of UTI.

Urinary tract infections are infections of the urinary tract which is from the kidneys to the urethra. Normally
the flow of urine and sloughing of cells will prevent microorganisms, but sometimes they may still cause
infection.
Classification of microorganisms causing UTI
i. Bacteria:
1. Gram-negative bacilli: E. coli, Proteus, Klebsiella, Enterobacter, Pseudomonas
2. Gram-positive cocci: S. aureus, S. epidermidis, S. saprophyticus, Enterococci
3. Gram-negative cocci: N. gonorrhea
4. Others: Mycobacterium tuberculosis, Salmonella, Gardnerella vaginalis
ii. Virus: Adenovirus
iii. Fungi: Candida albicans
iv. Parasites: Trichomonas vaginalis, Schistosoma hematobia, Enterobius vermicularis
Pathogenesis
i. UTI is either in the upper portion or lower portion of the urinary tract.
1. Upper tract:
a. acute pyelitis: infection of the pelvis of the kidney
b. acute pyelonephritis: infection of the parenchyma of the kidney
2. Lower tract:
a. Urethritis: infection of urethra
b. Cystitis: infection of the urinary tract
c. Prostatitis: infection of the prostate
ii. Infection may be precipitated by urinary obstruction due to prostatic enlargement, calculi, or
pregnancy.
iii. Infections of the lower urinary tract seem to be ascending infections caused by fecal coliforms while
pyelonephritis is due to hematogenous infections.
iv. Common symptoms of UTI include urgency, frequency of micturition, and associated pain with urine.
Pyelonephritis is characterized by loin pain, tenderness, high fever, and rigor.
v. Common presentation of cystitis or lower UTI is dysuria, fever with chills, and frequency. In upper UTI,
patients present with fever and flank pain.
Lab Diagnosis
i. Bacteriological diagnosis of UTI is carried out by significant bacteriuria using quantitative cultures.
Normal urine is sterile but is contaminated with genital commensals. Infection should further rise the
number of bacteria in the urine. A count of 100,000 bacteria per ml is significant and suggests infection
ii. Specimen
1. A clean-voided midstream sample of urine is used
2. In men and women the genital area is cleaned, and the first urine is discarded as it contains
more commensals. The midstream urine is collected in a sterile container.
iii. Methods of quantitative culture:
1. For routine diagnostic work, semiquantitative techniques are more convenient.
2. One loopful of urine is placed on a non-inhibitory medium (blood agar) and another loopful on
an indicatory medium (MacConkey)
3. The former gives a quantitative measurement of bacteriuria while the other gives diagnosis of
the bacterium
iv. Antimicrobial susceptibility test:
1. Bacteriological investigation of UTI is not complete w/o an antibiotic sensitivity test of isolate
2. E. coli and other common urinary pathogens develop drug resistance so frequently no
antibacterial therapy can be given until individual strains are tested.
3. Resistance is often to multiple drugs and is transferable

v. Screening test for UTI:

1. B/c urinary tract infection is such a common problem and bacteriological facilities are not
always available, several screening techniques have been introduced for the diagnosis of
significant bacteriuria
2. Griess nitrate test: Based on the absence of nitrate in normal urine. Presence of nitrate
indicates the presence of nitrate-reducing bacteria in urine
3. Catalase test: Presence of catalase is seen by frothing on addition of H2O2 indicating bacteriuria
4. Microscopic demonstration of bacteria in gram-stained films of urine
5. Dip slide culture methods: agar coated slides are immersed in urine, incubated, and growth is
estimated by colony counting or by color change of indicators
6. None of these are as sensitive or reliable as a culture

22. Enumerate the organisms causing diarrhea. Describe morphological, cultural characteristics,
antigens, toxins, and pathogenesis of E. coli.

Diarrhea is an increase in fluid frequency or volume of bowel movement relative to the usual habit of the
individual.
Classifications of organisms causing diarrhea
i. Bacteria: V. cholera, E. coli, Salmonella, Shigella, C. botulinum, C. difficile, S. aureus, Campylobacter,
Yersinia enterocolitica
ii. Virus: Rotavirus, Norwalk virus, Adenovirus, Calicivirus, Coronavirus, Astrovirus, Enterovirus
iii. Fungi: Candida albicans
iv. Parasites: E. histolytica, G. lamblia, B. coli, Cryptosporidium, Isospora belli, F. hepatica, T. saginata, T.
solium, H. nana, T. trichiura, A. duodenale
E. coli is gram-negative straight rod with peritrichous flagella. Capsules and fimbriae are found in some strains.
Cultural Characteristics
i. It is an aerobe and facultative anaerobe.
ii. Many strains are hemolytic on blood agar.
iii. On MacConkey agar colonies are bright pink due to lactose fermentation.
iv. Can also be grown on EMB agar. (Eosine methylene blue) Methylene blue inhibits gram-positive
bacteria. E. coli take a metallic green sheen in this medium.
v. IMViC: + + - Antigenic structure
i. E. coli and most enterics have three antigens: O antigen (LPS), capsular antigen K, and flagella antigen
H.
ii. O antigen: These are the external component of the LPS on gram-negative bacteria.
iii. K antigen: This is a polysaccharide antigen located in the microcapsule. It encloses O antigen which
prevents O antibodies from attacking the O antigen. K antigen also prevents phagocytosis.
iv. H antigen: This antigenic determinant makes up the subunits of the bacterial flagella. Only motile
bacteria will have this antigen.
v. F antigens: Fimbriae are important virulence factors
Virulence Factors
i. E. coli have two types of virulence factors: surface antigens and toxins
ii. Surface antigens
1. The O antigen prevents phagocytosis and protects the bacillus from bactericidal effects of
complement.
2. Fimbriae are important in the initial attachment and colonization
iii. Toxins: E. coli produce hemolysins and enterotoxins
1. Hemolysins do not appear to be relevant in pathogenesis
2. Enterotoxins

a. There are three toxins that E. coli produces:

b. Heat Labile Toxin (LT)
i. Heat labile toxin is similar to the cholera toxin.
ii. It has one A (A for active) subunit and five B subunits (B for binding)
iii. The toxin binds to the GM1 ganglioside receptor on the intestinal epithelial
cells by means of subunit B. The A subunit activates adenyl cyclase which
creates cAMP leading to increased outflow of water and electrolytes into the
lumen leading to diarrhea
c. Heat stable toxin (ST)
i. ST activates the cGMP in the intestine which induces fluid accumulation
d. Both LT and ST inhibit the reabsorption of Cl- and Na+ and stimulate the secretion of Cland HCO3e. Verotoxin/Shiga-like toxin
i. Similar to Shigella toxin. They are both cytogenic
ii. They both inhibit protein synthesis by inhibiting 60S ribosome which results in
intestinal epithelial cell death

Pathogenesis
i. E. coli is normally not pathogenic but when it gets conjugated, lysogenized, or there is direct
transposon DNA insertion, E. coli will acquire virulence factors which causes pathogenesis
ii. Urinary tract infection
1. Acquisition of pili allows E. coli to travel up urethra and infect the bladder (cystitis) or the
kidneys (pyelonephritis). E. coli is the most common cause of UTI in women and hospitalized
patients with catheters.
2. Symptoms include burning on urination (dysuria), increased frequency of micturition, and
feeling of fullness over the bladder.
iii. Diarrhea
1. E. coli diarrhea affects infants and adults. Death is due to dehydration. It is often called
travelers disease.
2. Severity of diarrhea is according to the virulence factors that E. coli possesses
3. There are five different types of E. coli diarrhea:
4. Enteropathogenic E. coli (EPEC)
a. The pathogenesis is not fully understood. These strains do not produce enterotoxins or
invade the cells.
b. It may be due to bacilli attaching to the enterocyte membrane and causing disruption
of the brush border microvilli
5. Enterotoxigenic E. coli (ETEC)
a. Severity varies from mild watery diarrhea to fatal disease similar to cholera.
b. First, bacilli adhere to intestinal mucosa by fimbriae called colonization factor antigens.
Then, ETEC produce enterotoxins LT and ST.
c. Stool looks like rice water just like V. cholera
6. Enterohemorrhagic E. coli (EHEC)
a. These E. coli produce Shiga-like toxin which can cause hemorrhagic colitis (bloody
diarrhea with severe abdominal cramps). It can possibly be fatal
b. Some of these strains called E. coli 0157:H7 can cause hemolytic uremic syndrome.
There is anemia, thrombocytopenia, renal failure (causes uremia)
c. Lab diagnosis can be made by demonstration of the bacilli in feces directly or in culture
7. Enteroinvasive E. coli (EIEC)
a. These E. coli have the capacity to invade interstitial epithelial cells.

