P8.35.2^35.2. Identification of Microorganisms from Specimens^853^863^,,^26056^26380%
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35.2
Identification of Microorganisms from Specimens

The clinical specimen is an unknown, and the microbiology laboratory scientists are the detectives who can identify the microorganism(s) in the specimen; they determine the cause of a patient's infection. The clinical microbiology laboratory provides preliminary or definitive identification of microorganisms using various tests and procedures that have the highest probability of rapid identification based on (1) microscopic examination of specimens, (2) study of the growth and biochemical characteristics of isolated microorganisms (pure cultures), (3) rapid and automated detection of unique microbial signatures, (4) bacteriophage typing (restricted to research settings and the CDC), and (5) molecular methods. Choosing the appropriate test or procedure is determined by the specimen and what is generically expected to be in the specimen based on the patient history.

Microscopy

Light microscopy is used to image organisms that are typically larger than 0.5 μm, for example, protozoa, fungi, and bacteria. Electron microscopy is used to see things that are much smaller, such as viruses. Some morphological and genetic features used in classification and identification of microorganisms are presented in section 17.3 and tables 17.1 and 17.2. Standard references, such as the Manual of Clinical Microbiology published by the American Society for Microbiology, provide details about reagents and staining procedures.

Bacteria

Wet-mount, heat-fixed, or chemically fixed specimens can be examined with an ordinary bright-field microscope. Examination can be enhanced with either phase-contrast or dark-field microscopy. The latter is the procedure of choice for the detection of spirochetes in skin lesions associated with early syphilis or Lyme disease. The fluorescence microscope can be used to identify any microorganism after it is stained with fluorochromes such as acridine orange, which stains nucleic acids, or any fluorochrome-labeled antibody that binds to specific microbial antigens. Many stains that can be used to examine specimens for specific microorganisms have been described. Two of the more widely used bacterial stains are the Gram stain and the acid-fast stain (see figure 2.18). Because these stains are based on the chemical composition of cell walls, they are not useful in identifying bacteria without cell walls (e.g., mycoplasmas).

Gram Stain

The use of fluorescently labeled monoclonal antibodies in microscopic techniques has been an important breakthrough in diagnostic microscopy. In the 1980s immunlogists created hybrid cells (hybridomas) that live a very long time and secrete antibodies (see Techniques & Applications 33.1). Recall that each hybridoma cell and its progeny normally produce a monoclonal antibody (mAb) of a single specificity that can bind or capture the antigen to which it was produced. These antigen-capture antibodies recognize a single epitope and are therefore used for diagnostics. One such cross-cutting method, known as immunofluorescence or immunohistochemistry, is used to detect a variety of microorganisms and results from the chemical attachment of fluorescent dyes called fluorochromes to mAbs; the mAb then binds to a single epitope and the fluorescent molecule “reports” that binding. This technique can be used to tag specific microorganisms in a clinical specimen. Examples of commonly used fluorochromes include rhodamine B and fluorescein isothiocyanate (FITC), which can be coupled to antibody molecules without changing the antibody's capacity to bind to a specific antigen. In the clinical microbiology laboratory, fluorescently labeled mAbs to viral or bacterial antigens have replaced polyclonal antisera for use in culture confirmation when accurate, rapid identification is required. With the use of sensitive techniques such as fluorescence microscopy, it is possible to perform antibody-based microbial identifications with improved accuracy, speed, and fewer organisms. Antibodies (section 33.7)

Monoclonal Antibody Production

Two main kinds of fluorescent antibody assays are used: direct and indirect. Direct immunofluorescence involves fixing the specimen (cell or microorganism) containing the antigen of interest onto a slide (figure 35.3a). Fluorochrome-labeled antibodies are then added to the slide and incubated. The slide is washed to remove any unbound antibody and examined with the fluorescence microscope (see figure 2.13) for fluorescence. The pattern of fluorescence reveals the antigen's location. Direct immunofluorescence is used to identify antigens such as those found on the surface of group A streptococci and to diagnose enteropathogenic Escherichia coli, Neisseria meningitidis, Salmonella spp., Shigella sonnei, Listeria monocytogenes, and Haemophilus influenzae type b.

