List of contents Microbiology

NO. Subject Page No.

Introduction

Section I – Microbiology

1. Sterilization and Disinfection

2. Selection of antibiotics

3. Gram-positive bacilli Aerobic non-spore forming bacilli

4. Gram positive cocci Genus Staphylococci

5. Genus Streptococcus

6. Genus campylobacter

7. Haemophilus

8. Sexually transmitted diseases (STD) Genus Neisseria

9. Legionella

10. Bordetella

11. (Part – one )Anaerobic Bacteriology

(Part – Two) Spore-forming gram-positive Bacilli: Bacillus and

Clostridium Species

12. Spore-forming gram-positive Bacilli: Bacillus and Clostridium

Species

13. Mycobacterium

14. Central Nervous System Diagnostic Microbiology

15. Gastrointestinal Tract Diagnostic Microbiology

16. Diagnostic Microbiology /Urinary tract infections (UTIs)

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 List of contents Microbiology

17. Eye Diagnostic Microbiology

Section II - Virology

18. General structure and classification of viruses

19. Virological Tests

20. Herpesviruses

21. paramyxoviruse virus

22. Hepatitis B Virus

23. Enteroviruses

24. Adenoviruses

25. Human Immunodeficiency Virus

26. Human Cancer Viruses

27. What is a vaccine

Section III – Parasitology

Section IV - Medical Mycology

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Section I– Microbiology Introductory By Dr. Mohammed Ayad

Introductory Lecture-Part One

Microorganisms can be found in every ecosystem and in close association with every type of multicellular

organism.

They populate the healthy human body by the billions as benign passengers (normal flora) and even as

participants in bodily functions. Those relatively few species of microorganisms that are harmful to

humans, either by production of toxic compounds or by direct infection, are characterized as pathogens.

Most infectious disease is initiated by colonization (the establishment of proliferating microorganisms on

the skin or mucous membranes). The major exceptions are diseases caused by introduction of organisms

directly into the bloodstream or internal organs.

Microbial colonization may result in:

1- elimination of the microorganism without affecting the host

2- infection in which the organisms multiply and cause the host to react by making an immune or other

type of response

3- Transient or prolonged carrier state.

Infectious disease occurs when the organism causes tissue damage and impairment of body function.

All prokaryotic organisms are classified as bacteria, whereas eukaryotic organisms include fungi,

protozoa, and helminths as well as humans.

Prokaryotic organisms are divided into two major groups: the eubacteria, which include all bacteria of

medical importance, and the archaebacteria, a collection of evolutionarily distinct organisms.

Most bacteria have shapes that can be described as a rod, sphere, or corkscrew. Prokaryotic cells are

smaller than eukaryotic cells. Nearly all bacteria, with the exception of the Mycoplasma, have a rigid cell

wall surrounding the cell membrane that determines the shape of the organism.

The cell wall also determines whether the bacterium is classified as gram positive or gram negative.

External to the cell wall may be flagella, pili, capsule.

Bacterial cells divide by binary fission. However, many bacteria exchange genetic information carried on

plasmids (small, specialized genetic elements capable of self-replication) including the information

necessary for establishment of antibiotic resistance.

Atypical bacteria include groups of organisms such as Mycoplasma, Chlamydia, and Rickettsiae that,

although prokaryotic, lack significant characteristic structural components or metabolic capabilities that

separate them from the larger group of typical bacteria.

Fungi are nonphotosynthetic, generally saprophytic, eukaryotic organisms. Some fungi are filamentous

and are commonly called molds, whereas others (that is, the yeasts) are unicellular.

Fungal reproduction may be asexual, sexual, or both, and all fungi produce spores. Pathogenic fungi can

cause diseases, ranging from skin infections (superficial mycoses) to serious, systemic infections (deep

mycoses).

Protozoa are single-celled, nonphotosynthetic, eukaryotic organisms that come in various shapes and

sizes. Many protozoa are free living, but others are among the most clinically important parasites of

humans. Members of this group infect all major tissues and organs of the body.

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They can be intracellular parasites, or extracellular parasites in the blood, urogenital region, or intestine.

Transmission is generally by ingestion of an infective stage of the parasite or by insect bite.

Helminths are groups of worms that live as parasites. They are multicellular, eukaryotic organisms with

complex body organization. They are divided into three main groups: tapeworms (Cestodes), flukes

(Trematodes), and roundworms (Nematodes).

Helminths are parasitic, receiving nutrients by ingesting or absorbing digestive contents or ingesting or

absorbing body fluids or tissues. Almost any organ in the body can be parasitized.