b. The resulting disease is very similar to Shigella

c. Since E. coli are in cells, the host tries to get rid of invading bacteria and this results in
immune-mediated inflammatory reaction with fever. White blood cells invade the
intestinal cells.
d. The diarrhea is bloody and also has WBCs
8. Enteroaggregative E. coli (EAEC)
a. These strains appear to aggregate in a stacked brick formation causing persistent
diarrhea
iv. Pyogenic infections
1. E. coli can cause infections such as peritonitis and abscesses intra-abdominally.
2. They are also the second most frequent cause of neonatal meningitis (behind Group B strep)
3. E. coli is a common cause of hospital acquired pneumonia
v. Septicemia
1. E. coli is the most common cause of gram-negative sepsis. It is when blood stream is invaded
by E. coli. Conditions like septic shock occur and can cause death

Enterobacteria-II Shigella
23. Pathogenesis and lab diagnosis of bacillary dysentery (shigellosis)

Shigellae are non-motile, non-sporing, non-capsulated Gram-negative bacilli. Fimbriae may be present.
Shigella is only pathogenic and not a regular inhabitant of the intestines
They are aerobes and facultative anaerobes. MacConkey agar can be used.
Shigella does not ferment lactose or produce H2S. This allows differentiation from E. coli which ferments
lactose and Salmonella which produces H2S
They have the O antigen. They sometimes have the K antigen and F antigen.
There are four subgroups of Shigella: S. dysenteriae, S flexneri, S. boydii, and S. sonnei
Pathogenicity
i. Shigellae cause bacillary dysentery. Infection is by ingestion.
ii. Invasive property: Their pathogenic properties are similar to enteroinvasive E. coli. They bind to M cells
and invade the lamina propria then the neighboring enterocytes. Shigellae continue to invade cells and
cause cell death and inflammation.
iii. The colon has shallow ulcers where cells have sloughed off.
iv. Exotoxin: Shigellae releases an exotoxin called shiga toxin which inhibits eukaryotic 60S ribosomal
subunit causing cell death. It has a B (binding) subunit and an A (active) subunit.
v. Endotoxin: Due to LPS
vi. Shigellosis is called bacillary dysentery
1. The main clinical features are loose feces with blood, mucus, and pus.
2. There is fever, abdominal pain/cramps.
3. Diarrhea is because damaged intestinal cells are unable to reabsorb water and electrolytes
Lab Diagnosis
i. Specimen
1. Diagnosis is made from isolating the bacillus from the feces.
2. Fresh feces should be inoculated without delay.
3. Gram-negative broth is a selective broth used to culture Shigella from stool specimens.
ii. Microscopy of the feces shows plenty of pus cells
iii. Culture
1. MacConkey and DCA or XLD plates are inoculated. Plates are inspected for pale or pink-colored
colonies.
Treatment

i. Shigellosis is usually self-limiting.

ii. Oral rehydration must be adequate
iii. To avoid bacterial resistance, only very severe cases should be given antibiotics. Fluoroquinolones like
norfloxacin can be given.

Enterobacteria-III Salmonella
24. Classify the organisms causing fever. Give the morphology, cultural characteristics, pathogenesis,
and lab diagnosis of Salmonella typhi.

The genus Salmonella consists of bacilli that parasitize the intestines leading to enteric fever, gastroenteritis,
septicemia, and the carrier state.
Salmonella are Gram-positive rods which are motile with peritrichous flagella.
Salmonella are aerobic and facultatively anaerobic. On MacConkey agar they are non-lactose fermenting. On
deoxycholate citrate (DCA) media and xylose lysine deoxycholate (XLD) colonies show black heads due to H2S
production.
Antigenic structure
i. Salmonella possess the flagellar antigen H, LPS antigen O, and surface antigen Vi.
ii. H antigen: Antigen is present on the flagella. It is strongly immunogenic and induces antibody
formation rapidly.
iii. O antigen: Part of the LPS which is an integral part of the cell wall.
iv. Vi antigen: it is a polysaccharide capsule that surrounds the O antigen thus protecting the bacteria
from antibody attack on the O antigen. (Basically the same as the K antigen but named differently for
Salmonella)
Pathogenicity
i. Salmonella are strict parasites of animals or humans.
ii. Salmonella cause 4 conditions in humans: Enteric fever, Gastroenteritis, Septicemia with or without
local suppurative lesions, and a carrier state.
iii. Enteric Fever includes typhoid fever caused by S. typhi
iv. Typhoid fever is acquired by ingestion of bacteria. Incubation time takes 1-3 weeks.
1. On reaching the gut, the bacilli attach themselves to the microvilli of the ileal mucosa and
penetrate the lamina propria and submucosa.
2. They are phagocytosed by macrophages but resist intracellular killing and multiply in the cell
3. They enter the mesenteric lymph nodes where they multiply and via the thoracic duct enter
the bloodstream.
4. Transient bacteremia occurs where the bacilli are seeded in the liver, gallbladder, spleen, bone
marrow, lymph nodes, lungs, and kidneys where further multiplication takes place.
5. At the end of the incubation period, there is massive bacteremia from the sites of
multiplication and clinical disease starts.
6. As bile is a good medium for the bacilli, the bacteria grows well in the gallbladder and is
discharged into the intestine where it inflames the Peyers patches and lymphoid follicles of
the ileum. They undergo necrosis and slough off leaving behind the characteristic typhoid
ulcers. Ulceration leads to intestinal perforation and hemorrhage.
v. Clinical features
1. Disease may range from mild undifferentiated pyrexia to a rapidly fatal disease
2. Onset is usually gradual with headache, malaise, anorexia, a coated tongue, and abdominal
discomfort with either constipation or diarrhea
3. Typical features are step-ladder pyrexia with relative bradycardia and toxemia
4. A soft, palpable spleen and hepatomegaly is common.
5. Rose spots that fade on pressure are common on light skinned patients

vi. Carriers are patients who continue harbor the bacteria in their gallbladder.
1. They can continue to shed bacteria for a couple weeks (convalescent carrier), more than 3
months but less than a year (temporary carrier), and those who shed the bacilli for over a year
are called chronic carriers. (Typhoid Mary a cook)
vii. Salmonella choleraesuis can cause sepsis. S. choleraesuis will infect the lungs, brain, and bone but does
not involve the GIT.
viii. Salmonella is encapsulated by the Vi capsule so the immune system opsonizes them via antibodies
then the macrophages and neutrophils in the spleen phagocytize them. So people who have no spleen
due to trauma or sickle-cell disease are particularly susceptible to Salmonella
ix. Gastroenteritis from Salmonella is very common and not very dangerous. It causes nausea, abdominal
pain, and diarrhea with or without trace blood. Fever occurs in some patients. Diarrhea is caused by a
cholera-like toxin.
Lab Diagnosis
i. Bacteriological diagnosis of enteric fever consists of isolation of the bacilli from the patient and
demonstration of antibodies in his serum
ii. Specimen
1. Blood, stool, and urine is collected. Serum is needed for the Widal test
iii. Blood Culture
1. A positive blood culture is diagnostic
2. Blood cultures are positive in approx. 90% of cases in the first week of fever. Antibiotic therapy
reduces the bacteria levels in the blood.
3. Blood is collected and inoculated into a culture bottle containing ble broth. Broth is necessary
otherwise the blood has bactericidal action. After incubation overnight the bile broth is
subcultured on MacConkey agar.
4. Serotyping slide by agglutination is also possible via typhoid O antiserum.
iv. Clot culture
1. 5 ml of blood is withdrawn and allowed to clot. The clot is broken up and added to a bottle of
bile broth.
2. Serum from this is used for Widal test. This allows the Widal test to be performed and also
removes the bactericidal action of serum.
v. Feces Culture
1. Salmonella is shed in feces during the disease and even after sometimes.
2. Useful b/c even if antibiotics have been given, they dont affect bacteria in the gut as much as
the blood
3. Fecal samples are placed directly on MacConkey, DCA, XLD, and Wilson-Blair (most selective)
media.
vi. Urine Culture
1. Salmonella is shed in urine irregularly and infrequently. So urine culture is less useful than the
culture of blood or feces.
2. Urine samples are inoculated into enrichment and selective media.
vii. Other materials for culture
1. Bone marrow and bile can be used but are generally not employed
viii. Serology/Widal Test
1. This is a test for the measurement of H and O agglutinins for typhoid bacilli in the patients sera
2. Two tubes are used for the test
a. A narrow tube with a conical bottom for H agglutination (Dreyers tube)
b. A short round-bottomed tube for O agglutination (Felix tube)
3. Procedure