 
FIGURE 35.3
Direct and Indirect Immunofluorescence.(a) In the direct fluorescent antibody (DFA) technique, the specimen containing the antigen is fixed to a slide. Fluorescently labeled antibodies that recognize the antigen are then added, and the specimen is examined with a fluorescence microscope for fluorescence. (b) The indirect fluorescent antibody technique (IFA) detects antigen on a slide as it reacts with an antibody directed against it. The antigen-antibody complex is located with a fluorescent antibody that recognizes antibodies. (c) A photomicrograph of cells infected with cytomegalovirus (green fluorescence) and adenovirus (yellow fluorescence) using an indirect immunofluorescent staining procedure.
Figure 35.3 Micro Inquiry

Which type of immunofluorescence would most likely be used to test a clinical sample for the presence of an intracellular pathogen such as Hepatitis C virus?

Indirect immunofluorescence (figure 35.3b) is used to detect the presence of antibodies in serum following an individual's exposure to microorganisms. In this technique, a known antigen (e.g., a virus) is fixed onto a slide. The patient serum containing antibodies is then added, and if the specific antibody is present, it reacts with antigen to form a complex. When a second, fluorescein-labeled antibody (not from the patients' serum) is added, it reacts with the fixed antibody. After incubation and washing, the slide is examined with the fluorescence microscope. The occurrence of fluorescence shows that the antibody specific to the (viral) antigen is present in the serum and that its presence is reported by the fluorescence of the secondary antibody that has attached to the serum antibody (figure 35.3c). A common application of indirect immunofluorescence is the identification of Treponema pallidum antibodies in the diagnosis of syphilis.

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Chlamydiae can be demonstrated in tissues and cell scrapings with Giemsa staining, which detects the characteristic intracellular inclusion bodies (see figure 19.12). Immunofluorescent staining of tissues and cells with monoclonal antibody reagents is a more sensitive and specific means of diagnosis of chlamydial disease.

Fungi

Direct microscopic examination of most specimens suspected of containing fungi can be made by light microscopy as well. Identification of hyphae in clinical specimens is a presumptive positive result for fungal infection. Definitive identification of most fungi is based on the morphology of reproductive structures (spores). Lactophenol aniline (cotton) blue is typically used to stain fungi from cultures. Fungal infections (i.e., mold and yeast infections) often are diagnosed by direct microscopic examination of specimens using fluorescence. For example, the identification of molds often can be made if a portion of the specimen is mixed with a drop of 10% Calcofluor White stain on a glass slide. Light microscopes (section 2.2)

Parasites

Identification and characterization of ova, trophozoites, and cysts in the specimen result in the definitive diagnosis of protozoa infection. This is typically accomplished by direct microscopic evaluation of the clinical specimen. Typical histological staining of blood, negative staining of other body fluids, and immunofluorescence staining are routinely used in the identification of parasites. Concentrated wet mounts of blood, stool, or urine specimens can be examined microscopically for the presence of eggs, cysts, larvae, or vegetative cells of parasites. D'Antoni's iodine (1%) is often used to stain internal structures of parasites. Blood smears for apicomplexan (malaria) and flagellate (trypanosome) parasites are stained with Giemsa.

Viruses

Most viruses are too small to be visualized by light microscopy alone. Fluorescence microscopy using fluorescently labeled antibodies is used for some virus detection. For high magnification and resolution, viruses must be imaged by electron microscopy. Specialized fixing and staining procedures are also used to prepare viruses for electron microscopy. However, very few clinical laboratories have access to electron microscopes, except those associated with large hospitals or research departments.

Growth and Biochemical Characteristics

For those microorganisms that can be grown in culture and identified by particular growth patterns and biochemical characteristics, specific tests are used. These tests vary, depending on whether the clinical microbiologist is dealing with viruses, fungi (yeasts, molds), parasites (protozoa, helminths), common gram-positive or gram-negative bacteria, rickettsias, chlamydiae, or mycoplasmas.

Bacteria

Isolation and growth of bacteria are required before many diagnostic tests can be used to confirm the identification of the pathogen. The presence of bacterial growth usually can be recognized by the development of colonies on solid media or turbidity in liquid media. The time needed for visible growth to occur is an important variable in the clinical laboratory. For example, most pathogenic bacteria require only a few hours to produce visible growth, whereas it may take weeks for colonies of mycobacteria or mycoplasmas to become evident. The clinical microbiologist as well as the clinician should be aware of reasonable reporting times for various cultures.

