BASIC PRINCIPLES OF BACTERIOLOGY (Dr.

Sia-Cunco)
June 21 & 23, 2011

EUKARYOTES VS. PROKARYOTES

o o o o

Diplococci: in pairs (Neisseria gonorrhoeae) Streptococci: in chains (Streptococcus pyogenes which causes tonsillitis) Staphylococci: in clusters Tetrads or Octads: packets of four or eight; usually not pathogenic

BACILLI - rod-shaped. Ave. size = 3m o Diplobacilli: in pairs

o o

Streptobacilli: in chains Coccobacilli: short rod; resembles an elongated cocci (Haemophilus influenza causes otitis media, respiratory infections, and meningitis in children) Palisade formation: usually seen in Vibrio cholere

o
The 3 Domain System Of Classification of Organisms o EUKARYOTES: more complex cells with a true nucleus and membrane bound organelles. o PROKARYOTES: less complex cells w/o a nucleus or membrane-bound organelles. 10x smaller than eukaryotes. a) ARCHAEA Most live in extreme environments and are termed EXTREMOPHILES Pyrolobus fumarii in volcanoes (thrives in a temperature of 110 degrees Celsius) Cell walls have NO Peptidoglycan
Main differentiating factor between Archaea and Bacteria

SPIROCHETE o Spiral-shaped o Moves by spinning around its long axis o Example: Treponema pallidum causative agent of syphilis VIBRIO o Curved or comma-shaped bacilli May occur singly or in pairs (gull-wing morphology like Campylobacter jejuni) Campylobacter jejuni which causes diarrhea brought about by poultry (chicken)

o o

b) BACTERIA Single celled organisms that divide by binary fission Round, Rod and Spiral Most with rigid cellwalls containing Peptidoglycan peptidoglycan is a supporting structure of cell wall The genus Mycoplasma has NO cell wall. It only has a cytoplasmic membrane; Mycoplasma pneumonia causes the atypical pneumonia; Ureaplasma urealyticum causes UTI)
Note: Since the prokaryotes are further divided into two subdivision, the three domain systems are the ff: Eukaryote, Archaea, and Bacteria.

Cell-Wall-Deficient (CWD) Bacteria o Pleomorphic: exists in a variety of shapes o Mycoplasma pneumoniae (cause of atypical pneumonia), and Ureaplasma urealyticum (cause of UTI)

MORPHOLOGY OF BACTERIAL CELLS

COCCI - round/spherical; Average size= 1m

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Gram-Positive Cell Walls

Thick layer of Peptidoglycan (30 layers of glycan

chains) but fully permeable to sugars, amino acids & ions.

Teichoic Acid chains of ribitol or glycerol phosphate, sugars & alanine attached to NAM; this is antigenic Lipoteichoic Acid Reaches the cytoplasmic
BACTERIAL CELL STRUCTURE Cell Wall
Rigid structure holding the cell together and prevents it from bursting. Determines the shape of the organism Antigenic Strength is due to PEPTIDOGLYCAN membrane

Gram-Negative Cell Walls

Peptidoglycan

Backbone:

alternating N- acetylglucosamine and N acetylmuramic acid connected by 1 4 linkages (Glycan Chain) Tetrapeptide side chains attached to NAM Identical Peptide Cross bridges connecting the tetrapeptide chains. Glycan chains are joined together
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OUTER MEMBRANE o Lipid bilayer; the outer leaflet is made up of lipopolysaccharides (the outer membrane is also called the lipopolysaccharide layer) o Protective barrier against toxic compounds including antibiotics (Gram negatives are more resistant to antibiotics) o With porins that allow passage of small molecules and ions in and out of the periplasm. o Lipopolysaccharide (or LPS) is also called the endotoxin. Lipid A: anchors the LPS to the lipid bilayer; Antigenic O-Specific Polysaccharide side chain: portion directed away from the
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membrane opposite that of Lipid A. Varies among different strains of bacteria.(E.coli )157:H7 this specific strain causes epidemic diarrhea, traced to inadequately cooked beef/burgers) PERIPLASM o Gel-like fluid between the outer membrane and the cytoplasmic membrane (or Inner Membrane) o Contains proteins involved in nutrient degradation and transport