a. Equal volumes of serum dilutions and the H and O antigens are mixed in Dreyers and
Felix tubes and incubated in a water bath at body temperature overnight.
b. Control tubes containing the antigen and normal saline are set to check for
autoagglutination. Agglutination titers of the serum are read. H agglutination leads to
the formation of loose, cotton-wooly clumps while O agglutination is seen as a disc-like
pattern at the bottom of the tube.
4. Interpretation
a. Agglutination titer will depend on the stage of the disease. Agglutinins usually appear
by the end of the first week so blood taken earlier may give a negative result
b. Results of a single test should be interpreted with caution as it is necessary to obtain
information on the distribution of antibody levels in normal populations
c. Agglutinins may be present on the account of prior disease or immunization
Treat with ciprofloxacin or ceftriaxone

25. Diagnosis of carriers

Identification of fecal carriers is by isolation of the bacillus from feces or from bile.
The frequency and intensity of bacillary shedding is varied so multiple samples must be taken.
Widal test is of little value because it can be used for screening but confirmation should be made by a culture
Sewer-swab methods can be used. Pads left in sewers and drains are cultured and by tracing positive swabs,
one may be led to the house containing the carrier.

26. Prophylaxis of Enteric Fever

Typhoid fever can be controlled by general measures such as improvements in sanitization and provision of
protected water supply. It is mostly eradicated in developed countries.
TAB vaccine: Specific prophylaxis with head killed typhoid bacillus. The vaccine is given in two doses at an
interval of 4-6 weeks. Given mostly only in endemic areas. Recommended for troops and medical personnel
Live oral vaccine: a mutant of S. typhi that cannot induce any illness as it self-destructs after four or five cell
divisions
Vi vaccine: Injectable vaccine containing purified Vi polysaccharide antigen.
Both the oral and Vi vaccines are recommended only for those over five years of age. Protection lasts for at
least three years and afterwards a booster can be given.
Cell-mediated immunity seems to be more relevant than humoral immunity against S. typhi

27. Kauffman-White scheme for salmonella

A classification method for naming and identifying Salmonella

Inclusion in the genus is based on common biochemical properties
Classification within the genus takes place by antigenic characteristics
According to this, salmonellae are initially classified into serological groups based on the presence of distinctive
O antigen factors.

34. Yersinia, Pasteurella, Francisella

1. Yersinia Pestis: Morphology, Cultural Characteristics, Pathogenesis, and Lab Diagnosis

Yersinia is part of the Enterobacteriaceae. Y. pestis is a Gram-negative bacillus with rounded ends and convex
sides arranged in short chains or in small groups. In smears with Giemsa or methylene blue, it shows bipolar
staining with two ends densely stained and a clear central area
The bacillus is aerobic and facultatively anaerobic.
It is not nutritionally exacting and can grow on:
i. Nutrient agar: colonies are small, delicate, transparent discs
ii. Blood agar: dark brown due to the absorption of the hemin pigment
iii. Colorless on MacConkey agar

iv. In broth, flocculent growth occurs at the bottom with no turbidity. If grown in a flask of broth with oil
or ghee floated on top (ghee broth) a characteristic growth occurs which hangs down into the broth
from the surface resembling stalactites. (stalactite growth)
Virulence factors
i. Fraction-1 (F-1) antigen: a heat labile protein envelope which inhibits phagocytosis
ii. V and W antigens: they inhibit phagocytosis and intracellular killing of the bacillus
iii. Virulent strains produce bacteriocin, coagulase, and fibrinolysin.
iv. Endotoxin: an LPS which is similar to endotoxins of enteric bacilli
v. Murine toxins: role in pathogenesis not known
Pathogenesis
i. Y. pestis causes the bubonic plague. It is an endemic which has killed of the population of Europe in
1400s and killed 10 million in India between late 1800s and early 1900s.
ii. Bacteria reside in rats but spread by fleas. Once all the rats in an area are killed, the fleas will then
attack humans and give them the bacteria.
iii. There are three forms of the plague: bubonic, pneumonic, and septicemic
iv. Bubonic plague:
1. Lymph nodes draining the site of entry of the bacillus become infected. Commonly the inguinal
lymph nodes. The glands become enlarged, red, and suppurate. Fever and headache start.
2. In some, the infection remains localized at the site of the flea bite with only minor symptoms.
3. Bacilli then enter the bloodstream and produce septicemia. Sometimes there are hemorrhages
into the skin and mucosa which causes blackish discoloration (Black Death). DIC is common and
may lead to gangrene of the skin, fingers, and penis. Case fatality is 75% in untreated cases.
v. Pneumonic Plague
1. Rarer than bubonic plague.
2. Spread by droplet infection. The bacilli spread through the lungs producing hemorrhagic
pneumonia. Cyanosis is prominent.
3. Bloody mucoid sputum that is coughed out contains bacilli
4. Highly contagious and in untreated patients is almost always fatal
vi. Septicemic plague
1. The terminal event in the bubonic or pneumonic plague but sometimes may occur primarily.
Lab diagnosis
i. Lab should be able to diagnose plague in humans as well as rats, because timely detection of infection
in rats may help prevent epidemic spread.
ii. Humans
1. Specimen: For bubonic plague, buboes are collected. For pneumonic plague, sputum us
collected. For septicemic plague, blood is collected.
2. Direct demonstration: Bacilli may by readily demonstrated in buboes and sputum by
microscopy, culture, or animal inoculation.
a. Microscopically: Smears are stained with methylene blue to show the bipolar stained
bacilli.
b. Culture: Cultures may be made from the buboes, spleen, heart blood, and from bone
marrow
c. Animal inoculation: Guinea pig can be inoculated
3. Serological tests: Antibodies to the F-1 antigen can be detected by passive hemagglutination.
4. Molecular methods: PCR can be used for presumptive diagnosis of plague
iii. In rats: Autopsy are done on the dead rats and buboes are found.
Treat with gentamycin