The initial identity of a bacterial organism may be suggested by (1) the source of the culture specimen; (2) its microscopic appearance and gram reaction; (3) its pattern of growth on selective, differential, or metabolism-determining media; and (4) its hemolytic, metabolic, and fermentative properties on the various media (table 35.1; see also table 20.7). For example, methylene blue is often used to inhibit the growth of gram-positive bacteria, whereas phenylethyl alcohol is often used to inhibit gram-negative bacteria. Sheep blood–supplemented agars can be used to determine hemolytic capabilities (see figure 21.20).

 
Table 35.1
Isolation of Pure Bacterial Cultures from Specimens
Selective Media
A selective medium is prepared by the addition of specific substances to a culture medium that will permit growth of one group of bacteria while inhibiting growth of some other groups. These are examples:
Salmonella-Shigella agar (SS) is used to isolate Salmonella and Shigella species. Its bile salt mixture inhibits many groups of coliforms. Both Salmonella and Shigella species produce colorless colonies because they are unable to ferment lactose. Lactose-fermenting bacteria will produce pink colonies.
Mannitol salt agar (MS) is used for the isolation of staphylococci. The selectivity is obtained by the high (7.5%) salt concentration that inhibits growth of many groups of bacteria. The mannitol in this medium helps in differentiating the pathogenic from the nonpathogenic staphylococci, as the former ferment mannitol to form acid while the latter do not. Thus this medium is also differential.
Bismuth sulfite agar (BS) is used for the isolation of Salmonella enterica serovar Typhi, especially from stool and food specimens. S. enterica serovar Typhi reduces the sulfite to sulfide, resulting in black colonies with a metallic sheen.
Hektoen enteric agar is used to increase the yield of Salmonella and Shigella species relative to other microbiota. The high bile salt concentration inhibits the growth of gram-positive bacteria and retards the growth of many coliform strains.
Differential Media
The incorporation of certain chemicals into a medium may result in diagnostically useful growth or visible change in the medium after incubation. These are examples:
Eosin methylene blue agar (EMB) differentiates between lactose fermenters and nonlactose fermenters. EMB contains lactose, salts, and two dyes—eosin and methylene blue. E. coli, which is a lactose fermenter, will produce a dark colony or one that has a metallic sheen. S. enterica serovar Typhi, a nonlactose fermenter, will appear colorless.
MacConkey agar is used for the selection and recovery of Enterobacteriaceae and related gram-negative rods. The bile salts and crystal violet in this medium inhibit the growth of gram-positive bacteria and some fastidious gram-negative bacteria. Because lactose is the sole carbohydrate, lactose-fermenting bacteria produce colonies that are various shades of red, whereas nonlactose fermenters produce colorless colonies.

Blood agar: addition of citrated blood to tryptic soy agar makes possible variable hemolysis, which permits differentiation of some species of bacteria. Three hemolytic patterns can be observed on blood agar.

  1. α-hemolysis—greenish to brownish halo around the colony (e.g., Streptococcus gordonii, Streptococcus pneumoniae)

  2. β-hemolysis—complete lysis of blood cells resulting in a clearing effect around the colony (e.g., Staphylococcus aureus and Streptococcus pyogenes)

  3. Nonhemolytic—no change in medium (e.g., Staphylococcus epidermidis and Staphylococcus saprophyticus)

Media to Determine Biochemical Reactions
Some media are used to test bacteria for particular metabolic activities, products, or requirements. These are examples:
Urea broth is used to detect the enzyme urease. Some enteric bacteria are able to break down urea, using urease, into ammonia and CO2. Media turns pink if urease is present.
Triple sugar iron (TSI) agar contains lactose, sucrose, and glucose plus ferrous ammonium sulfate and sodium thiosulfate. TSI is used for the identification of enteric organisms based on their ability to metabolize glucose, lactose, or sucrose, and to liberate sulfides from ammonium sulfate or sodium thiosulfate. Acid reactions turn media yellow. Basic reactions turn media orange to red.
Citrate agar contains sodium citrate, which serves as the sole source of carbon, and ammonium phosphate, the sole source of nitrogen. Citrate agar is used to differentiate enteric bacteria on the basis of citrate utilization. Citrate utilization turns media blue.
Lysine iron agar (LIA) is used to differentiate bacteria that can either deaminate or decarboxylate the amino acid lysine. LIA contains lysine, which permits enzyme detection, and ferric ammonium citrate for the detection of H2S production. Lysine metabolism is indicated by red-purple media.
Sulfide, indole, motility (SIM) medium is used for three different tests. One can observe the production of sulfides, formation of indole (a metabolic product from tryptophan utilization), and motility. This medium is generally used for the differentiation of enteric organisms. Sulfide production turns media black. Indole formation results in a red color on top of media upon addition of Kovacs reagent.