Embedded with protein gates that permit nutrients to enter and wastes to exit. H20, O2 and CO2 pass thru by simple diffusion depending upon the concentration of molecules on each side of the membrane (Osmosis) Excretes enzymes that degrade Protein, sugars and lipids so it can pass through. Functional analog of the Mitochondria (which are absent in bacteria)

Glycocalyx

This is where the peptidoglycan is embedded

Gelatinous

PEPTIDOGLYCAN o Thin layer sandwiched outer and inner membrane

between

the

material secreted by the cell membrane and secreted outside the cell wall. o Slime Layer: loosely attached to the cell wall. Enables bacteria to glide on surfaces. o Capsule:

LIPOPROTEIN o Stabilizes the outer membrane attaches it to the peptidoglycan.

and

Firmly attached to the cell wall.

Differences in the cell wall composition of bacteria account for the typical staining characteristics. o Gram (+) VIOLET

Antiphagocytic (Enables bacteria to survive longer) - Used to differentiate among bacteria (H. influenza b the pathogenic form of H.
influenza)

Detected using a capsule stain (stands

Has a thick peptidoglycan layer, thus it takes up the crystal violet dye. The alcohol decolorizer dehydrates the peptidoglycan layer, acting as a permeability barrier. Hence, the dye is retained.

out like a halo)

Gram (-) RED

The alcohol decolorizer destroys the outer membrane. Since there is only a thin layer of peptidoglycan, it is decolorized immediately. Thus, it takes up the secondary stain, safranin, which is reddish in color.

Peptidoglycan is broken by: o PENICILLIN: prevents

Encapsulated bacteria: S-Colonies (smooth) Non Encapsulated: R-Colonies (rough)

Flagella

cross linking of glycan chains; more effective for Gram(+) o LYSOZYME: breaks the bond that links NAM and NAG. Found in tears, saliva, and sweat.

Appendage of twisted threads of Flagellin arising

from the cell membrane & projects out of the cell wall and capsule. For motility.

Acid Fast Cell Wall

There are only 3 groups that have acid fast cell wall: Nocardi, actinomycetes, and mycobacteria; commonly we encounter mycobacteria; mycobacteria is the cause of TB and leprosy Mycobacteria have a waxy material on their cell wall called mycolic acid. Carbolfuschin introduced into the cell wall by heating or by treatment with a detergent cannot be removed by HCl

4 kinds: o Peritrichous flagella are all over the bacteria o Lophotrichous flagella are found only as a tuft on one portion of the bacteria o Amphitrichous flagella on both ends o Monotrichous a single flagellum on the bacteria

Cytoplasmic Membrane/Inner Membrane

Lipid bilayer that surrounds the cytoplasm

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 Small, circular, double stranded DNA w/c is not a

part of the chromosome. With 10 to several hundred genes Can be transferred via the sex pilus Bacteria may contain one plasmid, multiple copies of the same plasmid or multiple plasmids.

Ribosomes
70S from 30S + 50S For Protein synthesis

Pili/Fimbriae

Storage Granules

Hair-like Not

structures arising from the cytoplasm and made up of Pilin. Mostly on Gram-negative bacteria for motility. Enables bacteria to anchor on surfaces. (Piliated strains are pathogenic) o Example: Neisseria gonorrhoeae, there are piliated and non-piliated strains; if what you get is the piliated strain, then it will attach your urethral mucosa and will cause you gonorrhea; the non-piliated ones cannot anchor into you r mucosa so you just flush it out when you urinate

Accumulated

products nutrient (Glycogen).

synthesized

from

Endospores

Dormant

Sex

cell type produced by sporulation within cells (e.g. by Bacillus anthracis and cereus, & Clostridium tetani, perfringens, and botulinum) whenever nutrients are lacking. May germinate into a vegetative cell when nutrients are available.

Pilus: enables transfer of genetic material (plasmid) from one bacterial cell to another (Conjugation).

Resistant to heat, chemicals and UV light Can be killed off by by the Autoclave at 121 C, 15 psi
for 15 mins; or glutaraldehyde

SPORULATION
Initiated when there are low levels of nutrients. Bacteria can sense when it is facing starvation conditions.