Viruses are obligate intracellular parasites that do not have a cellular structure. Rather, a virus consists of

molecule(s) of DNA (DNA virus) or RNA (RNA virus), but not both, surrounded by a protein coat.

A virus may also have an envelope derived from the plasma membrane of the host cell from which the

virus is released. Viruses contain the genetic information necessary for directing their own replication but

require the host’s cellular structures and enzymatic machinery to complete the process of their own

reproduction.

The fate of the host cell following viral infection ranges from rapid lysis and release of many progeny

virions to gradual prolonged release of viral particles.

The human body is continuously inhabited by many different microorganisms (mostly bacteria, but also

fungi and other microorganisms), which, under normal circumstances in a healthy individual, are harmless

and may even be beneficial.

These microorganisms are termed “normal flora.” The normal flora is also termed commensals, which

literally means “organisms that dine together.” Except for occasional transient invaders, the internal

organs and systems are sterile, including the spleen, pancreas, liver, bladder, central nervous system, and

blood.

A healthy newborn enters the world in essentially sterile condition, but, after birth, it rapidly acquires

normal flora from food and the environment, including from other humans.

The human microbiome is the total number and diversity of microbes found in and on the human body.

In the past, the ability to cultivate organisms from tissues and clinical samples was the gold standard for

identification of normal flora and bacterial pathogens.

However, the recent application of culture-independent molecular detection methods based on DNA

sequencing indicates that the human body contains a far greater bacterial diversity than previously

recognized.

Unlike classic microbiologic culture methods, molecular detection requires neither prior knowledge of an

organism nor the ability to culture it. Thus, molecular methods are capable of detecting fastidious and

nonculturable species. Even using advanced molecular techniques, it is difficult to define the human

microbiome because microbial species present vary from individual to individual as a result of

physiologic differences, diet, age, and geographic habitat. Despite these limitations, it is useful to be

aware of the dominant types and distribution of resident flora, because such knowledge provides an

understanding of the possible infections that result from injury to a particular body site.

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Microbial Flora

The most common sites of the body inhabited by normal flora are, as might be expected, those in contact

or communication with the outside world, namely, the skin, eye, and mouth as well as the upper

respiratory, gastrointestinal, and urogenital tracts.

I- Skin can acquire any bacteria that happen to be in the immediate environment, but this transient

flora either dies or is removable by washing. Nevertheless, the skin supports a permanent bacterial

population (resident flora), residing in multiple layers of the skin. The resident flora regenerates even

after vigorous scrubbing.

Estimate of the skin microbiome using classical culture techniques: Staphylococcus epidermidis and other

coagulase-negative staphylococci (CON) that reside in the outer layers of the skin appear to account for

some 90 % of the skin aerobes. Anaerobic organisms, such as Propionibacterium acnes, reside in deeper

skin layers, hair follicles, and sweat and sebaceous glands.

Skin inhabitants are generally harmless, although S. epidermidis can attach to and colonize plastic

catheters and medical devices that penetrate the skin, sometimes resulting in serious bloodstream

infections.

While estimate of the skin microbiome using molecular sequencing techniques: The estimate of the

number of species present on skin bacteria has been radically changed by the use of the 16S ribosomal

RNA gene sequence to identify bacterial species present on skin samples directly from their genetic

material.

Previously, such identification had depended upon microbiological culture, upon which many varieties of

bacteria did not grow and so were not detected. Staphylococcus epidermidis and Staphylococcus aureus

were thought from culture-based research to be dominant. However DNA analysis research finds that,

while common, these species make up only 5 % of skin bacteria. The skin apparently provides a rich and

diverse habitat for bacteria.

II- Eye

The conjunctiva of the eye is colonized primarily by S. epidermidis, followed by S. aureus, aerobic

Corynebacteria (diphtheroids), and Streptococcus pneumoniae. Tears, which contain the antimicrobial

enzyme lysozyme, help, limit the bacterial population of the conjunctiva.

III- Buccal cavity and Nasal passages

The mouth and nose harbor many microorganisms, both aerobic and anaerobic. Among the most common

are diphtheroids (aerobic Corynebacterium species), S. aureus, and S. epidermidis. In addition, the teeth

and surrounding gingival tissue are colonized by their own particular species, such as Streptococcus

mutans.

S. mutans can enter the bloodstream following dental surgery and colonize damaged or prosthetic heart

valves, leading to potentially fatal infective endocarditis.

