Medical Bacteriology: Thursday, May 26, 2022

MEDICAL BACTERIOLOGY
Thursday, May 26, 2022 1

CHAPTER ONE: INTRODUCTION TO MICROBIOLOGY
What is Microbiology?
Micro - too small to be seen with the naked eye
Bio – life
Logy - study of
Microbiology: Is a subject which deals with living organisms that
are individually too small to be seen with the naked eye.
Considers the microscopic forms of life and deals about their:
Reproduction
Physiology
 Participation in the process of nature
 Helpful and harmful relationship with other living things
and
 Significance in science and industry

Subdivision of Microbiology
 Bacteriology: The study of structure, functions and activities of bacteria
 Mycology: Is the scientific study of fungi.
 Virology: Is the scientific study of viruses.
 Immunology: Is the scientific study of body defense.
 Parasitology: Is study of parasite & their interaction of the host.
History of Microbiology
Hippocrates: Father of medicine;
Ill health resulted due to changes in air, winds, water, climate,

food, nature of soil and habits of people.
Varro: Disease was caused by animate particles invisible to naked eye but
which were carried in the air through the mouth and nose into the body.

Fracastorius
Agents of communicable disease were living germs, which are
transmitted by
• Direct contact with humans and animals
• Indirectly by objects; but no proof because of lacking
experimental evidence.
Antony Van Leeuwenhoek (1632-1723) - Father of
Microbiology
• He was the first to describe “animalcules” (single celled
organism) using simple microscope with one lens.
• He was the first who properly described the different
shapes of bacteria.
• He was not concerned about the origin of microorganism

But, many other scientists were searching for an explanation for
spontaneous appearance of living things from
Decaying meat
Stagnating ponds
Fermenting grains and
Infected wounds.
On the basis of these observations, two major theories were
formulated.
• Abiogenesis (Greek a-bio-genesis, “non biological origin”
(theory of spontaneous generation)
• Biogenesis- life comes from preexisting cells

Abiogenesis (Theory of Spontaneous Generation)
Living organisms are generated by decaying organic substances.
Example:
 Mice spontaneously appear in stored grain
 Maggots spontaneously appear in meat.
Generated by Greek philosopher- Aristotle

An experiment to disprove spontaneous generation;
 Francesco Redi (1626-1697) : Proved that no maggots appeared
in meat when flies were prevented from laying eggs.
 Used three flasks and placed meat in each of the three flasks
One of the flasks open- maggots appear
Another one tightly sealed- no maggots observed
The last one covered with gauze- maggots appear
Conclusion: abiogenesis is not acceptable theory

Biogenesis
Louis Pasteur (1822-1895 GC)
French microbiologist and chemist
Was the scientist who disproved the theory of abiogenesis
once and for all, by his experiment.
Experiment of Louis Pasteur:
Two vessels
Exposed freshly boiled broths to air in vessels contained a
filter to stop all particles passing through
The other broth was accessible to dust as the vessel was left
open

Observation
The broth in open vessel became turbid
No turbidity was observed in broth in a sealed vessel
Conclusion
Organisms that grew on broths came from outside, as spores
on dust, rather than being generated within the broth
• Microorganisms are present in air but not created by air
Refuses the concept of spontaneous generation
Strong justification for development of germ theory of
disease
Other contributions of Louis Pasteur
Concept of pasteurization (Milk)
The concept of sterilization
Development of broth for growing microorganisms
Developed the first vaccine for anthrax and rabies
The Germ Theory of Diseases
The work of a German scientist, Robert Koch (1843-1910).
Germ theory of disease: “States that specific disease is caused by
specific pathogen”
Major achievements of Robert Koch
1. Discovery and use of solid medium in bacteriology
2. Discovery of causative agents of tuberculosis, anthrax and
cholera.
3. Koch’s phenomenon
4. Koch’s postulates
Koch’s postulates: Proof of Germ Theory of Disease
A microorganism can be accepted as a causative agent of an
infectious disease only if the following conditions are satisfied.

1. The microorganism should be found in every case of the disease
and under conditions which explain the pathological changes and
clinical features
Problem: Some bacteria colonize without causing symptomatic
disease.
2. Bacteria must be isolated in pure culture.
Problem: Some bacteria cannot be cultivated.
3. Isolated bacterium must produce disease when inoculated in to
human or animal. Most difficult postulate to satisfy, for diseases
serious to use in human volunteers
4. It should be possible to re-isolate the bacterium in pure culture
from the lesion produced in the experimental animal.
 Now a days additional postulate is mentioned i.e. Specific
antibody to the bacterium should be detectable in the serum
during the course of the disease.

Generally, germ theory of disease proposes that
microorganisms are the cause of many diseases.
Exceptions to Koch’s postulate
Many healthy people carry pathogens but do not exhibit
symptoms of the disease.
Some microbes are very difficult or impossible to grow in
vitro (in the laboratory) in artificial media. Eg. Treponema
pallidum.
Certain diseases develop only when an opportunistic
pathogen invades immunocompromised host.

THE MICROBIAL WORLD
Classification of microorganism is as follows.
I. Eukaryotes
 Algae
 Protozoa
 Fungi
II. Prokaryotes
 Bacteria
EUKARYOTIC CELL
 Eu- true, Karyote- nucleus
 The eukaryotic cell has a true membrane bound
nucleus, usually containing multiple chromosomes, a
mitotic apparatus, a well defined endoplasmic
reticulum and mitochondria

PROKARYOTIC CELL
Pro- primitive
Karyote- nucleus
The prokaryotic cell possesses naked DNA without associated
basic proteins, divides amitotically by binary fission and
bounded by a semi rigid cell wall.

The Distinguishing Features Between Eukaryotic Cell And Prokaryotic Cell
Feature Prokaryotic Cell Eukaryotic Cell

Size 1µm 10µm
Nuclear membrane Absent Present
Chromosome Single Multiple
Nucleolus Absent Present
Histones Absent Present
Sexual reproduction Absent Present
Mitochondria Absent Present
Endoplasmic reticulum Absent Present
Lysosomes Absent Present
Peptidoglycan Present Absent
CM Composition Phospholipids and Protein Sterols
Nucleoid Present Absent

Bacteriology
Definition:
Bacteriology: The study of structure, functions and activities
of bacteria.
Medical Bacteriology: - The study of medically important
bacteria
Bacterial Cell
General property
Typical prokaryotic cell
Contain both DNA and RNA
Most grow in artificial media
Replication is by binary fission
Contain rigid cell wall
Sensitive to antimicrobial agent

STRUCTURE OF BACTERIA
Bacterial structure is considered at three levels.
1. Cell Envelope Proper:
 Cell wall and
 Cell membrane.
2. Cellular Elements Enclosed Within the Cell Envelope:
 Mesosomes
 Ribosomes
 Nuclear apparatus, and
 Cytoplasmic granules.
3. Cellular Elements External to the Cell Envelope:
 Flagellum
 Pillus and
 Glycocalyx

1. Cell Envelope Proper
A. Cell Wall
Multi layered structure and constitutes about 20% of the
bacterial dry weight.
Average thickness is 0.15-0.5 µm
Main structural component – Peptidoglycan
• Peptidoglycan
Repeating disaccharide units
Polypeptides

Components of cell wall of Gram negative bacteria
Peptidoglycan
Lipoprotein
Phospholipid
Lipopolysaccharide
Components of cell wall of Gram positive bacteria
Peptidoglycan(Thicker)
Teichoic acid

Functions of Cell Wall
Provides shape to the bacterium
Gives rigidity to the organism
Protects from environment
Provides staining characteristics to the bacterium
Contains receptor sites for phages
Site of action of antibody
Contains components toxic to host
B. Cell Membrane
Also named as cytoplasmic membrane
It is a delicate trilaminar unit membrane.
It accounts for 30% of the dry weight of bacterial cell.
It is composed of 60% protein, 20-30% lipids and 10-20%
carbohydrate

Function of Cell Membrane
Regulates the transport of nutrients and waste products into
and out of the cell.
Synthesis of cell wall components
Assists DNA replication
Secretes proteins
Carries on respiration
Captures energy in ATP
2. Cellular Elements Enclosed Within The Cell Envelope:
A. Mesosomes
Convoluted invagination of cytoplasmic membrane often at sites
of septum formation.
It is involved in DNA segregation during cell division and
respiratory enzyme activity.

B. Ribosomes
Cytoplasmic particles which are the sites of protein synthesis.
It is composed of RNA (70%) and proteins (30%) and constitutes
90% of the RNA and 40% of the total protein.
C. Cytoplasmic granules
Represent accumulated food reserve
D. Nuclear apparatus
Well defined nucleus and nuclear membrane, discrete
chromosome and mitotic apparatus are not present in bacteria;
So nuclear region of bacteria is named as nuclear body, nuclear
apparatus and nucleoid.
Bacterial genome consists of single molecule of double stranded
DNA arranged in a circular form.
 Bacteria may have extra chromosomal genetic material named
as plasmids, which may facilitate survival and propagation of the
microorganism, conferring virulence and drug resistance.
3. Cellular Elements External to the Cell Envelope
A. Glycocalyx (capsule and slime layer)
 Capsule is gel firmly adherent to cell envelope, composed of
polysaccharide.
 Slime is gel easily washed off from cell envelope.
 AlI bacteria have at least a thin slime layer.
Features of Capsule
1. Usually weakly antigenic.
2. Not necessary for viability.
3. Endow virulence.
4. Protects from phagocytosis.
5. Visualized by negative staining and capsule staining

B. Flagellum
It is the organ of locomotion in bacterial cell.
Size: 3-20m in length and 0.01-0.013m in diameter.
It is composed of protein named as flagellin.
The flagellar antigen in motile bacterium is named as H antigen
Consists of three parts. These are.
Filament
Hook and
Basal body
The basal body and hook are embedded in the cell surface while
the filament is free on the surface of bacterial cell.
• Their presence in bacterial cell is detected by
Hanging drop preparation
Swarming phenomenon on surface of plate agar.
Motility media.

Flagellar Arrangements
Atrichous: Bacteria with no flagellum.
Monotrichous: Bacteria with single polar flagellum.
Lophotrichous: Bacteria with bunch of flagella at one pole.
Amphitrichous: Bacteria with flagella at both poles.
Peritrichous: Bacteria with flagella all over their surface.

C. Pili (fimbriae)
It is hair like structure located on bacterial surface and important
for adherence to bacterial cell surface.
Two types
Common pili: The structure for adherence to cell surface.
Sex pili: The structure for transfer of genetic material from the
donor to the recipient during the process of conjugation.
D. Spores
Structures which are capable of surviving under adverse
environmental conditions like heat, drying, freezing, action of
toxic chemicals and radiation.
Bacterial spore is smooth walled and oval or spherical in shape.
It does not take up ordinary stains.
It looks like areas of high refractivity under light microscope.
It is significant in spread of disease and sterility of materials.

Cellular Element External to the Cell Envelope: Flagellum, Pilus
and Glycocalyx

CLASSIFICATION OF BACTERIA
Based on
A. Morphology and Arrangement
B. Staining
C. Cultural Characteristic
E. Biochemical Reaction
F. Molecular Characteristics
A. Morphology
When bacteria are visualized under light microscope, the following
morphology are seen.
1. Cocci - Round or oval bacteria measuring about 0.5-1.0µm in
diameter. They are found in single, pairs, chain or cluster
1.1 Micrococci:- Cocci occurring single.

1.2 Diplococci:- Arranged in group of two
1.3 Streptococci :- Chain forming cocci
1.4 Staphylococci:- Bunches of cocci or irregular groups or grapes of cocci
Spherical Bacteria

2. Bacilli(Rod shaped):- stick- like bacteria with rounded- tapered
square with a site measuring 1-10mm in length by 0.3 - 1.0 mm in
width.

3. Spiral shaped bacteria
 It is a regular or irregular distance between twisting
Borrelia, Treponema leprospira
Spirochete:
burgdorferi
Borrelia

B. Staining
Bacteria staining are the process of coloring of colorless object using stains
(dyes)
Uses of staining
To observe the morphology, size and arrangement of bacteria
To differentiate one group of bacteria from the other group.
Type of staining methods
Simple staining method
Differential staining method
Special staining method
1. Simple staining method
It is a type of staining method in which only a single dye is used.
Two kinds of simple stains
A.Positive staining:
 The bacteria or its parts are stained by the dye
Example: Methylene blue stain, Crystal violent stain

B. Negative staining:
 The dye stains the background and the bacteria remain unstained
Example: India ink stain
2. Differential staining method
 Multiple stains (dyes) are used to distinguish different group of bacteria
 React differently with different types of bacteria
 2 Most Common
Gram Stain
•
• Acid-Fast Stain
A. Gram stain
 Gram Stain: Named after Christian Gram who invented it
 Most bacteria are differentiated by their Gram reaction due to difference on
their cell wall structure.

A. Gram stain
Principle:
 Bacteria are stained with Crystal violet and Iodine, then subjected
to decolorization with alcohol/acetone.
Bacteria that retain the dye are: Gram-positive
Bacteria that loose the dye are: Gram-negative
Difference - due to structure of cell wall

Gram Positive---------Thick cell wall
Gram Negative--------Thin cell walls

Principle of staining technique:
1.Crystal violet: all cells are stained violet.
2. Iodine acts as a mordant (fixes the dye) all cells remain violet.
3.Alcohol acetone decolorizes gram negative cells only: Gram-
positive remains violet, gram-negative becomes colorless.
4.Counter stain with Safranin: Gram-positive remains violet while
gram negative becomes red.
Result
 Gram (+) Purple
 Gram (-) Red
Gram-Positive Gram-Negative
B. Ziehl- Neelson staining Method
Ziehl- Neelson stain (Acid fast stain) is used to stain mycobacterium
and other acid fast organisms which cannot be stained with gram
stain.
Principle- Once the mycobacterium is stained by the primary stain it
can not be decolorized with acid. So named as Acid fast bacteria
Acid - Fast Stain
1. Carbol fuchsin (Red)
2. Acid Alcohol
3. Counterstain with Methylene Blue
Acid - Fast Cells Red
Non Acid - Fast Blue
Background- Blue

3. Special Stains
A. Capsule Staining method
B. Spore staining method
C. CULTURAL CHARACTERISTIC
EXAMPLE: -Colorless white, pink and pigmented colony
-Smooth, (Mucoid) and shinny rough colony
D. Biochemical reaction
 Catalase positive/negative
 Oxidase positive/negative
 Lactose fermenter/lactose non-fermenter

BACTERIAL GENETICS
Most of the genetic information in a bacterial cell is contained with
in chromosome.
A single molecule of DNA arranged as a double in closed
loop.
Bacterial inherited characteristics are encoded in DNA. These are:
• Chromosome
• Extra chromosome (Plasmid)
Chromosome - Bacterial chromosome is circular double stranded
DNA attached to bacterial cell membrane
Plasmids: are self replication extra chromosomal DNA molecules.
 Plasmids are not essential to the life of the cell but they may have
selective advantage for these organism
R-factor (Resistance)
Extra cellular toxin
Genetic Variation in Bacteria
Genetic Variation in Bacteria can occur by
Mutation
Gene Transfer
Mutation
Bacterial mutations occur when the information in a bacterial chromosome
may alter by different means
Mutation rate in bacteria is determined by
• Accuracy of DNA replication
• Occurrence of damage to DNA
• Effectiveness of mechanisms for repair of damaged DNA
There are two types of mutation:
Spontaneous mutation
Induced mutation
Gene Transfer
The transfer of genetic information can occur by either of the three methods:
• Conjugation
• Transduction
• Transformation

Conjugation
A process where by DNA is transferred from one bacterium to another by cell to cell
physical contact (through sex pilli)
Plasmids are the genetic elements most frequently transferred by conjugation
Fig. Transfer/replication process of a conjugative plasmid.

