Collection Transportation and Processing of Specimens for Culture

The perception that anaerobes have little or no role in many infections originates from the fact that many past studies did not attempt to identify such a role or used improper methods for collecting specimens for anaerobes. Therefore, carefully assessing studies for methodological properties before judging their ability to determine the role of anaerobes in an infectious process is essential. Multiple examples of differences in the rate of recovery of anaerobic bacteria between studies that used proper techniques and those that used improper techniques can be found.

Earlier studies of chronic otitis media (1) and human and animal bites (2), which did not employ methods for anaerobes found these organisms in a small number of cases. However, when better techniques were used, anaerobes were recovered in the majority of the cases (3,4). Because anaerobes may invade any body site, and they have been recovered in a variety of infections in children, anaerobes' potential role in an infectious site should be assessed individually. The prevalence of anaerobic bacteria in an infection is a major factor in deciding which clinical specimens should be processed for anaerobes.

The proper management of anaerobic infection depends on appropriate documentation of the bacteria causing the infection. Without such an approach, the patient may be exposed to inappropriate, costly, and undesirable antimicrobial agents and their adverse side effects.

Anaerobic infections present special bacteriologic problems not encountered in other types of infections, and such problems may make the therapeutic approach even more difficult. Generally, bacteriologic results will not be available so quickly as in aerobic infections, particularly if the infection is mixed (as are more than one-half of the cases). Some laboratories may fail to recover certain or all of the anaerobes present in a specimen. This situation can occur particularly when the specimen is not promptly put under anaerobic conditions for transport to the laboratory. If care is not taken to avoid contamination of the specimen with normal flora, anaerobes may be recovered which have little to do with the patient's illness. As all laboratories are not equipped to identify anaerobes accurately, presumptive results maybe very misleading.

Appropriate cultures for anaerobic bacteria are especially important in mixed aerobic and anaerobic infections. Techniques or media that are inadequate for isolation of anaerobic bacteria, either because of a lack of an anaerobic environment or because of an overgrowth of aerobic organisms, can mislead the clinician to assume that the aerobic organisms recovered are the only pathogens present in an infected site, therefore causing the clinician to direct therapy toward only those aerobic organisms.

The nature of the various organisms in a mixed infection will also influence the choice of drugs. Drugs active against anaerobic bacteria may be quite inactive against the accompanying aerobic or facultative organisms. When mixed infections involve several organisms, two or more drugs may be required to provide effective coverage for each of the organisms in the mixture.

Because anaerobic bacteria frequently can be involved in various infections, ideally, all properly collected specimens should be cultured for these organisms. The physician should make special efforts to isolate anaerobic organisms in infections in which these organisms are frequently recovered, such as abscesses, wounds in and around the oral and anal cavities, chronic otitis media and sinusitis, aspiration pneumonia, and intraabdominal and obstetrical and gynecological infections among others.

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

Aspirates obtained under computed tomography or ultrasound guidance (e.g., abdominal

abscesses)

Tissue or bone

Surgical specimen using tissue biopsy scraping or curette

Sinuses or mucus surface

Aspiration after decontamination or surgical specimen

abscesses

Ear

Aspiration after decontamination of ear canal and membrane; in perforation: cleanse ear

canal and aspirate through perforation

Pulmonary

Transtracheal aspiration, lung puncture or bronchial lavage,3 and bronchial brushing3

Pleural

Thoracentesis

Urinary tract

Suprapubic bladder aspiration

Female genital tract

Culdocentesis following decontamination, surgical specimen, transabdominal needle

aspirate of uterus, and intrauterine brush3

a Using double-lumen catheter and quantitative culture.

a Using double-lumen catheter and quantitative culture.

The most acceptable documentation of an anaerobic infection is through culture of anaerobic microorganisms from the infected site. Three elements requiring the cooperation of the physician and the microbiology laboratory are essential for appropriate documentation of anaerobic infection: collection of appropriate specimens, expeditious transportation of the specimen, and careful laboratory processing.