35. Hemophilus
2. Hemophilus Influenzae

H. influenzae is a small, Gram-negative, non-motile, non-sporing bacillus.

Cultural characteristics
i. H. influenzae requires specific nutrients for growth. The accessory growth factors named X and V
(found in blood) are essential for growth.
ii. The X factor is hemin or other porphyrins required for the synthesis of cytochrome and other enzymes
like catalase and peroxidase involved in aerobic respiration.
iii. The V factor is a co-enzyme called NAD (nicotinamide adenosine dinucleotide) which acts as a
hydrogen acceptor in the metabolism of the cell.
iv. H. influenzae is aerobic but can also grow anaerobically.
v. It grows on blood agar well if a source of V factor is also added.
vi. Often S. aureus is streaked on a blood agar plate containing H. influenzae because S. aureus supplies
NAD to the H. influenzae
Antigenic properties
i. Has three major surface antigens
ii. Capsular polysaccharide: There are six capsular types but Type B is most important. The capsule is
composed of polyribitol ribose phosphate. The non-capsulated H. influenzae can also be pathogenic
and are called non-typeable strains.
iii. Outer membrane protein antigens (OMP)
iv. Lipooligosaccharides
Pathogenicity
i. Diseases of H. influenzae can be classified into invasive and non-invasive
1. Invasive: The bacillus acts as a primary pathogen causing acute invasive infections. Bacilli
spread through blood being protected from phagocytes by their capsule. Meningitis is the most
common disease of these type of infection. Laryngoepiglottitis, conjunctivitis, bacteremia,
pneumonia, arthritis, endocarditis, and pericarditis can also occur.
2. Non-invasive: Bacillus spreads by local invasion along mucosal surfaces and causes secondary
infections usually of the respiratory tract. Otitis media, sinusitis, bronchiectasis, and
exacerbations of chronic bronchitis can occur.
ii. Meningitis
1. Most serious disease caused by H. influenzae
2. Bacilli reach the meninges from the nasopharynx through the bloodstream
3. Disease is most common from the age of 6 months and 3 years because maternal antibodies
are gone and the babys antibodies are still not well developed. Older children gain resistance
from infections from subclinical H. influenzae. (takes 3-5 years of subclinical infections to gain
resistance)
4. Infants show non-specific symptoms like fever, vomiting, and altered mental status
5. While fatality is low with antibiotic treatment, when antibiotics are used the bacteria are lysed
and release endotoxins which cause a violent immune response which destroys bacteria as
well as neurons. This leads to mental retardation, seizures, language delay, or deafness.
6. It has been noted that giving corticosteroids before giving antibiotics prevents destruction of
neurons because the steroids will inhibit the immune system.
iii. Epiglottitis
1. Acute inflammation of the epiglottis with obstructive laryngitis.
2. Tracheostomy might be necessary to relieve respiratory obstruction caused by a grossly
enlarged uvula

3. Children will develop sore throat, fever, severe upper airway wheezing, and is unable to
swallow. Excess saliva will drool out of the childs mouth as it is unable to pass the swollen
epiglottis.
iv. Pneumonia: Typically occurs in infants and is accompanied by empyema and sometimes meningitis.
v. Suppurative lesions
1. Lesions such as arthritis, endocarditis, and pericarditis may result from hematogenous
dissemination. Otitis media occurs by direct spread from the nasopharynx.
2. Septic arthritis from H. influenzae is the most common arthritis in infants. Usually, a single joint
is infected resulting fever, pain, swelling, and decreased mobility of the joint.
Lab diagnosis
i. Specimen: CSF, blood, or sputum is collected depending on the site of infection.
ii. Microscopy: Presence of pleomorphic, Gram-negative bacilli in CSF should indicate H. influenzae
iii. Direct antigen detection: Capsular polysaccharide antigen may be present in CSF in meningitis and in
urine in systemic infection.
iv. Culture
1. Chocolate agar: When blood is heated the V factor is released from erythrocytes
2. Blood agar: S. aureus must be streaked
3. Nutrient agar with discs of X and V factors
4. Fildes agar: Adding peptic digest of blood to nutrient agar. Best agar for H. influenzae growth
Active immunization with HiB PRP vaccine for the polysaccharide capsule is given at 2, 4, 6, and 15 months.
For serious infections, cefotaxime or ceftriaxone are given. For milder infections, ampicillin or amoxicillin can
be given.

3. Koch-Weeks Bacillus

This bacilli is H. aegyptius. It is identical to non-capsulated H. influenzae.

Causes a highly contagious form of conjunctivitis (pink eye)
Common in tropics but responds well to local sulfonamides or gentamicin.

36 Bordetella
4. Bordetella Pertussis

B. pertussis is a small, ovoid coccobacillus. Non-motile, non-sporing, and capsulated.

It is an obligate anaerobe. Complex media are needed for isolation. Regan-Lowe media is the currently used
media.
Virulence factors
i. Surface agglutinogens: They are associated with fimbriae. They help bacteria to attach to respiratory
epithelial cells.
ii. Pertussis toxin (PT): Plays an important role in the pathogenesis of whooping cough. It causes
lymphocytosis, increases sensitivity to histamine, and induces excessive insulin secretion by the
pancreatic islet cells.
iii. Filamentous hemagglutinin (FHA): Adheres to the cilia of the respiratory epithelium and to
erythrocytes. Also promotes secondary infection by aiding H. influenzae and S. pneumoniae to bind to
respiratory epithelium.
iv. Adenylate cyclase: Acts by catalyzing the production of cAMP by various types of cells.
v. Heat Labile toxin: It is dermonecrotic and lethal.
vi. Tracheal cytotoxin: Induces ciliary damage
Pathogenicity
i. B. pertussis causes whooping cough/pertussis
ii. The disease has three stages: catarrhal, paroxysmal, and convalescent with each stage lasting two
weeks.

iii. Onset is insidious with low-grade fever, catarrhal symptoms, and a dry, irritating cough.
iv. Clinical diagnosis during the catarrhal stage is hard but antibiotics are most effective during this stage.
v. In the paroxysmal stage, cough increases in intensity. Patient experiences violent spasms of continuous
coughing followed by a long rush of air into almost empty lungs with a characteristic whoop
vi. Final stage is convalescence during which the frequency and severity of cough gradually decrease
vii. Complications include bronchopneumonia, lung collapse, convulsions, coma, paralysis, retardation,
blindness.
Lab diagnosis
i. Respiratory samples can be collected by a nasal swab or cough plate methods.
ii. In cough plate method, a culture plate is held in front of the patients mouth during a bout of cough so
the droplets of respiratory exudate will fall onto the medium.
iii. Culture is done on the Regan-Lowe medium
Antibiotics are only useful within the first ten days of the disease. Erythromycin is the drug of choice.
Prophylaxis is given via the DPT vaccine which includes pertussis, diphtheria, and tetanus.

37. Brucella
5. Brucella: Morphology, Cultural Characteristics, Pathogenesis, and Lab Diagnosis.

Brucella is a non-motile, aerobic, Gram-negative coccobacilli. They are zoonosis.

Many strains of Brucella require 5-10% CO2 for growth.
i. Simple media: growth is scanty and slow. Liver infusion media is widely used for the cultivation of
brucellae.
ii. Solid media: Colonies are small, moist, translucent and glistening.
Classification:
i. Classified based on CO2 requirements, H2S production, phage lysis, oxidative metabolic tests etc.
ii. Names of Brucella are also based on the animals they infect
1. Brucella melitensis: (goats)
2. Brucella abortus (causes abortions in cows)
3. Brucella suis (pigs)
4. Brucella canis (Dogs)
Pathogenicity
i. The three major species are pathogenic. B. melitensis is the most pathogenic. B. abortus and B. suis are
of intermediate pathogenicity
ii. Organisms from an infected animal enter the human body through a wound, the conjunctiva, by
inhalation, or by ingestion of products from infected animals. The brucellae spread from the initial site
to the lymph nodes and there facultative intracellular growth in macrophages.
iii. They then spill into the bloodstream and are disseminated throughout the body.
iv. Fever, sweats, extreme fatigue, loss of appetite, backache, and headache occur. The fever is undulant.
(Peaks in the evening and returns to normal by the morning. Slowly raises during the day and then
peaks at night)
v. Brucellosis can be acute or chronic.
vi. Acute brucellosis has undulant fever, prolonged bacteremia, asthmatic attacks, nocturnal drenching
sweats, exhaustion, anorexia, constipation, chills, nervous irritability, and muscular pains
vii. Chronic brucellosis lasts for years and the patient has bouts of sweating, joint pain, and minimal
pyrexia
viii. There is a formation of a granuloma in response to brucella.
Lab diagnosis
i. Blood culture is the most definitive method for the diagnosis of brucellosis but often unsuccessful.
Castaneda method is used. Cultures can also be obtained from bone marrow or lymph nodes.

ii. Serological methods are also important in diagnosis. The ones used are agglutination, complement
fixation, and ELISA. They detect anti-Brucella antibodies.

38. Mycobacterium I: M. tuberculosis

6. Classify mycobacterium. Give the morphology, cultural characteristics, pathogenesis, and lab
diagnosis of M. tuberculosis.