Transduction
Is a method of gene transfer in which a virus (phage) acts as a
vehicle for carrying DNA from a donor bacterium to recipient
bacterium.

Transformation
A process by which a bacterium acquires DNA fragments or
genes from surroundings.
Usually this occurs in microbial culture.
Fig. Transformation process in bacteria

BACTERIAL NUTRITION
Bacteria, like all cells, require nutrients for the maintenance of their metabolism and for
cell division.
Bacterial structural components and the macromolecules for the metabolism are
synthesized from the elements.
The four most important elements of bacteria are carbon, hydrogen, oxygen and nitrogen.
1. Nutrition Requirement
Autotrophs: Free-living, non-parasitic bacteria which use carbon dioxide as

carbon source.
The energy needed for their metabolism can be obtained from:
Sun light - Photoautotrophs

Inorganic compounds by oxidation - Chemoautotrophs

Heterotrophs: Parasitic bacteria that require more complex organic compounds as their
source of carbon and energy.
Human pathogenic bacteria are heterotrophs.
The principal source of carbon is carbohydrate which are degraded either by oxidation,
in the presence of oxygen, or by fermentation, in the absence of oxygen, to provide
energy in the form of ATP.
Hydrogen and Oxygen
Obtained from water.
Essential for the growth and maintenance of cell.
Nitrogen
Constitutes 10% of dry weight of bacterial cell.
Obtained from organic molecules like proteins and inorganic molecules like ammonium
salts and nitrates.
NB: Main source of nitrogen is ammonia, in the form of ammonium salt.

2. Temperature Requirement
Most pathogenic bacteria grow best at an optimum temperature of

370C.
Optimum temperature is the temperature at which growth occurs

best.
Based on temperature requirement, microorganisms can be broadly

classified into psycrophylic, mesophilic, thermophilic and
hyperthermophilic

Psycrophylic-
 Are those bacteria, which grow in the range of -5 to 200C
• These bacteria include those which cause spoilages
of food at refrigeration temperature (2-8oc).
Mesophilic
 Are those bacteria, which grow at 20-450C and show
optimum growth at 37oC.
• All medically important bacteria (pathogenic bacteria) belong to this group.

Thermophilic
 Are those organisms which prefer high temperature (50-800C)
 May cause spoilage of under processed canned food
Hyperthermophilic
Those which grow at a temperature of above 800C
Some of them grow even at 2500C
Are found in hot springs, geysers and industrial heated wastes
3. Oxygen requirement
On the basis of oxygen requirement, bacteria have been divided in to:
Obligate Anaerobes -These grow only in the environment devoid of oxygen
Example: Clostridium
Facultative aerobes -These can grow under both aerobic and anaerobic
conditions. Example: Enterobacteriaceae
Obligate aerobes -These cannot grow unless oxygen is present in the
medium. Example: Pseudomonas
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Thursday, May 26, 2022
3. Oxygen Requirement
Microaerophilic- These organisms can grow under conditions with low oxygen
tension Example. Clostridium tetani.
Aerotolerant anaerobes –These bacteria oxidize nutrient substrates without using
elemental oxygen.
• Unlike obligate anaerobes, they can tolerate the presence of oxygen .
4. PH requirement
Most pathogenic bacteria require a pH of 7.2-7.6 for their optimal growth. Based on
pH requirement bacteria can be classified as
Neutrophilic:- Bacteria grow best at neutral pH (pH=7)
Most pathogenic micro-organism best grow at neutral pH (pH=7)
Acidophilic
Bacteria that grow best at acidic pH (pH<7)
Example: Lactobacilli, fungi and yeast
Alkalophilic
Bacteria that grow best at Alkaline pH (pH>7)
Example: Vibrio cholerae grow at a pH of 8.6
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BACTERIAL GROWTH
It is an orderly increase in all the components of an organism. It is an
increment in biomass.
Generation time
It is the time taken for the size of a bacterial population to double.
Time required for a cell to divide, bacteria divide into two equal daughter
cells and double the number.
The generation time varies with
The species
The amount of nutrients
The temperature
The pH and
Other environmental factors.

Bacterial growth phases
 The normal bacterial growth curve has four phases:
1. Lag Phase
2. Log Phase
3. Stationary Phase
4. Death Phase
1. Lag Phase
The period of adaptation with active macro molecular synthesis like DNA,
RNA, various enzymes and other structural components.
It is the preparation time for reproduction; no increase in cell number
2. Exponential (log) Phase

The period of active multiplication of cells.
Cell division proceeds at a logarithmic rate, and determined by the medium
and condition of the culture.

The number of bacteria during log phase growth can be calculated by the
following equation
Nt= No x 2 t/d
o Nt = is the number of bacteria after time (t)
o t/d = is the amount of time divided by the doubling time
o No = the initial number of bacteria
3. Stationary Phase
The period when the bacteria have achieved their maximal cell density or
yield.
There is no further increase in viable bacterial cell number.
The growth rate is exactly equal to the death rate.
A bacterial population may reach stationary growth when one of the
following conditions occurs:
• The required nutrients are exhausted
• Inhibitory end products are accumulated
• Physical conditions do not permit a further increase in population size

4. Decline Phase
The period at which the rate of death of bacterial cells exceeds the rate of
new cell formation.
There is drastic decline in viable cells.
Few organisms may persist for so long time at this period at the
expense of nutrients released from dying micro-organisms.
Factors influencing bacterial growth in vitro
Not all bacterial species grow under identical environmental conditions.
Each bacterial species has a specific tolerance range for specific
environmental parameters.
Outside the tolerance range, it may
Survive in dormant state or
Lose viability.

Rates of bacterial growth are greatly influenced by the
following environmental parameters:
Nutrition
Temperature
Oxygen
PH
Salinity
Pressure
Light radiation

STERILIZATION AND DISINFECTION
Sterilization: Destruction of all forms of microbial life including
spores.
Disinfection: Disinfection of microbes that cause disease; may not be
effective in killing spores
Antiseptic: Destruction or inhibition of microorganisms in living
tissue there by limiting or preventing the harmful effect of infection.
• Bactericidal vs Bacteriostatic actions of sterilization and disinfection
• Bactericidal - kills bacteria
• Bacteriostatic - inhibits bacterial growth
Sterilization and disinfecting agents are divided into two groups.
These are:
• Chemical means of sterilization and disinfection.
• Physical means of sterilization and disinfection

I. Chemical Means of Sterilization & Disinfection
These chemical agents destroy any type of microbes with out showing any form of
selectivity unlike antibiotics.
The efficacy of these agents depends on the following factors:
Concentration of the agent
Time of exposure
PH of the medium
Temperature
Nature of the organism
Presence of extraneous materials
Classification of Chemical Means of Sterilization and Disinfection
1. Chemical agents that damage the cell membrane
 Surface Active Agents
 Phenols
 Organic solvents

2. Chemical agents that denature proteins
 Acids and alkalis

3. Chemical agents that modify functional groups of proteins
and nucleic acids
 Heavy metals
 Oxidizing agents
 Dyes
 Alkylating agents
II. Physical Means of Sterilization and Disinfection
1. Heat:
The most reliable and universally applicable method of sterilization.
Mechanism of Action
 Dry heat - denatures protein.
 Moist heat - denatures and coagulates protein

1.1. Dry heat
 Incineration:
 Disposable wastes (paper cups, bags, dressings)
Red heat: heating materials until they look red
Flaming: passing materials through flame
 Inoculating Loop and Needles
Hot Air Sterilizer (Oven): It is done by applying 160 0C for 1 hour
or 180 0C for 30 minutes
Used on substances that would be damaged by moist heat
sterilization like gauzes, dressings or powders
1.2. Moist heat
Boiling - Maximum temperature is 100 0C and will there for not kill
all the spores
Tyndallization - Steaming of the material is done at 100 0C for 30
minutes on three consecutive days.
Pasteurization- It is the process of application of heat at
temperature of 620c for 30 minutes or 720c for 15 seconds
followed by rapid cooling to depress bacterial growth.
Uses:
Pasteurization of milk.
Preparation of bacterial vaccines.
Autoclave (Steam under pressure)
It is based on the principle that when microorganism is boiled at increased
pressure (in closed container), hot saturated steam will be formed which
penetrates cells of microorganisms.
Hot saturated steam in autoclaving acts as an excellent agent for sterilization
because of high temperature
- It destroys bacterial spores and vegetative cells.
Table: Time-Temperature-Pressure Level Relationship of moist
sterilization(Autoclaving)
Temperature Time Pressure Level
121ºC 15 minutes
15 lb/inch2
126ºC 10 minutes
20 lb/inch2
134ºC 3 minutes
30 lb/inch2
2. Filtration
Removes microorganisms from solutions that might be damaged

by heat
Culture media
Enzymes
Vaccines
Antibiotics

3. Radiation
1. Ionizing Radiation
Gamma rays & x-rays
• Penetrates most substances
Used on substances that could be damaged by heat like;
Plastic petri dishes
Plastic syringes
Catheters
Surgical gloves
2. Non-Ionizing Radiation
• UV Light
 Does not penetrate plastic, glass or proteinaceous matter
• Used to reduce microbial populations in;
• Hospital rooms
• Nurseries
• Operating rooms

ANTIMICROBIAL AGENTS AND DRUG
RESISTANCE MECHANISMS

Antimicrobial agent: A general term for drugs, chemicals, or other
substances that either kill or slow the growth of microbes.
Among the antimicrobial agents are antibacterial drugs, antiviral agents,
antifungal agents, and antiparasitic drugs.
Most microbiologist distinguish two groups of antimicrobial agents used
in the treatment of infectious disease:
Antibiotics: Are natural substances produced by certain groups of
microorganisms
Chemotherapeutic agents: are chemically synthesized.
Characteristics of Antibiotics
Antibiotics are low-molecular weight substances that are produced as
secondary metabolites by certain groups of microorganisms, especially
Streptomyces, Bacillus, and a few molds (Penicillium and
Cephalosporium) that are inhabitants of soils.
Antibiotics may have a cidal (killing) effect or a static (inhibitory)
effect on a range of microbes.

The range of bacteria or other microorganisms that are affected by a certain
antibiotic is expressed as its spectrum of action.
Antibiotics effective against prokaryotes which kill or inhibit a wide range of
Gram-positive and Gram-negative bacteria are said to be broad spectrum.
If effective mainly against Gram-positive or Gram-negative bacteria, they are
called narrow spectrum.
If effective against a single organism or disease, they are referred to as
limited spectrum.
A clinically-useful antibiotic should have as many of these characteristics as
possible.
It should have a wide spectrum of activity with the ability to destroy or
inhibit many different species of pathogenic organisms.
It should be nontoxic to the host and without undesirable side effects.
It should not eliminate the normal flora of the host
It should be able to reach the part of the human body where the infection is
occurring.
It should be inexpensive and easy to produce.
It should be chemically-stable (have a long shelf-life).
Microbial resistance is uncommon and unlikely to develop.

Antimicrobial Modes of Action
Antimicrobials have different modes of action like;
 Inhibition of cell wall synthesis - Penicllins, cephalosporins
 Inhibition of cell membrane function - Polymixin B and colistin
 Inhibition of protein synthesis - Aminoglycosides, macrolides
 Inhibition of nucleic acid synthesis - Quinolones, Metronidazole
Mechanisms of Antimicrobial Resistance

1. Reduced Permeability
 Gram Negative bacteria possess: LPS and Lipid A (slow drug penetration)
 Drugs diffuse through porin proteins: changes in porin copy number, size or selectivity alter
the rate of diffusion of these antibiotics
For example, Neisseria gonorrhoea to penicillin
2. Enhanced Efflux
 Efflux of the drug occurs through an export protein
 These export proteins are membrane-associated proteins which export drugs from the cell
• E.coli and other Enterobacteriaceae against tetracycline

3. Enzymatic Inactivation
Many Gram Negative and Gram Positive bacteria are resistant to chloramphenicol
due to chloramphenicol transacetylase.
Chloramphenicol acetyltransferase acetylates hydroxyl groups
Acetylated chloramphenicol binds less well to the ribosome and protein synthesis
continues normally.
4. Alteration or over expression of the drug target

Over expression of target decreases competitive inhibition
In Gram Negative, DNA gyrase seems to be the primary target for all quinolones
Mutations change the binding-site conformation resulting reduced binding affinity to

quinolones

5. Loss of enzymes involved in drug activation
In this case, the antibiotic itself is a prodrug, which has no direct activity
against the bacteria, rather it relies on the activation by a bacterial enzyme
 For example; Metronidazole is activated through nitroreductase
Thus, mutations in this enzyme cause resistance to
Metronidazole
Genetics of Antimicrobial Resistance
Resistance can be an intrinsic property of the bacteria themselves or it can be acquired
Intrinsic resistance is the innate ability of a bacteria to resist a certain drug due to absence
of target, low affinity target, low permeability, tolerance and efflux mechanisms.
Acquired bacterial antibiotic resistance can result from a mutation/gene transfer of cellular
genes.

Control of Resistance
Certain principles can help keep the drug resistance problem under
control:
Use antimicrobials conservatively and specifically in therapy.
Use an adequate dosage and duration of therapy to eliminate the infecting
organism.
Select antimicrobials according to the proved or anticipated known
susceptibility of the infecting strain whenever possible.
Use narrow-spectrum rather than broad-spectrum antimicrobials when the
specific etiology of an infection is known.
Avoid environmental contamination with antimicrobials.
Rigidly apply careful, aseptic and hand washing procedures to help prevent
spread of resistant organisms
Restrict the use of therapeutically valuable antimicrobials for nonmedical
purposes.

Host-Parasite Relationships
A symbiont either harms or lives at the expense of another organism (the
host),
For a parasitic organism, and the relationship is called parasitism.
In this relationship the body of the host can be viewed as a microenvironment
The most common forms of symbiotic relationships are;
Parasitism
Commensalism
Mutualism
Parasitism
An infectious disease is any change from a state of health in which part or all
of the host body is not capable of carrying on its normal functions due to the
presence of an organism or its products.
Any organism or agent that produces such a disease is a pathogen.
Its ability to cause disease is called pathogenicity.
A primary (frank) pathogen is any organism that causes disease in a
healthy host by a direct interaction.
Conversely, an opportunistic pathogen is an organism that is either normally free-
living, or a part of the host’s normal microbiota, but which may adopt a pathogenic
role under certain circumstances, such as when the immune system is compromised

The outcome of most host-parasite relationships is dependent on
three main factors:
The number of organisms present in or on the host
The virulence of the organism
The host’s defenses or degree of resistance.
o Usually the greater the number of organisms within a given host, the greater
the likelihood of disease.
o However, a few organisms can cause disease if they are extremely virulent
or if the host’s resistance is low.
o A host’s resistance can drop so much that its own microbiota may cause
disease.
o Such a disease is sometimes called an endogenous disease because the
agent originally comes from within the host’s own body.
o Endogenous diseases can be a serious problem among hospitalized patients
with very low resistance.

