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Section I– Microbiology Introductory By Dr. Mohammed Ayad

Host mediated pathogenesis

The pathogenesis of many bacterial infections is caused by the host response rather than by bacterial

factors. Examples of host response–mediated pathogenesis are seen in diseases such as gram-negative

bacteria sepsis, tuberculosis, and tuberculoid leprosy.

The tissue damage in these infections is caused by various cytokines released from the lymphocytes,

macrophages, and polymorphonuclear leukocytes at the site of infection or in the bloodstream. Often the

host response is so intense that host tissues are destroyed, allowing remaining bacteria to proliferate.

Another microbial virulence factor is the antigenic variation as a successful pathogen must evade the

host’s immune system that recognizes bacterial surface antigens. One important evasive strategy for the

pathogen is to change its surface antigens.

This is accomplished by several mechanisms. One mechanism, called phase variation, is the genetically

reversible ability of certain bacteria to turn off and turn on the expression of genes coding for surface

antigens. The second mechanism, called antigenic variation, involves the modification of the gene for an

expressed surface antigen by genetic recombination with one of many variable unexpressed DNA

sequences. In this manner, the expressed surface antigen can assume many different antigenic structures.

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Section I– Microbiology Introductory By Dr. Mohammed Ayad

Introductory Lecture-Part Two

Identifying the organism causing an infectious process is usually essential for effective antimicrobial and

supportive therapy.

Initial treatment may be empiric, based on the microbiologic epidemiology of the infection and the

patient’s symptoms. Definitive microbiologic diagnosis of an infectious disease usually involves one or

more of the following five basic laboratory techniques:

1- Direct microscopic visualization of the organism

2- Cultivation and identification of the organism

3- Detection of microbial antigens

4- Detection of microbial DNA or RNA

5-Detection of an inflammatory or host immune response to the microorganism

A clinical history is the most important part of patient evaluation. For example, a history of cough points

to the possibility of respiratory tract infection, whereas dysuria (painful or difficult urination) suggests

urinary tract infection.

A history of travel to developing countries may implicate exotic organisms. For example, a patient who

recently swam in the Nile has an increased risk of schistosomiasis. Patient occupations may suggest

exposure to certain pathogens, such as brucellosis in a butcher or anthrax in farmers. Even the age of the

patient can sometimes guide the clinician in predicting the identity of pathogens. For example, a gram

positive coccus in the spinal fluid of a newborn infant is unlikely to be Streptococcus pneumoniae

(Pneumococcus) but most likely to be Streptococcus agalactiae (group B) which is sensitive to penicillin

G.

By contrast, a gram-positive coccus in the spinal fluid of a 40year-old patient is most likely to be S.

pneumoniae. This organism is frequently resistant to penicillin G and requires treatment with a third

generation cephalosporin (such as Cefotaxime or ceftriaxone) or vancomycin.

In many infectious diseases, pathogenic organisms (excluding viruses) can often be directly visualized by

microscopic examination of patient specimens, such as sputum, urine, and CSF.

The organism’s microscopic morphology and staining characteristics can provide the first screening step in

arriving at a specific identification. The organisms to be examined do not need to be alive or able to

multiply. Microscopy yields rapid and inexpensive results and may allow the clinician to initiate treatment

without waiting for the results of a culture, as in the spinal fluid specimens.

Gram stain

As unstained bacteria are difficult to detect with the light microscope, most patient material is stained prior

to microscopic evaluation. The most common and useful staining procedure is the Gram stain, which

separates bacteria into two classifications according to their cell wall composition. If a clinical specimen on

a microscope slide is treated with a solution of crystal violet and then iodine, the bacterial cells will stain

purple. If the stained cells are then treated with a solvent, such as alcohol or acetone, gram positive

organisms retain the stain, whereas gram-negative species lose the stain, becoming colorless.

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Section I– Microbiology Introductory By Dr. Mohammed Ayad

Addition of the counter stain safranin stains the clear, gram-negative bacteria pink or red. Most, but not all,

bacteria are stainable and fall into one of these two groups ((microorganisms that lack cell walls, such as

Mycoplasma, cannot be identified using the Gram stain)).

Gram stain applications

The Gram stain is important therapeutically because gram-positive and gram-negative bacteria differ in their

susceptibility to various antibiotics, and the Gram stain may, therefore, be used to guide initial therapy until

the microorganism can be definitively identified.

In addition, the morphology of the stained bacteria can sometimes be diagnostic. For example, gramnegative intracellular diplococci in urethral pus provide a presumptive diagnosis of gonorrhea. Gram

stains of specimens submitted for culture are often invaluable aids in the interpretation of culture results. For

example, a specimen may show organisms under the microscope but appear sterile in culture media. This

discrepancy may suggest the presence of either fastidious organisms (bacteria with complex nutrient

requirements) that are unable to grow on the culture media employed or fragile organisms, such as

gonococcus or anaerobic organisms, which may not survive transport.

In these cases, direct visualization with the Gram stain may provide the only clue to the nature, variety, and

relative number of infecting organisms.

Gram stain limitation is due to the need for high number of microorganisms required to be of benefit; as

visualization with the Gram stain requires greater than 104

organisms /ml of the specimen.

Liquid samples with low numbers of microorganisms (for example, in CSF), require centrifugation to

concentrate the pathogens, the sediment is then examined after staining.