STAINING
Organisms are made to contrast with the background. o Basic Dyes: Positively charged and stains negatively charged parts of the cell. o Acidic Dyes: Negatively charged and stains the background. o Simple Stains: Puts contrast between colorless (e.g. methylene blue) o Differential Stain: Distinguishes one group of organism from another. Gram Stain: GM (+) & GM (-) Acid Fast Stain: Detects Mycobacteria Nocardi, and Actinoomycetes o Special Stains: Stains specific structures inside or outside the cell. Capsule Stain: The capsule does not take up a stain, so it stands

Chromosome

Single,

long, supercoiled, double-stranded, circular DNA containing 850 to 6,500 genes is packaged in the nucleoid.

Plasmid/Extrachromosomal DNA
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out against a stained background. (Cryptococcus) Endospore Stain: Malachite green + heat Flagella Stain: Stain coats the flagella and is visible under a light microscope.

Non Selective: Supports the growth of many different bacteria. Numerous colonies are seen. (Nutrient Agar) o Selective Media: Has ingredients that will inhibit some bacteria while allowing the growth of others. (Mannitol o Salt Agar for S. aureus, also changes
color of agar from red to yellow due to fermentation)

CLASSIFICATION OF BACTERIA
Taxonomy - arranges organisms into groups with similar properties. o Identification: Process of characterizing organisms. (Microscopic exam, culture characteristics, biochemical tests, nucleic acid analysis)
Examples of Biochemical Tests:

All

micrococci and staphylococci are catalase-positive, and all streptococci are catalase-negative (catalase deactivates hydrogen peroxide into water and oxygen) Staphylococcus aureus clots plasma in four hours (coagulase-positive)

Classification: Arranging organisms into similar or related groups (nucleotide sequencing determines the genetic relatedness) Nomenclature: Assigning of names

Criteria for Classification of Bacteria

Differential Medium: Contains chemicals to indicate which microbes possess and which lack a particular biochemical process. (MacConkey agar E.coli is pink) o Biochemical Tests : detects characteristic metabolic function. (Catalase Test) o Serology: Use of antibodies that react with specific bacterial surface structures (Capsule) Remember: Taxonomic classification is arranged in a hierarchical order. o Kingdom: Prokaryotae o Division o Class o Order o Family: Enterobacterioceae o Genus: Escherichia o Species: coli o Subtype: E. coli O157:H7 Note: Family, genus, and species are commonly used The Three Domain System o Comparison of the nucleotide sequence of 16S rRNA revealed evolutionary relationships among organisms. o Eukarya, Bacteria & Archaea Bergeys Manual of Systematic Bacteriology o Definitive work on taxonomic classification of bacteria o The Eubacteria: Gm(+), Gm(-), those without cell walls o The Archaebacteria

Microscopic Morphology o Size and shape: whether organism is a fungus, protozoan, etc. (e.g. Wet mount Trichomonas) o Gram Stain: narrows down the possible identity of the organism. o Special Stains: Capsule, Acid Fast Metabolic Differences o Growth on Media : Agar + Nutrients. The media will distinguish among various bacteria in a clinical sample. (e.g. Blood Agar Plate/BAP Streptococcus pyogenes produces
beta-hemolysis in this media, while Streptococcus pneumoniae produces partial/greenish/alphahemolysis)

Nomenclature
Uses the Binomial System of Carolus Linnaeus The 1st letter of the Genus is capitalized and the word is underlined or italicized The specific epithet is NOT capitalized but underlined or italicized o Species= Genus + specific epithet o Escherichia coli o Escherichia coli The Genus may be designated by a single letter abbreviation o E. coli Other acceptable terms o Staphylococci Staphylococcus spp.
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 Slang terms o GC N. gonorrhea o Staph o Strep Genus + sp. epithet +subspecific epithet o H. infuenzae ssp. aegyptus

Bacteria are named for the disease they cause o Bacillus anthracis- Anthrax o Vibrio cholerae- Cholera o Mycobacterium leprae Leprosy o Clostridium tetani Tetanus