COLLECTION OF SPECIMENS

Specimens must be obtained free of contamination so that saprophytic organisms or normal flora are excluded, and culture results can be interpreted correctly (Table 1). Because indigenous anaerobes often are present on the surfaces of skin and mucous membranes in large numbers, even minimal contamination of a specimen with the normal flora can give misleading results. On this basis, specimens can be designated according to their acceptability for anaerobic culture to either the acceptable or unacceptable category. Materials that are appropriate for anaerobic cultures should be obtained using a technique that bypasses the normal flora. Unacceptable or inappropriate specimens can be expected to yield normal flora also and therefore have no diagnostic value. Sites that are normally inhabited by a rich indigenous flora, such as the oral cavity, intestinal tract, or vagina, should not be cultured for anaerobes except under specific circumstances. Unacceptable specimens include coughed sputum, bronchoscopy aspirates, gingival, and throat swabs, feces, gastric aspirates, voided urine, and vaginal swabs (Table 2). Exceptions to these guidelines can be made when the clinical condition warrants such a culture. For example, selective media may be used to detect only a possible pathogen, such as Clostridium difficile, in stool obtained from a patient with colitis.

Acceptable specimens 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 (TTA), direct lung puncture,

TABLE 2 Specimens that Should Not Be Cultured for Anaerobes

Feces or rectal swabs Throat or nasopharyngeal swabs Sputum or bronchoscopic specimens Routine or catheterized urine Vaginal or cervical swabs

Material from superficial wound or abscesses not collected properly to exclude surface contaminations Material from abdominal wounds obviously contaminated with feces, such as an open fistula

Collection, Transportation, and Processing of Specimens TABLE 3 Specimens Appropriate for Anaerobic Culture

All normally sterile body fluids other than urine, such as blood, pleural, and joint fluids Urine obtained by suprapubic bladder aspiration

Percutaneous transtracheal aspiration, direct lung puncture, or double-lumen catheter bronchial brushing and bronchoalveolar lavage (both cultured quantitatively) Culdocentesis fluid obtained after decontamination of the vagina Material obtained from closed abscesses Material obtained from sinus tracts or draining wounds or double-lumen catheter bronchial brushing and bronchoalveolar lavage (Table 3). Direct needle aspiration is probably the best method of obtaining a culture, while use of swabs is much less desirable. Specimens obtained from sites that normally are sterile may be collected after thorough skin decontamination, as is for the collection of blood, spinal joint, or peritoneal fluids.

Cultures of coughed sputum and specimens obtained from bronchial brushing or bronchoscopy except for those done via a protective double-lumen catheter generally are contaminated with normal oral and nasal aerobic and anaerobic flora and are therefore unsuitable for culture. Acceptable respiratory specimens include: percutaneous or TTA, bronchial brushing collected via a double-lumen protected catheter, "protected" bronchoalveolar lavage, direct lung puncture, thoracentesis fluid, and lung tissue. Because the trachea below the thyroglossal area is sterile in the absence of pulmonary infection, TTA, which is done below this site, is a reliable procedure for obtaining suitable culture material for the diagnosis of pulmonary infection (5,6). TTA is usually not recommended in patients with severe hypoxia, hemorrhagic diathesis, or severe cough (7). Rare complications, such as hypoxia, bleeding, subsequent emphysema, or arrhythmia, have been reported in adult patients (8). In children, side effects of this procedure included mild hemoptysis and, in rare instances, subcutaneous emphysema. TTA has been used successfully also for the diagnosis of aspiration pneumonia and lung abscess in children (6). Cultures obtained by TTA contained fewer pathogens than did cultures of expectorated sputum.

Some clinical situations may present the clinician with difficult issues regarding obtaining an adequate culture, such as a tracheal culture of an intubated patient with tracheobronchitis, endometrial culture in patients with suspected endometritis after delivery, or a tonsillar surface culture searching for beta-lactamase-producing bacteria. In all these instances, the cultures from surrounding areas of the infected sites show similar isolates to those isolated from the infectious condition. Therefore, selective search for virulent organisms only, such as anaerobic gram negative bacilli (AGNB) or beta-lactamase-producing bacteria, may be helpful.