After the microscopic and growth characteristics of a pure culture of bacteria are examined, specific biochemical tests can be performed. Some of the most common biochemical tests used to identify bacterial isolates are listed in table 35.2. Classic dichotomous keys are coupled with the biochemical tests for the identification of bacteria from specimens. Generally, fewer than 20 tests are required to identify clinical bacterial isolates to the species level (figure 35.4).

 
Table 35.2
Some Common Biochemical Tests Used by Clinical Microbiologists in the Diagnosis of Bacteria from a Patient's Specimen
Biochemical Test Description Laboratory Application
Carbohydrate fermentation Acid and/or gas are produced during fermentative growth with sugars or sugar alcohols. Fermentation of specific sugars used to differentiate enteric bacteria as well as other genera or species
Casein hydrolysis Detects the presence of caseinase, an enzyme able to hydrolyze milk protein casein. Bacteria that use casein appear as colonies surrounded by a clear zone. Used to cultivate and differentiate aerobic actinomycetes based on casein utilization. For example, Streptomyces uses casein and Nocardia does not.
Catalase Detects the presence of catalase, which converts hydrogen peroxide to water and O2 Used to differentiate Streptococcus (−) from Staphylococcus (+) and Bacillus (+) from Clostridium (−)
Citrate utilization When citrate is used as the sole carbon source, this results in alkalinization of the medium. Used in the identification of enteric bacteria. Klebsiella (+), Enterobacter (+), Salmonella (often +); Escherichia (−), Edwardsiella (−)
Coagulase Detects the presence of coagulase. Coagulase causes plasma to clot. This is an important test to differentiate Staphylococcus aureus (+) from S. epidermidis (−).
Decarboxylases (arginine, lysine, ornithine) The decarboxylation of amino acids releases CO2 and amine. Used in the identification of enteric bacteria
Esculin hydrolysis Tests for the cleavage of a glycoside Used in the differentiation of Staphylococcus aureus, Streptococcus mitis, and others (−) from S. bovis, S. mutans, and enterococci (+)
β-galactosidase (ONPG) test Demonstrates the presence of an enzyme that cleaves lactose to glucose and galactose Used to separate enterics (Citrobacter +, Salmonella −) and to identify pseudomonads
Gelatin liquefaction Detects whether or not a bacterium can produce proteases that hydrolyze gelatin and liquify solid gelatin medium Used in the identification of Clostridium, Serratia, Pseudomonas, and Flavobacterium
Hydrogen sulfide (H2S) Detects the formation of hydrogen sulfide from the amino acid cysteine due to cysteine desulfurase Important in the identification of Edwardsiella, Proteus, and Salmonella
IMViC (indole; methyl red; Voges-Proskauer; citrate) The indole test detects the production of indole from the amino acid tryptophan. Methyl red is a pH indicator to determine whether the bacterium carries out mixed acid fermentation. VP (Voges-Proskauer) detects the production of acetoin. The citrate test determines whether or not the bacterium can use sodium citrate as a sole source of carbon. Used to separate Escherichia (MR+, VP−, indole +) from Enterobacter (MR−, VP+, indole−) and Klebsiella pneumoniae (MR−, VP+, indole−); also used to characterize members of the genus Bacillus
Lipid hydrolysis Detects the presence of lipase, which breaks down lipids into simple fatty acids and glycerol Used in the separation of clostridia
Nitrate reduction Detects whether a bacterium can use nitrate as an electron acceptor Used in the identification of enteric bacteria, which are usually +
Oxidase Detects the presence of cytochrome c oxidase that is able to reduce O2 and artificial electron acceptors Important in distinguishing Neisseria and Moraxella spp. (+) from Acinetobacter (−) and enterics (all −) from pseudomonads (+)
Phenylalanine deaminase Deamination of phenylalanine produces phenyl-pyruvic acid, which can be detected colorimetrically. Used in the characterization of the genera Proteus and Providencia
Starch hydrolysis Detects the presence of the enzyme amylase, which hydrolyzes starch Used to identify typical starch hydrolyzers such as Bacillus spp.
Urease Detects the enzyme that splits urea to NH3 and CO2 Used to distinguish Proteus, Providencia rettgeri, and Klebsiella pneumoniae (+) from Salmonella, Shigella and Escherichia (−)
 
FIGURE 35.4
Classic Dichotomous Keys for Clinically Important Genera.(a) Schematic outline for the identification of gram-positive bacteria. (b) Schematic outline for the identification of gram-negative bacteria.
Figure 35.4 Micro Inquiry

You have detected aerobic, gram-positive cocci that are catalase positive but do not ferment glucose in a clinical sample from a skin wound. What is it?

