Overview and General Considerations

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Overview and General Considerations

Objectives

This chapter provides an overview of the methods used to identify anaerobic microorganisms. The detailed technical procedures discussed should be used in conjunction with specifics provided in Chapter 42 to develop a clear understanding of the full process, from specimen collection to identification. However, readers should consider the following general objectives for the information and methods provided.

1. State the specific diagnostic purpose for the test methodology.

2. Briefly describe the test principle associated with the test methodology.

3. Outline limitations and describe a process for trouble-shooting or reporting results if a test result is equivocal or indistinguishable.

4. State the appropriate quality control organisms and results used with each testing procedure.

5. Define and differentiate obligate (strict), moderate, facultative, and aerotolerant anaerobes.

6. List suitable specimens for isolation of anaerobic bacteria and characteristics of these specimens that might suggest the presence of an anaerobic infection.

7. Explain the proper techniques for collecting, transporting, and processing clinical specimens for anaerobic bacteriology.

8. Explain the use of antigen detection methodologies in the diagnosis of anaerobic infections.

9. List the media used for cultivation of anaerobic bacteria.

10. Describe the appropriate incubation conditions for cultivation of anaerobic bacteria.

11. Describe the procedures for the identification of and antibiotic susceptibility testing for anaerobic bacteria.

General Characteristics

The organisms described in this chapter and in Chapter 42 usually do not grow in the presence of oxygen (O2); they are obligate, or strict, anaerobes (0% O2). Obligate anaerobes are killed upon brief exposure (less than a few minutes) to atmospheric oxygen. Obligate anaerobes include Prevotella sp., Fusobacterium sp., and Bacteroides spp. These chapters also include some aerotolerant organisms (5% O2), such as Actinomyces spp., Bifidobacterium spp., and Clostridium spp., which are capable of growth in the presence of either reduced or atmospheric oxygen but grow best under anaerobic conditions. Finally, facultative anaerobes do not require atmospheric oxygen but are capable of growth in oxygen and anaerobic environments.

Anaerobic organisms lack superoxide dismutase and catalase, the enzymes required to breakdown reactive oxygen species produced during respiration or aerobic metabolism. In addition, oxygen has a high affinity for organic compounds containing nitrogen, hydrogen, carbon, and sulfur, which interferes with normal biologic activity. Because they are unable to protect themselves against the action of oxygen, anaerobes require an environment free of oxygen to survive and grow.

Specimen Collection and Transport

The importance of proper collection and transport of specimens for anaerobic culture cannot be overemphasized. Because indigenous anaerobes are often present in large numbers as normal flora on mucosal surfaces, even minimal contamination of a specimen can produce misleading results. Box 41-1 shows the specimens acceptable for anaerobic culture; Box 41-2 presents specimens that are likely to be contaminated and therefore are unacceptable for anaerobic culture. In general, material for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe. Use of swabs is a poor alternative because of excessive exposure of the specimen to the deleterious effects of drying, the possibility of contamination during collection, and the easy retention of microorganisms in the fibers of the swab. If a swab must be used, it should be from an oxygen-free transport system.

A crucial factor in obtaining valid results with anaerobic cultures is the transport of the specimen; the lethal effect of atmospheric oxygen must be nullified until the specimen can be processed in the laboratory. Recapping a syringe and transporting the needle and syringe to the laboratory is no longer acceptable because of safety concerns involving needle stick injuries. Therefore, even aspirates must be injected into an oxygen-free transport tube or vial.

Three kinds of anaerobic transport systems are shown in Figures 41-1 to 41-3. Figure 41-1 shows is a rubber-stoppered collection vial containing an agar indicator system. The vial is gassed out with oxygen-free carbon dioxide (CO2) or nitrogen. The specimen (pus, body fluid, or other liquid material) is injected through the rubber stopper after all air has been expelled from the syringe and needle. If only a swab specimen can be obtained, a special collection device with an oxygen-free atmosphere is required (see Figure 41-2). When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO2 or nitrogen to spill out and be displaced by ambient air. A tissue specimen can be immersed in a small amount of liquid to prevent it from drying and then placed in an anaerobic pouch (see Figure 41-3). All specimens should be held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen.