Diagnosis and Cultures

Cultures are helpful but nonessential for diagnosis in some infections such as tetanus, botulism, and gas gangrene. In some infections, such as minor skin and soft-tissue infections or ruptured appendix anaerobes are part of the infectious flora, but their presence does not need to be documented. However, their identification may be necessary when complication occurs (i.e., generalized peritonitis, bacteremia) in very young or very old patients with underlying serious illnesses, in those who require prolonged therapy, or in infections that failed to respond to empirical therapy. Even in these instances, it is not always necessary to identify all isolates, and it may be sufficient to search for antibiotic-resistant ones such as the Bacteroidesfragilis group.

Even though it is important to obtain cultures prior to therapy, it may be still important to get them after the patient has been treated for a while. Since it may take at least several days and sometimes even longer to obtain definite bacterial information, generation of interim reports may assist in the management of seriously ill patients.

TRANSPORTATION OF SPECIMENS

The ability to recover anaerobes is influenced by the care applied to transportation and laboratory processing of specimens. Unless proper precautionary measures are taken during collection, transport, and laboratory processing, pronounced changes can occur in the aerobic and anaerobic microbial population of a clinical specimen (9). Sensitivity to oxygen causes some obligate anaerobes to die rapidly when exposed to air. In clinical samples, obligate anaerobes can be overgrown by facultative anaerobes unless the sample is processed rapidly after collection. The organisms must be protected, therefore, from the deleterious effects of oxygen during the time between the collection of the specimen and the inoculation of that specimen into the proper anaerobic medium in the microbiology laboratory. Failure to take proper precautions may result in misleading data, which may be detrimental to the patient (9-13).

Anaerobes vary in the conditions they require for survival. In accordance with their oxygen sensitivity, some organisms are classified as "moderate" and some as "fastidious." The moderate group is capable of growing in a 2% to 8% oxygen concentration. B. fragilis, Prevotella oralis, Prevotella melaninogenica, Fusobacterium nucleatum, and Clostridium perfringens belong to this group. Some fastidious anaerobes will grow at 0.5% oxygen levels, and some are extremely oxygen sensitive, such as some strains of B. fragilis and peptostreptococci (14). Low oxidation-reduction potential is another basic requirement for growth of certain anaerobic bacteria, as for Bacteroides vulgatus and Clostridium sporogenes (15). Such conditions usually exist in areas where anaerobes are present as part of the normal flora and at infected sites. The implication of these observations is that specimens must be carefully and rapidly handled in both transporting and processing to ensure good recovery of anaerobes.

The specimens should be placed into an anaerobic transporter containing anaerobic transport medium with an oxidation-reduction indicator as soon as possible after their collection. Aspirates of liquid specimen or tissue are always preferred to swabs, although systems for the collection of all three culture forms are commercially available (Fig. 1). Several versions of the anaerobic transport media also are commercially available (Baltimore Biological Laboratories, Cockeysville, Maryland, U.S.A.; Anaerobe Systems, Morgan Hill, California, U.S.A.).

These transport media are very helpful in preserving the anaerobes until the time of inoculation. Liquid specimen is best aspirated into a syringe through a needle and injected

Portacul Bacteria Collection
FIGURE 1 Commercial transport media used for the transportation of anaerobic specimens. Left, swab; middle, vial; right, syringe and needle.

into the anaerobic (oxygen-free) transport vial containing an oxidation-reduction indicator. Contrary to past recommendations, syringes used for aspiration should not be utilized for transportation because spillage of their contents could be hazardous, there is a potential danger of needle stick injuries, and because oxygen diffuses into plastic syringes (within 30 minutes). Body fluids can be transported in sterile tubes, especially if they contain more than 1 mL, with as small an airspace above the fluid level as possible, and kept upright to avoid mixing with air.