Exponential increase in mass until nutrients become exhausted or toxic metabolites accumulate and inhibit growth. Stationary Phase o Exhaustion of nutrients & accumulation of toxic products causes growth to cease completely Decline Phase o Also known as death phase o Number of dying cells exceeds the number of new cells formed. o

PHYSIOLOGY AND CONTROL OF BACTERIA

Factors that Influence Bacterial Growth

Bacteria divide by binary fission Microbial growth is defined as an increase in the

1.

number of cells in a population. Generation or Doubling Time: Time it takes for a population to double in number o Staphylococcus 30 mins o E. coli- 20 minutes o Myobacterium tuberculosis 24 hrs

Bacterial Growth Curve

Nutrition: Needed to synthesize cellular components Carbon, O2, Hydrogen, Nitrogen, Sulfur,Phosphorus, K. Mg, Ca Carbon Sources o Autotrophs: Use CO2 as their sole source of Carbon. o Heterotrophs: Use organic compounds other than co2 as their carbon source. Most bacteria are heterotrophs Energy Source o Phototrophs: use light as an energy source. (e.g. Cyanobacteria) o Chemolithotrophs: use inorganic chemicals as energy source. o Chemoorganotrophs: use organic chemicals as an energy source. Nitrogen Source o Assimilated in the form of ammonia by Nitrogen fixation o Nitrogen is a component of protein and nucleic acids Mineral Source: K, Mg, Ca, Fe and other minerals are usually provided in tap water or as contaminants of other ingredients Growth Factors: vitamins and amino acids that the bacteria cannot synthesize are taken from the environment (e.g. they get iron from the blood)

The number of viable cells is determined and

plotted Lag Phase o Cells are adapting to a new environment. o Enzymes & intermediates are formed until they are present in concentrations that permit growth to resume. Log / Exponential Phase

2. Temperature Bacteria have a well-defined UPPER and LOWER temperature limit within which it grows and outside of which growth stops. Optimum Growth Temperature: Organisms multiply rapidly. o Psychrophiles: Prefer cold T, between -5 to 15 o Psychrotrophs: Refrigerator T of 4 Causes food spoilage o Psychroduric: Prefers warm T but can survive freezing (E. coli in commercially bought ice) o Mesophiles: Grow best at normal body temperature. Include most pathogenic bacteria (Disease causing bacteria) Optimum at 25 to 45 C
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o o

Thermophiles: Found in hot springs, water heaters 45 to 70 C Hyperthermophiles: Usually Archaea 70 to 110

3. Oxygen It is useful to classify bacteria on the basis of their relationship to O2 and CO2 Obligate Aerobes: require O2 as hydrogen acceptor o Require an atmosphere containing O2 at room air concentration (20-21% O2) o Ex: Mycobacteria (usually stay on

Bacteria grow in complex communities and in association with other microbes. o Ex: Mouth- Aerobes and Anaerobes Bacteria may attach to surfaces and live in a polysaccharide-encased community of unrelated cells called a BIOFILM. (e.g. slime on drainpipes ) o Has channels through which nutrients and waste materials pass o Protect against harmful chemicals like disinfectants

Principles of Bacterial Control

the apex of lungs due to high oxygen tension)

Microaerophiles: O2 concentrations lower than

room air (5%)

Washing or scrubbing can minimize the time

o Ex: N. gonorrheae Obligate Anaerobe: Can only grow in the

absence of O2 Ex: is Bacteroides fragilis which is a normal flora of the large intestine Aerotolerant Anaerobe: Grows better in the absence of O2 but can survive in atmospheres containing O2 (Strep pyogenes) Facultative Anaerobe: Survives in the presence or absence of O2. o Ex: Enterobacteriaceae

necessary to sterilize or disinfect a product Dirt, grease, blood, pus can interfere with heat penetration and the action of disinfectants. Antimicrobial Agents o Biocide: chemical or physical agents w/c inactivate microbes o Bacteriostatic: inhibits bacterial multiplication (e.g.
Chloramphenicol)

o o

Bactericidal: able to kill

bacteria Sterilization: renders a surface/product free from organisms and spores. o Disinfectant: reduces the number of organisms on a product or surface to a level specified for its intended use. NOT FOR LIVING TISSUE. o Antiseptic: destroys or inhibits microorganisms on LIVING TISSUES. (e.g.
povidone iodine)

Capnophiles: Grow better in increased

concentration of CO2 (Bacteroides ) 4. pH Bacterial Species survive within a range of pH values Microorganisms regulate their pH by pumping protons in and out of the cell. Neutralophiles: Grow best at pH 6 -8

Acidophiles: pH 3 Alkaliphiles: pH 10.5

5. Water All microbes require H2O for growth Osmotic pressure & salt concentration will have to be controlled o Increased salt in the environment plasmolysis Osmolality is regulated by the active transport of K+ ions into the cell.