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Certain bacteria require special considerations. For instance, the rickettsias, chlamydiae, and mycoplasmas differ from other bacterial pathogens in a variety of ways. They can be diagnosed by immunoassays or by isolation of the microorganism. Because isolation is both hazardous and expensive, immunological methods are preferred. Isolation of rickettsias and diagnosis of rickettsial diseases are generally confined to reference and specialized research laboratories.

Fungi

Fungal cultures remain the standard for the recovery of fungi from patient specimens; however, the time needed to culture fungi varies anywhere from a few days to several weeks, depending on the organism. For this reason, fungal cultures demonstrating no growth should be maintained for a minimum of 30 days before they are discarded as a negative result. Cultures should be evaluated for rate and appearance of growth on at least one selective and one nonselective agar medium, with careful examination of colonial morphology, color, and dimorphism. Typically, the isolation of fungi is accomplished by concurrent culture of the specimen on media that is respectively supplemented and unsupplemented with antibiotics and cycloheximide. Antibiotics inhibit bacteria that may be in the specimen and cycloheximide inhibits saprophytic (living on decaying matter) molds. However, a number of media formulations are routinely used to culture specific fungi (table 35.3).

 
Table 35.3
Examples of Media Used to Isolate Fungi
Culture Medium Target Fungi
Antibiotic agar Fungi in polymicrobial specimens
Caffeine agar Aspergillus, Rhizopus, and others
Chlamydospore agar Dimorphic fungi
Cornmeal agar Most fungi including pathogens
Malt agar Ascomycetes
Malt extract agar Basidiomycetes
Potato dextrose-yeast extract agar Mushrooms
Sabouraud dextrose agar (SAB) Most fungi
SAB + chloramphenicol + cyclohexamide Dimorphic fungi and dermatophytes
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Parasites

Culture of protozoa from clinical specimens is not routine. Thus detection and identification of these microorganisms is by microscopy, molecular techniques, or both.

Viruses

It is important to note that the culture of virus is not typically done in hospital labs. Viruses are grown and identified by isolation in conventional cell (tissue) culture in specialized labs equipped to provide the biosafety and biocontainment required for the specific virus. Hopsital labs, rather, identify virus in clinical specimens by immunodiagnosis (fluorescent antibody, enzyme immunoassay, radioimmunoassay, latex agglutination, and immunoperoxidase) and by molecular detection methods such as nucleic acid probes and PCR amplification assays. Several types of systems are available for virus cultivation: cell cultures, embryonated hen's eggs, and experimental animals; these are discussed shortly.

Cell cultures are divided into three general classes: (1) Primary cultures consist of cells derived directly from tissues such as monkey kidney and mink lung cells that have undergone one or two passages (subcultures) since harvesting. (2) Semicontinuous cell cultures or low-passage cell lines are obtained from subcultures of a primary culture and usually consist of diploid fibroblasts that undergo a finite number of divisions. (3) Continuous or immortalized cell cultures, such as HEp-2 cells, are derived from transformed cells that are generally epithelial in origin. These cultures grow rapidly, are heteroploid (having a chromosome number that is not a simple multiple of the haploid number), and can be subcultured indefinitely.

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Each type of cell culture favors the growth of a different array of viruses, just as bacterial culture media have differing selective and restrictive properties for growth of bacteria. Viral replication in cell cultures is detected in two ways: (1) by observing the presence or absence of cytopathic effects (CPEs) and (2) by hemadsorption. A cytopathic effect is an observable morphological change that occurs in cells because of viral replication. Examples include ballooning, binding together, clustering, or even death of the culture cells. During the incubation period of a cell culture, red blood cells can be added. Several viruses alter the plasma membrane of infected culture cells so that red blood cells adhere firmly to them. This phenomenon is called hemadsorption. Viral multiplication (section 5.3)

Embryonated chicken eggs can be used for virus isolation. There are three main routes of egg inoculation for virus isolation as different viruses grow best on different cell types: (1) the allantoic cavity, (2) the amniotic cavity, and (3) the chorioallantoic membrane. Egg tissues are inoculated with clinical specimens to determine the presence of virus; virus is revealed by the development of pocks on the chorioallantoic membrane, by the development of hemagglutinins in the allantoic and amniotic fluid, and by death of the embryo.

