Three major groups of anaerobic bacteria account for approximately 80% of the diagnosed anaerobic infections. These are Bacteroides spp, Peptostreptococcus spp and Clostridium spp. The most frequently encountered species in each genus are: Bacteroides fragilis, Peptostreptococcus magnus, and Clostridium perfringens. Specific methods for the isolation and identification of these three genera and species should also serve as a model for isolating and initiating the identification process of other clinically important anaerobic bacteria.
Specimen collection and transport to the laboratory
This has been covered thoroughly in previous sections. Swabs are to be avoided for the collection of specimens, as anaerobes are very sensitive to air and drying. Specimens for the culture of anaerobes should be taken carefully from the active site of infection. The services of a surgeon may be required for the collection of some specimens. This is particularly true of needle aspiration of pus, obtaining tissue and/or pus specimens from infected wounds, empyema, or draining abscesses.
The specimen should be placed in a sterile, tightly closed container, or if one of these is not available, the entire aspirated specimen should be transported immediately to the laboratory in the syringe, with the needle capped or protected by a rubber stopper.
Establishing an anaerobic environment for incubating cultures
A variety of methods exist for creating an anaerobic environment. One that is simple and inexpensive is the use of an anaerobic jar made of thick glass or polycarbonate, with a capacity of 2.5 - 3.5 litres, and equipped with a secure gasproof lid that is easily removed and replaced. After putting the inoculated Petri dishes into the jar, an anaerobic atmosphere is generated by introducing a commercially available disposable anaerobiosis-generating device, and closing the lid. A simple and dependable device is the Anaerocult A (Merck, Darmstadt, Germany, Cat. No. 13829). This is a paper tray containing dry reagents. Just before use, 35 ml of water are poured evenly over the surface of the paper, using a graduated cylinder. This activates the binding of oxygen and the release of carbon dioxide. The moistened tray is then placed in the jar along with the inoculated plates. The lid is tightly secured and the jar is placed in the incubator at 35 °C for 48 hours. If necessary one or more plates may be observed for growth or colony type after 24 hours, but a new Anaerocult A must be activated and placed in the jar before reincubation. Other disposable devices for generating anaerobiosis take the form of flat, sealed, foil envelopes that release hydrogen and carbon dioxide after addition of water. These devices, however, require a palladium catalyst fixed on the underside of the lid of the jar. The catalyst becomes inactivated during use and should be regenerated or replaced at regular intervals, as recommended by the manufacturer. Disposable redox-indicator strips, which change from blue (or red) to colourless in an anaerobic atmosphere, are available from a number of manufacturers.
Tubes of broth cultures for anaerobes, such as thioglycollate broth or cooked meat broth, need not be incubated under anaerobic conditions, because their formulations contain reducing substances that will create an anaerobic environment. When the volume of broth is sufficient (10 - 12 ml per 15-mm diameter standard screw-top tube) and the medium freshly prepared, anaerobic conditions are produced in the lower part of the tube. If not used on the day of preparation, the tubes should be heated with the screw-top loosened for about 15 minutes in a boiling water-bath to remove dissolved oxygen, the screw-top tightened, and the medium allowed to cool before inoculation.
Anaerobic culture media
Anaerobic cultures should be performed only when requested by the clinician, when the specimen has a foul smell, or when the result of the Gram-stained smear indicates the possibility of an anaerobic infection, e.g., the presence of a pleomorphic mixed flora of Gram-positive and Gram-negative rods and cocci, the presence of Gram-negative fusiform rods, or the presence of square-ended thick Gram-positive rods that may be Clostridium.
Routine anaerobic cultures should not be done on urine, genital secretions, faeces, or expectorated sputum; the presence of anaerobic bacteria in these specimens is indicative of contamination with the normal commensal flora of the respective specimen site. Clinicians should be informed that specimens containing normal flora are not acceptable for anaerobic cultures unless there is strong justification.
Ordinary blood agar is a good plating medium for isolation of the most important anaerobic pathogens. For isolation of the more fastidious species, a blood agar base enriched with growth factors (haemin and menadione) is recommended. Such a base is commercially available as Wilkins-Chalgren anaerobe agar.
Anaerobic bacteria are often part of a complex microflora, also involving aerobic organisms, and anaerobic blood agar may be made selective by the addition of one or more specific antibiotics. For example, the addition of an aminoglycoside (neomycin, kanamycin) in a final concentration of 50 μg/ml inhibits the majority of aerobic and facultative bacteria. A solution of the aminoglycoside is prepared by dissolving 500 mg in 100 ml of distilled water. Melt 100 ml of anaerobic agar base and when it has cooled to 56 °C, add aseptically 5 ml of defibrinated blood and 1 ml of the antibiotic solution. Mix well, and aseptically pour about 15 - 18 ml into sterile Petri plates. These plates should be used as soon as possible, or kept in a refrigerator preferably in a plastic bag or sleeve.