Septic: presence of pathogenic microbes in living tissue. o Aseptic: free of microorganisms o Antibiotics: inhibit or destroy selective bacteria at low concentrations. Physical Agents

Moist Heat: boiling for 10 minutes destroys most microorgaisms and viruses except spores. Autoclave : 121 C at 15psi x 15 min kills spores o Dry Heat: wire loops are heated red hot Petri Dishes : ovens

Bacterial Growth in Nature

Chemical Agents o Alcohol: rapid, broad spectrum, against bacteria, viruses and fungi. NOT sporocidal o Glutaraldehyde: sterilization of endoscopes and surgical equipment. SPOROCIDAL (Instruments are immersed for
30 minutes. But glutaraldehyde is toxic, hence Angulo, Aover, Apale, Aquino, Aquino 7

the need to rinse instruments with sterile water afterwards.)

Aldehydes: Chlorhexidine is used in handwashing and oral products. o Bisphenols: antiseptic soaps. Triclosan & Hexachlorophene are bactericidal and sporostatic. o Na Hypochlorite: bactericidal & virucidal. Sporicidal at higher concentrations. o Iodine: bactericidal, fungicidal, tuberculocidal, virucidal and sporocidal. Iodophores or Povidone Iodine (Betadine) are complexes of Iodine and a carrier. o Heavy MetalSilver Sulfadiazine: Broad spectrum combination of 2 antibacterials
Used for burns, but not in the facial area, since it can produce dark scars

NORMAL FLORA/MICROBIOTA
Population of microorganisms that inhabit the skin and mucous membranes of healthy normal persons. A human first becomes colonized by normal flora at the moment of birth, during passage through the birth canal, and after the first feeding. Resident Flora o Fixed type of microorganisms found at a given area. If disturbed, promptly re-establishes themselves. o Maintains health and normal function. GIT Flora Vitamin K and B o May become pathogenic if transplanted from its normal habitat. E. coli (normal flora of large
intestine, but is the most common cause of UTI especially in females)

H2O2: for bacteria, viruses, yeast. Sporicidal at 10-30% and in longer contact time. o Phenol/Carbolic Acid: antiseptic/ disinfectant

Not o

used anymore, but is still the industry standard

Transient Flora o Non pathogenic or potentially pathogenic (especially if

immunocompromised)

Quarternary Ammonium Compound: Sterillium has this as added component. Can be used as an antiseptic and for cleaning surfaces.

ECOLOGY Microbial Interactions

o Inhabits the skin, mucous membrane for hours, days or weeks o Derived from the environment and does not establish itself permanently on the surface o May proliferate and produce disease if resident flora is disturbed

Neutral: few species are neutral because they

e.g. Methicillin-resistant S. aureus (MRSA) can be acquired from contact with patients

use up the food supply and excrete toxic waste products. Antagonistic: elaboration of Bacteriocins

Normal Flora of the Skin

The high moisture (axilla, groin, and areas between the toes) supports the growth of bacterial cells. Found in the epidermis and the upper parts of the hair follicles o Gram-positive cocci (Staphylococcus epidermidis and Micrococcus sp.) o Corynebacteria such as Propionibacterium sp. These are generally nonpathogenic and considered to be commensal Sometimes potentially pathogenic Staphylococcus aureus is found on the face and hands in individuals who are nasal carriers

substances elaborated by some bacteria, which will kill off other bacteria

Synergism: cooperative effort of 2 or more

microbe species that produces a result that could not be achieved individually. (e.g. Bacterial vaginosis In which Lactobacillus acidophilus, the
normal flora of the vagina that maintains acidity, disappears in certain cases of hormonal imbalance or topical use of antibiotics Vaginal pH becomes alkaline Overgrowth of Gardnerella and Mobiluncus Synergistic effect)

