Microbiology & Antimicrobial Susceptibility Testing of Anaerobes

DIAGNOSTIC MICROBIOLOGY

Collection of specimens for anaerobic bacteria

The proper management of anaerobic infection depends on appropriate documentation of the bacteria causing the infection. Three essential elements require the physician’s cooperation with the microbiology laboratory for appropriate documentation of anaerobic infection: collection of appropriate specimens, their expeditious transportation and careful laboratory processing.^1,2

Specimens must be obtained free of contamination so that saprophytic organisms or normal flora are excluded and culture results can be interpreted correctly. Because indigenous anaerobes are often present on the surfaces of skin and mucous membranes in large numbers, even minimal contamination of a specimen with normal flora can give misleading results. On this basis, specimens can be designated according to their acceptability for anaerobic culture to the acceptable or the unacceptable category. Materials that are appropriate for anaerobic cultures should be obtained using a technique that bypasses the normal flora (Table 1). Unacceptable or inappropriate specimens can yield normal flora also and therefore have no diagnostic value. ^2,3 (Table 2)

Acceptable specimens (Table 3) include blood specimens, aspirates of body fluids (pleural, pericardial, cerebrospinal, peritoneal and joint fluids), urine collected by percutaneous suprapubic bladder aspiration, abscess contents, deep aspirates of wounds, and specimens collected by special techniques, such as transtracheal aspirates or direct lung puncture. Specimens of the lower respiratory tract are difficult to obtain without contamination. Double lumen catheter bronchial brushing and bronchoalveolar lavage, cultured quantitatively, are useful. Direct-needle aspiration is probably the best method of obtaining a culture, and the use of swabs is much less desirable. Specimens obtained from sites that are normally sterile may be collected after thorough skin decontamination, as is the case for the collection of blood, or spinal, joint or peritoneal fluids. ^1,2 

                                                Needle aspirate of an abscess

Appropriate cultures for anaerobes are especially important in polymicrobial infections.1 Techniques or media that are inadequate for isolation of anaerobes, because of a lack of an anaerobic environment or because of an overgrowth of aerobic organisms, can be misleading. This may cause the clinician to direct therapy toward only the isolated aerobic organisms.

Transportation of specimens

Prompt delivery of specimens to the laboratory is essential. Various transport devices are available that generate an oxygen-free environment. These systems generally contain an oxygen-free environment provided by mixture of carbon dioxide, hydrogen and nitrogen, plus an indicator that illustrates aerobic conditions. The specimens should be placed into an anaerobic transporter as soon as possible after collection. Aspirates of liquid specimen or tissue are always preferred to swabs, although systems for the collection of all culture forms are commercially available.

Liquid specimens are inoculated into a commercially available anaerobic transport vial. A plastic or glass syringe and needle are also used for transport. After collection all air bubbles are expelled from the syringe. Plastic syringe should be processed within 30 minutes. Swabs are placed in sterilized tubes containing carbon dioxide or prereduced, sterile Carey and Blair semisolid media. Tissue specimens are transported in an anaerobic jar or in a Petri dish inside a sealed plastic bag that can be rendered anaerobic by use of a catalyzer.

                                 

                                Port A-Cul (BBL) swab transportation system

Laboratory diagnosis

Certain findings are suggestive of anaerobic infection (Table 4). However, laboratory diagnosis begins with the examination of a Gram-stained smear of the specimen. The appearance of the Gram-stained organisms will give important preliminary information regarding types of organisms present, suggest appropriate initial therapy and serve as a quality control on the final culture analysis. The laboratory should be able to recover all of the morphologic types in the ratio in which they are seen.

The techniques for cultivation should provide optimal anaerobic conditions throughout processing. Detailed procedures of these methods can be found in microbiology manuals.1 These could be the prereduced tube method, the anaerobic glove-box technique, or the anaerobic jar or bag systems.

Anaerobic jar (GasPack BBL)

Anaerobic glove-box

Specimens should be inoculated onto enriched blood agar medium (containing vitamin K1 and hemin) and a selective medium (for Bacteroides spp.), such as laked sheep blood agar with kanamycin and vancomycin. The use of selective media along with nonselective one increases the recovery rate and can shorten the time to identification of organisms. Prereduced vitamin K1 enriched thioglycolate broth is used as a backup. However, this media alone should never be used as a substitute for a solid media. ³

Cultures should be placed immediately under anaerobic conditions and incubated for 48 hours or longer. Plates should then be examined for approximate number and types of colonies present. Each colony type should be isolated, tested for aerotolerance and identified.

An additional period of 36–48 hours is generally required to identify completely the anaerobic bacteria to a species or genus level using biochemical tests. Kits containing these biochemical tests are commercially available. These are good with fast growing anaerobes (i.e. Bacteroides fragilis and Clostridium perfringens). Rapid enzymatic tests are also available. This panel test allows identification after only 4 hours incubation. Other rapid tests are the direct fluorescent microscopy and gas liquid chromatography. Direct cellular fatty acid analysis using gas liquid chromatography with capillary column can also be useful. Nucleic acid probes and polymerase chain reaction methods are developed for rapid identification of anaerobes.

Most clinical microbiology laboratories are able to identify the major anaerobic bacteria. Peptostreptococci are generally not speciated because they are generally susceptible to commonly used antimicrobials. Clostridium spp. can be identified by the presence of spores and their ability to survive 30 minutes exposure to ethanol or heating to 80°C for 10 minutes. Nonspore-forming Gram-positive bacilli can be speciated by gas liquid chromatography and biochemical tests. Propionibacterium spp., which are often a contaminant, can be separated from other nonspore-forming Gram-positive bacilli by a catalyze test and indole reaction. B. fragilis group grows on 20% bile and is generally catalase positive. 
Pigmented Prevotella and Porphyromonas spp. produce black or brown pigment within a week when growing on rabbit blood agar medium. Fusobacterium spp. have distinct morphology on Gram stain and, in contrast to Bacteroides spp., are susceptible to kanamycin.

