Formaldehyde-inactivated toxins, called toxoids, are useful in preparing vaccines.

Exotoxin proteins are, in many cases, encoded by genes carried on plasmids or temperate bacteriophages.

An example is the diphtheria exotoxin that is encoded by the tox gene of a temperate bacteriophage that

can lysogenize Corynebacterium diphtheriae. Strains of C. diphtheriae that carry this phage are

pathogenic, whereas those that lack the phage are nonpathogenic.

b. Endotoxins: These are heat-stable, LPS components of the outer membranes of gram-negative (but

not gram-positive) bacteria. They are released into the host’s circulation following bacterial cell lysis.

LPS consists of polysaccharide composed of repeating sugar subunits (O antigen), which protrudes from

the exterior cell surface; a core polysaccharide; and a lipid component called lipid A that is integrated into

the outer leaflet of the outer membrane.

The lipid A is responsible for the toxicity of this molecule. The main physiologic effects of LPS

endotoxin are fever, shock, hypotension, and thrombosis, collectively referred to as septic shock. These

effects are produced indirectly by macrophage activation, with the release of cytokines, activation of

complement, and activation of the coagulation cascade. Death can result from multiple organ failure.

Elimination of the causative bacteria with antibiotics can initially exacerbate the symptoms by causing

sudden massive release of endotoxin into the circulation. Although gram-positive bacteria do not contain

LPS, their cell wall peptidoglycan and teichoic acids can elicit a shock syndrome similar to that caused

by LPS but usually not as severe.

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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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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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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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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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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.

Thayer-Martin agar is another selective medium composed of chocolate agar supplemented with

several antibiotics that suppress the growth of nonpathogenic Neisseria and other normal and abnormal

flora. This medium is normally used to isolate Gonococci.

When submitting samples for culture, the physician must alert the laboratory to likely pathogens

whenever possible, especially when unusual organisms are suspected. This allows inclusion of

selective media that might not be used routinely.

Bacterial Identification

The most widely used identification scheme involves determining the morphologic and metabolic

properties of the unknown bacterium and comparing these with properties of known microorganisms.

Alternate identification schemes using nucleic acid–based methods are used also. It is essential to start

identification tests with pure bacterial isolates grown from a single colony.

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Single enzyme test for bacterial identification

Different bacteria produce varying spectra of enzymes; e.g., some enzymes are necessary for the

bacterium’s individual metabolism, and some facilitate the bacterium’s ability to compete with other

bacteria or establish an infection.

Tests that measure single bacterial enzymes are simple, rapid, and generally easy to interpret. They can

be performed on organisms already grown in culture and often provide presumptive identification.

1) Catalase test: The enzyme catalase catalyzes the degradation of hydrogen peroxide to water

and molecular oxygen (H2O2 → H2O + O2). Catalase positive organisms rapidly produce

bubbles when exposed to a solution containing hydrogen peroxide. The catalase test is key in

differentiating between many gram-positive organisms; e.g., Staphylococci are catalase positive,

whereas Streptococci and Enterococci are catalase negative. The production of catalase is an

important virulence factor because H2O2 is antimicrobial, and its degradation decreases the

ability of neutrophils to kill invading bacteria.

2) Oxidase test: The enzyme cytochrome c oxidase is part of electron transport and nitrate

metabolism in some bacteria. The enzyme can accept electrons from artificial substrates (such as a

phenylenediamine derivative), producing a dark, and oxidized product. This test assists in

differentiating between groups of gram-negative bacteria. Pseudomonas aeruginosa; e.g., is

oxidase positive.

3) Urease: The enzyme urease hydrolyzes urea to ammonia and carbon dioxide (NH2CONH2 +

H2O → 2NH3 + CO2). The ammonia produced can be detected with pH indicators that change

color in response to the increased alkalinity. The test helps to identify certain species of

Enterobacteriaceae, Corynebacterium urealyticum, and Helicobacter pylori.

4) Coagulase test: Coagulase is an enzyme that causes a clot to form when bacteria are

incubated with plasma. The test is used to differentiate Staphylococcus aureus (coagulase

positive) from coagulase-negative Staphylococci.