Laboratory animals, especially suckling mice, also may be used for virus isolation. Inoculated animals are observed for specific signs of disease or death. Several serological tests for viral identification make use of mAb-based immunofluorescence. These tests detect viruses such as herpes simplex virus in tissue cultures.

bc_background:yellow
  1. Name two specimens for which microscopy would be used in the initial diagnosis of an infectious disease.

  2. Name three general classes of cell cultures.

  3. Explain two ways by which the presence of viral replication is detected in cell culture.

  4. What are the advantages of using monoclonal antibody (mAb) immunofluorescence in the identification of viruses?

  5. How can a clinical microbiologist determine the initial identity of a bacterium?

  6. Describe a dichotomous key that could be used to identify a bacterium.

  7. How can fungi and protozoa be detected in a clinical specimen? Rickettsias? Chlamydiae? Mycoplasmas?

Rapid Methods of Identification

Clinical laboratory scientists (medical technologists) are the trained and certified workforce that is the front line in laboratory-based disease detection. They staff the sentinel laboratories that receive patient specimens. The production of faster and more specific detection technologies has enabled medical technologists to rapidly and accurately identify disease agents. In this way, clinical microbiology laboratories provide rapid, accurate, and timely microbial identification and antimicrobial susceptibility results that assist clinicians in the diagnosis and treatment of infectious disease. Clinical microbiology has also benefited greatly from technological advances in equipment, computer software and databases, molecular biology, and immunochemistry. With new technology, it has been possible to shift from the multistep methods previously discussed to unitary procedures and systems that incorporate standardization, speed, reproducibility, miniaturization, mechanization, and automation. These rapid identification methods can be divided into three categories: (1) manual biochemical “kit” systems, (2) mechanized/automated systems, and (3) immunologic systems.

One example of a “kit approach” biochemical system for the identification of members of the family Enterobacteriaceae and other gram-negative bacteria is the API 20E system. It consists of a plastic strip with 20 microtubes containing dehydrated biochemical substrates that can detect certain biochemical characteristics (figure 35.5). The biochemical substrates in the 20 microtubes are inoculated with a pure culture of bacteria evenly suspended in sterile physiological saline. After 5 to 12 hours of incubation, the 20 test results are converted to a seven- or nine-digit profile. This profile number can be used with a computer or a book called the API Profile Index to identify the bacterium.

FIGURE 35.5
A “Kit Approach” to Bacterial Identification.The API 20E manual biochemical system for microbial identification. (a) Positive and (b) negative results.

In addition to traditional techniques and kits, laboratories also use semiautomated microbial identification and susceptibility instruments. These instruments automate the incubation, reagent addition, and evaluation of test results from miniaturized versions of classical bench tests. Examples of these identification and susceptibility instruments include the Phoenix™ automated microbiology system, the VersaTREK®, and the MicroScan® WalkAway. These systems use carriers, reagents, and reporting components that are uniquely made for them. The typical identification and susceptibility test begins after the primary isolation of the organisms in pure culture. A saline suspension of the pure culture is adjusted to a specific density and is coded so as to be tracked throughout the various stages of evaluation. Aliquots of the bacterial suspension are then injected into various, miniaturized test chambers containing biochemical reagents. Colorimetric or fluorometric reactions resulting from each test are then evaluated against controls to report on the specific capabilities of the organism. The results of the individual reactions are evaluated together to predict organism identification, within a calculated confidence interval. Antimicrobial susceptibility is determined by evaluating organism growth when exposed to various antibiotics and reported by similar means. Thus the organism's identity and its sensitivity to a panel of antibiotics (a profile called its antibiogram) are determined, often within hours.

