Anaerobic Bacteria Overview

Anaerobic Bacteria

• Anaerobic bacteria do not need oxygen to grow
• They vary in their tolerance to oxygen
• Obligate anaerobes require anaerobic conditions for growth; oxygen is toxic to them and kills them
• Aerotolerant anaerobes can grow in the presence of oxygen but grow best in anaerobic environments
• Facultative anaerobes do not require oxygen but will use it if it is available

Oxygen Reduction

• Superoxide anion and hydrogen peroxide are toxic
• Strict aerobes and facultative anaerobes have superoxide dismutase and/or catalase to neutralize these toxic substances

Oxidation-Reduction (Redox) Potential

• Anaerobic bacteria require low redox potential
• High redox environments contain oxygen
• Normal human tissue and aerobic culture media have high redox potential
• Reducing agents like thioglycollate, cysteine, and dithiothreitol are included in anaerobic media

Normal Habitat

• Soil, water, and animals
• Human normal flora (NF) includes oral cavity, upper respiratory tract (URT), intestinal tract, genitourinary tract, and skin.
• Facultative organisms use oxygen in protected areas, to reduce redox potential and inactivate harmful oxygen molecules

Diseases

• Exogenous infections are caused by organisms, spores, or toxins entering the body through ingestion or trauma (e.g., tetanus, botulism)
• Endogenous infections occur near mucosal surfaces (e.g., bacteremia, abscesses, gas gangrene, pneumonia)

Clues to Anaerobic Diseases

• Deep or puncture wounds; animal or human bites
• Foul-smelling, gaseous discharge
• Necrotic tissue, vascular stasis
• Presence of sulfur granules (actinomycosis)
• Black or fluorescent red color
• Previous therapy with aminoglycosides (aminoglycosides are ineffective against anaerobes)
• Failure to grow organism on a Gram stain

Specimen Collection

• Appropriate specimens include blood, sterile body fluids (CSF), bone marrow, cavity fluid, urine (when collected as a suprapubic aspirate), abscesses, ulcers, and draining wounds.
• Biopsy material.
• Inappropriate specimens include superficial skin sites; voided or catheterized urine; expectorated sputum, throat, or nasopharyngeal swabs, bronchial washings; vaginal, cervical, and urethral swabs; and stool or rectal swabs.

Transport and Processing

• Inoculate media and place immediately in an anaerobic environment
• Limit exposure to room air; do not allow to dry out
• Do not refrigerate specimens
• Minimize time at room temperature.
• PRAS media, anaerobically sterilized transport medium is needed; modified Cary-Blair or Amies agar is used
• Rezasurin indicator, used to show the amount of oxygen. Reducing substances help protect against oxygen and inhibit NF.
• A sterile container is required

Culture

• Specimens for anaerobic culture are cultured aerobically on BAP, CHOC, MAC, and on anaerobic media to correlate aerotolerance
• Anaerobes require vitamin K, hemin, and yeast extract.

Nonselective Media

• CDC (or anaerobic blood agar plates) support the growth of obligate and facultative anaerobes
• Anaerobic broth, supplemented with thioglycollate or chopped (cooked) meat, supports the growth of all obligate and facultative anaerobes, with anaerobes growing towards the bottom and facultative organisms growing throughout.

Aerotolerance

• Testing determines whether an isolate is strict or facultative by incubating the isolate in both aerobic and anaerobic environments.
  • Growth only on anaBAP suggests a strict anaerobe
  • Growth on both anaBAP and CHOC suggests a facultative anaerobe
  • Growth only on CHOC suggests an aerotolerant anaerobe

Fluorescence

• Fluorescence occurs under ultraviolet light (366 nm) with colors of red, orange, pink, and chartreuse

Rapid Identification Tests

• Catalase test
• Motility test
• Urease test
• Indole
• Esculin hydrolysis
• Aerotolerance
• Fluorescence
• Disk tests
• Lecithinase
• Naglar test
• Lipase
• Reverse CAMP

Specimen Collection (Mycobacteria)

• Sputum, bronchial washings, gastric contents, urine, stool, and tissue
• First morning sputum, on 3 consecutive mornings, 5-10 ml volume, refrigerate overnight.
• If sputum isn't produced, bronchoscopic washings (BAL) or biopsy.
• Gastric aspirate or washing
  • Young children < 3 years cannot produce their own sputum, often swallow sputum. The mycobacteria can be destroyed by stomach acid. If this is the specimen, neutralize the aspirates or urine with sodium bicarbonate.
• Urine
  • Neutralize with sodium bicarbonate if refrigerated overnight.
• Stool
• AIDS patients need M. avium screening.
• Inappropriate specimens, such as swabs, may not contain enough material or the mycobacteria may not dislodge. 24-hour pooled sputum or urine specimens may hinder the analysis due to contamination or organism inhibition from the interference.