Swabs may be placed in the sterilized tubes containing carbon dioxide or prereduced, anaerobically sterile Carey and Blair semisolid media. A preferred method is to use a swab that has been prepared in a prereduced anaerobic tube. However, this is not commercially available.

Tissue specimens or swabs can be transported anaerobically in a Petri dish placed in a sealed plastic bag that can be rendered anaerobic by use of an anaerobic generator (BBL Microbiological Systems, Cockeysville, Maryland, U.S.A.) (Fig. 2). Alternatively, small pieces of tissue may be placed into the anaerobic transporter by removing the cap and pushing the tissue into the agar. Most of the common and clinically important anaerobic bacteria are moderate anaerobes, as shown by the examination of various types of clinical specimens for anaerobes (14,15).

Syed and Loesche (16) studied the survival of human dental plaque flora in various transport media and concluded that because numbers and kinds of microorganisms in clinical materials vary widely, no transport device should be expected to give optimal protection for all anaerobes that may be encountered in specimens. Even though some of the transport systems can support the viability of anaerobic organisms for up to 24 hours (17,18), all specimens should be transported and processed as rapidly as possible after collection to avoid loss of fastidious oxygen-sensitive anaerobes and overgrowth of facultative bacteria.

Anaerobic Bags Pouches For Anaerobes
FIGURE 2 Commercial anaerobic bag system used for transportation of tissue or other specimens.

When delay in transportation is expected, specimen should be kept at room temperature, as cold temperature enhances oxygen diffusion, and incubator temperature cause loss of some bacterial strains and overgrowth of others.

We have observed significant differences in the recovery rate of anaerobic bacteria from abscesses when we compared two commercially available transport media. One system was far superior to the other, although both were licensed for use (19). Because many studies that document the efficacy of transport systems for anaerobes use stock cultures (18) and not clinical specimens, the clinical microbiology laboratory should evaluate the performance of each system in clinical specimen before accepting the system for clinical use.

PROCESSING OF SPECIMENS IN THE LABORATORY

Laboratory diagnosis of anaerobic infections begins with observing the gross appearance (necrosis, pus) and odor, as well as the examination of a Gram-stained smear of the specimen. Putrid or fetid smell in a clinical sample is almost always associated with the presence of anaerobes and is due to the production of volatile short-chain fatty acids and amines by these organisms. The appearance and relative number of the Gram-stained organisms will give important preliminary information regarding types of organisms present, suggest the need for special selective media, suggest appropriate initial therapy, preserve the relative proportions of organisms present at the time of specimen collection, and serve as a quality control on the final culture analysis. The laboratory should be able to recover all of the morphological types in the approximate ratio in which they are seen.

When necessary, phase-contrast or dark-field microscopy can help detect the presence of motile organisms, spores, and morphotypes (i.e., spirochetes) that do not grow on ordinary media.

Immunofluorescence staining can assist in detecting special organisms such as Actinomyces spp. and Propionibacterium propionicus. Unfortunately, this method is not specific enough for B. fragilis group and other AGNB.

The techniques for cultivation of anaerobes should provide optimal anaerobic conditions throughout processing. Detailed procedures of these methods can be found in microbiology manuals (12,13). Briefly, these methods could be the prereduced tube method (i.e., the VPI roll tube method) or the anaerobic glove box technique, which provides an anaerobic environment throughout processing, or the GasPak system (Becton Dickinson Co., Cockeysville, Maryland, U.S.A.) or the Bio-Bag system (Pfizer Diagnostics Division, Groton, Connecticut, U.S.A.), which is a more simplified method.

As a minimum requirement for the recovery of anaerobes, specimens should be inoculated onto enriched nonselective blood agar medium (containing vitamin Kj and hemin) such as Brucella, trypticase soy, or schaedler agar; for anaerobic gram-negative bacilli, a selective medium such as laked sheep blood agar with kanamycin and vancomycin. Bacteroides bile esculin agar allows the growth of B. fragilis group and Bilophila wadsworthia; phenylethylalcohol agar excludes swarming Proteus spp. and other aerobic gram-negative bacilli. For Clostridium, egg yolk-neomycin agar may be used.