The term virulence refers to the degree or intensity of pathogenicity.
o It is determined by three characteristics of the pathogen: invasiveness,
infectivity, and pathogenic potential.
Invasiveness is the ability of the organism to spread to adjacent or other tissues.
Infectivity is the ability of the organism to establish a focal point of infection.
Pathogenic potential refers to the degree that the pathogen causes damage.
 A major aspect of pathogenic potential is toxigenicity.
Toxigenicity is the pathogen’s ability to produce toxins, chemical substances

that will damage the host and produce disease.
 Virulence is often measured experimentally by determining the lethal dose 50
(LD50) or the infectious dose 50 (ID50)

These values refer to the dose or number of pathogens that will either kill or infect,
respectively, 50% of an experimental group of hosts within a specified period.
It should be noted that disease can result from causes other than toxin production
Bacterial Toxins
i. Lipopolysaccharides of Gram-Negative Bacteria

The lipopolysaccharides (LPS, endotoxin) of gram-negative bacteria are derived from
cell walls and are often liberated when the bacteria lyse.
The substances are heat-stable, have molecular weights between 3000 and 5000
(lipooligosaccharides, LOS) and several million (lipopolysaccharides), and can be
extracted (eg, with phenol-water).

ii. Exotoxins
Many gram-positive and gram-negative bacteria produce exotoxins of
considerable medical importance.
Some of these toxins have had major roles in world history.
For example, tetanus caused by the toxin of C tetani killed as many as 50,000
soldiers of the Axis powers in World War II; the Allied forces, however,
immunized military personnel against tetanus, and very few died of that disease.
Exotoxin Endotoxin
Produced by Gram negative and Gram Produced by Gram negative
positive
Formed within the cell and released Derived from cell walls and are often
into the environment liberated when the bacteria lyse.
Heat labile Heat stable
Less potent

SYSTEMIC BACTERIOLOGY

GRAM POSITIVE COCCI
There are two most important genera of gram-positive cocci
o The Genus Staphylococcus
o The Genus Streptococcus
GENUS STAPHYLOCOCCUS
Characteristics:
• Gram positive,
• non spore-forming,
• non-motile,
• spherical cells,
• usually arranged in grape-like irregular clusters (in group)
• Single cocci,
• pairs,
• tetrads and chains are seen in liquid cultures
• Produces catalase
Contain 3 main Species of Clinical importance:
Staphylococcus aureus
Staphylococcus epidermis
Staphylococcus saprophyticus
A) Staphylococcus aureus
 The most pathogenic of the group
 Coagulase production differentiate it from the others
 They are normal flora of the skin and mucosal membrane
 S. aureus infection results from direct contamination of a wound or nosocomial
infection
 Person to person spread is possible through direct contact or exposure to contaminated
fomites.
Virulence factor:
 Peptidoglycan (Mucopeptide)
 Teichoic acid
 Protein A
 Capsule
 Catalase
 Coagulase and clumping factor
 Hyaluronidase
 Proteinases and lipases
 Staphylokinase
 -Lactamase
 Toxins

Virulence Factors of S. aureus
Enzymes:
Coagulase – Coagulates plasma and blood; produced by 97% of human isolates;
diagnostic
Hyaluronidase – Digests connective tissue
Staphylokinase – Digests blood clots
DNase – Digests DNA
Lipases – Digest oils; enhances colonization on skin
Penicillinase – Inactivates penicillin
Toxins:
Hemolysins (α, β, γ, δ) – Lyses red blood cells
Leukocidin – Lyses neutrophils and macrophages
Enterotoxin – Induce gastrointestinal distress
Exfoliative Toxin – Separates the epidermis from the dermis
Toxic Shock Syndrome Toxin (TSST) - Induces fever, vomiting, shock, systemic
organ damage

Enterotoxins
6 (A-F) soluble toxins produced by S.aureus strains
Inhibit T- cell activation thus preventing cell mediated immunity
Heat stable (Resist boiling for 30 minutes)
Resistant to gut enzymes
Responsible for food poisoning
Produced when S.aureus grows on carbohydrate & proteineous food
Plasmid may carry a protein that regulates toxin production
Epidemiology and Pathogenesis
Present in most environments frequented by humans
Readily isolated from fomites
Carriage rate for healthy adults is 20-60%.
Carriage is mostly in anterior nares, skin, nasopharynx, intestine.
Predisposition to infection include: poor hygiene and nutrition, tissue injury,
preexisting primary infection, diabetes, immunodeficiency.
Increase in community acquired methicillin resistance - MRSA

Pathogenesis
Staphylococci produce disease through:-
 Their ability to multiply & invade tissue
 Production of extracellular enzymes & toxins
Diseases produced by S. aureus

A. Focal suppuration and abscess formation:
1. Superficial Infection:
 Folliculitis - Superficial infection of hair follicle usually resolved with
no complications
 Carbuncles - Larger and deeper lesion created by aggregation and
interconnection of a cluster of furuncles
 Boils
 Abscess formation

2. Deep-seated lesions:
 Osteomyelitis
 Septic arthritis
 Endocarditis
 Meningitis
 Bronchopneumonia, etc.
3. Bacteraemia with multiple abscesses in tissues

 If S. aureus disseminates and bacteremia follows
 Endocarditis
 Acute hematogenous osteomyelitis
 Meningitis, or
 Pulmonary infection can result.

B. Toxin-mediated staphylococcal diseases
1. Food poisoning
 Results from ingestion of preformed enterotoxin in contaminated food that
is improperly cooked and kept unrefrigerated for some time.
2. Toxic Shock Syndrome (TSS):
 This is associated with TSST-1.
 TSS has an abrupt onset of fever, vomiting, diarrhoea, muscle pains, rash
 Hypotension, heart and renal failure may occur in severe cases
3. Staphylococcal scalded skin syndrome (SSSS):
 Occurs due to the exfoliative toxin that desquamates skin
 Produced by phage II strains of S. aureus.
 The syndrome occurs in babies and young children.
S. aureus is different from other Staphylococci due to;
 Coagulase positivity
 Mannitol positivity
 Beta hemolysis

B. S. epidermidis and S. saprophyticus
They are opportunistic pathogens.
They are coagulase negative
S. epidermidis is frequently associated with
• Endocarditis
• Bacteremia
• Wound infection and
• Urinary tract infection (UTI).
S. saprophyticus is now recognized as an important cause of UTI in
sexually active females
Laboratory Diagnosis:
Specimen: Surface swabs, pus, blood, sputum, cerebrospinal fluid, stool…
A. Microscopy:
 Staphylococcus species are non-motile, gram positive cocci of uniform
size that occur characteristically in groups.

B. Culture
Blood agar plates give typical colonies in 18 hours at 370C
Media containing 7.5% NaCl (mannitol salt agar) inhibits the growth of other
bacteria, but not S. aureus
C. Catalase Test
A drop of H2O2 (+) small amount of bacterial growth on a slide
Formation of bubbles (release of O2) ----- Positive test
D. Coagulase Test
Human/citrated rabbit/plasma diluted (+) equal volume of growth from colonies
from broth culture
Incubated at 370C & If clots form in 1- 4 hours=positive test

Treatment
Penicillin sensitive staphylococci
• Penicillin
• Ampicillin
Penicillin resistant staphylococci
• Cloxacillin
• Oxacillin
• Floxacillin
Methicillin resistant staphylococci
• Vancomycin
Prevention and control

Source of infection is shedding human lesions, the human respiratory tract
and skin
Contact spread of infection occur in hospitals
Treatment of nasal carriers with topical antiseptics or Rifampicin and anti-
staphylococcal drug

GENUS STREPTOCOCCUS
Properties
 Gram positive in reaction
 Forms pairs/chains during growth
 Found everywhere
 Contain normal flora & pathogenic species
 Non – motile, non – spore forming
 Catalase negative
 Produce variety of infections, ranging from pharyngitis & cellulitis to sepsis
Classification of Streptococci
 They are heterogeneous group of bacteria, and no one system sufficient to classify
them.
 Currently Streptococci can be classified based on
1. Hemolytic reactions on blood agar
 Alpha, beta - hemolysis or non – hemolysis
2. Serologic properties:
 It is designated A -H and K -V
 Clinically important are A,B,C,D,F,G

Table : Hemolytic Pattern of Streptococci
Hemolysis
Appearance Designation Example
Partial (alpha) Incomplete S. Pneumonia
α hemolytic
hemolysis (green (no group)
zone)
Complete (beta) Clear, Complete S.agalactiae (Group B)

β hemolytic
lysis of RBC S.Pyogene (Group A)
None No hemolysis Enterococci (G_D)

γ hemolytic

Lancefield Grouping of Streptococci
Streptococci produce group specific carbohydrates identified
using group specific antiserum.
It is designated A-H and K-V.
Example:
For group A streptococci: Rhamnose N - acetyl glucosamine
For group B: Rhamnose- glucosamine polysaccharide

Group A Streptococci (S. pyogenes)
Characteristics
 Gram positive cocci usually in chain
 Some strains produce capsule and pathogenic strain contain M protein (attachment
factor, antigenic and anti-phagocytic)
 The most pathogenic member of the genus
 Produces a large number of powerful enzymes and toxins.
 Present as a commensal in the nasopharynx of healthy adults, and more commonly in
children (10% carriage)
Transmission
 Person to person from droplets during coughing, on the hand of health personnel,
or from fomites like towels.

Virulence factors:
Capsule: is antiphagocytic
Lipotiechoic acid: binds to epithelial cells
M-protein: adhesion, antiphagocytic, degrade complement component C3b.
M-like protein: binds to Ig M and IgG and alpha-2 microglobulin
F-protein: mediates adherence to epithelial cells
Pyrogenic exotoxin: mediate pyrogenecity, enhancement of delayed type
Hemolysins: Two types
Hemolyze RBCs in vitro
A. Streptolysin-S: - It is non immunogenic due to its small size
- Resist Oxygen(Oxygen stable)
B. Streptolysin-O: - Oxygen labile
- Hemolytically active in a reversibly reduced state
- Antigenic
Streptokinase: Lyses blood clots (Fibrinolysin) and facilitate spread of
bacteria.

DNase: Depolymerises DNA
Hyaluronidase: Spreading Factor
 It degrades the ground substance of connective tissue (hyaluronic acid) and
aids in spreading infectious microorganisms.
Pathogenesis
 Causes disease by three main mechanisms:
1. Inflammation
 Tonsilitis
 Pharyngitis
 Cellulites-inflammation of subcutaneous loose connective tissue
 Otitis media - inflammation of middle ear, tympanum
 Impetigo - most commonly in children
2. Exotoxin and hemolysin production
 Scarlet fever and toxic shock syndrome
3. Immunogenic disorder (sequelae)
 Rheumatic fever (cross reaction of heart tissues with anti-bacterial
Antibody) (Type IV)
 Acute glomerulonephritis (Type III reaction)

Group B streptococci (S. agalactiae)
 They are commensals found in the oral cavity, intestinal tract and vagina.
 Transmission occurs from an infected mother to her infant at birth, and
venereally (propagated by sexual contact) among adults.
 The sialic acid capsule is the most important virulence factor and
inhibits the alternative complement path way.
 The pathogenesis is based on the ability of the organism to induce an
inflammatory response.
 However, unlike S. pyogenes, no enzyme or toxin and evidence for any
immunologically induced disease.
 In neonates it causes two forms of diseases
1.Early on set:
 Results from vertical transmission of the microorganism from mother to
infant in the uterus or during passage through the birth canal.
 It has high mortality as a result of neonatal sepsis or pneumonia.

2. Late onset:
Can be acquired from mothers, hospital personnel, etc.
 Meningitis is the most frequent complication of late on set.
Group B streptococci can also cause endocarditis, arthritis, osteomyelitis and sexually
transmitted diseases.
Viridans Streptococci
They are commensals of oral cavity
They don’t react with Lancefield grouping sera
There are seven species
They aren’t primarily pathogenic, but act as opportunistic challengers.
They cause sub acute bacterial endocarditis (SBE).
They usually enter the blood stream from oropharynx

Group D Streptococci
 They are commensals of the GIT
 They can be enterococci( S. faecalis) or non-enterococci (S. bovis)
Transmitted nosocomially
 Enterococci have the ability to grow in 6.5% NaCl and at PH 9.6, able to
survive at 600c for 30 min.
 Enterococci are now the third most common cause of infective endocarditis;
they also cause urinary tract infection in hospitalized patients.
Laboratory diagnosis
Specimen
S. pyogenes- Throat swab, pus, blood depending on the site of infection
S. agalactiae- vaginal swab, blood, ear swab and cerebrospinal fluid of
new born
Enterococci- Blood, pus and urine
Viridans streptococci- Blood

Microscopy
Streptococci are gram positive and non-motile
Some strains of group A streptococci and other streptococci are capsulated
In fluid media they can form long chains
Culture
Most streptococci grow aerobically and anaerobically
They can grow at a temperature range of 22-42˚C with an optimum temperature of 35-37˚C
The temperature range for enterococci is 15-45˚C
 Colonies are usually
 Less than 1mm in diameter,
 Grey white, or colorless,
 Dry or shiny and
 Irregular.

Treatment:
S.pyogenes and S.agalactiae
Penicillin and
Erythromycin
Enterococci
 Ampicillin
Viridans
Penicillin
Erythromycin and
Cotrimoxazole

S. pneumoniae (Pneumococci)
 Are fastidious lancet shaped gram positive cocci arranged in pairs(diplococci)
 They are facultative anaerobes
 They have an autolytic enzyme
 Possess a capsule of polysaccharide that permits typing with specific antisera

(more than 80 serotypes)
Epidemiology
 S. pneumoniae can be found as a commensal in the upper respiratory
tract.
 Most infections are caused by endogenous spread from the colonized

nasopharynx.
nasopharynx

Pathogenesis
Pneumococcus cause pneumonia, meningitis, endocarditis, Otitis media,
bacteremia and infection of the upper respiratory tract.
The following factors lower host resistance and predispose to pneumococcal
infection
Alcohol or drug intoxication or other cerebral impairment that can

depress the cough reflex and aspiration or secretion.
Abnormality of the respiratory tract
Abnormal respiratory dynamics( e.g. pulmonary congestion or heart

failure)
Chronic disease such as sickle cell anemia and nephrosis
Trauma to the head that causes leakage of spinal fluid through the
nose predisposes to pneumococcal meningitis.