Mycobacteria are slender rods that sometimes show branching, filamentous forms resembling fungal
mycelium. They are acid-fast (along with nocardia), aerobic, non-motile, non-capsulated, and non-sporing.
Mycobacteria consists of three groups: obligate parasites, opportunistic pathogens, and saprophytes.
Obligate parasites
i. M. tuberculosis, M. bovis, M. africanum, M. microti (cause tuberculosis in various mammals)
ii. M. leprae causes leprosy
Opportunistic pathogens
i. Non-tuberculous mycobacteria: Mixed groups of isolates from various sources which are opportunistic
pathogens and cause many types of diseases.
Saprophytes
i. Saprophytic mycobacteria: M. phlei, M. smegmatis (frequently contaminates urine cultures)
M. tuberculosis is a straight or slightly curved rod which occurs singly, in pairs, or in small clumps. Long
filamentous and branching forms may sometimes be seen.
After staining with basic dyes they resist decolorization by alcohol. With the Ziehl-Neelsen method (acid-fast
stain) they resist decolorization by 20% sulfuric acid so they are acid fast. It is because they have mycolic acid,
which is a lipid, in their cell wall.
Cultural characteristics
i. The bacilli grow slowly with the generation time being 14-15 hours. Colonies appear in about 2 weeks
but can be longer.
ii. Solid media: Lowenstein-Jensen (LJ) culture is the most routine one.
1. It consists of coagulated hen eggs, mineral salt solution, asparagine, and malachite green.
(malachite green inhibits other bacteria)
2. M. tuberculosis forms dry, rough, raised, irregular colonies that are white/yellow.
iii. Liquid media: Generally not used for routine cultivation but for sensitivity testing, chemical analysis,
and preparation of antigens and vaccines.
1. Diffuse growth is obtained in Dubos medium containing Tween-80.
2. Growth begins at the bottom and creeps up the sides.
Antigenic properties
i. Mycolic acid is an important part of M. tuberculosis cell wall. It is a large fatty acid that is responsible
for the acid-fastness of the bacteria.
ii. Mycoside is a mycolic acid bound to a carbohydrate making it a glycolipid
iii. Cord factor is mycoside formed by the union of 2 mycosides with a disaccharide. Cord factor inhibits
neutrophil migration and damages mitochondria.
iv. Sulfatides are mycosides that resemble cord factors with sulfates attached to the disaccharide. They
inhibit phagosomes from fusing with lysosomes that contains bacteriocidal enzymes. The facultative
intracellular nature of M. tuberculosis is due to sulfatides
v. Wax D is a complicated mycoside that acts as an adjuvant (enhances antibody formation to an antigen)
and activates the protective cellular immune system
vi. Proteins are responsible for tuberculin reaction and induce delayed type hypersensitivity
Pathogenesis

i. The mode of infection is by direct inhalation of aerosolized bacilli contained in the droplet nuclei of
expectorated sputum. Coughing, sneezing, and speaking releases numerous droplets from people with
M. tuberculosis.
ii. Majority of inhaled bacilli are arrested by the natural defenses of the upper respiratory tract. Bacilli
reaching the lungs are ingested by alveolar macrophages and multiply inside them.
iii. Infection with M. tuberculosis induces cell-mediated immunity which manifests as delayed
hypersensitivity and resistance to infection.
iv. The first exposure to M. tuberculosis is called primary tuberculosis and is usually subclinical.
1. Bacilli are inhaled by aerosolized droplet nuclei which land in the most air-filled areas of the
lung: middle and lower lung zones
2. There is a small area of pneumonitis with neutrophils and edema. The bacteria enter
macrophages, multiply, and spread via the bloodstream and lymphatics to regional lymph
nodes, lungs, and other organs
3. The infection will now be contained or become symptomatic.
4. Asymptomatic primary infection
a. Cell-mediated immunity starts and the foci of bacteria become walled off in a caseous
granuloma. (Caseous center containing macrophages, multinucleated giant cells,
fibroblasts, and collagen deposits with a periphery of lymphocytes.)
b. The granulomas heal by fibrosis, calcification, and scar formation
c. Organisms in these lesions are decreased in number but remain viable.
d. Calcified tubercles/granulomas in the middle or lower lung zone is called a Ghon focus.
A Ghon focus accompanied by a perihilar lymph node calcified granulomas is called a
Ghon/Ranke complex.
5. Symptomatic primary tuberculosis
a. Occurs less frequently. Mostly in people who have weak cell-mediated system like
children, elderly, and immunocompromised.
b. Most of these people will also eventually contain the infection and form granulomas.
c. In very severe cases, the lung infiltrates will advance to lung necrosis, forming a hole in
the lungs or cavities. The cavities can become fluid filled and these fluid filled cavities
can be visualized on chest radiographs or CT scans.
v. Secondary tuberculosis/reactivation tuberculosis
1. When tuberculosis occurs after the bacteria have been dormant for some time.
2. Infection can occur in any of the organs that have been seeded during the primary infection.
3. Infection may be precipitated by a temporary weakness of the immune system
4. Pulmonary tuberculosis: Is the most common site of secondary tuberculosis. Infection usually
occurs in the apical areas of the lung around the clavicles because the oxygen tension is highest
there. Patients present with chronic low-grade fever, night sweats, weight loss, and a
productive cough that may have blood in it.
5. Pleural and pericardial infection: Infection of fluid collections around the lungs or heart
6. Lymph node infection: Most common extrapulmonary site. Cervical nodes usually affected.
They become swollen, mat together, and drain.
7. Kidney: Patients will have red and white blood cells in the urine but no bacteria will grow
because M. tuberculosis takes weeks to grow.
8. Skeletal: involves the thoracic and lumbar spine, destroying the intervertebral discs and then
the adjacent vertebral bodies
9. Joints: chronic arthritis of one joint
10. Central nervous system: There is subacute meningitis and granuloma formation in the brain

11. Miliary tuberculosis: tiny millet-seed-sized tubercles are disseminated all over the body.
Kidneys, liver, lungs, and other organs are riddled with the tubercles.
Lab diagnosis
i. Involves demonstrating the bacillus in the lesion by microscopy, isolating it in culture, transmitting the
infection to experimental animals, demonstrating hypersensitivity to tuberculoprotein, and using
molecular diagnostic methods.
ii. Specimen: Sputum is best collected in the morning before any meal. When sputum is not available,
laryngeal swabs or bronchial washings may be collected.
iii. Decontamination and concentration of specimens
1. Specimens from sputum need prior treatment so that other microorganisms dont overgrow
mycobacteria. Sputum samples also contain an organic matrix which may trap mycobacterial
cells. Therefore, liquefaction, decontamination, and concentration improve the yield.
2. Petroffs method or NALC combined with 2% NaOH can be used
iv. Microscopy
1. Ziehl-Neelsen/Acid-fast stain: Most reliable method in the diagnosis. Smear is covered with
strong carbol fuchsin and heated to steaming. Slide is then washed and decolorized with 20%
sulfuric acid and then with 95% ethanol. After washing, the smear is counterstained with
Loefflers methylene blue. Under oil immersion, acid fast bacilli are seen as bright red rods on a
blue background.
v. Culture
1. Very sensitive diagnostic technique
2. Solid media: Concentrated material is inoculated into at least two bottles of Lowenstein-Jensen
Medium. Any growth is smeared and tested by microscopy. If specimen is positive by
microscopy, then drug sensitivity test is set up.
vi. Anti-tubercular sensitivity tests:
1. As drug resistance is an important problem in tuberculosis, it is desirable to test the sensitivity
of isolates as an aid to treatment.
2. There is absolute concentration methods, resistance ratio method, proportion method, and
automated systems.
vii. Animal inoculation: Inoculated into two guinea pigs and clinical symptoms and autopsy done.
viii. Molecular Methods: Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are used.
ix. Immunodiagnosis
1. Serological tests are not useful in diagnosis anymore. Demonstration of hypersensitivity to
tuberculoprotein (tuberculin testing) is a standard procedure
2. Mantoux test (Tuberculin test): 0.1 ml of PPD is injected intradermally on flexor side of the
forearm. The site is examined 48-72 hours later and induration is measured at its widest point.
Induration of 10 mm or more is positive, less than 5 mm is negative, and 6-9 mm is equivocal.
3. Tuberculin test: After induction of cell-mediated immunity against M. tuberculosis, any
additional exposure will cause a localized delayed hypersensitivity (Type IV). Intradermal
injection of purified protein derivative (PPD) causes redness and localized skin swelling. It can
be used to detect if a person has been infected with M. tuberculosis because only infected
people will have the reaction. It works when macrophages take up the PPD and present the
antigen to T cells who travel to the injection site and release lymphokines which causes
redness and induration
4. A positive tuberculin test indicates hypersensitivity to tuberculoprotein, denoting infection
with the tubercle or BCG vaccination in the past, with or without clinical disease. A negative
test occurs in people who have never had contact with tubercle bacilli

5. False positives occur when there is infection by atypical mycobacteria or a person had the
BCG vaccine. False negatives occur when people have decreased cell-mediated immunity due
to malnutrition, steroids, immunosuppressed etc. because cell-mediated immunity causes the
delayed hypersensitivity
6. Tuberculin testing can be used as an aid in diagnosis of active infection.
x. Extrapulmonary tuberculosis specimen will depend on the site of infection. It can be urine, CSF, joint
fluid, biopsy material, blood, etc.