Automated systems for bacterial identification

Microbiology laboratories are increasingly using automated methods to identify bacterial pathogens;

as in the Vitek System, small plastic reagent cards containing micro liter quantities of various

biochemical test media in 30 wells provide a biochemical profile that allows for organism

identification. An inoculum derived from cultured samples is automatically transferred into the card,

and a photometer intermittently measures color changes in the card that result from the metabolic

activity of the organism.

The data are analyzed, stored, and printed in a computerized database. There are many commercial

variants of these automated systems and several can be used for simultaneous identification and

antimicrobial susceptibility determination.

Tests based on the presence of metabolic pathways (API; analytical Profile Index)

These tests measure the presence of metabolic pathways in a bacterial isolate, rather than a single

enzyme. Commonly used assays include those for oxidation and fermentation of different

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carbohydrates, the ability to degrade amino acids, and use of specific substrates. A widely used manual

system for rapid identification of members of the family Enterobacteriaceae and other gram-negative

bacteria makes use of twenty micro tubes containing substrates for various biochemical pathways. The

test substrates in the micro tubes are inoculated with the bacterial isolate to be identified, and, after 5

hours incubation, the metabolic profile of the organism is constructed from color changes in the micro

tubes. These color changes indicate the presence or absence of the bacteria’s ability to metabolize a

particular substrate. The results are compared with a data bank containing test results from known

bacteria. The probability of a match between the test organism and known pathogens is then

calculated.

Immunological bacterial identification

In the diagnosis of infectious diseases, immunologic methods take advantage of the specificity of

antigen–antibody binding, as known antigens and antibodies are used as diagnostic tools in

identifying microorganisms.

Serologic detection of a patient’s immune response to infection, or antigenic or nucleic acid evidence

of a pathogen in a patient’s body fluids, is frequently useful. Immunologic methods are useful when

the infecting microorganism is difficult or impossible to isolate or when a previous infection needs

to be documented.

I- Detection of microbial antigen with known antiserum

These methods of identification are often rapid and show favorable sensitivity and specificity; unlike

microbial culturing techniques, these immunologic methods do not permit further characterization of

the microorganism, such as determining its antibiotic sensitivity or characteristic metabolic patterns:

1. Quellung reaction: Some bacteria having capsules can be identified directly in clinical specimens

by a reaction that occurs when the organisms are treated with serum containing specific antibodies.

The Quellung reaction makes the capsule more refractile and thus more visible, but the capsule does

not actually swell. This method can be used for all serotypes of S. pneumoniae, H. influenzae type b,

and Neisseria meningitidis groups A and C.

2. Slide agglutination test: Some microorganisms, such as Salmonella and Shigella species, can be

identified by agglutination (clumping) of a suspension of bacterial cells on a microscopic slide.

Agglutination occurs when a specific antibody directed against the microbial antigen is added to the

suspension, causing cross-linking of the bacteria.

II-Identification of serum antibodies

Detection in a patient’s serum of antibodies that are directed against microbial antigens provides

evidence for a current or past infection with a specific pathogen; and it characterized by:

1) Antibody may not be detectable early in an infection

2) The presence of antibodies in a patient’s serum cannot differentiate between a present and a prior

infection

3) A significant rise in antibody titer over a 10 to14-day period does distinguish between a present or

prior infection

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Techniques such as complement fixation and agglutination can be used to quantitate antimicrobial

antibodies.

1. Complement fixation: It is the older method but still useful method for detecting serum

antibody directed against a specific pathogen employs the ability of antibody to bind complement.

A patient’s serum is first incubated with antigen specific for the suspected infectious agent,

followed by the addition of complement. If the patient’s serum does contain immunoglobulin

(IgG or IgM) that target the specific antigen (indicating past or current infection), then the added

complement will be sequestered in an antigen–antibody–complement complex (“complement

fixation”).

Then the sensitized (antibody-coated) indicator sheep RBCs are added to the solution. If

complement has been fixed (because the patient’s serum contained antibodies against the added

antigen), then little complement will be available to bind to the antibody–RBC complexes, and the

cells will not lyse.

If complement has not been depleted by initial antigen–antibody complexes (because the patient’s

serum does not contain antibodies to the specific antigen), the complement will bind to the antibody–

RBC complexes, causing the cells to lyse. As hemolyzed RBCs release hemoglobin, the reaction can

be monitored with a spectrophotometer.