Digestion and Decontamination

• Specimens from sterile sites can be concentrated and directly inoculated. Specimens with normal flora and mucus need digestion (like N-acetyl-L-cysteine, or NALC) then decontamination with NaOH.
• Concentration increases the number of organisms per mL of sample.
• Centrifuge, then make AFB smear and inoculate media with the sediment. Use both liquid and solid media in the testing.

Specimen Processing

• Digestion frees bacteria from protein clumps using NALC (mucolytic agent)
• Decontamination removes normal flora (NF)
• Mycobacteria resist many bactericidal effects due to their high lipid concentration
• Decontaminate to kill contaminants without harming mycobacteria.

Staining for AFB Smear

• Ziehl Neelsen (hot stain) and Kinyoun stain (cold stain) use heat or the reagent, respectively, to force the stain into the cell, due to a high lipid cell wall content

Mycobacteria Identification

• Slow-growing mycobacteria are distinguished from rapid-growing mycobacteria by their growth rate, which takes longer to complete.
• Different colony morphologies will vary (e.g., ruffled, buff colored, cording, or cauliflower shape)
• The presence or absence of pigmentation differs between species.
• Photoreactivity tests evaluate the production of carotenoid pigments in different species.
• Several biochemical tests will differentiate species (like catalase, niacin, nitrate reduction, Tween 80 hydrolysis, and tellurite reduction

Culture Examination

• Examine twice weekly for the first month, once per week thereafter.
• Examine agar-based cultures (microcolonies) with a microscope at 4x-10x.
• Examine egg-based cultures (hand-lens)

Other Pathogenic Species

• These include M. genavense, M. simiae, M. xenopi, M. szulgai.

Newer Identification Tests

• These tests use different methods to identify mycobacteria (e.g., chromatography and molecular identification)

Antimicrobial Susceptibility Tests

• Susceptibility tests of M. tuberculosis include isoniazid. rifampin, ethambutol, and streptomycin
• Susceptibility methods include absolute concentration methods, resistance ratio, and proportional testing procedures

Classification of Mycobacteria

• M. tuberculosis Complex (including M. tuberculosis)
• Nontuberculous mycobacteria (NTM) (also known as mycobacteria other than tuberculosis or MOTT) such as those in the M. avium complex
• Uncultivable mycobacteria (which are difficult to grow in the laboratory)

Anaerobic GNR

• Normal flora of mucous membrane. This group includes Bacteroides, Prevotella, Prophyromonas, Fusobacterium, and Veillonella
• The Bacteroides fragilis are a subgroup

Anaerobic GPC

• includes Peptostreptococcus, which are chains, and Peptococcus, which are clusters. Specific tests like the sodium polyanethel sulfonate test differentiate these genera.

Anaerobic GPR

• includes Actinomyces spp, Bifidobacterium spp, Propionibacterium spp, Eggerthella spp. and Eubacterium, and all can present in actinomycosis

Other Non-spore-forming GPR

• includes Corynebacterium, Listeria, Erysipelothrix, Acranobacterium, Lactobacillus, and Gardnerella

Spirochetes

• Examples include Borrelia, Leptospira, and Treponema. There are many subgroups, which can have diverse characteristics.

Borrelia

• The Borrelia genus is responsible for Lyme disease, which is transmitted by arthropods. Some Borrelia cause relapsing fever.

Treponema

• T. pallidum, including the subtypes pallidum, pertenue, endemicum, and carateum, are responsible for syphilis infections. There are various stages of disease.

Leptospira

• This genus is responsible for Leptospirosis, often a zoonotic spread from animals, like dogs, rats, or cattle, through contact with contaminated water from urine.

Explore the fascinating world of anaerobic bacteria, which thrive without oxygen. This quiz covers their growth conditions, types, and the biochemical mechanisms that allow them to survive in oxygen-rich environments. Test your knowledge of these essential microorganisms and their habitats!