7. Prophylaxis of M. tuberculosis.

General measures are adequate nutrition, good housing, and health education
The BCG (bacille Calmette-Guerin) vaccine is a live attenuated vaccine administered intradermally at the
insertion of the deltoid immediately after birth or shortly after
A tuberculin negative recipient becomes positive after receiving the BCG vaccine
Immunity lasts for 10-15 years.
There are stringent safety measures in the production of BCG vaccine so there are few complications but
abscess, lymphadenopathy, fever, keloid, and other lesions can occur
The consensus is that BCG may not protect from tuberculosis infection but protects children against more
serious types of the disease such as meningitis and disseminated tuberculosis

39. Mycobacterium II Atypical Mycobacteria

8. Atypical mycobacteria

Mycobacteria other than tubercle bacteria that can occasionally cause human disease are called nontuberculous mycobacteria (NTM) or atypical mycobacteria
They cause opportunistic infections in immunocompromised patients
The most common incidence is an AIDS patient with Mycobacterium avium-complex (MAC). Patients present
with unexplained fevers, weight loss, diarrhea, general malaise, and elevation in alkaline phosphatase.
MAC is also the most common cause of atypical mycobacterium lung disease. They present either as:
i. An upper lung cavitary disease, predominantly in male smokers
ii. As lower and middle lung involvement with bronchiectasis and nodular infiltrates in middle aged nonsmoking women.
NTM can also cause pulmonary disease, lymphadenitis, skin lesions, bone and joint infections etc.

40. Mycobacterium III Mycobacterium leprae

9. Discuss the morphology, pathogenesis, and lab diagnosis of M. leprae.

M. leprae is an acid-fast rod with some branching although it shows considerable morphological variation. It is
less acid fast so 20% sulfuric acid is used instead of 5% sulfuric acid.
It is not possible to grow this bacterium on artificial media so it is grown on the footpads of mice, in armadillos,
and in monkeys. Its generation time is 12-13 days.
Pathogenesis
i. M. leprae causes leprosy which is chronic granulomatous disease of humans involving the skin,
peripheral nerves, and nasal mucosa usually.
ii. India is one of the six countries that has a high occurrence of leprosy
iii. The clinical manifestations of leprosy depend on:
1. Its tendency to grow better in cooler body temperatures so it grows closer to the skin surface
2. The cell-mediated immunity of the host because M. leprae is a facultative intracellular bacteria.
Cell-mediated immunity is the system to keep the bacteria in check but also causes
inflammation and granuloma formation particularly in the skin and nerves

iv. There are five types of leprosy ranging in severity of the disease. Lepromatous leprosy (LL) is the most
severe and tuberculoid leprosy (TL) is the least. The three remaining classes represent ranges between
these two: Borderline lepromatous (BL), borderline (BB), and borderline tuberculoid (BT)
v. Lepromatous leprosy
1. Cell-mediated immunity is the least in LL so the patient cannot mount a delayed
hypersensitivity reaction.
2. LL primarily involves the skin, eyes, and nerves but any organ can be infected by bacilli
3. Superficial nodular lesions (lepromata) develop which consist of granulation tissue containing a
dense collection of vacuolated cells. The nodules ulcerate become secondarily infected and
cause distortion and mutilation.
4. Facial skin can become so thickened that the face looks lion-like. (leonine facies) The nasal
cartilage can be destroyed causing saddle-nose deformities. Internal testicular damage can
lead to infertility. Anterior segment of eyes can be involved leading to blindness
5. Most peripheral nerves are thickened and demyelinated leading to loss of sensation in the
extremities.
vi. Tuberculoid leprosy
1. Patients mount a formidable cell-mediated response so the damage is minimal.
2. Patient demonstrates localized, superficial, unilateral skin and nerve involvement.
3. There are only 1-2 lesions that are well-defined, hypopigmented, elevated blotches. The area
within the rash is hairless with diminished or absent sensation

Number of lesions
Hair growth on skin lesions
Sensation in lesions of the
extremities
Acid fast bacilli in skin
scrapings
Lepromin skin test

Tuberculoid
Single
Absent
Completely lost

Borderline
Several
Slightly decreased
Moderately lost

None

Several

Lepromatous
Many
Not affected
Not affected but there
is some numbness
Innumerable

Strongly positive

No reaction

No reaction

Lab Diagnosis
i. Bacteriological diagnosis is easy in LL but may be difficult in TL
ii. Specimens
1. Nasal smear: A blunt scalpel is introduced into the nose and the internal septum is scraped
sufficiently to remove a piece of mucus membrane
2. Skin smear: Samples from the skin should be obtained from the edges of the lesion
3. Silt skin smear: Skin over the earlobe is cut about 5 mm long with a scalpel to get the infiltrated
layers. Other sites samples are the buttocks, forehead, chin, and cheek.
4. Nerve biopsy: sample collected from thickened nerves
iii. Microscopy
1. Diagnosis consists of demonstration of acid fast bacilli in the lesions by the Ziehl-Neelsen
technique using 20% sulfuric acid.
iv. Culture: difficult because M. leprae do not grow in artificial cultures
1. Mouse foot pad and armadillo can be used but are impractical
v. Serology: Detection of antibody against M. leprae

10. Lepromin Test

Lepromin test is a method to measure the ability of the host to mount a delayed hypersensitivity reaction
against antigens of M. leprae, lepromins.
Early reaction:

i. Early reaction of Fernandez occurs which consists of erythema and induration developing in 24-48
hours. They are poorly defined and of little significance. It is similar to the tuberculin test.
Late reaction:
i. Late reaction of Mitsuda occurs in 1-2 weeks, reaching a peak in 4 weeks.
ii. Reaction consists of an indurated skin nodule, which may ulcerate. There is infiltration of lymphocytes,
epitheloid cells, and giant cells which is a cell-mediated immune response
Applied importance
i. Lepromin test is not used to diagnose leprosy or indicate prior contact with M. leprae.
ii. Test is used to classify the lesions of leprosy patients. Positive indicates tuberculoid and negative in
lepromatous.
iii. To assess the prognosis and response to treatment. Positive indicates good prognosis and negative
indicates a bad prognosis.
iv. Assess resistance of an individual to leprosy.

41. Spirochetes
11. Enumerate the sexually transmitted disease. Give the morphology, pathogenesis, and lab
diagnosis of Treponema pallidum.