Specimen -depending on the site of infection, specimens include
Sputum,
Ear discharge
Sinus drainage for Microscopy And Culture,
Blood for Culture And
CSF for Culture, Microscopy And Chemistry.
Microscopy
S. pneumoniae is elongated lancet shaped gram positive diplococci.
It is capsulated and non-motile

Culture
Blood agar: Following overnight incubation, pneumoniae forms
translucent or mucoid colonies, 1–2 mm in diameter.
In young cultures the colonies are raised but later become flattened with raised
edges, giving them a ringed appearance
Pneumococci show alpha-haemolysis, i.e. colonies are surrounded by an area of
partial haemolysis with a green-brown discoloration in the medium.
Quelling Reaction- Swelling of pneumococcal capsules when pneumococcal
colonies are treated with antibody in culture.
Treatment, prevention and control
 Drugs of choice are penicillin, erythromycin and cotrimoxazole
 Immunization with polyvalent polysaccharide vaccine provides long lasting
immunity (at least 5 years ).

Name Lancifield Hemolysis Habitat Important Laboratory Common and Important
Group Criteria Diseases
Streptococcus A Beta Throat, skin Large colonies (> 0.5 mm), Pharyngitis, impetigo, rheumatic
3
pyogenes PYR test positive, inhibited by fever, glomerulonephritis
bacitracin
Streptococcus B Beta Female genital Hippurate hydrolysis, CAMP- Neonatal sepsis and meningitis
agalactiae tract positive4
Enterococcus D None, Colon Growth in presence of bile, Abdominal abscess, urinary tract
faecalis alpha hydrolyze esculin, growth in infection, endocarditis
6.5% NaCl, PYR-positive
Streptococcus D None Colon Growth in presence of bile, Endocarditis, common blood
bovis hydrolyze esculin, no growth in isolate in colon cancer
6.5% NaCl, degrades starch
Viridans Usually not Alpha, Mouth, throat, Optochin-resistant. Colonies not Dental caries (S mutans),
streptococci typed or none colon, female soluble in bile. Carbohydrate endocarditis, abscesses (with many
untypable genital tract fermentation patterns other bacterial species)
Streptococcus None Alpha Throat Susceptible to optochin. Pneumonia, meningitis,
pneumoniae Colonies soluble in bile, endocarditis
quellung reaction-positive

GRAM POSITIVE RODS
Three medically important genera:
Non spore Forming
1. Genus Corynebacterium
Spore Forming
2. Genus Bacillus
3. Genus Clostridium
Genus Corynebacterium
An individual organism is small club shaped rod
They are aerobic or facultative anaerobic, non-motile, unencapsulated
and Catalase positive
They normally colonize the
o Skin
o URT
o GIT and
o Urogenital tract

Comprises the toxin-producing pathogens
C. diphtheriae
C. ulcerans and
Several commensals (diphtheroides) which normally colonize the skin, nasopharynx,
oropharynx, GIT and UGT
Corynebacterium diphtheriae
 Is an obligate aerobe found in the URT of humans and is non-motile.
 C.diphtheriae is the most important member of the group, as it can produce a powerful exotoxin that
causes diphtheria in humans
Epidemiology
Humans are the only reservoir hosts
Is found in the throat and nasopharynx of carriers and patients with diphtheria. This disease is a
local infection, usually of the throat.
Spread from person to person is by exposure to respiratory droplets or skin contact

Virulence factor
A-B toxin that inhibits protein synthesis is the major virulence factor
Pathogenesis
The toxicity observed in diphtheria is directly attributed to an exotoxin
production which is coded by the tox-gene.
C.diphtheriae causes
Diphtheria (respiratory or cutaneous)- fever, weakness, and severe inflammation of the affected
membranes.
Pharyngitis and
Endocarditis- Inflammation of endocardium
Specimen - throat or nasopharyngeal swab for microscopy and culture for throat
diphtheria and skin swab for cutaneous diphtheria.
Microscopy
• Corynebacterium species are gram positive non-motile rods
• They are non-capsulated and don’t form spores
• They are markedly pleomorphic.

…
Culture
C. diphtheriae grow aerobically.
On tellurite whole blood agar:- grey black colonies are developed.
On modified Tinsdale’s medium (which contains potassium tellurite, an
inhibitor of other respiratory flora) :-colonies will be grey black raise and
surrounded by a dark brown area.
Serology
Toxigenicity (virulence) testing of C. diphtheriae can be demonstrated by
guinea pig inoculation or by the gel electrophoresis precipitation technique
Treatment
Diphtheria anti-toxin neutralizes the exotoxin before binding to host
cell.
Penicillin and erythromycin are drugs of choice
The cornerstone of prevention of diphtheria is immunization with toxoid,
usually administered in the DPT triple vaccine together with tetanus
toxoid and pertussis antigens.
GRAM POSITIVE SPORE FORMING RODS
Genus Bacillus and Genus Clostridium
Genus Bacillus
Species of the genus Bacillus are gram-positive, form endospores, and are either
strictly or facultative aerobic.
Most of the species of Bacillus are found in soil and water, and are usually
encountered in the medical laboratory as airborne contaminants.
Bacillus anthracis and Bacillus cereus are medically important
Bacillus anthracis
Anthrax bacillus is non motile and facultative anaerobe.
It is a spore former, capsulated when cultivated outside the host
organism
Epidemiology
B .anthracis primarily infects herbivores with humans being accidental
hosts.

Anthrax affects principally domestic herbivores--sheep, goats, and horses--and is
transmitted to humans by contact with infected animal products or contaminated dust.
Infection can be acquired by inoculation, ingestion or inhalation.
B. anthracis spores may remain viable for many years in contaminated pastures, or in
bones, wool, hair, hides, or other animal materials.
These spores, like those of clostridia, are highly resistant to physical and chemical
agents.
Virulence factors
1. Capsule - Is made of a polypeptide D-glutamic acid (unique
capsule).
• It is antiphagocytic.
2. Protein exotoxin (unique toxin)- it has three parts
Protective antigen (PA) binds to host cell
 Edema factor (EF) is adenylate cyclase that catalyses the production of cAMP
 Lethal factor (LF) is cytotoxic that can kill cells

Pathogenesis and Clinical Feature
 The pathogenesis of B. anthracis is by the interaction of three parts of
the toxin. To cause disease:
 The PA will attach to host cell.
 Binding of EF to PA produces edema
 The binding of the LF to the PA produces cell death.
There are three forms of anthrax.
1. Cutaneous anthrax (Malignant pustule):
• Bacilli enter damaged skin/innoculation
• 95 % of anthrax presentation
• Characterized by a black necrotic lesion with a definite edematous margin on hands,
arms, face or neck with regional lymphadenitis associated systemic symptoms.
2. Pulmonary anthrax (Wool sorter’s disease):
 5% of anthrax presentation
 Caused by inhaling large numbers of B. anthracis spores
 Presents with substernal pain, cough;
 Fatal if not treated early.

3. Intestinal anthrax:
Due to ingesting infected meat
Patients with abdominal pain, vomiting, and bloody diarrhea
Septicaemia often develops
Rare to occur
Specimen:
Fluid or pus from skin lesion
Blood
Sputum
Smear: Capsulated gram-positive rods with centrally located spores
from culture
• Large capsulated gram-positive rods without spores from primary
specimen

Culture
Grows aerobically in ordinary media over wide range of temperature.
When grown on blood agar plates, the organisms produce non-hemolytic
gray to white mucoid colonies with a rough texture.
Comma-shaped outgrowths may project from the colony.
Positive for Nitrate reduction test, Catalase test and ferment glucose, maltose,
sucrose, with acid production but not gas
Treatment: Ciprofloxacin, Penicillin+ gentamycin or streptomycin
Prevention and Control
Disposal of animal carcasses by deep burial or burning
Decontamination (autoclaving) of animal product
 Protective clothing and gloves for handling potentially infected materials
Active immunization of domestic animals with live attenuated vaccine
Immunize high occupationally risk persons with anthrax

Bacillus cereus
Spore forming facultative anaerobe
It has heat stable enterotoxin, heat labile enterotoxin as virulence factor
B. cereus is predominantly responsible for food poisoning in humans
B. cereus is an important cause of eye infections
Cause device associated disease
Clinical Manifestation
Food poisoning caused by B. cereus (usually rice) is manifested by nausea,
vomiting, abdominal cramps, and occasionally diarrhea and is self-limiting,
with recovery occurring within 24 hours.
Lab Diagnosis
Smear from colonies show large gram positive sporulating bacilli
B. cereus, unlike B. anthracis, is motile, non-capsulated, and produces
haemolytic colonies on blood agar.
The organism is non-lactose fermenting, producing pale colonies on
MacConkey agar.

Genus Clostridium
General characteristics
 Large, gram positive
 Majority are motile, but C. perfringens is non-motile
 Anaerobic, rod shaped, spore forming (central, sub-terminal or terminal
spores)
 The remarkable capacity of clostridia to cause disease is attributed to the
following features:
o Ability to survive adverse environmental conditions through spore formation.
o Rapid growth in nutritionally enriched, O2 deprived environment.
o Production of numerous histolytic toxins, enterotoxin and neurotoxins
There are four medically important species

 C. tetani
 C. perferingens
 C. botulinum
 C. difficle

Clostridium tetani
Is a small, motile, that can produce, round terminal spores
Strict anaerobe (vegetative cells are extremely oxygen sensitive)
Epidemiology
Ubiquitous; spores are found in moist soil and can colonize GIT of humans and
animals
Risk is greater for people with inadequate vaccine induced immunity.
Virulence factors
It produces two important toxins
An oxygen labile hemolysin (tetanolysin)
Plasmid encoded heat labile neurotoxin (tetanospasmin)
- blocking the release of neurotransmitters for inhibitory
synapses

Pathogenesis and Clinical manifestation:
Infection of devitalized tissue (wound, burn, injury, umblical stamp, surgical suture) by
spores of C. tetani → Germination of the spore and development of vegetative
organism→ Neurotoxin release from vegetative cells → The toxin binds to receptors on
the presynaptic membrane of motor neuron → Inhibition of secreting neurons (glycine)
→ Spastic paralysis, muscle spasms and hyper reflexia
Incubation Period= 4-5 days to several weeks
Tetanus clinical presentation:

Lock jaw
Arched back
Arm flexion and of leg extension
Fever and sweating
Muscle spasm and rigidity
The patient is fully conscious

Laboratory diagnosis:-
Can be cultured in anaerobic atmosphere and produces a terminal
spore having the characteristic appearance of a “tennis racket”.
Culture
• When isolated (only very occasionally), C. tetani produces a fine
film of feathery growth.
• Use a hand lens to examine the plate.
• On fresh blood agar, C. tetani is haemolytic (alpha first followed
by beta haemolysis)
Treatment requires debridement of wound, antibiotic therapy,
passive immunization with antitoxin globulin (TAT) and vaccination
with tetanus toxoid.

Clostridium perfringens
Is large rectangular gram positive bacillus
Is non motile and hemolytic
Epidemiology
Ubiquitous; present in soil, water and intestinal tract of humans and animals
Virulence factors
Pathogenicity determinants of C. perfringens include:
Enzymes:: Collagenase, Proteinase, Hyaluronidase and DNAse
Toxins: Phospholipase C (alpha toxin or lecithinase)
Has lethal, necrotizing and hemolytic effect on tissue
Cause cell lysis
Enterotoxin causes food poisoning

Pathogenesis
Sub–divided in to 5 types (A-E) on the basis of toxin production (human disease
is caused by type A and C)
Grow in traumatized tissue.
Cause different types of diseases
Soft tissue infection(cellulites, suppurative myositis, gas gangrene)
Food poisoning
Septicemia
Gas gangrene is associated with:-
 Crepitation in the subcutaneous tissue and muscle
 Foul-smelling discharge
 Rapidly progressing necrosis
 Fever
 Hemolysis,
 Toxemia,
 Shock, and death.

Specimen: Infected tissue, pus
Smear: Non-motile, capsulated, large gram-positive rods in smears
from tissue; spores are rarely seen.
Nagler reaction : Used for final identification of C. perfringens
It is about toxin production and neutralization by specific antitoxin.
In medium containing lecithin, the opacity that can be produced by C.
perfringens can be inhibited by applying specific antitoxic serum to
the medium which will inactivate the lecithinase.
High dose penicillin therapy is the antibiotic of choice.
Proper wound care/debridement and prophylactic antibiotics will prevent most
infections.
Food should be adequately cooked to kill the micro-organism.

Clostridium botulinum
Spores of C. botulinum are widely distributed in soil; they often contaminate vegetables,
fruits and other materials.
Produce a neurotoxin which is the most active known poison
There are seven serotypes (A-G) of which A, B and E are the principal causes of human
illness.
Pathogenesis and Clinical manifestation:
Botulism is intoxication rather than infection
Route of entry is under cooked consumption of C. botulinum toxin
contaminated food.
These toxins block the release of the neurotransmitter acetylcholine
resulting in double vision, slurred speech, decreased saliva, difficult
swallowing and general weakness and paralysis.
Death is secondary to respiratory failure or cardiac arrest.

Diseases:
1. Food borne botulism- Associated with home canned foods and preserved fish.
2. Infant botulism- C. botulinum causes infantile botulism in which the bacteria colonize the gut of infants
and produce toxin which is absorbed.
3. Wound botulism- This occurs when spores contaminate a wound, germinate,

and produce toxin at the site.
Laboratory diagnosis:
 Demonstration of toxin in patient’s serum and left over food.
 Death of mice after intra-peritoneal injection of toxin.
Treatment:
 Administration of intravenous trivalent antitoxin (A, B, E)
 Mechanical ventilator for respiratory support.

Prevention and control:
Sufficient heating of canned foods before consumption
Strict regulation of commercial canning
Proper home canning methods
Spore germination can be prevented by
• Maintaining food in acidic pH or
• By storing at 4oc or colder.
Heating at 80oc for 20 minutes can destroy the toxin.

Clostridium difficle
 Slender bacilli - with large, oval, subterminal spores
 It is ubiquitous and can colonize the intestine of a small proportion of healthy individuals.
 Nosocomial infection
Virulence factors
Two toxin: Toxin A (Enterotoxin ), Toxin B (Extremely lethal Cytopathic toxin)

Pathogenesis and disease
Administration of antibiotics like ampicillin, clindamycin and cephalosporins results in
killing of colonic normal flora and
Proliferation of drug resistant C. difficle and release of cytotoxins
Clinically presents with pseudo-membranous colitis (sever) and manifests with fever,
abdominal cramps, antibiotic associated watery or bloody diarrhea leading to dehydration,
septicemia and shock

The implicated antibiotic should be discontinued
Treatment can be made by metronidazole and vancomycin
NB: Specimens suspected to contain clostridium species should be

labeled as “HIGH RISK”

GENUS: NEISSERIA
Characteristics:
They are non-motile, gram negative intracellular diplococci
Rapidly killed by drying, sunlight, heat, and disinfectants
Ferment carbohydrates producing acid but not gas
Each cocci is kidney-shaped with adjacent concave sides
Grow best on complex media under aerobic conditions containing 5% CO2
Oxidase positive.
The main species of medical importance are:
N. meningitidis
N. gonorrhoeae.