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The rapidly growing discipline of immunology has greatly aided the clinical microbiologist. Numerous technologies now exist that exploit the specificity and sensitivity of monoclonal antibodies to detect and identify microorganisms. These are briefly discussed here and expanded upon in section 35.3. Monoclonal antibodies (mAbs) have many applications. For example, they are routinely used in the typing of tissue; in the identification and epidemiological study of infectious microorganisms, tumors, and other surface antigens; in the classification of leukemias; in the identification of functional populations of different types of T cells; and in the identification and mapping of antigenic determinants (epitopes) on proteins. Importantly, mAbs can be conjugated with molecules that provide colorimetric, fluorometric, or enzymatic activity to report the binding of the mAb to specific microbial antigens. Numerous microbial detection kits are available to screen clinical specimens for the presence of specific microorganisms (table 35.4). mAbs in these kits have been produced against a wide variety of bacteria, viruses, fungi, and protozoans, respectively. The mAbs are produced as cross-species or cross-genus isotypes, that is, they have reactivity against epitopes that are common to a number of species within a genus or a number of genera within a class, so as to be used as an adjunct method in the taxonomic identification of the microorganisms being detected. Importantly, those mAbs that define species-specific antigens are extremely valuable in diagnostic reagents, as they can detect the unique species of microorganism causing disease. mAbs that exhibit more restrictive specificity can be used to identify strains or biotypes within a species and in epidemiological studies involving the matching of microbial strains. Coupling sensitive visualization technologies such as fluorescence or scanning tunneling microscopy to mAb detection systems makes it possible to perform microbial identifications with improved accuracy, speed, and fewer organisms.

Table 35.4
Some Common Rapid Immunologic Test Kits for the Detection of Bacteria and Viruses in Clinical Specimens

Culturette Group A Strep ID Kit (Marion Scientific, Kansas City, Mo.)

The Culturette kit is used for the detection of group A streptococci from throat swabs.

Directigen (Hynson, Wescott, and Dunning, Baltimore, Md.)

The Directigen Meningitis Test kit is used to detect H. influenzae type b, S. pneumoniae, and N. meningitidis groups A and C.

The Directigen Group A Strep Test kit is used for the direct detection of group A streptococci from throat swabs.

Gono Gen (Micro-Media Systems, San Jose, Calif.)

The Gono Gen kit detects Neisseria gonorrhoeae.

OraQuick (OraSure Technologies, Bethlehem, Pa.)

Detects HIV antibodies in saliva in 10 minutes.

Staphaurex (Wellcome Diagnostics, Research Triangle Park, N.C.)

Staphaurex screens and confirms Staphylococcus aureus in 30 seconds.

SureCell Herpes (HSV) Test (Kodak, Rochester, N.Y.)

Detects the herpes (HSV) 1 and 2 viruses in minutes.

Importantly, the anthrax attacks in 2001 in the United States spawned a renewed demand for “better, faster, and smarter” microbial detection and identification technologies. While nucleic acid-based detection systems, such as PCR, have garnered much attention as the basis of newer detection systems, antibody-based identification technologies are still considered more flexible and easier to modify. Traditional antibody-based detection technologies are being linked to sophisticated reporting systems that provide “med techs” with an ever-increasing array of cutting-edge technology. Examples of more recent microbial identification technologies include biosensors based on: (1) microfluidic antigen sensors, (2) real-time (20-minute) PCR, (3) highly sensitive spectroscopy systems, and (4) liquid crystal amplification of microbial immune complexes. Some of these technologies are being used as part of military sentinel detection programs; others are awaiting approval by various licensing agencies before being deployed in clinical laboratories. Additional technologies are expected as the demand for immediate, highly sensitive microbial detection increases globally.

bc_background:yellow
  1. Describe in general how biochemical tests are used in the API 20E system to identify bacteria.

  2. Why might cultures for some microorganisms be unavailable?

  3. What are some of the benefits of monoclonal antibodies as diagnostic reagents?

Bacteriophage Typing

Bacteriophages are viruses that attack members of a particular bacterial species or strain within a species. Bacteriophage (phage) typing is based on the specificity of phage surface proteins for cell surface receptors. Only those bacteriophages that can attach to these surface receptors can infect bacteria and cause lysis. On a Petri dish culture, lytic bacteriophages cause plaques on lawns of sensitive bacteria. These plaques represent infection by the virus (see figure 5.20). Viruses and other acellular infectious agents (chapter 5)

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In bacteriophage typing, the clinical microbiologist inoculates the bacterium to be tested onto a Petri plate. The plate is heavily and uniformly inoculated so that the bacteria will grow to form a solid lawn of cells. The plate is then marked off into squares (15 to 20 mm per side), and each square is inoculated with a drop of suspension from the different phages available for typing. After the plate is incubated for 24 hours, it is observed for plaques. The phage type is reported as a specific genus and species followed by the types that can infect the bacterium. For example, the series 10/16/24 indicates that this bacterium is sensitive to phages 10, 16, and 24, and belongs to a collection of strains, called a phagovar, that have this particular phage sensitivity. Bacteriophage typing remains a tool of research and reference laboratories.