Direct Detection Methods

Antigen Detection

The cytotoxin (toxin B) of Clostridium difficile can be detected using a tissue culture assay. This assay, performed in various cell lines, is based on the neutralization of cytopathic effect when the cell-free fecal extract is adsorbed using either Clostridium sordellii or C. difficile antitoxins. Latex particle agglutination tests or enzyme-linked immunosorbent assays (ELISA) to detect toxin A or toxin B (or both) are also available.

Screening of stool samples for the production of glutamate dehydrogenase (GDH) using enzyme immunoassays is commonly used. GDH-positive samples should be examined for production of toxin A and B. Commercially available DNA-based methods are available. However, C. difficile colonization occurs, and therefore molecular testing does not indicate C. difficile enteric disease.

Gram Staining

The Gram stain is an important rapid tool for anaerobic bacteriology. Not only does it reveal the types and relative numbers of microorganisms and host cells present, it also serves as a quality control measure for the adequacy of anaerobic techniques. The absence of leukocytes does not rule out the presence of a serious anaerobic infection, however, because certain organisms, such as clostridia, produce necrotizing toxins that destroy white blood cells. A positive Gram stain with a negative culture may indicate (1) poor transport methods, (2) excessive exposure to air during specimen processing, (3) failure of the system (jar, pouch, or chamber) to achieve an anaerobic atmosphere, (4) inadequate types of media or old media, or (5) killing of microorganisms by antimicrobial therapy.

Standard Gram stain procedures and reagents are used, except that the safranin counterstain is left on for 3 to 5 minutes. Gram-negative anaerobes often stain poorly with safranin, resulting in failure to visualize pathogenic organisms. As an alternative, 0.5% aqueous basic fuchsin can be used as the counterstain to improve identification of gram-negative anaerobes. In addition, some gram-positive anaerobes (e.g., Clostridium spp.) stain pink. Enhanced Gram stain reagents are available that contain different concentrations in the reagents, in addition to a Gram Enhancer, which is applied after decolorization to suppress the red color in the background, aiding the differentiation of gram-negative anaerobes.

Table 41-1 presents the cellular morphology seen with Gram staining of common anaerobes.

TABLE 41-1

Gram Stain Morphology, Colonial Appearance, and Other Distinguishing Features of Common Anaerobic Bacteria