1. N.gonorrhoeae
Characteristics:
An obligate parasite of the human urogenital tract.
Antigenic structure: antigenically heterogeneous and capable of changing its surface
structures.
1. Pilli: Hair-like appendages extending from bacterial surface and
enhance attachment to host cells and evade human defense.
2. Por (Protein I) - Pores on the surface of bacteria through which
nutrients enter the cell.
3. Opa (Protein II) - Important for firm attachment and invasion of
bacteria to host cells.
4. RMP (Protein III): It is associated with por in the formation of
pores in the cell.
5. Lipooligosaccharide (LOS): Responsible to damage epithelial cells
 Is responsible for most of the symptoms/toxicity of gonorrhea
due to endotoxin effect of LOS.

6. Fbp(Iron binding protein): Expressed when there is limited iron
supply and directly acquire iron from human transferrin, does not
use siderophores unlike other bacteria.
7. IgA1 protease: Splits and inactivates major mucosal IgA (IgA1)
Epidemiology
Strictly a human infection
In top 5 STDs
Infectious dose 100-1,000
Does not survive more than 1-2 hours on fomites

Gonorrhea
Infection is asymptomatic in 10% of males and 50% of females.
Males – urethritis, yellowish discharge, scarring and infertility
Females – vaginitis, urethritis, salpingitis (PID) mixed anaerobic
abdominal infection, common cause of sterility and ectopic tubal
pregnancies
Extragenital infections – anal, pharygeal, conjunctivitis, septicemia,
arthritis
Route of transmission:
 Sexual contact for adults and infants during birth process.
Clinical manifestation:
Male: there is urethritis, with yellow, creamy pus and painful urination
Female: Gonococcal cervicitis. If complicated: Gonococcal ovarian abscess,
Pelvic peritonitis, Infertility.
Infant: (When delivered through the infected birth canal)- Gonococcal
ophthalmia neonatorum
 If untreated and complicated leads to blindness

Specimen: Urethral swab, cervical swab, eye swab
Smear: Gram-negative intracellular diplococci
Culture: Requires an enriched media like chocolate agar
 Thayer-Martin agar and/or Modified New York City medium.
 Grows best in carbon dioxide enriched aerobic atmosphere with optimal temperature
of 35-370c.
 Ferment carbohydrate and produce acid without gas
Biochemical reaction: Oxidase positive.
Serology: Antibodies to gonococcal pili can be detected by
immunoblotting, RIA or ELISA tests
Treatment
 Gonorrhoeae is difficult to treat because of resistance to lots of
antibiotics, especially in developing countries.
 Penicillinase-producing Neisseria gonorrhoeae (PPNG) strains are
resistant to penicillin.
 Drug of choices are third-generation cephalosporins and
Quinolones.
 Avoid multiple sexual partner
 Using mechanical protection methods (condom)
 Early diagnosis and prompt treatment of cases
 Screening of high risk population groups
 Ophthalmic ointment application of erythromycin or tetracycline to the
conjunctiva of all newborns
Neisseria meningitidis
Characteristics:
Gram-negative intracellular diplococci.
Present in the nasopharynx in 5-10% of healthy people.
Antigenic structure:
Capsular carbohydrate: It is important for serogrouping of meningo cocci and there
are 13 serogroups.
The most important serogroups associated with disease in
humans are A, B, C, Y and W135.
Lipopolysaccharide: Responsible for the toxic effects found in meningococcal disease
Pili : Allow to colonization of nasopharynx
Epidemiology and Pathogenesis

 Prevalent cause of meningitis; sporadic or epidemic
Human reservoir – nasopharynx; 3-30% of adult population; higher in institutional settings
High risk individuals are those living in close quarters, children 6months-3 years, children
and young adults 10-20 years.
Disease begins when bacteria enter bloodstream, pass into cranial circulation, and multiply
in meninges
Very rapid onset; neurological symptoms; endotoxin causes hemorrhage and shock; can be
fatal

Clinical manifestation:
Diseases caused by Neisseria meningitidis includes:
 Pyogenic (purulent) meningitis, Meningococcal bacteremia, Meningococcal
encephalitis, Pneumonia, Arthritis and endocarditis, Urethritis.
The most severe form of meningococcemia is the life threatening Water House
Friderichsen syndrome, which is characterized by high fever, shock, widespread
purpura, disseminated intravascular coagulation and adrenal insufficiency.
Laboratory Diagnosis
Gram stain CSF, blood, or nasopharyngeal sample
Culture for differentiation
Rapid tests for capsular antigen
Specimen: Cerebrospinal fluid, blood
Smear: Gram-negative intracellular diplococci
Culture: Transparent or grey, shiny, mucoid colonies in chocolate agar after
incubation at 35-37Oc in a CO2 enriched atmosphere.
Biochemical reaction: Oxidase positive

Treatment:
Penicillin
Penicillin-allergic patients are treated with third-generation cephalosporins or
chloramphenicol
Chemoprophylaxis (Rifampicin) for households or close contacts
Avoidance of over crowding
Vaccination with polyvalent conjugate vaccine to high risk groups
Rifampicin is used as prophylactic drug to reduce the carrier state
during epidemics and given to house hold and other close contacts.

GRAM NEGATIVE COCCOBACILLI
It includes:
1.Genus Haemophilus
2.Genus Bordetella
3.Genus Brucella

GENUS HAEMOPHILUS
Characteristics:
 This is a group of small gram-negative, non-spore forming, non-
motile, pleomorphic bacteria that require enriched media for
growth.
 Growth is enhanced in CO2 enriched atmosphere.
 Present in upper respiratory tract as a normal microbial flora in
healthy people.
 The group is fastidious requiring growth factors for isolation.
 The growth factors are X-factor (Hematin) and V-factor (NAD).
Growth factor required Haemophilus species
X and V factor H. influenzae, H. aegyptius, and H. hemolyticus
X factor H. ducreyii
V factor H. parainfluenzae, H. Parahemolyticus

The main species of medical importance are:
H. influenzae
H. ducreyii &
H. aegyptius.
Haemophilus influenzae
Characteristics:
Gram-negative Coccobacilli.
Fastidious bacteria requiring growth factors for isolation.
 Found in upper respiratory tract as normal flora in healthy people.
A carrier rate for H.fluenzae is as high as 80% in children, and 20 to 50% in healthy
adults.

Antigenic structure
1.Capsular polysaccharide: There are six serotypes of H.
influenzae, A-F.
Capsular antigen type b is composed of polyribose ribitol
phosphate.
H. influenzae type b is the most common cause of disease in
humans.
It is the main virulence factor which provides anti-phagocytic
property.
2. Outer membrane protein: Lipooligosaccharide
 Contribute in adhesion and invasion of host tissue
3. IgA protease - Facilitating attachment to the respiratory mucosa

Clinical features:
The bacteria causes disease most commonly in young children.
Acquisition occurs by close bodily contact, inhalation and the main
source is other children
Acute pyogenic meningitis- learning deficits
Acute epiglotitis- life threatening
Pneumonia
Otitis media – deafness
Sinusitis , Cellulitis , Acute pyogenic arthritis
• Specimen: CSF, sputum, blood, pus, synovial fluid
• Smear: Gram-negative short rods.
• Culture:
Chocolate agar (heated blood) contain both X and V factor the bacteria
grows
Blood agar contain only X factor but grows in the presence of S. aureus-
Satellitism test.

Treatment:
 Ampicillin
 Chloramphenicol
 Cotrimoxazole and
 Third generation cephalosporins

 Active immunization with conjugated PRP (polyrabitol phosphate) vaccines prevents most H.
influenzae type b infections.
 Rifampin can be used as prophylaxis
H. ducreyii
Slender, gram-negative, ovoid bacilli, slightly larger than H. influenzae.
It causes genital ulcer known as chancroid (soft chancre),
It is sexually transmitted and a common cause of genital ulceration
In addition to the painful genital lesions, inguinal lymphadenopathy may
occur.
• If untreated, this progresses to formation of a bubo (a swollen lymph node), which then suppurates
(forms pus).

 Chancroid increases the risk of infection with HIV and facilitates transmission of the virus.
 Cultured in special enriched media (20-30% rabbit blood agar) with colonic morphology of small grey
glistening colonies surrounded by zone of hemolysis.
 Needs only X factor
 It is treated by erythromycin, cotrimoxazole and third generation cephalosporins.
GENUS BORDETELLA
Characteristics:
 Minute strictly aerobic, non motile ,gram negative coccobacilli that appears singly or in pairs
 Bordetella species of medical importance: B. pertussis
Pilli: Adheres to ciliated epithelial cells of respiratory tract.
Filamentous haemagglutinin: Adheres to ciliated respiratory tract.
Pertussis toxin: Lymphocytosis promoting factor, Histamine sensitizing factor, Insulin
secretion enhancing factor, Adenyl cyclase toxin, Dermonecrotic toxin, Hemolysin

Tracheal cytotoxin:
Inhibits DNA synthesis in ciliated respiratory epithelial cells.
Lipopolysaccharide: Damages respiratory epithelial cells.
Clinical features:
 B. pertussis causes whooping cough, an infection of the mucosa of the upper
respiratory tract.
 Route of transmission is respiratory from early cases and possibly carriers.
 It has three stages: Catarrhal stage, Paroxysmal stage & Convalescence stage
• Catarrhal stage, the patient is highly infectious but not very ill manifesting with mild coughing and
sneezing (nonspecific symptoms, and then progresses to include a dry, nonproductive cough).
• Paroxysmal stage, the patient presents with explosive repetitive cough with characteristic ‘whoop’
upon inhalation leading to exhaustion, vomiting, cyanosis and convulsion. Large amounts of mucus are
typically produced.
• Convalescence stage, the patient presents with prolonged cough
 Frequency and severity of the coughing gradually decrease
 Secondary complications may occur

Specimen:
Saline nasal wash (Preferred specimen)
Nasopharyngeal swab or cough droplets on cough plate
Smear: Small, non-motile, capsulated, gram-negative Coccobacilli
singly or in pair, and may show bipolar staining.
Culture: A selective and enrichment medium such as charcoal
cephalexin blood agar is recommended for the primary isolation of
B. pertussis.
• Small, convex, smooth colonies

GENUS BRUCELLA
Characteristics:
• Gram-negative, non-motile, non-sporulating, zoonotic, obligate intracellular
aerobic Coccobacilli
• There are three major human pathogenic species
Species Primary animal host
B. abortus Cattle
B. melitensis Goat/Sheep
B. suis Swine
B. abortus cause abortion in cattle but not humans due to presence of erythritol
(growth factor of brucellae) in cattle placenta.
Natural habitats
 Animals – main reservoir
 Transmit from Animal to humans through
– Milk products
– Handling of animals
– Inhalation of aerosols

Lab. Diagnosis:
Specimen: Blood, Biopsy material (Bone marrow, Lymph nodes),
serum
Culture: Grow in blood agar, chocolate agar, or brucella agar
incubated in 10% CO2 at 35-370C for 3 wks
Biochemical reaction: Non-hemolytic, Catalase positive, Oxidase
positive and Urease positive

GRAM NEGATIVE RODS
It comprises the following bacterial groups
1. Oxidase Negative- Catalase Positive - Enterobacteriaceae
Lactose-fermenters
 Escherichia species
 Klebsiella species
 Enterobacter species &
 Citrobacter species
Non-lactose fermenters – Enteric bacteria
 Salmonella species
 Shigella species and
 Proteus species
2. Oxidase Positive
 Pseudomonas
 Vibrio
 Helicobacter &
 Campylobacter

Family Enterobacteriaceae
Introduction
Gram negative rods, found primarily in colon of humans and animals.
Many are part of the normal flora.
All are facultative anaerobes.
They have simple nutritional requirements and ferment glucose.
They are catalase positive, oxidase negative and reduce nitrates to nitrites.
Antigens
Commonly possess a wide variety of antigens which are used in serotyping,
particularly Salmonellae, Shigellae, and E.coli.
1. O antigens: found in the bacterial cell wall and are heat stable.
2. K antigens: capsular polysaccharide antigens surround the cell wall.
3. H antigens: flagellar protein antigens possessed by motile enterobacteria.
• They are heat labile (destroyed at 60-100 ºC)

Virulence factor
1.Antiphagocytic surface properties:
 Capsules
 K antigens and
 Lipopolysaccharides (LPS).
2. Adhesins:
 Fimbriae/pili ,
 Intimin (non-fimbrial adhesin)
3. Invasins:
 Hemolysin
 Siderophores and
 Siderophore uptake systems for intracellular invasion and spread.
4. Toxins:
 Heat labile(LT) toxin
 Heat stable (ST) toxin
 Shiga -like toxin
 Cytotoxins and
 Endotoxin (LPS).

Genus Escherichia: Escherichia coli
E. coli causes:
Urinary tract infections: E. coli is the commonest pathogen isolated
from patients with cystitis (bladder).
Most common cause of nosocomial UTI.
Meningitis and bacteraemia in neonates. E. coli capsular type K1 is
associated with neonatal meningitis.
Diarrhoeal disease: infantile gastroenteritis, traveller’s diarrhoea,
dysentery, and haemorrhagic diarrhoea which may progress to
haemolytic uremic syndrome.
E. coli strains associated with diarrhoeal disease
These E coli are classified by the characteristics of their virulence
properties
1. Enterotoxigenic E. coli (ETEC): Causes watery (secretory) diarrhoea due to the
production of plasmid mediated toxins in infants and adults.
Pathogenic serogroups includes O6,O8, O15,O25,O27, O63,O119,O125-O128 and
O142.

2.Enteropathogenic E. coli (EPEC): Causes vomiting, fever, and
prolonged diarrhoea mainly in infants (less than 2 year).
Due to bacteria adhering to epithelial cells and multiplying and causing
lesions.
Pathogenic serogroups includes O26,O55, O86,O111, O114, O125-
O128 and O142.
3. Enteroinvasive E. Coli (EIEC): Causes dysentery (similar to shigellosis), fever
and colitis, with blood, mucus, and many pus cells in faecal specimens.
Due to bacteria invading and multiplying in epithelial cells.
Pathogenic serogroups includes O78,O115, O148,O153, O159 and O167.
4.Enterohemorrhagic E. coli (EHEC): Causes life- threatening haemorrhagic
diarrhoea (colitis) in all ages, without pus cells, and often without fever due to
verotoxin.
It can progress to haemolytic uremic syndrome with renal failure.
EHEC is due to cytotoxins damaging vascular endothelial cells and is mainly
associated with the serogroup O157:H7.
Infection occurs by ingesting contaminated meat products, unpasteurized milk
and dairy products.

5. Enteroaggregative E. coli ( EAEC)
Causes chronic watery diarrhoea and vomiting mainly in children.
The bacteria aggregates on villi of small intestine and result in
malabsorption and excessive fluid secretion
There are more than 50 pathogenic serogroups responsible for the
infection.
Specimen: Urine, pus, blood, stool, body fluid
Smear: Gram-negative rods
Culture: Lactose-fermenting mucoid colonies on maconkey agar and
some strains are hemolytic on blood agar .
Biochemical reaction
Lactose positive (Ferment lactose)
Indole positive
Lysine decarboxylase (LDC) positive

Treatment
Trimethoprime-sulphamethoxazole
Ampicillin
Cephalosporines
Aminoglycosides and
Cefotaxime

Reducing rise of nosocomial infections such as restricting use of antibiotics and avoiding
use of urinary catheters.
Maintenance of hygienic standards to reduce gastroenteritis.
Proper cooking of beef reduces risk of EHEC infections.