Sexually transmitted infections are a group of contagious conditions transmitted predominantly by sexual or
close body contact.
Classification of organisms causing STI
i. Bacteria: Treponema pallidum, N. gonorrhea, Chlamydia trachomatis, Gardnerella vaginalis,
Hemophilus ducreyi, Klebsiella granulomatis
ii. Virus: Herpes simplex virus, Cytomegalovirus, Human papilloma virus, Human immunodeficiency virus,
Hepatitis B virus, Molluscum contagiosum
iii. Fungi: Candida albicans
iv. Parasites: Trichomonas vaginalis
Treponema is a thin, delicate, gram-negative spirochete with tapering ends. It is actively motile via its
endoflagella which are polar flagella wound along the helical protoplasmic cylinder situated between the outer
membrane and cell wall.
T. pallidum cytoplasm is surrounded by a cytoplasmic membrane enclosed by a cell wall containing
peptidoglycan. Next is a lipid-rich outer membrane layer.
T. pallidum cannot be seen under the light microscope in wet films because they are too small but can be
discerned by negative staining with India ink. It can be seen via darkfield microscopy.
Pathogenic treponemes do not grow in artificial culture media.
Pathogenesis
i. T. pallidum enters the body by penetrating intact mucus membranes or by invading through epithelial
abrasions. Skin contact with an ulcer infected with T. pallidum can result in infection.
ii. Syphilis has three stages, with a latent stage between the 2nd and 3rd stages, in untreated patients
iii. Primary syphilis
1. The primary lesion is a painless chancre that erupts at the site of inoculation about 3-6 weeks
after initial contact. Regional non-tender lymph node swelling occurs as well.
2. The chancre is a firm, ulcerated, painless lesion with a punched-out base and rolled edges. It is
highly infectious. The chancre resolves over 4-6 weeks without a scar.
iv. Secondary syphilis
1. Untreated patients enter the bacteremic stage which occurs 6 weeks after the chancre has
healed.

2. In secondary syphilis the bacteria multiply and spread in the blood through the body. It is a
systemic disease with widespread rash, generalized lymphadenopathy, and involvement of
many organs.
3. Rash of secondary syphilis consists of small red, macular lesions symmetrically distributed over
the body particularly involving the palms, soles, and mucus membranes of the oral cavity. The
lesions can become papular (bumpy) and even pustular.
4. A characteristic skin lesion is called condyloma latum which is a wartlike lesion that occurs in
warm, moist sites like the vulva or scrotum.
5. Skin infections in areas of hair growth results in patchy bald spots and loss of eyebrows.
6. Secondary syphilis resolves over 6 weeks and the disease enters the latent phase
v. Latent Syphilis
1. Most patients are asymptomatic but 25% relapse back into secondary syphilis
2. 1/3 of untreated patients will progress into tertiary syphilis while the rest will be asymptomatic
vi. Tertiary syphilis
1. Develops over 6-40 years with slow inflammatory damage to organ tissue, small blood vessels,
and nerve cells. There are three categories of disease:
2. Gummatous syphilis
a. Gummas are localized granulomatous lesions which eventually necrose and become
fibrotic. They are usually found in the skin and bones.
b. Skin gummas are painless, lesions with sharp borders
c. Bone gummas produce deep gnawing pain
3. Cardiovascular syphilis
a. Characteristically, an aneurysm forms in the ascending aorta or aortic arch. This is
caused by chronic inflammatory destruction of the small arterioles (vasa vasorum) that
supply the aorta itself leading to necrosis of the media layer of the aorta.
b. The wall of the aorta splits as blood splits through the weakened media layer.
c. Aortic valve insufficiency and occlusion of the coronary arteries can also occur.
4. Neurosyphilis: Five most common manifestations:
a. Asymptomatic neurosyphilis: Patient is clinically normal but CSF is positive for syphilis
b. Subacute meningitis: Patient has a fever, stiff neck, and headache. CSF analysis reveals
a high lymphocyte count, high protein, low glucose, and positive syphilis.
c. Meningovascular syphilis: Spirochetes attack blood vessels in the brain and meninges
resulting in cerebrovascular occlusion and infarction of the nerve tissue in the brain,
spinal cord, and meninges.
d. Tabes dorsalis: There is damage to the posterior columns and dorsal roots of the spinal
cord. Results in ataxia, loss of reflexes, loss of pain and temperature sensation
e. General paresis: Progressive disease of the nerve cells in the brain leading to mental
deterioration and psychiatric symptoms
vii. Congenital Syphilis
1. Occurs in the fetus of an infected pregnant woman because T. pallidum can cross the placental
blood barrier.
2. There is a high rate of death (stillbirth, spontaneous abortion, and neonatal death) and almost
all of those that survive have early or late congenital syphilis
3. Early congenital syphilis
a. Occurs within 2 years and is like severe adult secondary syphilis with widespread rash
and condyloma latum. There is lymph node, liver, and spleen enlargement and bone
infection.
4. Late congenital syphilis

a. Similar to tertiary syphilis except that cardiovascular involvement is rare

b. Neurosyphilis is the same as adults but deafness is more common
c. Bone and teeth are commonly infected. Saddle nose can occur due to damage of the
palate and nasal septum due to inflammation. Bowing of the tibia can lead to saber
shins. Upper central incisors are widely spaced with a central notch in each tooth and
molars have too many caps.
d. Eye disease such a corneal inflammation can occur

Lab diagnosis
i. Spirochetes must be demonstrated under the microscope and antibodies must be found in the serum
or CSF
ii. Visualization of spirochetes is only effective during the active primary and secondary syphilis
iii. Specimen: Specimens must be collected from the lesions carefully as they are highly contagious. Serum
is collected for serology and CSF if neurosyphilis is detected.
iv. Microscopy:
1. Dark ground examination is done.
2. Direct fluorescent antibody test is also done.
v. Serological tests
1. The main methods for laboratory diagnosis
2. Nonspecific treponemal tests:
a. Infections with syphilis results in cellular damage so cardiolipin and lecithin are
released into the serum. The idea is to measure the antibodies to these antigens and if
a person has these antibodies then he may have syphilis. The antibody reacting with
cardiolipin and lecithin is called reagin. The two tests used are venereal disease
research laboratory (VDRL) and rapid plasma reagin (RPR)
b. In VDRL, inactivated (heated) serum is mixed with cardiolipin antigen and rotated for
four minutes. Cardiolipin remains as uniform crystals in normal serum but forms visible
clumps on combining with reagin. VDRL can also be used to test CSF in neurosyphilis. A
microscope is used to read the reaction.
c. RPR is a modified VDRL test. This test uses the VDRL antigen (cardiolipin) containing
fine carbon particles which makes the result more clear and evident to the naked eye.
d. These are nonspecific because about 1% of patients will receive a false negative
(Biological false positive (BFP)) because of pregnancy, viral hepatitis, acute febrile
illness, use of IV drugs etc.
3. Specific treponemal tests:
a. Specific tests look for the antibody to the spirochete itself so they avoid false positives
b. The original test was T. pallidum immobilization (TPI) but it is too complex
c. The test used now is indirect immunofluorescent treponemal antibody-absorption
(FTA-ABS). Patients serum is mixed with nonpathogenic strains of T. pallidum which
removes the antibodies shared by pathogenic and nonpathogenic T. pallidum. The
remaining serum is then added to a slide covered with pathogenic T. pallidum so
antibodies specific to the pathogenic T. pallidum will bind and give a positive result
d. T. pallidum hemagglutination assay (TPHA) is also another test that is used. It uses
tanned erythrocytes sensitized with sonicated extract of T. pallidum as antigen. The
test sera for TPHA are absorbed with a diluent containing components of
nonpathogenic treponeme called Reiter treponeme, rabbit testis, and sheep
erythrocytes. Very simple and economical
e. Enzyme immunoassays are also now available for T. pallidum

4. Quantitative tests are useful in monitoring the patients response to treatment, indicating the
stage of the disease, and in detecting re-infection. VDRL and RPR are preferred because they
become negative after treatment.
vi. PCR can also be used to detect the bacterial DNA.
VDRL or RPR
+
+
-

FTA-ABS or TPHA
+
+
-

Interpretation
Indicates an active treponemal infection
Probably a false negative
Successfully treated syphilis
Syphilis unlikely although an AIDS patient
with syphilis may be seronegative or a patient
who has just been infected with T. pallidum
will not develop an immune response yet