Organism Gram Stain* Media Appearance
Actinomyces spp. Gram-positive, branching, beaded or banded, thin, filamentous rods Ana BAP Colonies of most species are small, smooth, flat, convex, gray-white, translucent, with entire margins; colonies of A. israelii and A. gerencseriae are white, opaque, and may resemble a “molar tooth”; A. odontolyticus turns red after several days in ambient air and may be beta-hemolytic
Anaerococcus spp. Gram-positive cocci arranged in short chains or tetrads Ana BAP Small, white, translucent, smooth
Atopobium spp. Elongated gram-positive cocci; occur singly, in pairs, or in short chains Ana BAP Resemble lactobacilli
Bacteroides distasonis Gram-negative, straight rods with rounded ends; occur singly or in pairs Ana BAP Gray-white, circular, entire, convex, smooth, translucent to opaque; nonhemolytic
BBE At 48 hr, colonies are >1 mm, circular, entire, raised, and either (1) low convex, dark gray, friable, and surrounded by a dark gray zone (esculin hydrolysis) and sometimes a precipitate (bile) or (2) glistening, convex, light to dark gray, and surrounded by a gray zone
B. fragilis Gram-negative, pale-staining, pleomorphic rods with rounded ends; occur singly or in pairs; cells often described as resembling a safety pin (see Figure 42-4) Ana BAP White to gray, circular, entire, convex, translucent to semiopaque; nonhemolytic (see Figure 42-5)
BBE At 48 hr, colonies are >1 mm, circular, entire, raised, and either (1) low convex, dark gray, friable, and surrounded by a dark gray zone (esculin hydrolysis) and sometimes a precipitate (bile) or (2) glistening, convex, light to dark gray, and surrounded by a gray zone (see Figure 42-6)
B. ovatus Gram-negative, ovoid rods with rounded ends; occur singly or in pairs Ana BAP Pale buff, circular, entire, convex, semiopaque; often mucoid; nonhemolytic
BBE At 48 hr, colonies are >1 mm, circular, entire, raised, and either (1) low convex, dark gray, friable, and surrounded by a dark gray zone (esculin hydrolysis) and sometimes a precipitate (bile) or (2) glistening, convex, light to dark gray, and surrounded by a gray zone
B. thetaiotaomicron Gram-negative, irregularly staining, pleomorphic rods with rounded ends; occur singly or in pairs Ana BAP White, circular, entire, convex, semiopaque, shiny, punctiform; nonhemolytic
BBE At 48 hr, colonies are >1 mm, circular, entire, raised, and either (1) low convex, dark gray, friable, and surrounded by a dark gray zone (esculin hydrolysis) and sometimes a precipitate (bile) or (2) glistening, convex, light to dark gray, and surrounded by a gray zone
B. ureolyticus Gram-negative, pale-staining, thin, delicate rods with rounded ends; some curved Ana BAP Small, translucent or transparent; may produce greening of agar on exposure to air; colonies corrode (pit) the agar (see Figure 42-9) or may be smooth and convex or spreading
B. vulgatus Gram-negative, pleomorphic rods with rounded ends; occur singly, in pairs, or in short chains; swellings or vacuoles may be seen Ana BAP Gray, circular, entire, convex, semiopaque; nonhemolytic
BBE At 48 hr, colonies are >1 mm, circular, entire, raised, glistening, convex, light to dark gray but with no gray zone (esculin not hydrolyzed)
Bifidobacterium spp. Gram-positive diphtheroid; coccoid or thin, pointed shape; or larger, highly irregular, curved rods with branching; rods terminate in clubs or thick, bifurcated (forked) ends (“dog bones”) Ana BAP Small, white, convex, shiny, with irregular edge
Bilophila wadsworthia Gram-negative, pale-staining, delicate rods Ana BAP Small, translucent
BBE Grows at 3-5 days; colonies are usually gray with a black center because of production of hydrogen sulfide (H2S); black center may disappear after exposure to air
Clostridium botulinum Gram-positive, straight rods; occur singly or in pairs; spores usually subterminal and resemble a tennis racket Ana BAP Gray-white; circular to irregular; usually beta-hemolytic
C. clostridioforme Gram-positive rod that stains gram negative; long, thin rods; spores usually not seen; elongated football shape with cells often in pairs Ana BAP Small, convex, entire edge; nonhemolytic
C. difficile Gram-positive straight rods; may produce chains of up to six cells aligned end to end; spores oval and subterminal Ana BAP Large, white, circular, matte to glossy, convex, opaque; nonhemolytic; horse stable odor; fluoresces yellow-green
CCFA Yellow, ground-glass colony (see Figure 42-2)
C. perfringens Gram-variable straight rods with blunt ends; occur singly or in pairs; spores seldom seen but if present are large and central to subterminal, oval, and swell cell; large boxcar shapes (see Figure 44-6) Ana BAP Gray to grayish yellow; circular, glossy, dome shaped, entire, translucent; double zone of beta-hemolysis (see Figure 42-3)
C. ramosum Gram-variable straight or curved rods; spores rarely seen but are round and terminal; more slender and longer than C. perfringens Ana BAP Small, gray-white to colorless; circular to slightly irregular, smooth, translucent or semiopaque; nonhemolytic