Molecular Genetic Methods

Some of the most accurate approaches to microbial identification are through the analysis of proteins and nucleic acids. Examples include comparison of proteins; physical, kinetic, and regulatory properties of microbial enzymes; nucleic acid–base composition; nucleic acid hybridization; and nucleic acid sequencing (see figures 15.6 and 16.2 to 16.6). Other molecular methods being widely used are nucleic acid probes, real-time PCR amplification of DNA, and DNA fingerprinting. Techniques for determining microbial taxonomy and phylogeny (section 17.3)

Polymerase Chain Reaction

Nucleic acid–based diagnostic methods for the detection and identification of microorganisms have become routine in clinical microbiology laboratories. For example, DNA hybridization technology can identify a microorganism by probing its genetic composition. The use of cloned DNA as a probe is based on the capacity of single-stranded DNA to bind (hybridize) with a complementary nucleic acid sequence present in test specimens to form a double-stranded DNA hybrid. These hybrids are more sensitive than conventional microbiological techniques, give results in 2 hours or less, and require the presence of fewer microorganisms. DNA probe sensitivity can be increased by over 1 million times if the target DNA is first amplified using PCR. PCR can also be monitored in “real time” so as to detect microbial DNA amplification after each replicative cycle, identifying the microorganisms after only 25 to 30 amplification cycles (figure 35.6). In other words, DNA amplification can be measured continuously using real-time (rt) PCR (see figure 15.7). The most sensitive methods for demonstrating chlamydiae in clinical specimens involve nucleic acid sequencing and PCR-based methods. Polymerase chain reaction (section 15.2); Molecular characteristics (section 17.3) DNA Hybridization

FIGURE 35.6
PCR Kit Results.Real-time detection of avian influenza is possible using a PCR kit. Note that sufficient DNA amplification occurs after 25 to 30 cycles.

The nucleotide sequence of small subunit ribosomal RNA (rRNA) can be used to identify bacterial genera (see figure 17.3). Usually the rRNA encoding gene or gene fragment is amplified by PCR. After nucleotide sequencing, the rRNA gene nucleotide sequence is compared with those on international databases. This method of bacterial identification, called ribotyping, is based on the high level of 16s rRNA conservation among bacteria. As discussed in chapter 17, multiple “housekeeping” genes, instead of 16S rRNA genes, may be sequenced and analyzed in a technique called multilocus sequence typing (MLST) (see p. 453).

Genomic fingerprinting is also used in identifying pathogens. This does not involve nucleotide sequencing; rather, it compares the similarity of specific DNA fragments generated by restriction endonuclease digestion. BOX-, ERIC-, and REP-PCR are described in section 17.3 (see figure 17.4). Plasmids—autonomously replicating extrachromosomal molecules of DNA—can also be used in DNA fingerprinting. Plasmid fingerprinting identifies microbial isolates of the same or similar strains; related strains often contain the same plasmids (figure 35.7a). In contrast, microbial isolates that are phenotypically distinct have different plasmid fingerprints. Plasmid fingerprinting of many E. coli, Salmonella, Campylobacter, and Pseudomonas strains and species has demonstrated that this method often is more accurate than phenotyping methods such as biotyping, antibiotic resistance patterns, phage typing, and serotyping. Just as in genomic fingerprinting, isolated plasmid DNA is cut with specific restriction endonucleases (see table 15.2). The DNA fragments are then separated by gel electrophoresis to yield a pattern of fragments, which appear as bands in the gel. The molecular weight of each plasmid species can then be determined and patterns of restriction fragments compared. Gel electrophoresis (section 15.3)

 
FIGURE 35.7
Plasmid Fingerprinting.Agarose gel electrophoresis of plasmid DNA. A, B, C: plasmids that have not been digested by endonucleases; a, b, c: the same plasmids following restriction enzyme digestion.
Figure 35.7 Micro Inquiry

How many different plasmids are present in this analysis?

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  1. What is the basis for bacteriophage typing?

  2. How can nucleic acid–based detection methods be used by the clinical microbiologist?

  3. How can a suspect bacterium be plasmid fingerprinted?