1.2. Genus Salmonellae
Gram-negative, facultative rod-shaped bacteria
Motile, non-spore forming
Salmonellae live in the intestinal tracts of warm and cold blooded animals.
Some are ubiquitous but other species are specifically adapted to a particular host.
Figure . …. Antigenic structure of Salmonella typhi

Medically important species includes :
Salmonella typhi, Salmonella paratyphi, Salmonella choleriasis, Salmonella
typhimurium and Salmonella enteritidis.
Salmonella strain produce a thermolabile enterotoxin that bears a limited
relatedness to cholera toxin both structurally and antigenically.
Virulence Factors
Lipopolysaccharide(endotoxin)-released into the bloodstream
resulting in septicemia.
Invasins–proteins that mediate adherence to and penetration of
intestinal epithelial cells.
Factors involved in resistance to phagocytosis.
A. Catalase and superoxide dismutase–protect the bacteria from
intracellular killing by neutralizing oxygen radicals.
B. Defensins–small cationic proteins that prevent killing of bacteria
by phagolysosomes.

Factors involved in resistance to acid pH
• Salmonellae are protected from stomach acid and acid pH phagosome by
acid tolerance response (ATR) genes of chromosome.
Vi (virulence ) antigen – This surface antigen of Salmonella typhi has
anti-phagocytic properties.
Capsule (K antigen)– inhibit phagocytosis
Pathogenesis and clinical manifestations
The bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no
lesion), and are stopped in the mesenteric lymph nodes.
Bacterial multiplication occurs and part of the bacterial population lyses.
From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) may be released
into the bloodstream resulting in septicemia.
Release of endotoxin is responsible for cardiovascular problems.

The following diseases are caused by Salmonella:
 Salmonellosis: enteric fever (typhoid) - resulting from bacterial invasion of the
bloodstream.
 Acute gastroenteritis-resulting from a food borne infection/intoxication.
Salmonella strain produce a thermo labile enterotoxin that bears a limited relatedness to cholera
 Septicemia -is feature of enteric fever caused by Salmonella typhi and
Salmonella paratyphi toxin in blood
Specimen:
Blood
Bone Marrow
Stool
Urine
 Serum
Left over food , And
Duodenal Aspirates.

Blood -80% positive in the first week.
Stool (gastroenteritis) - 70-80% positive in the second and third week.
Urine- 20% positive in the third and fourth week.
Serum -For widal test- positive after the second week of illness.
Gram reaction - Gram-negative rods
Culture
1.Differential medium-for rapid isolation of lactose fermenter from non-
fermenter. Example: EMB agar, MacConkey agar and Deoxycholate
Citrate agar.
2. Selective medium-Favour growth of salmonella and shigella over other
Enterobacteriaceae e.g. SS agar
Widal test
The diagnostic value of the Widal test remains controversial.
Most agree that the test is not sufficiently sensitive or specific to be
clinically useful

Treatment
Ampicillin
Cephalosporin
Chloramphenicol
Plasmids mediated drug resistance is a problem of concern currently.
Prevention and Control
Personal hygiene.
Proper storage of food
Use of pasteurized milk and milk products.
Proper cooking of Vegetables and fruits
Health education
1.3. Genus Shigella
Shigellosis is an infectious disease caused by various species of Shigella.
Natural habitat: Intestinal tracts of humans and other primates.
Shigellae are slender gram-negative rods; coccobacillary forms occur in young
cultures.
Non-motile, non-spore forming, rod-shaped bacteria

Based on antigenic structure and biochemical reactions, Shigella organisms are
divided into four:
Subgroup A: Shigella dysenteriae- causes bacillary dysentery
- Serotype 1 was formerly called S. shiga
- Serotype 2 was formerly called S. schmitzii
Subgroup B: Shigella flexneri
- Contains 6 related serotypes and 4 serotypes
Subgroup C: Shigella boydii: Contains 18 distinct serotypes
Subgroup D: Shigella sonnei: Contains one serotype
Virulence factors
1. Endotoxin: irritate the bowel wall
2. Exotoxin: Enterotoxin and neurotoxin
3. S. dysenritiae type 1(shiga bacillus) produce heat labile exotoxin
(shigatoxin) mediated diarrhoea.
4. Long chain LPS - preventing the effect of serum complement.

Shigella infections are almost always limited to the gastrointestinal tract,
bloodstream invasion is quite rare.
 Shigellae are highly communicable with low infective dose (103 bacteria
enough to cause disease).
The essential pathologic process is invasion of the mucosal epithelial
cells (M cells) by induced phagocytosis, escape from the phagocytic
vacuole, multiplication and spread within the epithelial cell cytoplasm,
and passage to adjacent cells.
Result in abscess and bleeding of the intestine
Cause invasive disease similar to EIEC
 Specimen: Fresh stool or rectal swabs
 Gram reaction: Gram-negative non-motile rods.
 Culture

The following media can be used:
 MacConkey
 Eosin - methylene Blue agar
 Hektoen entric agar or Salmonella Shigella agar
Treatment
Ciprofloxacin,
Ampicillin,
Tetracycline,
Trimethoprim sulfamethoxazole and
Chloramphenicol
Prevention & control
 Sanitary control of water, food, and milk.
 Proper sewage disposal.
 Disinfection of excreta.
 Early detection and treatment of carriers.
e.g : Food handlers

Genus Klebsiella
Characteristics
Non-motile, lactose-fermenting, capsulated, gram-negative rods.
Main species of medical importance: K. pneumoniae, K. rhinoscleromatis & K.
ozenae
Klebsiella pneumoniae
It is found as a commensal in the intestinal tract, and also found in moist
environment in hospitals.
It is an important Nosocomial (hospital acquired) pathogen.
It causes: Pneumonia, Urinary tract infection, Septicemia and meningitis (especially
in neonates), Wound infection and peritonitis
Klebsiella rhinoscleromatis
• It causes rhinoscleroma of nose and pharynx to extensive destruction of
nasopharynx.
Klebsiella ozenae
• It causes ozaena manifesting with foul smelling nasal discharge leading to chronic
atrophic rhinitis.

Laboratory diagnosis of klebsiella species
• Specimen: Sputum, urine, pus, CSF, body fluid
• Smear: Gram-negative rods
• Culture: Large, mucoid, lactose-fermenting colonies on MacConkey agar
Genus Proteus
Are found in the intestinal tract of humans and animals, soil, sewage and water.
They are gram-negative, motile, non-capsulated, pleomorphic rods.
 Produce the enzyme urease and phenylalanine diaminase.
Certain species are very motile and produce a striking swarming colonies on blood
agar plate
Cell wall O antigens of certain strains of Proteus (such as OX -2, OX-19 and
OX-K) cross react with antigens of several species of rickettsia
 Species of medical importance are:
• P. mirabilis
• P. vulgaris
P. mirabilis causes:
 Urinary tract infection, Septicemia, Abdominal and wound
infection
P. vulgaris
 Isolated in wound infection and urinary tract infection.
 Important Nosocomial pathogen.
 Specimen: Urine, pus, blood, ear discharge
 Smear: Gram-negative rods
 Culture: Produce characteristic swarming colonies over the surface
of blood agar.
 Are non-lactose fermenting colonies on MacConkey agar.

GENUS PSEUDOMONAS
 Gram-negative motile strictly aerobic rods having very simple growth
requirement.
 Can be found in water, soil, sewage, vegetation, human and animal intestine.
 Species of medical importance:
- P. aeruginosa
- P. pseudomallei
Pseudomonas aeruginosa
 Found in human and animal intestine, water, soil and moist environment in
hospitals.
 Primarily a nosocomial pathogen.
 Invasive and toxigenic, produces infections in patients with abnormal host
defences.

Produces 3 diffusible pigments:
Pyocyanin – which can color the pus in a wound blue.
Fluorescein - a yellow pigment that fluoresces under ultraviolet light.
Pyorubin- red-brown pigment
Clinical features
Infections caused by P. aeruginosa include:
Skin infections
Urinary infections
Respiratory infections
External ear and eye infections
Specimen: pus, urine, sputum, blood, eye swabs, surface swabs
Smear: Gram-negative rods
Culture: Obligate aerobe, grows readily on all routine media over a wide range of
temperature (5-42OC).
Bluish-green pigmented large colonies with characteristic “fruity” or grape like odor on
culture media.

2. Genus Vibrio
The genus Vibrio consists of Gram-negative straight or curved rods, motile by means of
a single polar flagellum and facultative anaerobes.
They are distinguished from Enterics by being oxidase-positive and motile by means of
polar flagella.
Of the vibrios that are clinically significant to humans, Vibrio cholerae, the agent of
cholera, is the most important.
Found in fresh water, shellfish and other sea food.
 Man is the major reservoir of V. cholerae-O1 which causes epidemic cholera.
Readily killed by heat and drying; dies in polluted water but may survive in clean
stagnant water especially if alkaline or sea water for 1-2 weeks.

Vibiro cholerae
More than 130 different O serogroups have been described.
The classical cause of epidemic cholera possess the O1 antigen,
and it is known Vibiro cholera O1.
Vibiro cholerae O1
Virulence factors
1.The ability to produce cholera toxin.
2. Expression of toxin – co- regulated pilli
3.Colonization factor
4.Co-regulatory protein

Cholera toxin (CT)
 CT is the most important of the virulence factors produced by V. cholerae.
 CT is encoded by a lysogenic filamentous bacteriophage CTXФ which is found as a
prophage in the bacterial chromosome.
 Strains of V. cholerae that do not produce CT do not produce disease in human
volunteers
 Such strains may cause a milder form of diarrhea because of the presence of other toxins
 CT consists of a number of subunits: one A or enzymatic subunit and five identical B or
binding subunits.
 After ingestion of the V.cholerae O1, the bacteria adheres to the intestinal
wall with out invasion then produces an exotoxin causing excessive fluid
secretion and diminished fluid absorption resulting in diarrhoea (rice
water stool/stool+salt)

 CT decreases the net flow of sodium into the tissue. This produces a
net flow of chloride and water out of the tissue into the lumen causing
the massive diarrhea and electrolyte imbalance
 It is characterized by passage of voluminous watery diarrhoea (20
L/day) with out abdominal pain
Clinical features of cholera
• The presentation of cholera is diverse with many asymptomatic cases and others with
severe symptoms.
• The incubation period varies from >1 day to 5 days
• Symptoms are frequently sudden and the infection can be rapidly progressive.
• Patients may lose as much as 90% of their body weight in diarrhea over 3 to 4 days,
• This brings death in a few hours if not adequately treated

Specimen: Stool flecks
Smear: Gram-negative motile curved rods
 Motility of vibrio is best seen using dark-field microscopy.
Presumptive diagnosis: Inactivation of vibrio in a wet preparation
after adding vibrio antiserum.
Culture
• Thiosulphate citrate bile salt sucrose agar (TCBS) - selective media for
primary isolation of V.cholera
Biochemical Reaction
 Oxidase-positive.
 Ferment sucrose and maltose(acid; no gas).
 Do not ferment L-arabinose
Treatment
 Fluid and electrolyte replacement.
 Occasionally short-course antibiotic therapy, e.g. with tetracycline (but resistance is
common) or deoxycycline.

 Prevention mainly achieved by clean water and food supply.
 Use of tetracycline for prevention is effective during close contact
with infected patients.
3. Genus Campylobacter
 Campylobacters are motile, curved, oxidase-positive, thermophillic, Gram- negative rods
similar in morphology to vibrios.
 Campylobacters cause both diarrhoeal and systemic diseases and are among the most
widespread causes of infection in the world.
 The cells have one polar flagella and attached at their ends giving pairs “S” shapes or a
“seagull” appearance.
 C. jejuni, and C. coli are by far the most common
 Domestic animals such as dogs may also carry the organisms and probably play a
significant role in transmission to humans
 The most common source of human infection is undercooked poultry, but outbreaks have
been caused by contaminated rural water supplies and unpasteurized milk

Species of medical importance
Campylobacter jejuni
Campylobacter coli
Virulence factors
Lipopolysaccharides with endotoxic activity.
Cytopathic extra cellular toxins.
Enterotoxins.
Campylobacter jejuni accounts for 90 to 95% of human campylobacter
infections in most parts of the world.
Campylobacter jejuni and Campylobacter coli cause enteritis which
may take the form of toxigenic watery diarrhoea or dysentery.
The organisms are able to produce enterotoxins and cytotoxins.
In developing countries, C. jejuni and C. coli cause disease mainly in
children under 2 years.

The jejunum and ileum are the first sites to be come colonized followed
by the colon and rectum.
In well developed infections, mesenteric lymph nodes are enlarged.
Diarrhoea is due to disruption of intestinal mucosa due to cell invasion
by campylobacters and the production of toxins.
The toxin blocks the cell cycle of host cells but its precise role is not yet
clear.
• Specimen: Fresh diarrhoeal or dysenteric specimens containing blood,
pus and mucus
• Microscopy: Typical ‘S’ shaped gram-negative rods.
• Typical darting motility (swift and sudden) of the bacteria under dark
field microscopy or phase contrast microscope.

Culture
Campylobacter species are strictly microaerophilic, requiring incubation
in an atmosphere of reduced oxygen (5–10%) with added carbon dioxide
(about 10%).
C. jejuni and C. coli are thermophiles, i.e. they will grow at 42-43 ºC and 36–37 ºC
but not at 25 ºC.
Incubation at 42-43 ºC helps to identify C. jejuni and C. coli.
Blood agar: C. jejuni and C. coli produce non-haemolytic spreading,
droplet-like colonies on blood agar.
Campylobacter species are oxidase and catalase positive.
Treatment
Erythromycin or ciprofloxacin are drugs of choice for C. jejuni
enterocolitis.

1. Proper cooking of foods.
2. Avoiding contact with infected human or animals and their excreta.
3. Pasteurizing of milk and milk products.
4. No vaccine available.
4. Genus Helicobacter
 Spiral-shaped gram negative, microaerophilic, motile rods with polar
flagella.
 Nearly 20 species Helicobacter are now recognized.
 One group, the gastric Helicobacters colonize stomach and others the
enterohepatic group colonize the intestine and liver.
Species of medical importance: Helicobacter pylori

Helicobacter pylori
Infection with H. pylori (formerly Campylobacter pylori) is widespread.
Transmission is by person to person contact and probably also by
contaminated water and food.
H. pylori is thought to be the cause of most gastric and duodenal/peptic
ulcers.
In developing countries, H. pylori may also contribute to diarrhoea,
malnutrition and growth failure in young children
Virulence factors
1. Vaculating toxin (Vac A): has been associated with pore formation in
host cell membranes, the loosening of the tight junctions between
epithelial cells, thus affecting mucosal barrier permeability.
2. Cytotoxin A (Cag A): a marker of increased risk of both peptic
ulceration and gastric malignancy.