Although vitamin Kj-enriched thioglycolate broth (steamed before use) is generally used as a backup culture, this media alone should never be used as a substitute for a solid media. Interestingly, however, many clinical laboratories still use liquid media. The major limitation of such media is the probability of overgrowth of slow-growing strict anaerobes by rapid-growing aerobic and facultative organisms.

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 aero-tolerance, and identified.

An additional period of 36 to 48 hours is generally required to completely identify the anaerobic bacteria to a species or genus level, using biochemical tests. Kits containing these biochemical tests are commercially available (Pfizer Diagnostics Division, Groton, Connecticut, U.S.A.). Rapid kits that detect preformed enzymes are commercially available. They require a heavy inoculum, take a short incubation period (four hours in air), and have a 60-90% identification capability (20). Other rapid tests that have potential use and can also be used directly on clinical isolates are the direct fluorescent microscopy and direct gas liquid chromatography. Gas liquid chromatography has been used to assist in the identification of anaerobes (13) and has also been used for presumptive rapid and direct identification of these organisms in pus specimens (21). Nucleic acid probes have been developed for identification of indicator bacteria of periodontal disease. Currently, polymerase chain reaction methods with sequencing of the 16S RNA gene has become the new "gold standard" for identification of anaerobes (22,23).

Blood Cultures

It is advisable to inoculate two bottles in a ratio of 1 mL of blood to 10 mL of media; one bottle should be vented to optimize recovery of strict aerobes and the other unvented for the isolation of anaerobes. Care should be taken not to introduce air to the anaerobic bottle when inoculating with the blood, and avoid shaking the bottle to avoid further aeration. Bottles showing growth should be subcultured anaerobically, and negative culture bottles should be held for a week.

There are several commercially available blood culture media that are adequate for recovery of anaerobes (13). Automated system enabling detection of anaerobes in blood culture bottles that detect released radioactive CO2 (13).

Identification of an anaerobe to a species level is often cumbersome, expensive, and time-consuming, taking up to 72 hours. The decision of what level of testing is necessary for identifying an anaerobic organism is often a controversial issue. Usually, the clinician has to make such a decision. Occasionally, species identification of an organism will provide the diagnosis, as is the case with C. difficile in a patient with colitis or Clostridium botulinum in infants with botulism (11). However, because the origin of most anaerobes is endogenous, there are rarely epidemiological reasons to obtain their complete identification. Identifying the B. fragilis group that is more often causing bacteremia and septic complications has significant prognostic value.

Identification of an anaerobe is most helpful in determining what antibiotic to use in these species whose antibiotic susceptibility is predictable. Until the late 1970s, most clinically significant anaerobes except B. fragilis group were susceptible to penicillin (11). Therefore, extensive identification and antibiotic susceptibility testing were unnecessary. In the last decade, however, there have been significant changes, and now there is more variability in antimicrobial susceptibility patterns (see chapters 37 & 38). These changes have necessitated more extensive identification as well as antimicrobial susceptibility testing for some anaerobic bacteria. Organisms that should be identified include the following:

  1. Isolates from sterile body sites (i.e., blood, cerebrospinal fluid, joint).
  2. An organism with particular epidemiological or prognostic significance (e.g., C. difficile).
  3. An organism with known variable or unique susceptibility.

ANTIMICROBIAL SUSCEPTIBILITY OF ANAEROBIC BACTERIA (SEE ALSO CHAPTERS 37 & 38)

The susceptibility of anaerobic bacteria to antimicrobial agents has become less predictable. Resistance to several antimicrobial agents by B. fragilis group and other AGNB has increased over the past decade (24). A decrease in susceptibility to penicillin of C. perfringens has been noted (25). And the susceptibility of Clostridium species (other than C. perfringens) is variable and often unpredictable. Anaerobic organisms to be selected for susceptibility testing should include these organisms.

