Download LABORATORY ASSESSMENT OF ANAEROBIC BACTERIA

EDUCATIONAL COMMENTARY – LABORATORY ASSESSMENT OF ANAEROBIC BACTERIA
Learning Outcomes
Upon completion of this exercise, the participant will be able to:
•
list 8 indications that an infection is caused by anaerobic bacteria.
•
discuss factors to consider when deciding whether to presumptively or definitively identify
anaerobic isolates.
•
discuss circumstances that warrant susceptibility testing on anaerobic isolates.
Anaerobic bacteria occur both in the environment and as normal inhabitants of humans and other
animals, and can cause disease in 2 ways. A person can become ill either by ingesting toxins produced
by anaerobic bacteria (as occurs in botulism food poisoning), or by infection with the organism itself.
Infection, which causes most disease, occurs in 3 ways. First, anaerobic infections can occur when an
anatomic barrier is compromised, thus allowing normal anaerobic flora to enter a sterile site. This can
happen either when a physical barrier is broken during surgery or other trauma, or when other host
defenses are weakened by malignancy, diabetes, burns, immunosuppressive therapy, or aspiration.
Infections caused in this manner are termed endogenous infections.
Second, infections can result when anaerobic bacteria native to the environment contaminate wounds;
these are termed exogenous infections. Clostridium species cause most exogenous infections, but
infection caused by Sarcina ventriculi, Fusobacterium ulcerans, Desulfovibrio desulfuricans, and
Desulfomonas species also sometimes occur. Finally, infections by anaerobic bacteria can spread
through person-to-person contact. For example, nosocomial infections caused by Clostridium difficile are
transmitted person-to-person.
Normal anaerobic flora cause most anaerobic infections. Also, many anaerobic infections are
polymicrobial, which means that cultures grow multiple species of bacteria. These 2 factors can make it
hard to determine whether an anaerobic isolate is a contaminant, a contributor to infection along with
other bacteria, or the exclusive cause of infection.
Indications of Infection With Anaerobic Bacteria
One sign that an infection is caused by anaerobic bacteria is growth of an anaerobic isolate from a
suitable specimen that has been properly collected, transported, and processed. To successfully isolate
anaerobes, specimens must be collected in an oxygen-free system. In general, specimens suitable for
anaerobic culture are those that are collected by tissue biopsy or aspirated by needle and syringe
(Table), because these specimens are unlikely to be contaminated by normal anaerobic flora. Most
specimens collected by swab, on the other hand, are unsuitable because they are likely to be
contaminated by normally occurring anaerobes (Table).
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EDUCATIONAL COMMENTARY – LABORATORY ASSESSMENT OF ANAEROBIC BACTERIA
(cont.)
Table. Specimens Suitable and Unsuitable for Anaerobic Culture.*
Anatomic Site
Suitable Specimen
Unsuitable Specimen
Respiratory
Bronchial washings (only if obtained
with double-lumen plugged
catheter)
Percutaneous lung aspirate or biopsy
Transtracheal aspirate
Bronchial washings or brushings
Expectorated sputum
Nasopharyngeal swab
Secretions obtained by
nasotracheal or orotracheal
suction
Throat swabs
Genitourinary
Endometrial biopsy tissue obtained
with endometrial suction curette
Suprapubic bladder aspirate
Uterine contents obtained by
protected swab
Culdocentesis aspirate
Urethral swabs
Vaginal or cervical swabs
Voided or catheterized urine
Gastrointestinal
Tissue obtained by biopsy
Gastric or small bowel contents (only
in blind loop syndrome)
Stool specimens
Rectal swabs
Gastric or small bowel contents
(except in blind loop syndrome)
Ileostomy or colostomy drainage
Normally sterile fluids
Bile
Blood
Cerebrospinal fluid
Aspirated synovial fluid
Peritoneal (ascetic) fluid
Thoracentesis (pleural) fluid
N/A
Wounds, abscesses, tissues
Bone marrow
Decubitus ulcer (from base of lesion
after debridement)
Material aspirated from abscesses
Sulfur granules from draining fistulas
Tissues obtained by biopsy or
autopsy
Swabs from superficial skin lesions
(wounds, abscesses, burns,
cysts, ulcers)
* Table originally appeared in the American Proficiency Institute’s 2004 3rd Test Event educational
commentary on anaerobic bacteria. Reprinted with permission. ASCP©2004.
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EDUCATIONAL COMMENTARY – LABORATORY ASSESSMENT OF ANAEROBIC BACTERIA
(cont.)
Seven additional clues that an anaerobic bacterium is the cause of infection are:
1. The infection is near a mucosal surface. (Mucosal surfaces are colonized predominantly with
anaerobes.)
2. Aminoglycoside therapy does not resolve the infection. (Most anaerobes are resistant to
aminoglycosides.)
3. A foul odor is present. (Porphyromonas, Fusobacterium, and Clostridium difficile produce a foul
odor.)
4. A large amount of gas is present. (Clostridium species produce gas.)
5. A black color or brick-red fluorescence occurs. (Some Porphyromonas and Prevotella species
produce a pigment that turns black or fluoresces brick red.)
6. The organism has characteristic Gram stain morphology. (Bacteroides and Fusobacterium are
pleomorphic; Fusobacterium nucleatum is fusiform, and Clostridium species are large, grampositive rods with or without spores.)