Inoculation and isolation procedures
Specimens from suspected anaerobic infections should be inoculated without delay on to the following media:
• an anaerobic blood agar to be incubated in an anaerobic jar;
• an aerobic blood agar to be incubated in a candle jar;
• a plate of MacConkey agar;
• a tube of anaerobic broth (thioglycollate or cooked meat).
The aerobic cultures should be inoculated and processed as usual and examined after 24 and 48 hours for aerobic and facultative organisms. A small area of the anaerobic blood agar plate should be inoculated and the inoculum streaked out with a loop. The plates should be incubated and the anaerobic jar opened after 48 hours for inspection. If growth is insufficient, the plates may be reincubated for a further 24 or 48 hours. The broth culture should be heavily inoculated with a Pasteur pipette so as to distribute the inoculum throughout the medium in the tube.
After 48 hours, the growth on the anaerobic blood agar should be inspected and compared with the growth on the aerobic plating media. Each colony type should be examined with Gram stain. Bacteria with the same microscopic appearance that grow on aerobic and anaerobic agar are considered to be facultative anaerobes. Colonies that appear only on anaerobic agar are probable anaerobes and should be subcultured on two blood agar plates, one to be incubated anaerobically and one in a candle jar. If growth appears only in anaerobiosis, identification of a pure culture of the anaerobe should be attempted.
If growth is observed in the depths of anaerobic broth, it should be subcultured to aerobic and anaerobic blood agar and examined in the same way as the primary culture plates. As the liquid culture is inoculated with a larger volume of pus, it may be positive when the primary plates remain sterile.
Identification of medically important anaerobes
Bacteroides fragilis group
This group includes several related species that belong to the normal flora of the intestine and the vagina. They are frequently involved in abdominal and pelvic mixed infections and may also cause bacteraemia. B. fragilis is a non-motile Gram-negative rod, often showing some pleomorphism, growing rapidly on anaerobic blood agar. After 48 hours, the colonies are of moderate size (up to 3 mm in diameter), translucent, grey-white, and non-haemolytic. Rapid identification is possible with the bile stimulation test. A pure culture of the suspected organism is inoculated in the depth of two tubes of thioglycollate broth, with one of the tubes containing 20% (2 ml in 10 ml) sterile (autoclaved) ox bile. After 24 hours’ incubation, the growth in the two tubes should be compared: the growth of B. fragilis is clearly stimulated in the broth supplemented with bile.
The genus Clostridium contains many species of sporulating Gram-positive rods, some of which belong to the normal gut flora while others are found in dust and soil. The clinically most significant species is C. perfringens. It is commonly associated with gas gangrene and may also cause bacteraemia and other deep infections. Unlike most other species of Clostridium, it is nonmotile and does not form spores in the infected tissues or in young cultures.
C. perfringens grows rapidly in anaerobic broth with the production of abundant gas. On anaerobic blood agar, colonies of moderate size (2 - 3 mm) are seen after 48 hours. Most strains produce a double zone of haemolysis: an inner zone of complete clear haemolysis, and an outer zone of partial haemolysis.
Rapid identification is possible with the reverse CAMP test, which is performed as follows (see Fig. 6):1
1 HANSEN, M. V. & ELLIOT, L. P. New presumptive identification test for Clostridium perfringens: reverse CAMP test. Journal of clinical microbiology, 12: 617-619 (1980).
• Prepare a blood agar plate with 5% thrice-washed sheep blood.
• Streak a pure culture of Streptococcus agalactiae along the diameter of the plate. Streak the suspected Clostridium culture in a line perpendicular to, but not touching, the S. agalactiae.
• Incubate in the anaerobic jar for 24 hours.
C. perfringens forms an arrowhead-shaped area of enhanced haemolysis at the junction of the two streaks. CAMP-negative clostridia may be reported as “Clostridium sp. not C.
Several species of obligate anaerobic Gram-positive cocci belong to the commensal flora of the respiratory, digestive, and urogenital tracts. They are involved, usually in association with other aerobic or anaerobic bacteria, in anaerobic abscesses, wound infections, and even bacteraemia. The growth of anaerobic cocci in laboratory media is usually slower than that of Bacteroides or Clostridium and colonies are usually not apparent on blood agar until after 48 hours of incubation.
Species identification is not needed in routine bacteriology. Gram-positive cocci that produce small, convex, white colonies on anaerobic blood agar, but do not grow in aerobic conditions, may be presumptively identified as Peptostreptococcus spp.
Fig. 6. Reverse CAMP test
Antibiotic susceptibility tests
Antibiotic susceptibility tests should not routinely be performed on anaerobic bacteria, in view of the present lack of agreement on a standardized disc diffusion test.
Most anaerobic infections are caused by penicillin-sensitive bacteria, with the exception of infections originating in the intestinal tract or the vagina. Such infections generally contain Bacteroides fragilis, which produces β-lactamase and is resistant to penicillins, ampicillins, and most cephalosporins. Such infections can be treated with clindamycin, metronidazole, or chloramphenicol. Aminoglycosides have no activity against anaerobes, but they are often used for the treatment of patients who have mixed infections, because of their effectiveness against aerobic bacteria, which are often part of the complex flora.