The tests most useful for individual isolates are the Etest (AB Biodisk, Solna, Sweden) which is relatively expensive and the microbroth dilution test (these commercial trays do not always contain all the appropriate antimicrobials) (26). In addition to susceptibility testing, screening of anaerobic isolates (particularly Bacteroides species) for beta-lactamase activity may

TABLE 4 Anaerobic Infections for which Susceptibility Testing Is Indicated

Serious or life-threatening infections (e.g., brain abscess, bacteremia, or endocarditis)

Infections that failed to respond to empiric therapy

Infections that relapsed after initially responding to empiric therapy

Infections where an antimicrobial will have a special role in the patients outcome

When an empirical decision is difficult because of absence of precedent

When there are few susceptibility data available on a bacterial species

When the isolate(s) is often resistant to antimicrobial

When the patient requires prolonged therapy (e.g., septic arthritis, osteomyelitis, undrained abscess, or infection of a graft or a prosthesis)

be helpful. We routinely screen AGNB for beta-lactamase production using the nitrocefin disc. Such beta-lactamase screening of these isolates rapidly provides information regarding their penicillin susceptibility. It should be borne in mind that a longer-than-usual period (up to one hour) may be required for some organisms to show a positive reaction. Occasional bacterial strains may resist beta-lactamase antibiotics through mechanisms other than the production of beta-lactamase.

It is important to perform susceptibility testing to isolates recovered from sterile body sites, those that are recovered in pure culture or those that are clinically important and have variable or unique susceptibility.

The fact that routine susceptibility testing of all anaerobic isolates is time-consuming and in many cases unnecessary must be recognized. Therefore, susceptibility testing should be limited to selected anaerobic isolates (Table 4) (27). Antibiotics tested should include penicillin, a broad-spectrum penicillin, a penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, cefoxitin, a third-generation cephalosporin, metronidazole, tigecycline, a carbapenem (i.e., imipenem), and an extended spectrum quinolone (i.e., moxifloxacin) (28).

Correlation of the results of in vitro susceptibility and clinical and bacteriological response is not always possible. This discrepancy occurs because of a variety of reasons: individuals may improve without antimicrobial or surgical therapy, infections vary in duration, severity, and extent; failure can occur because of lack of needed surgical drainage; response depends on individual patients status such as underlying condition, age, and nutritional status; and the antimicrobial may not be effective because of enzymatic inactivation or a low Eh or pH at the infection site, low concentration at the site of infection; and because of variations or imperfections in the susceptibility testing. It is not necessary to eliminate all of the infecting organisms because reduction in counts or modification of the metabolism of certain isolates alone may be sufficient to achieve a good clinical response. Synergy between two or more infecting organisms, which is a common event in anaerobic infections, may confuse the clinical picture.

CONCLUSION

The physician treating a patient with suspected anaerobic infection must use appropriate methods of obtaining samples of the infected site. Proper procedure allows the physician to bypass areas of the normal flora and assures appropriate and rapid transportation of the sample. Reliable microbiological data can be obtained only when proper procedures are followed.

REFERENCES

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  10. Dowell VR, Jr., Hawkins TM. Laboratory methods in anaerobic bacteriology, CDC laboratory manual. U.S. Department of Health, Education, and Welfare. Atlanta: Center for Disease Control (publ. no. (CDC) 74-8272).
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  19. Brook I. Comparison of two transport systems for recovery of aerobic and anaerobic bacteria from abscesses. J Clin Microbiol 1987; 25:2020-2.
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Responses

  • mandy wechsler
    How is was collected when an aerobic organisms are suspected?
    9 months ago
  • donnie thompson
    How to process a specimen for anaerobic culture?
    4 months ago
  • LARA
    What samples are suitable to culture for anaerobes?
    3 months ago

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