2. Direct agglutination: Direct bacterial agglutination testing is sometimes ordered when a suspected

pathogen is difficult or dangerous to culture in the laboratory. This test measures the ability of a

patient's serum antibody to directly agglutinate specific killed (yet intact) microorganisms. This test is

used to evaluate patients suspected of being infected by Brucella abortus or Francisella tularensis.

3. Direct hemagglutination: Antibodies directed against RBCs can arise during the course of various

infections; such antibodies are typically found during infectious mononucleosis caused by EpsteinBarr virus. When uncoated (native) animal or human RBCs are used in agglutination reactions with

serum from a patient infected with such an organism, antibodies to RBC antigens can be detected. The

patient’s antibodies cause the RBCs to clump. This test is, therefore, a direct hemagglutination

reaction. In the case of some diseases, including pneumonia caused by Mycoplasma pneumoniae, IgM

auto antibodies may develop that agglutinate human RBCs at 4o C but not at 37o C ((termed the “cold

agglutinins” test))

Other tests used to identify serum antigens or antibodies

1. Latex agglutination test:

Latex and other particles can be readily coated with either antibody (for antigen detection) or antigen

(for antibody detection). Addition of antigen to antibody-coated latex beads causes agglutination that

can be visually observed; such methods are used to rapidly test CSF for antigens associated with

common forms of bacterial or fungal meningitis. When antigen is coated onto the latex bead,

antibody from a patient’s serum can be detected. Latex agglutination tests are widely used for the

identification of β-hemolytic Streptococci group A.

2. Enzyme-linked immunosorbent assay:

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Elisa is a diagnostic technique in which antibody specific for an antigen of interest is bound to the

walls of a plastic micro titer well. Patient serum is then incubated in the wells, and any antigen in the

serum is bound by the antibody on the well walls. The wells are then washed, and a second antibody is

added. This one is also specific for the antigen but recognizes epitopes different from those bound by

the first antibody. After incubation, the wells are again washed, removing any unattached antibody.

Attached to the second antibody is an enzyme, which, when presented with its substrate, produces a

colored product, the intensity of the color produced being proportional to the amount of bound antigen.

Elisa can also be used to detect or quantitate antibody in a patient’s serum; the wells are coated with

antigen specific for the antibody in question. The patient’s serum is allowed to react with the bound

antigen, the wells are washed, and a secondary antibody (that recognizes the initial antibody)

conjugated to a color product–producing enzyme is added to the well. After a final washing, substrate

for the bound enzyme is added to the well, and the intensity of the colored product can be measured.

3. Fluorescent-antibody tests: Organisms in clinical samples can be detected directly by specific

antibodies coupled to a fluorescent compound such as fluorescein. In the direct Immunofluorescence

antibody technique, a sample of concentrated body fluid (like CSF or serum), tissue scraping (like

skin), or cells in tissue culture is incubated with a fluorescein-labeled antibody directed against a

specific pathogen.

The labeled antibody bound to the microorganism absorbs ultraviolet light and emits visible

fluorescence that can be detected using a fluorescence microscope. A variation of the technique, the

indirect Immunofluorescence antibody technique, involves the use of two antibodies. The first is

unlabeled antibody (the target antibody), which binds a specific microbial antigen in a sample; and

this clinical sample is subsequently stained with a fluorescent antibody that recognizes the target

antibody. Because a number of labeled antibodies can bind to each target antibody, the fluorescence

from the stained microorganism is intensified.

Nucleic acid based tests

The most widely used methods for detecting microbial DNA fall into three categories:

1) Direct hybridization (non-amplified assay)

2) Amplification methods using the polymerase chain reaction (PCR)

3) DNA microarrays

Although not likely to completely replace culture techniques in the near future, nucleic acid–based

tests for the diagnosis of infectious diseases are gaining wider acceptance.

Direct detection of pathogens without target amplification

This highly specific method of pathogen detection involves identification of the DNA of the pathogen

in a patient sample or, more commonly, organisms isolated in culture. The basic strategy is to detect a

relatively short sequence of nucleotide bases of DNA sequence) that is unique to the pathogen. This is

done by hybridization with a probe, a single-stranded piece of DNA (usually labeled with a fluorescent

molecule) containing a complementary sequence of bases. In bacteria, DNA sequences coding for 16S

ribosomal RNA sequences (rRNA) are commonly used targets because each microorganism contains