Acid-fast stain

Stains such as Ziehl-Neelsen (the classic acid-fast stain) are used to identify organisms that have waxy

material (mycolic acids) in their cell walls. Most bacteria that have been stained with carbol- fuchsin can be

decolorized by washing with acidic alcohol. Certain acid-fast bacteria retain the carbol-fuchsin stain after

being washed with an acidic solution. The most clinically important acid-fast bacterium is Mycobacterium

tuberculosis, which appears pink, often beaded, and slightly curved. Acid fast staining is reserved for

clinical samples from patients suspected of having Mycobacterium infection.

Indian ink stain

This is one of the simplest microscopic methods. It is useful in detecting Cryptococcus neoformans in CSF.

One drop of centrifuged CSF is mixed with one drop of India ink on a microscope slide beneath a glass

cover slip. Cryptococcus is identified by their large, transparent capsules that displace the India ink

particles.

Potassium hydroxide preparation

Treatment with potassium hydroxide (KOH) dissolves host cells and bacteria, sparing fungi. One drop of

sputum or skin scraping is treated with 10 % KOH, and the specimen is examined for fungal forms.

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Section I– Microbiology Introductory By Dr. Mohammed Ayad

Bacterial Culturing

Culturing is routine for most bacterial and fungal infections but is rarely used to identify helminths or

protozoa. Culturing of many pathogens is straight forward, for example, streaking a throat swab onto a

blood agar plate in search of group A β-hemolytic Streptococcus.

Certain pathogens are very slow growing (for example, M. tuberculosis). Microorganisms isolated in

culture are identified using such characteristics as colony size, shape, color, and Gram stain, hemolytic

reactions on solid media, odor, and metabolic properties, also pure cultures provide samples for

antimicrobial susceptibility testing.

The success of culturing depends on appropriate collection and transport techniques and on selection of

appropriate culture media, because some organisms may require special nutrients. A. Specimen collection

Many organisms are fragile and must be transported to the laboratory with minimal delay. For example,

Gonococci and Pneumococci are very sensitive to heating and drying. Samples must be cultured promptly,

or, if this is not possible, transport media must be used to extend the viability of the organism to be cultured.

When anaerobic organisms are suspected, the patient’s specimen must be protected from the toxic effect of

oxygen.

All clinically important bacteria are heterotrophs (that is, they require organic carbon for growth).

Heterotrophs may have complex or simple requirements for organic molecules ((Organisms that can reduce

carbon dioxide and, therefore, do not require organic compounds for cell growth, are called autotrophs)).

Most bacteria require varying numbers of growth factors, which are organic compounds required by the cell

to grow, but which the organism cannot itself synthesize (for example, vitamins). Organisms that require

either a large number of growth factors or must be supplied with very specific ones are referred to as

fastidious.

Bacteria can be categorized according to their growth responses in the presence and absence of oxygen.

Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative

phosphorylation.

Strict anaerobes generate energy by fermentation or by anaerobic respiration and are killed in the presence

of oxygen.

Facultative anaerobes can grow in the absence of oxygen but grow better in its presence. Aerotolerant

anaerobes have mechanisms to protect themselves from oxygen (therefore, being able to grow in its

presence or absence) but do not use oxygen in their metabolism.

Microaerophiles require oxygen for their metabolism but cannot survive at atmospheric levels of oxygen,

microaerophiles are found in lakes and wet soil where the oxygen concentration is within an acceptable

range.

Two general strategies are used to isolate pathogenic bacteria, depending on the nature of the clinical

sample:

1-First method uses enriched media to promote the nonselective growth of any bacteria that may be present

2-Second approach employs selective media that only allow growth of specific bacterial species from

specimens that normally contain large numbers of bacteria (for example, stool, genital tract secretions, and

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Section I– Microbiology Introductory By Dr. Mohammed Ayad

sputum). Isolation of a bacterium is usually performed on solid medium. Liquid medium is used to grow

larger quantities of a culture of bacteria that have already been isolated as a pure culture.

 Enriched media: Media fortified with blood, yeast extracts, or brain or heart infusions are useful in

growing fastidious organisms. Blood agar contains protein sources, sodium chloride, and 5 % blood and

supports the growth of most gram-positive and gram-negative bacteria isolated from human sources.

Haemophilus influenzae and Neisseria gonorrhoeae are highly fastidious organisms. They require

chocolate agar, which contains red blood cells (RBCs) that have been lysed.

This releases intracellular nutrients, such as hemoglobin, hemin (“X” factor), and nicotinamide adenine

dinucleotide (“V” factor), required by these organisms.

Enriched media are useful for culturing normally sterile body fluids, such as blood and CSF, in which the

finding of any organisms provides reasonable evidence for infection by that organism. Failure to culture an

organism may indicate that:

1- The culture medium is inadequate

2- The incubation conditions do not support bacterial growth

 Selective media: The most commonly used selective medium is MacConkey agar, which supports the

growth of most gram-negative rods, especially the Enterobacteriaceae, but inhibits growth of gram-positive

organisms and some fastidious gram-negative bacteria, like Haemophilus and Neisseria species.

Growth on blood agar and chocolate agar but not MacConkey agar suggests a gram-positive isolate or a

fastidious gram-negative species. On the other hand, most gram-negative rods often form distinctive

colonies on MacConkey agar. This agar is also used to detect organisms able to metabolize lactose.

Clinical samples are routinely plated on blood agar, chocolate agar, and MacConkey agar. Hektoen

enteric agar is also a selective medium that differentiates lactose / sucrose fermenters and nonfermenters as well as H2S producers and non-producers. It is often used to culture Salmonella and

Shigella species.