Anaerobiosis in Microbiology

1. Definition

2. Classification by oxygen requirement

3. Why oxygen is toxic to anaerobes

Missing protective enzymes

1. Cytochrome system. This allows O2 to be used as a terminal electron acceptor. Anaerobes lack it (Jawetz 28e, Physiology and Growth).
2. Superoxide dismutase (SOD): 2 O2⁻ + 2H⁺ → H2O2 + O2.
3. Catalase: 2 H2O2 → 2 H2O + O2.

Nuances in the textbooks

• All strict anaerobes lack both SOD and catalase.
• Bacteroides fragilis group organisms have small amounts of both. This partly explains their oxygen tolerance and their virulence (Jawetz 28e).
• Some anaerobes (e.g., Peptococcus anaerobius) tolerate oxygen because they make NADH oxidase, which reduces oxygen to water.
• Tolerance varies between species and between strains of one species. For example, one strain of Prevotella melaninogenica grows at 0.1% O2 but not 1%, while another grows at 2% but not 4% (Jawetz 28e).
• Most human anaerobic infections are caused by "moderately obligate" anaerobes. Strict obligate anaerobes are rarely isolated from human infections (Jawetz 28e).
• The more virulent anaerobic pathogens often make catalase or SOD (Sherris & Ryan's Medical Microbiology, 8e).

Redox potential (Eh)

• Anaerobes grow only at a low or negative oxidation-reduction potential.
• Some need an extremely low value (about -300 mV). Key enzymes must stay in the reduced state to work, so aerobic conditions create a metabolic block (Sherris & Ryan's 8e).
• Anaerobes lack cytochromes, so they make energy only by fermentation.

4. Metabolism in anaerobiosis

• Fermentation (substrate-level phosphorylation). This gives only 2 ATP per glucose. End products are organic acids (lactic, acetic, butyric, propionic, succinic), alcohols and gases (H2, CO2). Gas chromatography of these end products is used for identification, and volatile fatty acids often give anaerobic pus a foul smell.
• Anaerobic respiration. Some bacteria use an alternative terminal electron acceptor such as nitrate, sulfate, fumarate or CO2 (Medical Microbiology 9e has a section on anaerobic respiration).
• Anaerobes also lack oxygen-dependent pathways, so they are resistant to aminoglycosides, which need oxygen-dependent transport into the cell.

5. Methods to achieve anaerobiosis in the laboratory

In liquid media

• Reducing agents such as sodium thioglycolate, cysteine or ascorbic acid.
  • Thioglycolate broth has oxygen tension that falls toward the bottom of the tube. This allows growth of obligate anaerobes and microaerophiles without special incubation (Quick Compendium of Clinical Pathology 5e).
  • A redox indicator (resazurin or methylene blue) shows oxygen penetration as a pink or blue band at the top.
• Robertson's cooked meat (RCM) medium. Heated meat particles contain unsaturated fatty acids that take up oxygen, and the medium also contains sulfhydryl groups. It is used for Clostridium and for storing cultures. A layer of sterile liquid paraffin on top gives extra protection.
• Tubes of agar can be sealed with a layer of petrolatum and paraffin (Jawetz 28e).

In solid media (plates)

1. Anaerobic jar (McIntosh and Fildes, GasPak system).
   • A gas-generating envelope plus water releases H2 and CO2.
   • The lid holds a palladium-coated alumina catalyst that combines H2 with O2 to form water.
   • A methylene blue indicator strip turns from blue to colorless when anaerobic.
   • Henry's textbook describes it as "oxygen (O2) is catalytically converted to water with palladium-coated alumina pellets contained in a lid chamber."
2. Anaerobic bags and pouches. A transparent bag holds its own gas generator and catalyst, e.g., the Bio-Bag.
3. Anaerobic glove box or chamber. This is a large airtight chamber filled with N2, H2 and CO2. Items pass through a gas interchange lock, work is done through sealed neoprene gloves, and palladium catalysts maintain anaerobiosis. Henry's states that all these systems are "equally effective in isolating clinically significant anaerobic bacteria."
4. Evacuation and replacement. Air is pumped out of the jar and replaced with an anaerobic gas mixture, typically 80 to 90% N2, 5 to 10% H2 and 5 to 10% CO2.
5. Pyrogallol or alkaline pyrogallate. Pyrogallol absorbs oxygen. This is an older, simple method (Fildes' method).
6. Candle jar. This gives only reduced O2 with raised CO2, which suits capnophiles. It does not give true anaerobiosis.
7. Gaspak with palladium, Anoxomat and similar commercial systems are variations on the same principles.