7. Sulfur granules are present. (Actinomyces often produces sulfur granules.)
Identification of Anaerobic Bacteria
Identification of anaerobic bacteria is important both for patient care and for epidemiological studies.
Knowing the organism’s identity can help the physician choose a therapy that is most likely to cure the
infection. Also, the identity of an anaerobic isolate often indicates the likely source of the infection. For
example, the presence of Porphyromonas suggests that the infection was caused by contamination with
bacteria from the oral cavity. Finally, identification of anaerobic isolates helps build a database of
information about the epidemiology of these organisms.
Most laboratories perform presumptive rather than definitive identification of anaerobic bacteria in most
situations. Presumptive identification is quick and economical, and it usually suffices to enable the
physician to choose an effective treatment. Definitive identification, on the other hand, is both expensive
and time-consuming, and it is usually not needed to implement an appropriate therapy.
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(cont.)
Presumptive identification of anaerobes is based on colony morphology, Gram stain appearance, and a
variety of tests. These include:
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Aerotolerance test to confirm that the organism is an anaerobe
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Fluorescence under long-wave ultraviolet light. Many pigmented Porphyromonas and Prevotella
species fluoresce brick red, F. nucleatum and C. difficile fluoresce chartreuse, and Veillonella
species fluoresce red.
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Special-potency antimicrobial disks (5-µg vancomycin, 1-mg kanamycin, 10-µg colistin) to
confirm the Gram stain and begin preliminary grouping
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Sodium polyanethol sulfonate (SPS) disk to presumptively identify a gram-positive coccus as
Peptostreptococcus anaerobius
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Nitrate disk to determine the bacterium’s ability to reduce nitrate
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Bile disk to presumptively identify an anaerobic gram-negative bacillus as Bacteroides fragilis
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Catalase test to differentiate aerotolerant Clostridium strains from Bacillus species
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Spot indole test to determine if an organism can produce indole from tryptophan
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Motility test to help identify motile gram-negative bacteria such as Campylobacter and
Mobiluncus
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Lecithinase and lipase reactions to help identify species of Clostridium
Sometimes, however, presumptive tests fail to identify an anaerobic isolate. When this happens, the
organism must be identified definitively. Techniques used for definitive identification include:
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Pre-reduced anaerobically sterilized (PRAS) and non-PRAS tubed biochemical test media
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Miniaturized biochemical-based and preexisting enzyme-based systems
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Gas-liquid chromatography (GLC) for end products of glucose fermentation
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High-resolution GLC analysis of whole-cell long-chain fatty acid methyl esters (FAME)
No single commercial system can reliably identify all anaerobic bacteria, so laboratories performing
definitive identification typically use a combination of PRAS biochemicals and GLC analysis.
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(cont.)
Susceptibility Testing of Anaerobic Bacteria
Compared with testing other bacteria, susceptibility testing of anaerobic bacteria is more costly and timeconsuming. Also, empiric therapy based on presumptive identification of the organism usually cures the
infection. For these reasons, laboratories usually forego susceptibility testing of anaerobic bacteria.
However, susceptibility testing is needed in the following situations:
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The isolate is known to be virulent or resistant to antibacterial drugs (Bacteroides spp, Prevotella
spp, Clostridium spp, Fusobacterium spp, Bilophila wadsworthia, Sutterella wadsworthensis).
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Empiric therapy fails to cure the infection.
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There is no precedent for empiric therapy.
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The infection is severe.
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Long-term therapy is required.
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The patient has a brain abscess, endocarditis, infection of a prosthesis or vascular graft, joint
infection, osteomyelitis, or a recurrent or refractory bacteremia.
Susceptibility testing of anaerobic bacteria is controversial, with experts debating which isolates to test,
which antibiotics to use, and which method to use. Even so, standard susceptibility testing methods have
been developed for anaerobic bacteria, and these should be followed when susceptibility testing is
needed. Currently accepted standards appear in document M11-A7, Methods for Antimicrobial
Susceptibility Testing of Anaerobic Bacteria; Approved Standard—7th ed. This document is published by
the Clinical Laboratory and Standards Institute (CLSI); it can be purchased at the CSLI website
(www.clsi.org).
Suggested Reading
Citron DM. Algorithm for identification of anaerobic bacteria. In: Murray PR, Baron EJ, Jorgensen JH, et
al, eds. Manual of Clinical Microbiology. 8th ed. Washington, DC: American Society for Microbiology;
2003:343-344.
Engelkirk PG, Duben-Engelkirk J. Anaerobes of clinical importance. In: Mahon CR, Lehman DG,
Manuselis G. Textbook of Diagnostic Microbiology. 3rd ed. St Louis, MO: Saunders Elsevier;
2007:587-640.
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EDUCATIONAL COMMENTARY – LABORATORY ASSESSMENT OF ANAEROBIC BACTERIA
(cont.)
Forbes BA, Sahm DF, Weissfeld AS. Overview and general considerations. In: Bailey & Scott’s
Diagnostic Microbiology. 12th ed. St Louis, MO: Mosby Elsevier; 2007:455-462.
Forbes BA, Sahm DF, Weissfeld AS. Laboratory considerations. In: Bailey & Scott’s Diagnostic
Microbiology. 12th ed. St Louis, MO: Mosby Elsevier; 2007:463-477.
© ASCP 2007
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American Proficiency Institute – 2007 3 Test Event