Host-Parasite Interaction

Commensalism: inconsequential association

between bacteria and higher organisms. o Example: Humans have Staphylococcus epidermidis as commensals Symbiosis: mutually beneficial relationships o Ex: E. coli and humans

Normal Flora of the Conjunctiva

Blinking mechanically washes away foreign

Parasitism: one organism derives benefits at

the expense of another.

objects including bacteria. Tears contain bactericidal lysozyme. Staphylococcus epidermidis and Propionibacterium acnes are dominant. Staphylococcus aureus, some streptococci,
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Haemophilus sp. occasionally found.

and

Neisseria

sp.

are

Normal Flora of the Respiratory Tract

Usually contain bacteria with melaninogenicus often dominant role.

mixtures of Bacteroides playing a

The nares (nostrils): o Staphylococcus epidermidis and

corynebacteria, o Often (20% of the population) with Staphylococcus aureus The pharynx (throat): o Streptococci and various Gramnegative cocci; Streptococcus mutans is the one that gives you your tooth decay o Sometimes pathogens such as S. pneumoniae, S. pyogenes, H. influenzae and N. meningitidis colonize the pharynx. The lower respiratory tract: o Free of microorganisms. o If the epithelium is damaged, it may become susceptible to infection by pathogens S. pneumoniae which is a pathogen and the most common cause of pneumonia in adults is normally found also in the upper respiratory tracts; it is normally present in this area; but it can cause you pneumonia if the epithelium of the respiratory tract is damaged by viral infection

Normal Flora of the Gastrointestinal Tract

Upper GI tract of adult humans: esophagus

contains only the bacteria swallowed with saliva and food May be colonized by a pathogenic bacterium, Helicobacter pylori - tears down the lining that coats
the gastric mucosa, making it susceptible to the action of hydrochloric acid may cause peptic ulcer disease o It is easly treated by the combination of antibiotic: amoxicillin and clarithromycin and something that may block off acid secretion like omeprazole

Large

intestine: predominant species are anaerobic Bacteroides and anaerobic lactic acid bacteria in the genus Bifidobacterium bifidum.

Bacteria found in the large intestine of humans

BACTERIUM Bacteroides fragilis Bacteroides melaninogenicus Bacteroides oralis Lactobacillus Clostridium perfring Clostridium septicum Clostridium tetani Bifidobacterium bifidum Staphylococcus aureus Enterococcus faecalis Escherichia coli Salmonella enteritidis Klebsiella sp. Enterobacter sp. Proteus mirabilis Pseudomonas aeruginosa RANGE OF INCIDENCE 100 100 100 20-60 25-35 5-25 1-35 30-70 30-50 100 100 3-7 40-80 40-80 5-55 3-11

Normal Flora of the Urogenital Tract

Urine is normally sterile since the urinary tract is flushed with urine every few hours. Staphylococcus epidermidis, Enterococcus faecalis and some alpha-hemolytic streptococci
usually found in periurethral area

The vagina becomes colonized soon after birth

with corynebacteria, staphylococci, streptococci, E. coli, and "Doderlein's bacillus" (Lactobacillus acidophilus). During reproductive life Lactobacillus acidophilus predominates. The lactic acid produced inhibits colonization by pathogens.

Normal Flora of the Oral Cavity

Oral bacteria include streptococci, lactobacilli,

staphylococci and corynebacteria, with a great number of anaerobes, especially bacteroides. The eruption of the teeth during the first year leads to colonization by S. mutans and S. sanguis.
o decay

Streptococcus mutans causes tooth

The oral flora: o Usual cause of various oral

diseases in humans, including abscesses, and periodontal disease.
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Mnemonic

All cocci are Gm Positive EXCEPT N, V, M o Neisseria o Veillonella o Moraxella All bacilli are Gram negative EXCEPT BECM BCLA P L o Bifidobacterium o Eubacterium o Corynebacterium o Mycobacterium o Bacillus o Clostridium o Listeria o Actinomycetes o Propionibacterium o Lactobacillus

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