Some normal residents of the nasopharynx can also cause disease like S. pneumoniae, found in the

nasopharynx of many healthy individuals, can cause acute bacterial pneumonia, especially in older adults

and those whose resistance is impaired ((Pneumonia is frequently preceded by an upper or middle

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respiratory viral infection, which predisposes the individual to S. pneumoniae infection of the

pulmonary parenchyma))

IV- Gastrointestinal tract

In an adult, the density of microorganisms in the stomach is relatively low (103

to 105

per gram of

contents) due to gastric enzymes and acidic pH.

The density of organisms increases along the alimentary canal, reaching 108

to 1010 bacteria per gram of

contents in the ileum and 1011 per gram of contents in the large intestine.

Some 20 % of the fecal mass consists of many different species of bacteria, more than 99 % of which are

anaerobes. Bacteroides species constitute a significant percentage of bacteria in the large intestine.

Escherichia coli, a facultative anaerobic organism, constitutes less than 0.1 % of the total population of

bacteria in the intestinal tract. However, this endogenous E. coli is a major cause of urinary tract

infections (UTIs).

V- Urogenital tract

The low pH of the adult vagina is maintained by the presence of Lactobacillus species, which are the

primary components of normal flora. If the Lactobacillus population in the vagina is decreased (for

example, by antibiotic therapy), the pH rises, and potential pathogens can overgrow.

The most common example of such overgrowth is the yeast-like fungus, Candida albicans which itself is

a minor member of the normal flora of the vagina, mouth, and small intestine. The urine in the kidney

and bladder is sterile but can become contaminated in the lower urethra by the same organisms that

inhabit the outer layer of the skin and perineum.

Normal flora benefits

Normal flora can provide some definite benefits to the host:

First, the sheer number of harmless bacteria in the lower bowel and mouth make it unlikely that, in a

healthy person, an invading pathogen could compete for nutrients and receptor sites.

Second, some bacteria of the bowel produce antimicrobial substances to which the producers

themselves are not susceptible.

Third, bacterial colonization of a newborn infant acts as a powerful stimulus for the development of

the immune system.

Fourth, bacteria of the gut provide important nutrients, such as vitamin K, and aid in digestion and

absorption of nutrients; although humans can obtain vitamin K from food sources, bacteria can be an

important supplemental source if nutrition is impaired.

Microbial flora problems

Clinical problems caused by normal flora arise in the following ways:

The organisms are displaced from their normal site in the body to an abnormal site. An example is

the introduction of the normal skin bacterium, S. epidermidis, into the bloodstream where it can colonize

catheters and heart valves, resulting in bacterial endocarditis.

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Potential pathogens gain a competitive advantage due to diminished populations of harmless

competitors; when normal bowel flora are depleted by antibiotic therapy leading to overgrowth by the

resistant Clostridium difficille , which can cause severe colitis.

Harmless, commonly ingested food substances are converted into carcinogenic derivatives by

bacteria in the colon; like the conversion by bacterial sulfatases of the sweetener cyclamate into the

bladder carcinogen cyclohexamine.

When individuals are immunocompromised, normal flora can overgrow and become pathogenic

((Colonization by normal, but potentially harmful, flora should be distinguished from the carrier state in

which a true pathogen is carried by a healthy (asymptomatic) individual and passed to other individuals

where it results in disease)). Typhoid fever is an example of a disease that can be acquired from a

carrier

Microorganism’s pathogenesis

A pathogenic microorganism is defined as one that is capable of causing disease. Some microorganisms

are unequivocally pathogenic, whereas others (the majority) are generally harmless.

An organism may invade an individual without causing infectious disease when the host’s defense

mechanisms are successful.

The occurrence of such asymptomatic infections can be recognized by the presence of antibody against

the organism in the patient’s serum. Some infections result in a latent state, meaning that the organism is

dormant but may be reactivated with the recurrence of symptoms. Moreover, some pathogens cause

disease only under certain conditions (for example, being introduced into a normally sterile body site or

infection of an immunocompromised host).

Bacterial mediated pathogenesis

The mechanism of infectious process may vary among bacteria, the methods by which bacteria cause

disease can, in general, be divided into several stages. Pathogenicity of a microorganism depends on its

success in completing some or all of these stages. The terms “virulence” and “pathogenicity” are often

used interchangeably. However, virulence can be quantified by how many organisms are required to cause

disease in 50 percent of those exposed to the pathogen (ID50, where I = Infectious and D = Dose), or to

kill 50 percent of test animals (LD50, where L = Lethal).

The number of organisms required to cause disease varies greatly among pathogenic bacteria, as less than

100 Shigella cause diarrhea by infecting the gastrointestinal (GI) tract, whereas the infectious dose of

Salmonella is approximately 100,000 organisms.