Practical points

• Place inoculated media in an anaerobic environment as quickly as possible (Henry's).
• Incubate at 35 to 37°C. Cultures are usually examined at 48 hours because anaerobes grow slowly, and incubation continues for at least 5 days (Henry's).
• Prereduced anaerobically sterilized (PRAS) media are used in reference labs.
• Set up an aerobic control plate to show the isolate does not grow in air. This confirms the isolate is an anaerobe.

Media commonly used

• Blood agar with hemin and vitamin K. This is enriched and supports most anaerobes.
• Brucella blood agar and Columbia blood agar are general anaerobic media.
• Kanamycin-vancomycin laked blood agar (KVLB) is selective for Bacteroides and Prevotella.
• Phenylethyl alcohol (PEA) agar is selective for anaerobic Gram-positive and Gram-negative organisms and inhibits facultative Enterobacteriaceae.
• Bacteroides bile esculin (BBE) agar is selective and differential for the B. fragilis group.
• Egg yolk agar shows lecithinase and lipase (C. perfringens).
• Cycloserine-cefoxitin fructose agar (CCFA) is used for C. difficile.

6. Specimen collection and transport

• Best specimens are pus aspirated with a needle and syringe, tissue and body fluids.
• Expel air from the syringe, then either cap it or inject the sample into an oxygen-free transport vial or tube.
• Avoid swabs, which dry out and expose organisms to air. Avoid contaminated sites such as expectorated sputum, voided urine, vaginal or cervical swabs, and feces (except for C. difficile and other specific tests).
• Deliver promptly to the lab. Transport should not be delayed beyond about 2 hours at room temperature.

7. Clinical clues to anaerobic infection

• Foul-smelling discharge.
• Infection located near a mucosal surface that carries normal anaerobic flora (mouth, gut, vagina, skin).
• Necrotic tissue, abscess, gas in tissues or crepitus.
• Gram stain of a purulent specimen shows organisms but aerobic culture is negative ("sterile pus").
• Failure to respond to aminoglycosides, trimethoprim-sulfamethoxazole or older quinolones.
• Black discoloration of exudate or fluorescence under UV light (pigmented Prevotella and Porphyromonas).
• Septic thrombophlebitis, bite wounds, aspiration pneumonia, or infection after bowel surgery.
• Infection after tissue trauma, vascular insufficiency, cancer, or foreign bodies.

8. Important anaerobic pathogens

9. Treatment points

• Surgical drainage and debridement are the mainstay for abscess and necrotic tissue. Antibiotics alone are often inadequate.
• Active drugs: metronidazole, clindamycin, beta-lactam/beta-lactamase inhibitor combinations, carbapenems, chloramphenicol, and tigecycline.
• Resistance: B. fragilis produces beta-lactamase and is often resistant to penicillin. Clindamycin resistance is rising, and C. difficile is a risk with clindamycin use.
• Anaerobes are intrinsically resistant to aminoglycosides, which are inactive without oxygen.

10. Quick revision summary

• Anaerobiosis means growth without O2. Anaerobes lack SOD, catalase and cytochromes, so reactive oxygen species accumulate and kill them. They also need a low Eh.
• Types: obligate, aerotolerant, facultative and microaerophilic.
• Lab techniques: thioglycolate broth, Robertson's cooked meat medium, GasPak jar (H2 plus palladium catalyst), glove box, pyrogallol.
• Collect pus or tissue by aspiration, avoid swabs, and transport quickly.
• Clinical clues: foul smell, gas, necrosis, and polymicrobial infection near mucosal flora.

Source notes

• Jawetz, Melnick & Adelberg's Medical Microbiology 28e: Glossary and Physiology and Growth (about p. 312-313) and Aeration. I did not retrieve exact page numbers for every passage.
• Sherris & Ryan's Medical Microbiology 8e: Chapter 29, anaerobiosis and selective media.
• Henry's Clinical Diagnosis and Management by Laboratory Methods: Culture Techniques.
• Quick Compendium of Clinical Pathology 5e: thioglycolate broth.