C. septicum Gram positive in young cultures but becomes gram negative with age; stains unevenly; straight or curved rods; occur singly or in pairs; spores subterminal, and oval and swell cells Ana BAP Gray; circular, glossy, translucent; markedly irregular to rhizoid margins resembling a “Medusa head”; beta-hemolytic; swarms over entire agar surface in less than 24 hr
C. sordellii Gram-positive rods; subterminal spores Ana BAP Large colony with irregular edge
C. sporogenes Gram-positive rods; subterminal spores Ana BAP Colonies firmly adhere to agar; may swarm over agar surface
C. tertium Gram-variable rods; terminal spores Ana BAP Resembles Lactobacillus spp.
C. tetani Gram positive, becoming gram negative after 24-hr incubation; occur singly or in pairs; spores oval and terminal or subterminal with drumstick or tennis racket appearance Ana BAP Gray; matte surface, irregular to rhizoid margin, translucent, flat; narrow zone of beta-hemolysis; may swarm over agar surface
Collinsella aerofaciens Gram-positive chains of coccoid cells Ana BAP Circular, entire, white center with translucent edge
Eggerthella lenta Gram-positive, small, straight rod with rounded ends Ana BAP Small, gray, translucent, circular, entire, convex
Eubacterium spp. Gram-positive pleomorphic rods or coccobacilli; occur in pairs or short chains; E. alactolyticum has a seagull-wing shape similar to Campylobacter spp.; E. nodatum is similar to Actinomyces spp. with beading, filaments, and branching Ana BAP Small, gray, transparent to translucent, raised to convex; colonies of E. nodatum may resemble A. israelii
Finegoldia magna Gram-positive cocci with cells > 0.6 µm in diameter; in pairs and clusters; resemble staphylococci Ana BAP Tiny, gray, translucent; nonhemolytic
Fusobacterium mortiferum Gram-negative, pale-staining, irregularly stained, highly pleomorphic rods with swollen areas, filaments, and large, bizarre, round bodies Ana BAP Circular; entire or irregular edge, convex or slightly umbonate, smooth, translucent; nonhemolytic
BBE >1 mm in diameter, flat and irregular
F. necrophorum subsp. necrophorum Gram-negative, pleomorphic rods with round to tapered ends; may be filamentous or contain round bodies; becomes more pleomorphic with age Ana BAP Circular, umbonate, ridged surface, translucent to opaque; fluoresces chartreuse; greening of agar on exposure to air; some strains beta-hemolytic
F. nucleatum subsp. nucleatum Gram negative; pale staining; long, slender, spindle-shaped with sharply pointed or tapered ends; occasionally cells occur in pairs end to end; resembles Capnocytophaga spp. (see Figure 42-11) Ana BAP Three colony types: bread crumb–like (white; see Figure 42-10), speckled, and smooth (gray to gray-white); greening of agar on exposure to air; fluoresces chartreuse; usually nonhemolytic
F. varium Gram negative, unevenly staining, pleomorphic; coccoid and rod shapes; occurs singly or in pairs Ana BAP Gray-white center with colorless edge resembling a fried egg; circular, entire, convex, translucent; nonhemolytic
BBE >1 mm in diameter, flat and irregular
Lactobacillus spp. Gram-variable pleomorphic rods or coccobacilli; straight, uniform rods have rounded ends; short coccobacilli resemble streptococci Ana BAP Resemble Lactobacillus spp. colonies on aerobic blood or chocolate agar, except colonies are usually larger when incubated anaerobically
Leptotrichia spp. Gram-negative, large, fusiform rods with one pointed end and one blunt end Ana BAP Large, raspberry-like colonies
Parvimonas micra Gram-positive cocci with cells < 0.7 µm in diameter; occur in packets and short chains Ana BAP Tiny, white, opaque; nonhemolytic
Mobiluncus spp. Gram-variable, small, thin, curved rods; the two species can be divided based on cell length Ana BAP Tiny colonies after 48 hr-incubation; after 3-5 days, colonies are small, low convex, and translucent
Peptococcus niger Gram-positive, spherical cells; occur singly or in pairs, tetrads, and irregular masses Ana BAP Tiny, black, convex, shiny, smooth, circular, entire edge; becomes light gray when exposed to air
Peptostreptococcus anaerobius Gram-positive, large coccobacillus; often in chains Ana BAP Medium, gray-white, opaque; sweet, fetid odor; colonies usually larger than most anaerobic cocci
Porphyromonas spp. Gram-negative coccobacilli Ana BAP Dark brown to black; more mucoid than Prevotella spp.; except for P. gingivalis, fluoresces brick red
Prevotella disiens Gram-negative rods; occur in pairs or short chains Ana BAP White, circular, entire, convex, translucent to opaque, smooth, shiny; nonhemolytic; fluoresces brick red
LKV Black pigment
P. melaninogenica Gram-negative coccobacilli Ana BAP Dark center with gray to light brown edges; circular, entire, convex, smooth, shiny; nonhemolytic; fluoresces brick red
LKV Black pigment
Propionibacterium spp. Gram-positive, pleomorphic, diphtheroid-like rod; club-shaped to palisade arrangements; called anaerobic diphtheroids Ana BAP Young colonies are small and white to gray-white and become larger and more yellowish tan with age; P. avidum is beta-hemolytic
Veillonella parvula Gram-negative, tiny diplococci in clusters, pairs, and short chains; unusually large cocci, especially in clusters, suggests Megasphaera or Acidaminococcus spp. Ana BAP Small, almost transparent; grayish white; smooth, entire, opaque, butyrous; may show red fluorescence under UV light (360 nm)