Identification of an anaerobe to a species level is often cumbersome, expensive and time-consuming, taking up to 72 hours. Identification is most helpful in selecting an antibiotic against a species that has predictable antibiotic susceptibility. Occasionally, species identification of an organism will provide the diagnosis, as with Clostridium difficile in colitis or Clostridium botulinum in botulism. Identifying B. fragilis group, which often cause bacteremia and septic complications, has significant prognostic value. However, because most anaerobes are endogenous, there are rarely epidemiologic reasons to perform complete identification.

ANTIMICROBIAL SUSCEPTIBILITY TESTING

The antimicrobial susceptibility of anaerobes has become less predictable. Resistance to several antimicrobials, especially by anaerobic Gram-negative bacilli (AGNB) and Fusobacterium spp., has increased.4 It is important to test susceptibility of anaerobes recovered from sterile body sites, those with particular epidemiologic or prognostic significance (e.g. C. difficile), or those that are clinically important and have variable susceptibilities.

Screening of AGNB isolates (particularly Prevotella, Bacteroides and Fusobacterium spp.) for beta-lactamase activity is helpful. However, occasional resistance is through other mechanisms.

Routine susceptibility testing is extremely time-consuming and often unnecessary. It should be limited to isolates from blood, bone, central nervous system and serious infections, as well as to those recovered in pure culture. Antibiotics tested should include penicillin, a  broad-spectrum penicillin, a penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, a second-generation cephalosporin (e.g. cefoxitin), metronidazole and a carbapenem . 5

The method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) includes agar dilution testing, microbroth and macrobroth dilution.4 Newer methods include the E-test and the spiral gradient end-point system.

                         E-test susceptibility testing method for anaerobes

REFERENCES

1.   Summanen P, Baron EJ, Ciron DM, et al. Wadsworth anaerobic bacteriology manual. 6th ed. Belmont, CA: Star Publishing; 2002.

2.   Finegold SM. Anaerobic bacteria in human disease. Orlando, FL: Academic Press Inc; 1977.

3. Brook. Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals. J Clin Microbiol. 1988 ;26:1181-8.

4. National Committee for Clinical Laboratory Standards Working Group on Susceptibility Testing of Anaerobic Bacteria.. Methods for antimicrobial susceptibility testing of anaerobic bacteria. 4th ed.; approved standard. NCCLS document M11-A4. Vol. 13, No. 26. Villanova, PA, 1997.

5. Snydman DR, Jacobus NV, McDermott LA, et al. National survey on the susceptibility of Bacteroides Fragilis Group: report and analysis of trends for 1997-2000. 

Clin Infect Dis. 2002 ;35(Suppl 1):S126-34.

Table 1:  Methods for collection of specimen for anaerobic bacteria

Infection site	Methods
Abscess or body cavity	Aspiration by syringe and needle
	Incised abscesses - syringe, or swab (less desirable); specimen obtained during surgery after cleansing the skin
Tissue or bone	Surgical specimen using tissue biopsy or curette
Sinuses or mucus surface abscesses	Aspiration after decontamination or surgical specimen
Ear	Aspiration after decontamination of ear canal and membrane; in perforation:  cleanse ear canal and aspirate through perforation
Pulmonary	Transtracheal aspiration, lung puncture, bronchoscopic aspirate*
Pleural	Thoracentesis
Urinary tract	Suprapubic bladder aspiration
Female genital tract	Culdocentesis following decontamination, surgical specimen Transabdominal needle aspirate of uterus intrauterine brush*

                            




*using double lumen catheter and quantitative culture.

Table 2: SPECIMENS THAT SHOULD NOT BE CULTURED FOR ANAEROBES

1)   Feces or rectal swabs

2)   Throat or nasopharyngeal swabs

3)   Sputum or bronchoscopic specimens

4)   Routine or catherized urine

5)   Vaginal or cervical swabs

6)   Material from superficial wound or abscesses not collected properly to exclude surface

      contaminations

7)   Material from abdominal wounds obviously contaminated with feces, such as an open fistula

Table 3: SPECIMENS APPROPRIATE FOR ANAEROBIC CULTURE

1)   All normally sterile body fluids other than urine, such as blood, pleural, and joint fluids

2)   Urine obtained by suprapubic bladder aspiration

3)   Percutaneous transtracheal aspiration, direct lung puncture, or double lumen catheter bronchial

      brushing and bronchoalveolar lavage (both cultured quantitavely).

4)   Culdocentesis fluid obtained after decontamination of the vagina

5)   Material obtained from closed abscesses

6)   Material obtained from sinus tracts or draining wounds

Table 4. Bacteriological finding suggestive of anaerobic infection.

1.   Inability to grow in aerobic cultures, organisms seen on Gram stain of the original material

2.   Typical morphology for anaerobes on Gram stain

3.   Anaerobic growth on proper media containing antibiotic-suppressing aerobes

4.   No growth or routine bacterial culture (“sterile-pus”)

5.   Growth in anaerobic zone of fluid or agar media

6.   Growth anaerobically on media containing puromomycin, Kanamycin, neomycin or   vancomycin.

7.   Gas, foul-smelling odor in specimen or bacterial culture

8.   Characteristic colonies on anaerobic plates

9.   Young colonies of Pigmented Prevotella and Porphyromonas may fluoresce red under ultraviolet light, and older colonies produce a typical dark pigment

10. Characteristic colonies on agar plates under anaerobic conditions (e.g. Clostridium perfringens, Fusobacterium nucleatum).