lecture 5 Anaerobes

• Anaerobes are the dominant members of normal human microbiota, predominantly residing on mucosal membranes of oral cavity, gastrointestinal tract, and female genital tract
• In the colon, the concentration of anaerobes reaches up to 99% of the total bacterial burden
• Clostridium, Pepetostreptococcus, Fusobacterium and Bacteroid species are the culturable anaerobes however many of the anaerobes are found to be uncultivable by conventional laboratory techniques known so far

  
• Collection and transport of materials for anaerobic culture

  1. Obtained with a needle and syringe

  2. Placed in an anaerobic transport tube containing oxygen-free carbon dioxide or nitrogen

  3. Injected through the rubber stopper in the transport tube and remains in the anaerobic environment until processed

  
• Anaerobic cultivation methods

  • Differ from the classic methods for aerobic cultivation
  • It is necessary to prevent molecular oxygen (O2) exposure as O2 is toxic to anaerobic micro-organisms to varying degrees
• Procedures and technical requirements for handling strictly anaerobic micro-organisms often prevent the cultivation of these organisms in most micro labs
• Anaerobic jar setup

  1. Commercially available hydrogen and carbon dioxide generator envelope (GasPak)

  2. Evacuation and replacement of air with an oxygen-free mixture of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide

  
• Pasteur effect

  The substrate uptake rate increases under anaerobic conditions compared to aerobic conditions to produce the same amount of biomass
• Hungate tubes, Balch tubes, Serum bottles

  • Commonly used culture vessels for anaerobes
  • Closed with butyl stoppers and sealed
• Larger volume cultivation vessels

  • Modified bottle with neck
  • Schott bottle
• Gassing of anaerobic cultures

  1. Repeated gassing is necessary as the gaseous substrate in the headspace is consumed

  2. Constant flow of oxygen-free gas over the surface of the medium is necessary when vials are opened to avoid exposure to oxygen
• Anaerobic glove box

  • Required for strict anaerobic manipulation of some organisms
• Roll tube method

  1. Stoppered test tube containing oxygen free gas and a thin layer of pre-reduced agar medium

  2. Inoculated with a loop while the tube is rotated to produce a spiral track on the agar surface

  3. Flushed with a stream of carbon dioxide to prevent entry of air
• Cultivation of strict anaerobes in microbiology laboratory

  • Challenging as it demands highly equipped systems with strict anaerobiosis and reduced culture media
  • Most anaerobic species are slow growing (can take up to 14 days to grow) and many are inactive for certain biochemical tests

  
• Quick anaero-system

  1. Disposable anaerobic gas pack

  2. Disposable culture envelope, a sealer and a reusable rack

  3. Catalyst unit with palladium-coated alumina pellets

  
• Anaerobic jar and gas pack

  1. Inoculated plates or tubes placed inside with gas generator sachet and indicator strip

  2. Chemicals in sachet react to produce hydrogen and carbon dioxide gas

  
• Replacement of atmospheric oxygen with O2-free gases and usage of reducing agents in culture media are useful steps to further facilitate the cultivation of anaerobes
• Six well plate method

  1. Works together with the AnaeroPack System

  2. Shown to be a better technique for culturing strict anaerobes like methanogens, sulfate reducing bacteria and hydrogen-producing syntrophs

  
• Comparing the growth of anaerobes, organisms showed better growth in Quick Anaero-system, but the anaerobic jar is adequate to recover clinically significant anaerobes
• The extremely oxygen-sensitive bacteria of the microflora apparently are not associated with infectious processes
• Non-culture methods for anaerobic identification


  • 16S rDNA sequencing
  • qRT-PCR
  • FISH
  • MALDI-TOF
  • Immunological assays (ELISA, Lateral flow)
• Antibiotics are a mainstay of treatment for anaerobic pathogens, and resistance is becoming a problem
• Anaerobic pathogens are generally more difficult to culture than aerobic bacteria, and most antimicrobials have been developed using aerobic assays
• In the past, anaerobic pathogens were often reported only with broad terms, but now they are often being reported using genus and species names
• Anaerobe susceptibility testing and reporting not only continue to remain relevant, but they are also likely to increase in demand as clinical microbiology laboratories improve technologically
• c jar


  Adequate to recover clinically significant anaerobes
• Non culture methods


  • 16S rDNA sequencing
  • qRT-PCR
  • FISH
  • MALDI-TOF
  • Immunological assays for ID of foodborne pathogens and toxins
  • ELISA
  • Lateral flow
• Antibiotics are a mainstay of treatment, and, as with aerobic pathogens, resistance is becoming a problem
• Reporting of anaerobic pathogens

  • In the past, they were often reported only with broad terms such as "Gram positive anaerobe"
  • Now they are often being reported using genus and species names
• Anaerobe susceptibility testing and reporting not only continue to remain relevant, but they are also likely to increase in demand as clinical microbiology laboratories improve technologically
• Why do anaerobic susceptibility testing (AST)

  • We have good empirical antibiotics
  • Anaerobes are often present in polymicrobial aerobic/anaerobic infections or mixed with other anaerobes, making it difficult to obtain pure cultures for AST
  • In such cases, it is helpful to know the predicted anaerobic susceptibility patterns based on information about strains circulating in the local region
• AST methods

  • Broth microdilution for the B. fragilis group
  • Agar dilution for all anaerobes, including the B. fragilis group
  • Disk diffusion is not suggested by the CLSI as a test method for anaerobes due to inaccurate results and poor correlation with the agar dilution method

  
• CLSI is now undertaking studies to re-evaluate whether broth microdilution can be performed for anaerobes other than the B. fragilis group, but results for other anaerobes, such as C. difficile, do not look promising

  
• AST interpretation

  Careful attention must be placed on the breakpoints applied to the datasets, because breakpoints can differ between breakpoint-setting organizations
• Patterns of resistance that deserve close attention due to rising rates

  • Resistance to the β-lactam-β-lactamase inhibitor combinations among the B. fragilis group
  • Increasing clindamycin resistance among all anaerobes
  • Metronidazole resistance is no longer limited to the B. fragilis group, as it now includes Gram-positive cocci and bacilli
  • Resistance of Clostridium to vancomycin

  
• In most cases, infections with anaerobic Gram-positive cocci are polymicrobial, thus providing AST results for them may not be as critical as for the more resistant anaerobic Gram-negative bacteria, such as B. fragilis
• Anaerobic Gram-positive cocci are generally considered susceptible to

  • Penicillin
  • Piperacillin-tazobactam
  • Meropenem
  • Metronidazole
• Resistance patterns in anaerobic Gram-positive cocci

  • Changes to penicillin-binding proteins may account for the elevated MICs to penicillin
  • Resistance to clindamycin is due to methylation of 23S rRNA at the site of drug action in the 50S subunit of ribosomes
• Anaerobic non-spore-forming Gram-positive bacilli


  • "Eubacterium" group
  • Actinomyces
  • Cutibacterium
  • Propionibacterium
  • Lactobacillus
  • Eggerthella lenta
  • Bifidobacterium

  
• Anaerobic non-spore-forming Gram-positive bacilli

  • They are usually susceptible to β-lactams (penicillins, cephalosporins, cephamycins, carbapenems, β-lactam- β-lactamase inhibitor combinations)
  • No CLSI clinical breakpoints exist for vancomycin with anaerobes, but it shows good in vitro activity against them
  • Many are intrinsically resistant to metronidazole
  • Surveys have shown approximately 7% resistance of C. acnes to clindamycin