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Ana BAP, Anaerobic blood agar plate; BBE, Bacteroides bile esculin agar; CCFA, cycloserine cefoxitin fructose agar; LKV, laked kanamycin-vancomycin blood agar; UV, ultraviolet.

*Typical Gram stain appearance is seen from broth (thioglycollate or peptone-yeast-glucose).

Specimen Processing

Specimens for anaerobic culture may be processed in the biologic safety cabinet, after which they are incubated in anaerobic jars or pouches or in an anaerobic chamber. The roll tube method developed at Virginia Polytechnic Institute is no longer widely used and is not discussed here.

Anaerobe Jars or Pouches

The most frequently used system for creating an anaerobic atmosphere is the anaerobe jar. Anaerobe jars are available commercially from several companies. For example, the GasPak (Figure 41-4) is made by Becton Dickinson (Sparks, Maryland); other companies that produce these devices include EM Diagnostic Systems (Gibbstown, New Jersey) and Oxoid U.S.A. (Columbus, Maryland). All of these systems use a clear, heavy plastic jar with a lid that is clamped down to make it airtight. Anaerobic conditions can be set up by two methods. The easiest method uses a commercially available envelope containing a hydrogen and CO2 generator that is activated either by adding water (GasPak) or by the moisture on the agar plates (EM Diagnostic Systems and Oxoid USA). The production of heat within a few minutes (detected by touching the top of the jar) and subsequent development of moisture on the walls of the jar are indications that the catalyst and generator envelope are functioning properly. Reduced conditions are achieved in 1 to 2 hours, although the methylene blue or resazurin indicators take longer to decolorize. Alternatively, the “evacuation-replacement” method can be used. Air is removed from the sealed jar by drawing a vacuum of 25 inches (62.5 cm) of mercury. This process is repeated two times, with the jar being filled with an oxygen-free gas, such as nitrogen, between evacuations. The final fill of the jar is made with a gas mixture containing 80% to 90% nitrogen, 5% to 10% hydrogen, and 5% to 10% CO2. Many anaerobes require CO2 for maximal growth.

The atmosphere in the jars is monitored using an indicator to check anaerobiosis. Anaerobe bags or pouches are useful for laboratories processing small numbers of anaerobic specimens. A widely used anaerobic pouch, the GasPak Pouch, is shown in Figure 41-3. Besides specimen transport, the pouch also can be used to incubate one or two agar plates.

Anaerobe Chamber

Anaerobic chambers, or glove boxes, are made of molded or flexible clear plastic. The flexible clear plastic chambers are the most widely used type. Specimens and other materials are placed in the chamber through an air lock. The technologist uses gloves (Forma Scientific, Marietta, Ohio) or sleeves (Sheldon Manufacturing, Cornelius, Oregon), to form airtight seals around the arms (Figure 41-5). Media stored in the chamber are kept oxygen free, and all work on a specimen, from inoculation through workup, is performed under anaerobic conditions. A gas mixture of 5% CO2, 10% hydrogen, and 85% nitrogen, plus a palladium catalyst, maintain the anaerobic environment inside the chamber.

Anaerobic Media

Initial processing of anaerobic specimens involves inoculation of appropriate media. Table 41-2 lists commonly used anaerobic media. Primary plates should be freshly prepared or used within 2 weeks of preparation. Plates stored for longer periods accumulate peroxides and become dehydrated; this results in growth inhibition. Reduction of media in an anaerobic environment eliminates dissolved oxygen but has no effect on the peroxides. Prereduced, anaerobically sterilized (PRAS) media are produced, packaged, shipped, and stored under anaerobic conditions. They are commercially available from Anaerobe Systems (Morgan Hill, California) (Figure 41-6) and have an extended shelf life of up to 6 months.

TABLE 41-2

Common Anaerobic Media

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Medium Components/Comments Primary Purpose
Anaerobic blood agar May be prepared with Columbia, Schaedler, CDC, Brucella, or brain-heart infusion base supplemented with 5% sheep blood, 0.5% yeast extract, hemin, L-cystine, and vitamin K1 Nonselective medium for isolation of anaerobes and facultative anaerobes
Bacteroides bile esculin agar (BBE) Trypticase soy agar base with ferric ammonium citrate and hemin; bile salts and gentamicin act as inhibitors Selective and differential for Bacteroides fragilis group; good for presumptive identification