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Chapter 4 : Anaerobic Bacteriology

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Abstract:

This chapter talks about the diagnosis of anaerobic bacterial infections involving various steps. Some of the most important considerations involved in the laboratory diagnosis of anaerobic infections include (i) selecting, collecting, and transporting specimens for microbiologic examination and (ii) processing and examining the specimens in the laboratory as rapidly as possible after they are received. Satisfactory commercially available primary isolation media and anaerobic incubation systems are discussed along with detailed recommendations for their use. The current software release (version 3.9) used in the authors' laboratory contains an extensive list of anaerobe genera and species. At present, simple-to-use commercial kits and equipment for specific molecular applications in clinical anaerobic bacteriology have been lacking. It appears that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) could be a powerful tool for rapid identification of bacteria that would otherwise be difficult to differentiate with other methods. This new technology holds promise for markedly decreasing the turnaround time for identification of anaerobes and other slow growing microorganisms, thereby enhancing the clinical relevance of this area of clinical microbiology.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4

Key Concept Ranking

Bacterial Classification
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Antimicrobial Susceptibility Testing
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Lower Respiratory Tract Infections
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Image of FIGURE 1
FIGURE 1

Inoculation of conventional PRAS biochemical test media.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 2
FIGURE 2

Presumpto quadrant plates 1, 2, and 3. One plate of CDC Anaerobe Blood Agar is also shown. See text and Table 8 for details.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 3
FIGURE 3

WEE-TABS stand-alone chromogenic tests. Each tube contains one tablet and a small volume of bacterial suspension.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 4
FIGURE 4

API 20A test strip. Miniaturized cupules are inoculated with a bacterial suspension and after 24 to 48 h of incubation, biochemical reactions are visualized.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 5
FIGURE 5

Minitek Anaerobe II tray containing paper disks impregnated with biochemical substrates. After inoculation with a bacterial suspension and 48 h of anaerobic incubation, positive reactions are indicated by color changes (see text).

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 6
FIGURE 6

API ZYM. Miniaturized cupules are inoculated with a turbid bacterial suspension. Nineteen enzymatic reactions are read and interpreted after a 4-h aerobic incubation.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 7
FIGURE 7

An-IDENT. This was one of the first commercially available 4-h packaged kits with a database for the identification of anaerobes.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 8
FIGURE 8

RapID ANA II. This 4-h rapid test system for the identification of anaerobes contains 10 wells for testing the performance of 18 preformed enzymatic tests.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 9
FIGURE 9

Vitek ANI card. This 4-h test system is incubated aerobically and comprises a single plastic card with 30 wells for performing various chromogenic and conventional biochemical tests.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 10
FIGURE 10

MicroScan Rapid Anaerobe Identification Panel. This 96-well microdilution tray contains dehydrated substrates and is incubated for 4 h aerobically.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 11
FIGURE 11

rapid ID 32A. A plastic strip with 32 cupules, 29 of which contain dehydrated substrates for rapid biochemical characterization of anaerobes, is incubated for 4 h aerobically.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 12
FIGURE 12

BBL Crystal ANR ID. Thirty wells in a plastic base are aligned and snapped together with a lid containing 29 biochemical and enzymatic substrates. Some reactions are visualized with a UV light reader (upper left-center), while others are read and interpreted using ambient light. (Photo courtesy of Becton Dickinson.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 13
FIGURE 13

Tox-B cytotoxin assay. Steps involved in the assay protocol are shown. (Photo courtesy of Tech Lab.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 14
FIGURE 14

Culturette Brand CDT latex agglutination test for glutamate dehydrogenase. (Photo courtesy of Becton Dickinson, Cockeysville, Md.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 15
FIGURE 15

ImmunoCard This rapid EIA detects glutamate dehydrogenase and is used as a screening test for

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 16
FIGURE 16

VIDAS immunoassay analyzer (see text).

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 17
FIGURE 17

Tox A/B EIA. Steps involved in the assay protocol are shown. (Photo courtesy of Tech Lab.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 18
FIGURE 18

Premier Cytoclone CD Toxin A/B. Refer to text for details. (Photo courtesy of Meridian Diagnostics.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 19
FIGURE 19

Triage Panel. This rapid membrane EIA detects glutamate dehydrogenase and toxin A. Positive results are indicated by bars appearing next to each corresponding antigen (center of panel). (Photo courtesy of Biosite Diagnostics.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 20
FIGURE 20

ColorPAC Toxin A rapid membrane EIA test kit. (Photo courtesy of Becton Dickinson.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 21
FIGURE 21

MIDI Sherlock Microbial Identification System, an automated GC analytical system. Included in the photo are the following: a Hewlett-Packard 6890 GC with an automatic sampler, a computer, and a printer. The GC contains a fused-silica capillary column and a flame ionization detector.

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Image of FIGURE 22
FIGURE 22

MALDI-TOF-MS. An example of a composite spectrum (bottom tracing in middle panel) that was produced from three bacterial mass spectral fingerprints is matched with an 89% probability to the reference spectrum of a strain of in the database. The lower panel shows the difference between the test spectrum and the database entry. (Photograph courtesy of Micromass UK Limited.)

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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References

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Tables

Generic image for table
TABLE 1

Examples of specimens and collection procedures in anaerobic bacteriology

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 2

Commercially available anaerobe transport devices

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 3

Commercially available anaerobic incubation systems: manufacturers and catalog list prices

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 4

Manufacturers and vendors of anaerobe chambers and GCs

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 5

Selection of conventional PRAS biochemical tests to differentiate anaerobe isolates

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 6

Commercially available products for anaerobe identification and list prices

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 7

Media and characteristics of anaerobe isolates that can be determined using the Presumpto quadrant plate procedure for anaerobe identification

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 8

Practical and simple manual methods for characterization and presumptive identification of anaerobes

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 9

Source of supplies for disk tests used in presumptive identification of anaerobes

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 10

Tests available in single-tablet format for assessing preformed enzymatic activity of anaerobes

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 11

Miniaturized and rapid commercial packaged kit systems for identification of anaerobes

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 12

Test substrates available in the API ZYM system for detecting preformed enzymatic activities

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 13

Data compiled from published commercial kit evaluations on the identification of commonly encountered anaerobes to genus and species level, correctly without the use of additional tests

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 14

Summary of biochemical test reactions or substrates available in the 4-h commercial packaged microsystems

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Table 15

Identification of anaerobic bacteria by rapid ID 32 A

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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Table 16

Identification of anaerobic bacteria using the BBL Crystal ANR ID System

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 17

Tests for the detection of antigens in stool specimens

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
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TABLE 18

Commercially available products related to testing

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4
Generic image for table
TABLE 19

Composite performance data compiled for six immunodiagnostic products marketed for detection of

Citation: Allen S, Emery C, Siders J. 2002. Anaerobic Bacteriology, p 50-83. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch4

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