The infectious dose of a bacterium depends primarily on its virulence factors. The probability that an

infectious disease occurs is influenced by both the number and virulence of the infecting organisms and

the strength of the host immune response opposing infection.

Virulence factors are those characteristics of a bacterium that enhance its pathogenicity, that is, properties

that enable a microorganism to establish itself and replicate on or within a specific host; they are as

follows:

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1. Entry into the host

The first step of the infectious process is the entry of the microorganism into the host by one of several

ports: by the respiratory, GI, or urogenital tract or through skin that has been cut, punctured, or burned.

Once entry is achieved, the pathogen must overcome diverse host defenses before it can establish itself.

These include phagocytosis; the acidic environments of the stomach and urogenital tract; and various

hydrolytic and proteolytic enzymes found in the saliva, stomach, and small intestine.

Bacteria that have an outer polysaccharide capsule (for example, Streptococcus pneumoniae and

Neisseria meningitidis) have a better chance of surviving these primary host defenses.

2. Adherence to host cells

Some bacteria (e.g. Escherichia coli) use pili to adhere to the surface of host cells. Group A Streptococci

have similar structures (fimbriae). Other bacteria have cell surface adhesion molecules or particularly

hydrophobic cell walls that allow them to adhere to the host cell membrane.

The adherence enhances virulence by preventing the bacteria from being carried away by mucus or

washed from organs with significant fluid flow, such as the urinary and the GI tracts. Adherence also

allows each attached bacterial cell to form a microcolony. An example of the importance of adhesion is

that of Neisseria gonorrhoeae in which strains that lack pili are not pathogenic.

3. Invasiveness

Invasive bacteria are those that can enter host cells or penetrate mucosal surfaces, spreading from the

initial site of infection. Invasiveness is facilitated by several bacterial enzymes, the most notable of which

are collagenase and hyaluronidase. These enzymes degrade components of the extracellular matrix,

providing the bacteria with easier access to host cell surfaces.

Many bacterial pathogens express membrane proteins known as "invasins" that interact with host cell

receptors, thereby eliciting signaling cascades that result in bacterial uptake by induced phagocytosis.

Invasion is followed by inflammation, which can be either pyogenic (involving pus formation) or

granulomatous (having nodular inflammatory lesions), depending on the organism.

The pus of pyogenic inflammations contains mostly neutrophils, whereas granulomatous lesions contain

fibroblasts, lymphocytes, and macrophages.

4. Iron sequestering

Iron is an essential nutrient for most bacteria. To obtain the iron required for growth, bacteria produce

iron-binding compounds, called siderophores. These compounds capture iron from the host by chelation,

and then the ferrated siderophores binds to specific receptors on the bacterial surface. Iron is actively

transported into the bacterium, where it is incorporated into essential compounds such as cytochromes.

The pathogenic Neisseria species are exceptions in that they do not produce siderophores but instead

utilize host iron-binding proteins, such as transferrin and lactoferrin, as iron sources.

Virulence factors that inhibit phagocytosis

1. The most important antiphagocytic structure is the capsule external to the cell wall, such as in S.

pneumoniae and N. meningitidis.

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2. The second group of antiphagocytic factors is the cell wall proteins of gram-positive cocci, such as

protein A of Staphylococcus and M protein of group A Streptococci.

5- Bacterial toxins

Some bacteria cause disease by producing toxic substances, of which there are two general types:

exotoxins and endotoxin. Exotoxins, which are proteins, are secreted by both gram-positive and gramnegative bacteria.

In contrast, endotoxin, which is lipopolysaccharide (LPS), is not secreted but instead is an integral

component of the cell walls of gram-negative bacteria.

a. Exotoxins: These include some of the most poisonous substances known. It is estimated that as little

as 1 μg of tetanus exotoxin can kill an adult human. Exotoxin proteins generally have two polypeptide

components. One is responsible for binding the protein to the host cell, and one is responsible for the toxic

effect.

In several cases, the precise target for the toxin has been identified. For example, diphtheria toxin is an

enzyme that blocks protein synthesis. It does so by attaching an adenosine diphosphate–ribosyl group to

human protein elongation factor EF-2, thereby inactivating it. Most exotoxins are rapidly inactivated by

moderate heating (60o C), notable exceptions being Staphylococcal enterotoxin and E. coli heat-stable

toxin (ST).

Also, treatment with dilute formaldehyde destroys the toxic activity of most exotoxins but does not affect

their antigenicity.