12. Relapsing fever: Borrelia recurrentis

Borrelia are large, motile, refractile spirochetes with irregular, wide, open coils.
Borrelia recurrentis is spread by louse or tick. Louse born B. recurrentis causes epidemics while tick born B.
recurrentis is endemic.
B. recurrentis is microaerophilic and Gram-negative. It stains well with Giemsa and bacterial stains.
Pathogenesis
i. B. recurrentis causes relapsing fever that is sudden in onset. During this period the Borrelia are
abundant in the patients blood. Fever subsides in 3-5 days. After an afebrile period of 4-10 days,
another bout of fever starts.
ii. If untreated, the disease ultimately subsides after 3-10 relapses with each relapse taking longer.
iii. Relapses are because B. recurrentis has the ability to change its antigens. When antibodies are formed
against the antigens of B. recurrentis to destroy it, the bacteria change its surface proteins so that the
antibody no longer recognizes it. Which causes the relapse. Eventually, the immune system creates
antibodies against the new antigens.
iv. The antigenic variation allows B. recurrentis to survive for several weeks
Lab diagnosis
i. Microscope: Borreliae are found in blood during the fever. Blood smears are stained with Giemsa or
Leishman stain.
ii. Culture: Not very successful and not used
iii. Serology: Demonstration of antibodies is too unreliable.
iv. Animal inoculation: can successfully inoculate blood from patient into white mice and then collect the
blood from the mice in two days and check for Borreliae
Treat with tetracyclines, penicillin, erythromycin

13. Lyme Disease: Borrelia Burgdorferi

Borrelia burgdorferi is a corkscrew shaped spirochete

It is a fastidious bacteria that can be grown in a modified Kelleys medium.
It is transferred by ticks but the reservoir is white-footed mouse or white-tailed deer. The disease only
manifests if the tick stays on the body for 24 hours.
Pathogenesis
i. Lyme disease occurs in three stages. It occurs mostly in Northeast, Midwest, and Northwestern USA
ii. Early localized stage
1. Consists of just a skin lesion (ECM) at the site of the bite along with flulike illness and regional
lymphadenopathy

2. Erythema chronicum migrans (ECM) starts off as a red (erythema) flat round rash, which
spreads out (migrates) over time (chronicum)
3. The outer border remains red while the center will be clear, turn blue, or even necrose.
iii. Early disseminated stage
1. Involves dissemination of B. burgdorferi to the skin, nervous system, hearts, and joints.
2. Skin lesions are more but smaller ECMs over the skin
3. B. burgdorferi can invade the brain, cranial nerves, and even motor/sensory nerves. Results in
meningitis, cranial nerve palsies, and peripheral neuropathies
4. Most common cardiac abnormalities include atrioventricular nodal block (heart block) and
sometimes myocarditis and left ventricular dysfunction
5. Migratory joint and muscle pain also occur. Large joints become hot, swollen, and painful.
iv. Late stage
1. About 10% of patients will develop chronic arthritis that lasts for more than a year.
2. There may be chronic neurologic damage. Encephalopathy may develop characterized by
memory impairment, irritability, and somnolence.
Lab diagnosis
i. Made by isolation of borrelia or by serology.
ii. Specimens include skin lesions, CSF, and blood.
iii. Culture is too impractical.
iv. ELISA and immunoblotting can be used to detect anti-Borrelia burgdorferi antibodies
Treat with doxycycline or penicillin

14. Leptospira

Leptospiras are actively motile, delicate spirochetes. They are too small to be seen under a light microscope.
They are helical rods that look like a tight coil. Their ends are hooked and resemble umbrella handles.
Cultural Characteristics
i. They can be grown in media enriched with Rabbit serum. They are aerobic and microaerophilic.
ii. Leptospiras can be grown on the chorioallantoic membrane of chick embryos
Leptospiras are divided into two species of which L. interrogans is pathogenic to humans
Pathogenesis
i. The spirochetes can penetrate abraded skin or mucus membranes when humans come in contact with
contaminated urine of dogs, rats, livestock, and wild animals.
ii. Clinically there are two phases:
iii. First or leptospiremic phase: the bacteria invade the blood and CSF, causing abrupt high fever,
headache, malaise, and severe muscle aches (thighs and lower back). The conjunctiva are red and the
patient experiences photophobia. After 1 week there is an afebrile period then the fever recurs
iv. Second or immune phase: There is appearance of IgM antibodies. Patients may develop meningismus
and CSF has WBCs.
v. L. interrogans can cause a more severe illness called Weils disease or infective jaundice. It involves
renal failure, hepatitis with jaundice, mental status changes, and hemorrhage in many organs.
Lab diagnosis
i. Specimen: Blood or urine is taken
ii. Microscopy:
1. Examination of blood: As leptospiras disappear from blood after the first week, blood
examination is helpful only in the early stages of the disease. A dark field or
immunofluorescent microscopy is used
2. Examination of urine: Leptospiras appear in urine in the second week of the disease and
intermittently thereafter for 4-6 weeks. Urine should be examined immediately after voiding.
Centrifuged deposit of urine is examined under dark ground illumination

iii. Culture
1. Four drops of blood are inoculated into bottles containing EMJH medium. They are incubated
for two days then left in the dark at room temperature for two weeks. Samples are examined
under dark ground illumination. CSF can also used
2. Direct culture of urine is not usually successful
iv. Animal inoculation
1. Blood from the patient is also inoculated into guinea pigs. The animals develop fever and die
within 8-12 days with jaundice and hemorrhage into the lungs.
v. Serological diagnosis
1. Antibodies appear in the serum towards the end of the first week.
2. Broadly reactive tests indicate leptospiral infections without indicating the exact serotype.

3.

Type-specific test indicate the serotype by demonstrating specific antibodies. Microscopic

agglutination test can be used, with agglutination indicating a positive reaction.

42. Mycoplasma
15. Mycoplasma pneumoniae

Mycoplasma are a group of bacteria that are devoid of cell walls making them highly pleomorphic
They have a membrane surrounding the cytoplasm that is rich in sterols
There are two pathological species: Mycoplasma pneumonia (causes atypical pneumonia) and Ureaplasma
urealyticum (causes non-gonococcal urethritis)
Mycoplasma does not possess spores, fimbriae, or flagella. They are gram-negative.
Cultural Characteristics
i. They are aerobic/facultative anaerobes that can be grown on solid or liquid media
ii. Media for cultivating mycoplasma are enriches with 20% horse/human serum and yeast extract. The
serum acts as a source for cholesterol and other lipids
iii. The colony has a fried egg appearance with a central opaque granular area of growth surrounded by a
flat translucent peripheral zone.
iv. Colonies are studied by Dienes method. A block of agar containing the colony is cut and placed on a
slide. It is covered with a cover slip which has an alcoholic solution of methylene blue
Pathogenesis
i. They produce surface infections by adhering to the mucosa of the respiratory, GI, and urinary tracts by
the protein P1 which is an adherence virulence factor
ii. M. pneumonia causes primary atypical pneumonia. It is atypical because it does not respond to
penicillins. It is self-limiting and resolves in 1-2 weeks.
iii. Onset is gradual with fever, malaise, headache, and sore throat. Paroxysmal cough may be present
with blood-tinged sputum.
iv. Rashes, meningitis, encephalitis, and hemolytic anemia are some complications.
v. Transmission is by droplets of nasopharyngeal secretions
Lab diagnosis
i. Specimens: Throat swabs or respiratory secretions are taken and placed transport media to prevent
dying or overgrowth by other bacteria
ii. Culture:
1. Growth is slow and may take 1-3 weeks.
2. Culture is made on complex media containing agar, broth, yeast, horse serum, and penicillin
(to inhibit growth of other bacteria)
3. Colonies have a fried egg appearance
iii. Molecular Methods
1. PCR is specific and rapid. Used where feasible.

Serology
i. Specific tests: Using mycoplasmal antigens such as immunofluorescence, hemagglutination, and
complement fixation test (patients serum is mixed with glycolipid antigens prepared from
Mycoplasma)
ii. Non-specific tests: Like Streptococcus MG and cold agglutination
iii. Cold Agglutination
1. Primary atypical pneumonia patients can develop monoclonal IgM antibodies directed at a
common red blood cell antigen called the I antigen. These antibodies bind to RBCs and cause
them to agglutinate.
2. Dilutions of the patients serum are mixed with RBCs and clumping is observed after leaving
overnight at 4oC
lactam antibiotics do not work with Mycoplasma. Treat with macrolides, tetracyclines, and quinolones