BIOL 2010 Lecture 13 - Anaerobic Methods

Lecture 13 - Anaerobic Methods

• Lecture for BIOL 2010
• References are Bailey and Scott's (Chapter 40) and Diagnostic Microbiology (Chapter 22)
• This lecture discusses anaerobic microorganisms, their characteristics, pathogenesis, and bacterial virulence. It also covers methods for collecting and processing specimens for anaerobic bacteriology.

Anaerobes Defined

• Anaerobes can replicate in the absence of oxygen.
• Ambient air contains approximately 21% oxygen and 0.04% carbon dioxide.
• Obligate anaerobes are killed in the presence of oxygen, and oxygen free radicals lead to immediate cell destruction.
• Obligate anaerobes require low oxidation-reduction (redox) potential.
• Capnophilic anaerobes require increased carbon dioxide (CO2) levels (5%-10%), and reduced oxygen (O2) to 15%.
• Microaerophilic anaerobes require oxygen reduced to 5% or less.
• Facultative anaerobes prefer oxygen as an electron acceptor but can grow without oxygen.
• Aerotolerant anaerobes tolerate oxygen but do not use it in metabolism. These can be considered moderate anaerobes which are bacteriostatic if exposed to oxygen.

Why Some Organisms Are Anaerobes

• Molecular oxygen has a direct toxic effect on some anaerobes. They lack enzymes to break down toxic products.
• Toxic products include superoxide anion, hydrogen peroxide, and hydroxyl radical.
• Strict aerobic and facultative anaerobic bacteria possess enzymes to break down toxic products, such as superoxide dismutase and catalase.

Where Anaerobes Are Found

• Exogenous anaerobes exist outside the bodies of animals. Infections develop when the agent is ingested or enters the body through trauma (e.g. Clostridium, Sarcina, Fusobacterium).
• Endogenous anaerobes exist inside animal bodies and become sources of infection (e.g. Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Clostridium, Propionibacterium, anaerobic cocci).

Important Concepts

• Proper collection technique for anaerobic samples is crucial to prevent misleading and non-useful clinical results. Sites of infection must be correctly identified.
• Rapid processing of specimens is necessary due to oxygen toxicity and specimen drying.

Specimens for Anaerobic Bacteriology

• Acceptable specimens include Cerebrospinal fluid (CSF), abscesses, aspirates, surgical specimens, body fluids, and biopsies.
• Unacceptable specimens include sputum, swabs of surface organs (e.g., throat, gingival, skin, perirectal), urine (midstream), large bowel content (e.g., stool, colostomy bags), and vaginal swabs.
• Ideal specimen collection methodology utilizes needles and syringes to prevent contamination and avoid air exposure.
• Specimens should be placed into pre-reduced anaerobically sterilized (PRAS) media immediately after collection to avoid oxygen exposure.

PRAS Transport Medium

• PRAS stands for pre-reduced anaerobically sterilized media
• This is the ideal medium for transporting samples.
• Two methods are acceptable, the tube and the eSwab system.

Processing of Clinical Samples

• Plates with inoculated anaerobic cultures require incubation at 35-37 degrees Celsius in anaerobic conditions, and must not be kept out of the anaerobic environment longer than 1 hour.
• Anaerobic incubation systems utilize a combination of catalyst (palladium pellets to remove residual oxygen), desiccant (absorbs formed water), and anaerobic gas (mixture of H2, CO2, and Nitrogen).
• Quality control for anaerobic systems should be in place including checking physical reactions (e.g. condensation), chemical indicators (e.g., methylene blue or resazurin strips), and biological indicators (e.g., obligate aerobe and anaerobes). The indicators should show changes after proper incubation timing.

Identification of Anaerobes

• The identification method of anaerobes depend on the size and capabilities of the laboratory, the clinical site(e.g., sterile site of infection), and the type of the infection.
• Presumptive identification is commonly used in most laboratories; it is quick, cost effective, and typically sufficient for treatment. Presumptive identification relies on observations of culture characteristics (color, smell) and Gram stain.
• Definitive ID testing is complex and expensive and often unnecessary for treatment.

Presumptive Identification of Anaerobes

• Cultural characteristics, aerotolerance tests, Gram stain observation, and quick biochemical tests (including catalase and spot indole tests) are used.

• Direct microscopic examination (Gram stain preparation) can help in ruling out or in identifying pathogens, establishing microbial count, and in selection of suitable culture media.

Presumptive ID - Culture

• Choice of anaerobic media is vital for accurate recovery.
• Anaerobes have special nutritional requirements which include vitamins (vitamin K and Hemin), yeast extract, and nutrient rich media (e.g., CDC blood agar).
• Specimens submitted for anaerobic culture often include requests for both aerobic and anaerobic cultures.
• Additional medias are required, including: chocolate agar, Brucella agar, Kanamycin-Vancomycin Laked blood agar (KVLB), Phenylethyl Alcohol Agar, Colistin Nalidixic acid Agar, and anaerobic broths (e.g. cooked meat, thioglycollate).

Media for Anaerobes

• Several media types are suitable for anaerobic growth including anaerobic blood agar (SBA), Brucella Blood Agar (BA), Kanamycin-Vancomycin Laked Blood Agar (KVLB), and other specialized medias.

Anaerobic Chamber, Jar, and Pouch

• Anaerobic chambers, jars, and pouches create anaerobic conditions.
• Water-generating systems for jars require a catalyst and water, while waterless systems do not.

Definitive ID Techniques

• Multitest systems, cellular fatty acid analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and 16S ribosomal RNA gene sequencing are definitive identification methods.

Specialized Potency Antimicrobial Disks

• These disks are used to confirm Gram stain reactions.
• These are NOT used for susceptibility testing. The purpose of the test is to rapidly differentiate types of Bacteria.

Other Special Potency Disks

• Nitrate disks test the ability to reduce nitrate.
• Sodium polyanethol sulfonate (SPS) disks test for susceptability within peptostreptococcus and peptoniphilus.
• Bile disks test for growth in bile.

Lecithinase and Lipase Reactions

• Lecithinase and lipase reactions can identify certain microorganisms including species such asC. perfringens and F. necrophorum.

Proteolytic Reactions

• Proteolytic reactions are used to identify organisms that produce proteolytic enzymes by observing a clear zone around their colonies . A strong light source can be used to facilitate observations.

Identifying Clues from Culture

• Foul odor, characteristic colony morphology, unique morphology on Gram stain, growth on specific media (e.g., BBE), double zone hemolysis on blood agar, and brick-red fluorescence on KVLB agar can point toward anaerobic presence.

Aerotolerance Testing

• Aerotolerance tests are used to determine if isolated microorganisms are strict or facultative anaerobes. Plates are inoculated and incubated to observe differences in bacterial growth with and without oxygen.

Summary of Presumptive ID

• Presumptions are based on Gram stain and colony morphology on different medias.
• Several simple, inexpensive, and rapid procedures are used.
• Reviewing these results can provide a presumptive identification.
• Presumptive identification is acceptable for treatment of anaerobes.