CASE NOTES
Anaerobic Culture - Why? What? How?
Ana I. S. Esteves & Mark Westman, Diagnostic Veterinary Bacteriology Laboratory, Elizabeth Macarthur Agricultural Institute, Department of Regional NSW, Menangle NSW
Posted Flock & Herd January 2023
Why should you consider anaerobic culture?
Anaerobic bacteria are very often unsuspected causative agents of infections that contribute significantly to animal morbidity and mortality. Anaerobes can be involved in infections such as suppurative arthritis, gas gangrene, blackleg, necrotising myocarditis, mastitis, generalised abscessation and other pyogenic infections.
What are anaerobic bacteria and what should you look out for?
Bacteria can be classified as either aerobes or anaerobes based upon their metabolic need for oxygen, which comprises approximately 20% of the atmospheric gases. Within the anaerobes, they can be further separated into categories based on their sensitivity to oxygen:
- Facultative anaerobes' metabolism is flexible; they grow well aerobically but also have the capacity to grow anaerobically if oxygen is not present. Staphylococci and Enterobacteriaceae are examples of facultative anaerobes.
- Aerotolerant anaerobes can tolerate atmospheric oxygen for a limited time and may even grow in its presence, but they do not require oxygen as part of their metabolism. Dichelobacter nodosus, the causative agent of footrot, is a known oxygen-tolerant bacteria.
- Obligate anaerobes cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated, otherwise they are harmed or killed by its presence. Clostridium and Bacteroides are examples of obligate anaerobes. Obligate anaerobes can further be subdivided into 2 types based on the percentage of oxygen that can prove toxic. Strict obligate anaerobes will not survive if there is more than 0.5% oxygen in the environment, while moderate obligate anaerobes can still withstand a 2 to 8% oxygen environment for a limited length of time.
Anaerobic bacteria can cause animal disease by one of three mechanisms:
- As invasive pathogens, when the infection results from the spread of normal microbiota from an adjacent mucosal or cutaneous surface into a normally sterile tissue through trauma or other changes to that particular tissue or structure.
- As toxigenic agents, when disease is caused as a result of toxin(s) produced by the microorganism. There are a variety of toxin-mediated diseases, ranging from mild diarrhoea to life threatening conditions, associated with enterotoxins produced by anaerobes like Clostridium perfringens, C. botulinum, C. tetani and Bacteroides fragilis.
- As participants in pyogenic infections, very often with purulent exudates and/or abscesses within any soft tissue of the affected animal, including the brain, lung, liver, spleen and other organs or tissues within the peritoneal cavity. While some organisms have the capacity of promoting abscessation on their own, abscesses are most often a result of a mixed infection that involves multiple microorganisms, with obligate anaerobes and facultative species acting in synergistic combinations. The microorganisms most commonly found within abscesses at specific anatomic sites tend to reflect the indigenous microbiota of adjacent mucosal surfaces, like abscesses found within the peritoneal cavity as a result of damage to the intestine often include Bacteroides spp., Enterococcus and a variety of other organisms that are part of the indigenous intestinal microbiota.
Anaerobe infections should be suspected when animals present with wounds or discharges that have a foul odour of bloody exudate, show tissue necrosis or gas production, or have a blackened discoloration. Anaerobic bacteria are often involved in tissue abscesses, bite wounds and other physical trauma, aspiration pneumonia and peritonitis.
How does anaerobic culture work?
All clinical material except specimens likely to be contaminated with normal flora should be routinely cultured for anaerobes. Because of the presence of anaerobes in all mucous membranes of mammals, specimen collection requires extreme caution to avoid contamination by the resident microbiota. Specimens that should not be cultured include nasal swabs, throat swabs, sputum, gastric contents, skin, faeces, voided or catheterized urine, and vaginal swabs. Whenever possible, tissue samples or fluid aspirates should be collected rather than swab samples. The material on swabs should never be allowed to dry out.
Specimens should be placed under anaerobic conditions immediately after collection for transport to the laboratory, since some anaerobes are quite oxygen sensitive and will die rapidly in an aerobic environment. If both aerobic and anaerobic culture are required, a separate specimen for anaerobic culture should be collected and appropriately stored/transported.
Specimen transport systems for fluids and aspirates can include sterile additive-free rubber stoppered transport vials/tubes containing an oxygen-free atmosphere (available commercially). Tissues should be collected into a sterile specimen container and the lid firmly closed. Swabs should immediately be placed into a swab system tube with Cary-Blair (preferentially) or Amies media. Our laboratory is currently developing an 'anaerobic kit' for the submission of fluid and tissue samples for anaerobic culture. Samples should not be refrigerated since chilling is detrimental to some anaerobes, and oxygen absorption is greater at lower temperatures. Specimens should be transported to the laboratory and processed for culture as soon as practicable.
Routine anaerobic testing will include two smears to be prepared directly from each specimen - one for Gram staining and one for carbol fuchsin - and inoculation of two anaerobic agar plates - a non-selective anaerobic agar plate and an anaerobic agar plate containing nalidixic acid and vancomycin (selective for Gram-negative anaerobes). A Gram stain examination of all specimens submitted for anaerobic culture can reveal the types and relative numbers of microorganisms and host cells present but also serves as a check for anaerobe recovery. Carbol fuchsin stain is performed in parallel as some of the Gram-negative anaerobes stain only faintly with the Gram stain.
Liquid media may be used as an optional pre-enrichment back-up source of culture material but are unsuitable for primary isolation and should only be used if the specimen originated from a site that would normally be sterile (e.g. joints, central nervous system). If no growth is detected in the primary anaerobic agar plates and anaerobic infection is suspected (either due to clinical signs or smear examination), incubated broth(s) may be inoculated into new anaerobic agar plates (both selective and non-selective) and checked for growth along with a wet-mount microscopy examination (wet-prep).
Anaerobic culture results are reported in a sequential manner as information becomes available. Reporting time for negative samples is eight working days; reporting time for samples requiring pre-enrichment and exhibiting suspect growth can be up to 14 working days.
Summary
| When to suspect involvement of anaerobic bacteria |
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| Common clinical presentations of anaerobic infections |
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| Submitting samples for anaerobic culture |
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References
- Clinical and Laboratory Standards Institute. 2014. Principles and Procedures for Detection of Anaerobes in Clinical Specimens; Approved Guideline. CLSI Document M56-A. Clinical and Laboratory Standards Institute, Wayne, PA
- Jorgensen et al. (Eds.) (2015) Manual of Clinical Microbiology 11th Edition, ASM Press
- Markey et al. (2014) Clinical Veterinary Microbiology 2nd Edition, Mosby Elsevier
- Quinn et al. (2011) Veterinary Microbiology and Microbial Disease 2nd Edition, Wiley-Blackwell
- Centres for Disease Control. 1987. Laboratory Methods in Anaerobic Bacteriology - CDC Laboratory Manual. HHS Publication No. (CDC) 87-8272, U.S. Department of Health and Human Services, Centres for Disease Control, Atlanta, Georgia