Initial colonization is facilitated by blockage of acid production by
a bacterial acid – inhibitory protein and neutralization of gastric
acids by the ammonia produced by bacterial urease activity.
The actively motile Helicobacter can then pass through gastric
mucus and adhere to the epithelial cells.
Localized tissue damage is mediated by urease by products,
mucinase, phospholipase and the activity of vaculating cytotoxin
that induce epithelial cell damage.
Diseases caused by H. pylori
1.Peptic ulceration-gastritis and hyper acidity.
2.Non -ulcer dyspepsia/disorder
3.Gastric cancer
4.Others – coronary heart disease and iron deficiency anaemia.

Specimen: Gastric biopsy and serum
Culture: Isolation of H. pylori in Skirrow’s media - translucent
colonies after 7 days
Urea Breath Test: The patient ingests 13C or 14C radio-labelled urea.
Any carbon dioxide produced by urease producing H. pylori is
detected in the breath using a mass spectrometer
Biochemical reaction: Catalase, Oxidase and Urease positive
Serology: Stool antigen test or serum antibody test
Treatment
Triple or quadruple therapy:
• Amoxicillin + clarithromycin/ metronidazole + Proton pump
inhibitors (PPI) + Omeprazole/lansoprazole OR
• Metronidazole + Bismuth subsalicylate/ Bismuth subcitrate +
Amoxicillin / Tetracycline + PPI.

Improving sanitary hygiene
Safe and clean water supply.
Proper storage of food.
No vaccine available.
GENUS MYCOBACTERIA
Characteristics:
Non-spore forming, non-motile, aerobic, Acid-fast bacilli.
Acid-fastness depends on the waxy envelope-mycolic acid of cell wall.
More resistant to chemical agents than other bacteria.
Once stained with primary stain, they resist decolorization by acid-
alcohol.
All bacteria are decolorized by acid-alcohol except Acid fast bacilli.

Members of the MTBC
• M. tuberculosis
• M. africanum
• M. canetti
• M. bovis
• M. bovis BCG vaccine variant
• M. leprae
• M. microti
• M. pinnepedii
Major pathogens are:

• Mycobacterium tuberculosis
• Mycobacterium leprae

Mycobacterium tuberculosis
Characteristics:
 Strictly aerobic acid-fast bacilli.
The main reservoir is an infected human.
Grows slowly and can be cultured in bacteriological media
Slow generation time (18-24 hours)
Antigenic structure
1. Lipids: Mycolic acid, waxes, phosphatides; responsible for acid-fastness
and granuloma formation
2. Proteins: Elicits the tuberculin reaction and antibody production.
3. Polysaccharides: Induce the immediate type of hypersensitivity.

Pathogenesis
Patients with active pulmonary tuberculosis shed large numbers of
organisms by coughing, creating aerosol droplet nuclei.
Because of resistance to desiccation, the organisms can remain
viable in the environment for a long time.
The principal mode of contagion is person-to-person transmission by
inhalation of the aerosol
• One droplet nuclei contains not more than 3 bacilli
• Coughing generates about 3000 droplet nuclei
• Talking for 5 minutes generates 3000 droplet nuclei
• Sneezing generates the most droplet nuclei by far, which can
spread to individuals up to 10 feet away
After being inhaled, mycobacteria reach the alveoli, where they multiply in the
pulmonary epithelium or macrophages.
Within two to four weeks, many bacilli are destroyed by the immune system, but
some survive and are spread by the blood to extra pulmonary sites.
The virulence of M. tuberculosis rests with its ability to survive and
grow within host cells.
The organism produces no demonstrable toxins; however, when engulfed
by macrophages, bacterial sulfolipids inhibit the fusion of phagocytic
vesicles with lysosomes.
Diseases
Primarily infection is lung
Tuberculosis meningitis and tuberculosis osteomyelitis are important
disseminated forms (extra pulmonary tuberculosis).
The disease generally manifests with
 low-grade persistent fever,
 coughing,
 night sweating,
 significant weight loss,
 fatigue and
 generalized weakness

Isolation and Identification of Mycobacteria
Unprocessed sputum 2 hours GeneXpert
Processing of Sputum or other sample MTB/RIF
Liquefaction/ NaOH-NALC
decontamination centrifugation
Sediment
1 day Smear (acid-fast microscopy)
Solid media Liquid media

colonies 7-14 days
Culture
3-6 weeks
Isolation Growth of AFB detected
•Conventional biochemical tests (4-8 weeks)

•Nucleic acid probes (2-3 hours)
Identification •Line-probe assay (5-6 hours)
•Antigen detection strip (30 minutes)
Thursday, May 26, 2022 CDC / NCID 186

Diagnosis of tuberculous infections:
Specimen: Sputum, body fluids, CSF
TB can affect every tissue in the body; thus the type of specimen for
examination varies widely.
Sputum is examined for diagnosis of PTB
Urine sample for renal tuberculosis,
CSF for meningeal tuberculosis,
Stools for intestinal tuberculosis, etc.
Treatment:
The policy in treatment of tuberculosis requires:
1. Prolonged Treatment: There is a slow response of TB treatment
and it should be continued for 6-12 months (DOTs = 6-8 months).
This is because:
a. Most bacilli are found intracellular
b. The caseous material interfere with the drug.
c. In chronic lesions TB bacilli are not dividing, i.e. "metabolically
inactive", hence resistant to drugs,
2. Combination of drugs due to:
a. The prolonged course of treatment needed, which may lead to toxicity,
and
b. The rapid emergence of resistant strains.
• Combination of drugs should be used to reduce toxicity and resistance.
• The drug of choice are isoniazide (INH), rifampicin, streptomycin, para-amino-salicylic acid &
ethambutol.
Prevention:
A.Public health measures
Early diagnosis of cases and their treatment until they become non-
infectious.
Control of infection from milk by pasteurization of milk.
B. Vaccination:
A live-attenuated vaccine – BCG commonly used.
It is prepared from bovine strain with a fixed very low virulence.
The vaccine is given in a single dose of 0.1ml containing 1-2 million organisms.
The aim is to create a controlled focus which stimulates hypersensitivity and CMI against
infection with both human & bovine types.

Characteristics:
Mycobacterium leprae
Typical acid-fast bacilli, arranged in singly, parallel bundles or in
globular masses.
Not grown in non-living bacteriologic media.
Characteristic lesions are grown in laboratory animals. Eg. Foot pads of
mice & Armadillos
Intracellular pathogens replicate within skin histocytes, endothelial
cells, and the Schwann cells of nerves.
Clinical features:
Incubation period is months to years.
Route of infection is through nasal mucus secretion.
Disease: Hansen’s disease or leprosy.
The lesion involves the cooler parts of the body example. Ear lobes.
Two major types of leprosy:
• Lepromatous leprosy &
• Tuberculoid leprosy
Comparison of the two types of leprosy
Characteristics Lepromatous leprosy Tuberculoid leprosy
Course Progressive Benign and non-progressive
Manifestation Nodular skin lesion Macular skin lesion
Involvement of nerve Slow Severe
Cell mediated immunity Weak Strong
AFB from skin lesion Abundant Scanty
Lepromin skin test usually negative Usually positive

Diagnosis:
Microscopic examination
Diagnosis depend on smears prepared from nasal scraping, ulcerated
skin nodules or material obtained by needle puncture of intact nodules.
These are stained with modified Ziehl-Neelsen in which 5% H2SO4 is
used for decolourization.
The presence of AFB in bunches intracellularly is diagnostic
Lepromin test:
This is an intradermal test similar to tuberculin.
The test is done by intradermal injection of heated exudate from lepromatous
nodules or from armadillo lesions.
A positive test indicates the presence of cell mediated immunity due to past or
recent exposure to lepra bacilli.
The test may be negative in severe cases of lepromatous leprosy.
Treatment:
• Sulphones - e.g. Dapsone (DDS) and rifampicin, are used for treatment.
• Clofazimine is given to sulphone resistant cases.
Prevention:
• No vaccine is available.
• Chemoprophylaxis may be used to contacts of case.
SPIROCHETES
Spirochetes are long, slender, motile, flexible, undulating, bacilli that
have a characteristic corkscrew or helical shape
Large heterogeneous group of spiral shaped organisms
The larger spirochetes (e.g. Borrelia spp) are gram-negative, others stain
poorly or not at all by the usual methods.
Better stained with Silver or Giemsa stains.
Genera:
3 major genera in the family spirochaetaceae are pathogenic to human:
1. Treponema causes syphilis, yaws & pinta.
2. Borrelia causes relapsing fever & Lyme disease
3. Leptospira causes leptospirosis (Weil's disease).
The genus treponema includes few pathogenic members

Treponema
Treponema pallidum is the most important & is the causative organisms
of syphilis.
Many commensal species occur in the mouth & genitalia.
Morphology:
Slender, with spiral coils regularly spaced 1μm apart
They are actively motile by means of an axial filament
The genus Treponema includes
• Treponema pallidum subspecies pallidum, which causes syphilis;
• Treponema pallidum subspecies pertenue, which causes yaws;
• Treponema pallidum subspecies endemicum, which causes endemic syphilis (also called bejel); and
• Treponema carateum, which causes pinta.

Treponema pallidum Cultural characteristics:
It has not been cultivated on artificial media.
SYPHILIS
1. Primary syphilis:
The bacteria enter the body through tiny breaks in the skin & mucous
membranes during sexual intercourse or lesion contact.
The treponemes enter the lymphatics, the regional lymph nodes become
involved, and bacteraemia occurs.
Chancre (hard type in which lymphocytes and plasma cells are
dominant) develops, at the site of inoculation, 6-12 weeks after
exposure to infection as a papule on the genitalia which ulcerates, not
painful, which heals spontaneously.

2. Secondary syphilis
This stage is characterized by the appearance of a red, maculopapular rash on
almost any part of the body, including the palms of the hands and soles of the
feet
Lesions occur 6-12 weeks after the appearance of the chancre e.g. skin rash,
condylomata of anus & vulva and mucous patches in the mouth.
T. pallidum is found in large numbers in the lesions of both primary &
secondary stages and highly infectious.
About 25% of patients are cured, 25% progress to the tertiary stage and the
rest under go latent stage.
3. Tertiary syphilis
Cellular immune response to T. pallidum & its metabolic products responsible
for the clinical cases.
CNS involvement as manifested by tabes dorsalis & paralysis.
Cardiovascular lesions causing aortic aneurism are common. Aneurism=bulge
in artery
“Tertiary stage,”characterized by the development of granulomatous lesions
(gummas)

4. Latent stage syphilis
This can last for many years.
Between secondary and tertiary
In many cases, this is followed by natural cure but in others, after
several years’, manifestation of tertiary syphilis appear
In approximately 40% of infected individuals, the disease progresses to
a tertiary stage
5. Congenital syphilis:
T. pallidum can be transmitted through the placenta to a fetus after the
first ten to fifteen weeks of pregnancy.
Infection can cause death and spontaneous abortion of the fetus or cause
it to be stillborn.

Lab Diagnosis:
I. Detection of Spirochetes in the lesion
These are examined by:-
a. Dark-ground microscope shows living motile spirochaetes with a
characteristic slow movement
b.Direct immunofluorescence using fluorescein labelled anti-
treponema antibodies.
II. Serologic diagnosis (STS): These are by using either non-
treponemal or treponemal antigens.
Non-treponemal tests: RPR, VDRL, CFT
Treponemal tests: FTAT, TPPAT, TPPHT
Treatment and prevention
 Penicillin G is curative for primary and secondary syphilis
 In cases of patient sensitivity to penicillin, alternate therapy with erythromycin or
tetracyclines may also be effective.
 There is no vaccine against T. pallidum; prevention depends on safe sexual practices

BORRELIAE
Many members of the genus are commensals.
Pathogenic Borrelia cause relapsing fever & lyme disease.
Lice or ticks become infected by feeding on patient's or rodent's blood
during the bacteraemia stage.
Morphology:
Large spirals with irregular coils. Motile
The coils are irregularly spaced 2-4um apart.
Cultural characters:
They can be cultured in the fluid media containing blood, serum or tissue
Borreliae are antigenically variable
B. recurrentis show antigenic variation in vivo

Relapsing Fever
A disease characterized by repeated attacks of fever alternating with
periods of apyrexia/afebrile state.
It is caused by B. recurrentis and its variants.
Epidemic relapsing fever is transmitted by lice while endemic RF is
transmitted by ticks. Rodents are reservoir for the organism
Borreliae multiplies in the blood of the insect & infection is transmitted
by their bite or by rubbing crushed lice or ticks into bite wounds.
After an IP of 3-10 days, there is sudden onset of fever which lasts for
about 4 days followed by afebrile period of 3-10 days when the patient
develops another attack of fever.
The organism is found in large numbers in the blood during the febrile
stage.
Antibodies against Borreliae appear during the febrile stage; these
agglutinate & destroy the organism and the attack is terminated.

Lab Diagnosis:
Blood is obtained during the rise in temperature for smear & animal
inoculation.
1.During the febrile stage: Blood films stained with leishman or Giemsa
stain reveal large numbers of loosely coiled spirochetes among red cells.
2. During the afebrile stage: The organism is scanty in the blood & blood
films are negative.
Diagnosis is done by injecting white mice with 1-2ml patient's blood.
After 2-4 days, films from tail blood are stained & examined for
presence of Borreliae.
The animal acts as in vivo enrichment medium.
Thursday, May 26, 2022 Borrelia are spiral bacteria (light microscopy (LM), 200
Giemsa stain
3. Serological diagnosis:
Complement fixation test (CFT) is used patients may have positive VDRL.
Treatment:
Penicillin, tetracyclines, erythromycin & chloramphenicol.
Jarisch Herxheimer reaction is produced following antibiotic treatment.
LYME DISEASE
• This illness is named after the town of Lyme, in USA where a number of cases were
first discovered in 1975.
• It is caused by Borrelia burgdorferi and is transmitted to man by the bite of a tick.
• Rodents & deer are the main animal reservoir.
• The disease has early & late manifestations:
• The early stages are characterized by a skin lesion called "erythema chronicum
migrans" associated with fever, chills, muscle pain & headache.
• Late manifestations which appear weeks or months later are arthritis,
myocarditis & neurologic manifestations.
Diagnosis:
• Detection of IgM antibodies by immunofluorescence or ELISA.
• Cultures are possible but difficult.

Treatment:
• Tetracyclines, penicillin & erythromycin.
• About 15% of patients develop Jarisch-Herxheimer reaction following
antibiotics.
LEPTOSPIRAE
• Two species are recognized: Leptospira biflexa & Leptospira interogans,
classified in 23 serogroups on the basis of common antigens, each
subdivided into one or more serovars (formerly called serotypes).
Habitat:
• They are found in moist environments.
• L. biflexa is a saprophyte present in pools & streams.
• L. interogans is a potential pathogen for man & animals & is harboured in
the kidneys of some rodents & domestic animals.

• Tightly coiled, thin, flexible spirochetes 5-15um long, with very fine spirals
Morphology:
0.1-0.2 um wide.
• One end of the organism is often bent, forming a hook.
• There is active rotational movement.
• It can be seen in fresh unstained preparation by the electron microscope. It can
be stained with Fontana or Giemsa stain.
Culture:
• They grow best under aerobic conditions at 28-30oC in protein-rich semisolid
media (Fletcher or Tween 80 albumin media).
• They produce round colonies 1-3mm in diameter in 6-10 days.
Pathogenesis
• L. interrogans is the cause of zoonotic disease leptospirosis.
• Human become infected by direct skin contact with the urine of infected animals or
with recently contaminated soil or water.
• The organism can enter through abrasions in skin or mucous membranes.
• Ingestion of contaminated water or food can cause human infection.
• Infection occurs mainly in sewage workers, miners& farmers.
• After an incubation period of 1-2 weeks there is leptospiraemia

Clinical Findings
1. Tissue involved: Bacteraemia occurs following inoculation, the
bacteria establish themselves in the kidneys, liver, spleen, meninges
and conjunctiva.
2. Symptoms associated with leptospirosis are usually non-specific.
Headache, fever, chills, myalgia & photophobia are common.
Jaundice & meningitis are more severe manifestations of the disease
3. Weil's disease: It is a serious form of Leptospirosis caused by L.
interogans serovar icterohaemorrhagiae. The disease is characterized
by severe jaundice and has a fatality rate about 25%.
4. Pretibial fever (also known as Fort Brags fever) is characterized
by skin rash on the front part of the legs below the knees or a
generalized rash.

Diagnosis of Leptospirosis:
Specimens:
Blood during the first week of illness and serum later for serology.
Urine during the second week of illness and continue to be excreted
intermittently after 4-6 weeks.
Microscopic examination
Dark field examination or thick smears stained by Giemsa stain
occasionally show leptospirae in fresh blood. Dark field examination
of centrifuged fresh urine may also be positive.
Culture: whole fresh blood or urine can be cultured in Flecher's
semisolid or Tween 80 albumin medium. Growth is slow, and
cultures should be kept for several weeks
Serology: ELISA shows Abs after a week

LEPTOSPIRAE
Treatment:
Penicillin is effective in large doses early in the infection. In
allergic patients streptomycin, tetracyclines or erythromycin can
be used. Penicillin may cause a temporary exacerbation of the
symptoms (Jarisch-Herxheimer reaction) but this should not
prevent continuation of treatment.
Control:
Preventing exposure to potentially contaminated water &
reducing contamination by rodent control. Doxycycline, 200mg
orally once weekly during heavy exposure, is effective
prophylaxis. Dogs can receive vaccination.

Mycoplasma
Properties of Mycoplasma
Previously considered as pleuropneumonia like organisms (PPLO)
Mycoplasma are among the smallest living MOs capable of independent
existence.
Pleomorphic bacteria occurring as cocci or long filaments which may appear
branched, 0.1-2µm in size, non-motile.
No rigid cell wall, but are composed of a small unit of cytoplasm enclosed in a
protein-lipid membrane.
The cytoplasm contains strand of DNA and ribosomes.
• Pathogenic mycoplasma require sterols for their growth and some also require
urea.
• The urea dependent mycoplasma form microscopic colonies on artificial
culture media.
• The mycoplasma that do not require urea are classified in the genus
mycoplasma?.

Normal habitat:
• Wide spread in nature being found in soil, plants and the mucous
surfaces of animals.
• In humans, M. hominis & U. urealyticum can be found in the lower
urogenital tract, mouth and throat
• M. pneumoniae can be found in the respiratory tract.
Species (main medically important)
1. Mycoplasma pneumoniae
2. Mycoplasma hominis
3. Ureaplasma urealyticum
Pathogenicity and clinical disease:
M. pneumoniae
• Causes pulmonary disease–upper respiratory illness, sore throat, ear infections,
atypical pneumonia (characterized by slow onset, fever, headache, malaise, non-
productive cough).
• Expectorated sputum is mucoid; contains neutrophils, bacterial cell are not
visible on Gram stain because very small.

Complications
• Hemolytic anemia (cold agglutinin auto- immunity), arthritis and
inflammatory muscles, skin rashes, myocarditis, pericarditis and GIT
disorders.
M. hominis
• Causes PID in women and pyelonephritis.
• Associated with spontaneous abortion & post partum fever
complications.
U. urealyticum
• One of the causes of non gonococcal urethritis in men.
• May also cause PID in women and possibly UTI

Lab diagnosis
Specimens:
• Consist of throat swab, sputum, inflammatory exudates, respiratory,
urethral, or genital secretions
Microscopic examination:
• They can not be detected directly in stained specimen preparations
(since they lack cell wall), but can be detected in preparations
following culture.
Culture:
• The material is inoculated on to:
1. PPLO agar (special solid media) supplemented with 20% serum
(as a source of cholesterol) yeast extract and DNA, incubated for 3-
10 days at 37oC with 5 % CO2 (under microaerophilic conditions),

Mycoplasma
2 - Special broth (beef heart infusion ,BHI, present in PPLO broth with serum) and
incubated aerobically at 37oC for 2-4 days and subculture on solid media.
• Colonies show the fried egg appearance embedded into the surface of the medium
and can be identified by growth inhibition using antisera & immunofluorescence.
Serology:
1. CFT performed with glycolipid Ags extracted with chloroform - methanol from
cultured mycoplasma.
2. Haemeagglutination inhibition test - applied to red cells with adsorbed
mycoplasma Ags.
3. Indirect immunofluorescence - IF
4. The test that measures growth inhibition by antibody is quite specific
Treatment
• Mycoplasma are sensitive to tetracyclines and erythromycin. Protein synthesis
inhibitor used!
• They are resistant to penicillin & cephalosporins because mycoplasma lack cell
walls and these drugs interfere with bacterial cell wall synthesis.

Chlamydiae
Chlamydiae consists of 4 species that cause a variety of disease
including genital and RT infections.
They are non-motile & form intracellular inclusions in the host cell
that can be seen by light microscope.
They reproduce in the cytoplasm of host cells and cannot survive
outside the cell due to their limited metabolic capacity.
They use ATP produced by the host cell to fuel their metabolic
reactions. energy parasite
They possess both RNA & DNA, differentiating them from viruses,
and have cell walls similar in structure to G negative bacteria.

The developmental cycle
• Chlamydiae have a unique life cycle, with morphologically distinct infectious
and reproductive forms
• The infectious and extracellular form of the bacteria is elementary body, and the
metabolically active replicating form is reticulate body (noninfectious and is
reliant on the host cell for survival).
• After attachment of the elementary body to a receptor on the host cell surface, it
enter to cells by endocytosis and alternated with reticulate body
• Morphologic change of reticulate body back to elementary body release the
infectious elementary bodies.
Species of chlamydiae
• Chlamydia trachomatis
• Chlamydia psittaci
• Chlamydia pneumoniae
Chlamydia trachomatis
• Chlamydia trachomatis is an obligate intracellular bacterium with 15 immunotypes,
as follows:

Chlamydia trachomatis
1. A- C cause trachoma endemic in Africa, Asia)
2. Serotypes L1, L2, L3
• Associated with lymphogranuloma venurum (LGV) - STI that results in
inflammation and swelling of genital lymph nodes - genital ulcer in tropical
countries.
3. Serotypes D-K: associated with genital tract infection (urethritis, cervicitis,
epididymitis, Reiter’s syndrome, proctitis, endometritis).
C. pneumoniae
Human pathogen that causes respiratory tract infection.
The genetic make-up of C. pneumoniae varies from the other 3 spp of chlamydia.
There is less than 10% similarity between C. pneumoniae and other spp
Pneumonia caused by C. pneumoniae is sometimes referred to as walking
pneumonia.
C. Psittaci
• It affects mainly birds and transmitted to humans through respiratory route and cause
psittacosis characterized like influenza

Rickettisiae
• Rickettisiae: obligate intracellular bacteria transmitted to humans by
arthropods vectors, e.g. lice, ticks & fleas, that play important roles
in their life cycles.
• They share some antigens proteins with other spp
Morphology:
• Rickettisiae are pleomorphic, G-ve, coccobacilli, better stained with
Giemsa (appear blue) & with macchiavello stain (appear red).
• They have cell walls made up of peptidoglycans resembling those of
G -ve bacteria
Cultural characters:
• Require - tissue culture cell lines or embryonated eggs
• R. quintana: exception - grow on blood agar in 10% CO2

Pathogenesis:
• Rickettsiae enter the body through the bite or feces of an infected
arthropod vector.
• They enter endothelial cells by induced phagocytosis, multiply
intracellularly & destroying their host cells.
• The bacteria invade vascular epithelial cells & become widely
disseminated.
• Human infection may occur also from inhalation of dust
Basic features of Ricketsiae and C. burnetii
• Rickettsiae & C. burnetii resemble viruses in that they are mostly
obligate parasites and are unable to survive as free living
organisms.
• They are about the size of the largest viruses & can be seen with the
light microscope.
• Unlike viruses, they contain both RNA and DNA, multiply by
binary fission, have cell walls that contain muramic acid, possess
enzymes, and show sensitivity to antiseptics & antibiotics.

Toxic properties:
• Rickettsiae contain toxins that produce death in animals within a few hours after
injection.
Disease caused by Rickettsiae:
1. Epidemic typhus:
• Occurs in epidemics & is transmitted by body louse.
• Initial symptoms of the disease are headache and fever 6-15 days after being exposed to
R. prowazekii.
• Macular rash start after 4-6 days of illness on the trunk and axillary folds & then spread
to the extremities
• The disease is fatal especially in old age.
• The Weil Felix reaction is positive with proteus OX-19.

Rickettsial Diseases
2. Brill-Zinsser disease
 Relapse of louse-borne typhus that commonly occurs many years after
the primary infection.
 R. prowazekii remains sequestrated in cells of reticuloendothelial
system.
 Antibody titre to proteus OX-19 are absent. Such individuals are
immune to a 2nd infection.
3. Murine typhus (endemic typhus):
 The causative organism is R. typhi.
 The vector for human infection is the rat flea. Human is accidental
hosts.
 The disease rambles louse-borne typhus in pathogenesis,
symptomatology & serology.

4. Tick-borne spotted fever
• The most severe is Rocky Mountain spotted fever. Causative
organism is R. rickettsii.
• The appearance of the rash on the pales & palms of the feet is
considered diagnostic.
5. Rickettsial Pox:
• Caused by R. akari, a mild infection transmitted by mites.
• The rash is first maculopapular but becomes vesicular. The rash
resembling that of Varicella.
• Patients usually recover;
• The disease is transmitted from animal to human by a rodent mite.
• The major reservoir of rickettsial pox is the house mouse
• Control of mice is best method of disease prevention

6. Scrub typhus:
• It is caused by R. tsutsugamushi, and humans become infected from
larvae of rodent mites called chiggers.
• Generalized lymphadenopathy & lymphocytosis are common. Cardiac
& central involvement may be severe.
• Antibody titers to proteins OX-K can be detected.
7. Q fever
• caused by Coxiella burnetii, an atypical rickettsial disease.
• The bacteria are resistant to dryness, survive for months outside the due to
endospore formation.
• Human disease is acquired by the respiratory route rather than by the bite of an
arthropod.
8. Trench fever
• Caused by Rochalimaa quintana, which are closely resemble Rickettsiae.
• They can grow in cell-free media, grow on blood agar in 10% CO2.
• Patients have fever, chills, headache, myalgia & is apparent when individuals are
severely stressed & infested with large numbers of body lice.
Lab diagnosis:
Microscopy:
• In tissue preparation, rickettsiae can be stained with Giemsa or
Macchiavello stains.
Culture:
• Embryonated egg inoculation techniques are used.
Serology:
• Most rickettsial disease are usually diagnosed serologically -
specific IgM Abs are produced followed in the later stages by an
IgG response.
• The IgG persist in the serum for several years.
• Immunity against louse-borne typhus and spotted fever lasts for
about one year after infection.

1. The most reliable and useful serological method for diagnosing
rickettsial infection is the indirect fluorescent antibody test.
2. CFT with Rickettisial Ags like the IFA has the advantage that it is
often positive at the end of the 1st wk of infection
3 - ELISA tests have been performed with rickettsial Ags.
4 - Agglutination of Rickettsiae: Rickettsiae are agglutinated by
specific antibodies.
5 -Latex agglutination test: Latex particles adsorb soluble
rickettsial Ags and can then be agglutinated by Ab
6 - Weil- Felix reaction:
During rickettsial infection, pts develop Abs that agglutinate certain
strains of P. vulgaris. Eg:
 Proteus strain OX19 - agglutinated strongly (1: 500 or more) by sera from persons
infected with epidemic or murine typhus,
 Weakly by sera from those infected with RMSF
 Not at all by those infected with Q fever.

• False positive reactions may also occur in proteus infections, relapsing fever, brucellosis,
rat bite fever/tularwmia, infectious monoucleosis, and other acute febrile illness
Antibiotic therapy:
• The most commonly used are tetracycline, erythromycin and
chloramphenicol.
Control:
• Control depending on breaking the infection chain (avoiding vectors),
treating patients with antibiotics, and immunizing when possible

Prevention
1. Prevention of transmission by breaking the chain of infections.
a. Epidemic typhus: Delousing with insecticide.
b. Murine typhus: Rat- proofing building and using rat poisons-to
avoid rat flea.
c. Scrub typhus: Clearing from campsites the secondary jungle
vegetation in which rats and mites live.
d. Spotted fever: clearing of infested land, personnel prophylaxis in
the form of protective clothing such as high boots, socks worn over
trousers; tick repellents; and frequent removal of attached ticks.
e. Rickettsial pox: Elimination of rodents and their parasites from
human houses.

2. Prevention of transmission of Q fever:
• By adequate pasteurization of milk. "High temp, short time"
pasteurization at 71.5oC for 15 sec are adequate to destroy viable
coxiella.
3. Prevention by vaccination:
• Active immunization has been used with formalinized antigens
prepared from the yolk sacs of infected chick embryos or from
cell cultures.
• These vaccines have been prepared for:
• Epidemic typhus (R. prowazekii)
• Rocky Mountain (R. rickettsi)
• Q fever (Coxiella burnetti)

Medical Bacteriology: Thursday, May 26, 2022

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MEDICAL BACTERIOLOGY

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Ill health resulted due to changes in air, winds, water, climate,

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Living organisms are generated by decaying organic substances.

Generated by Greek philosopher- Aristotle

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Feature Prokaryotic Cell Eukaryotic Cell

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Example: Methylene blue stain, Crystal violent stain

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Bacteria that retain the dye are: Gram-positive

Bacteria that loose the dye are: Gram-negative

Difference - due to structure of cell wall

Gram Negative--------Thin cell walls

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Fig. Transfer/replication process of a conjugative plasmid.

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Fig. Transformation process in bacteria

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Autotrophs: Free-living, non-parasitic bacteria which use carbon dioxide as

Sun light - Photoautotrophs

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Most pathogenic bacteria grow best at an optimum temperature of

Optimum temperature is the temperature at which growth occurs

Based on temperature requirement, microorganisms can be broadly

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2. Exponential (log) Phase

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