NON-SPOREFORMING ANAEROBIC BACTERIAA
Non-sporeforming anaerobic bacteria cause a great variety of bacterial infections. Usually they affect human tissues together with facultative anaerobic and aerobic microflora. Anaerobic bacteria dominate in several body compartments, primarily in the oral cavity and gastrointestinal tract but even the infections of respiratory tract are proven to be of polymicrobial origin, where anaerobes occur in more than 50% of cases.
Since most of non-sporeforming anaerobes are normal representatives of human flora, they can induce infectious inflammation only in high concentrations, or within normally sterile body cavities and compartments, in immunocompromised patients, etc. Nosocomial strains of these bacteria demonstrate enhanced virulence and multidrug antibiotic resistance.
Classification of Pathogenic Gram-negative Non-sporeforming Anaerobes
Most of human anaerobic non-sporeforming pathogens pertain to related microbial families
- Desulphovibrionaceae with their major genera and species:
- Bacteroides (major species are B. fragilis, В. ovatus, В. vulgatus, В. thetaiotaomicron);
- Porphyromonas (species P. gingivalis, P. endodontalis),
- Prevotella (species P. intermedia, P. melaninogenica, P. heparinolytica);
- Tannerella (major species is T. forsythia);
- Fusobacteria (species F. nucleatum, F. necroforum);
- Bilophila (species B. wadsworthia).
Structure and Properties
Bacteroides and related bacteria are gram-negative rods, albeit coccobacteria may occur. Certain microbial species can possess capsule and/or flagella. Fusobacteria render characteristic rod-like shapes with tapered ends.
- All of these agents are anaerobic bacteria, but they develop different levels of oxygen tolerance. Some strains of bacteroids can survive at 1-2% concentration of oxygen. Low oxidation-reduction potential promotes bacteroidal growth.
- Usually they are cultivated on blood agar with yeast extract and other growth factors (e.g., Schaedler anaerobe agar) within anaerobic jars better in atmosphere of 5-10% СО2. They grow slowly within 4-5 days. Some species (e.g. P. melaninogenica) produce black pigment.
- The representatives of genus Bacteroides are resistant to bile salts and penicillin that distinguishes them from other similar bacteria.
- Strict an aerobes have no cytochrome systems and possess neither catalase, no superoxide dismutase enzymes. Nevertheless, bacteroides members express small amounts of catalase and superoxide dismutase that partially preserve bacteria from reactive oxygen radicals.
- Anaerobic bacteria gain energy from fermentation of different substrates. Bacteroids utilize vast number of carbohydrates producing the broad spectrum of acid end products. Also they metabolize pepton.
- Antigenic structure is variable, depending on cell wall composition, capsule and flagella presence.
- Bacteroides produce destructive enzymes (hyaluronidase, collagenase, plasmin, heparinase, etc.) that cause tissue damage.
- B.fragilis produces exotoxin fragilysin. This toxin exhibits potent proteolytic activity destroying intercellular junctions between enterocytes.
- Cell wall lipopolysaccharides can activate leukocyte chemotaxis, but they are almost lack of endotoxin activity. Capsule layer of bacteroides preserves them from phagocytosis.
- Enzymes destroying antibacterial drugs (e.g., beta-lactamases) confer microbial resistance to antibiotics. Also bacteroides are resistant to aminoglycosides and demonstrate growing resistance to tetracyclines.
- Non-sporeforming anaerobic bacteria demonstrate generally low resistance to external influences. They are killed by heating at 65oС in 15 min, and in 1 min by boiling; when exposed to the opened air, they are irreversibly inactivated in 24-48 h. However, in feces they may stay viable up to 1 month.
Pathogenesis of Infections caused by Non-sporeforming
- Bacteroides species are the major part of normal microflora of large intestine. Bowel injury and/or the increase of permeability of intestinal wall induce microbial spread towards peritoneal cavity that results in abdominal purulent inflammation followed by peritonitis or intestinal abscessing.
- B. fragilis demonstrates markedly enhanced virulence in comparison with other bacteroidal species. This pathogen represents only 0.5% of all microorganisms of large intestine, but it can be isolated in 30-60% of cases of anaerobic infections, especially in their intra-abdominal and wound localizations.
- This bacterium possesses a number of potent virulence factors. Among them are agressive enzymes hyaluronidase, proteases, and hemolysin. Together with enterotoxin fragilysin that destroys intercellular contacts of enterocytes they promote microbial leakage across the intestinal wall thus stimulating microbial invasiveness and spread from their primary sites.
- Capsular polysaccharide of B. fragilis triggers local pathological inflammatory response that leads to formation of tissue abscesses.
- As the result, B. fragilis takes an active part in peritonitis, intestinal and liver abscesses, appendicitis, abscesses of subcutaneous adipose tissue, endometritis, vulvar abscesses, trophic ulcers in diabetes patients, lung abscesses, anaerobic infections of central nervous system (brain abscesses and subdural empyema),
- Similar pathology can be caused by other bacteroidal species (B ovatus, B vulgatus, B. thetaiotaomicron, etc.).
- Severe anaerobic infections are also caused by the members of Bilophila and Fusobacterium genera. For instance, Bilophila wadsworthia is isolated in 50% of cases of appendicitis.
- Fusobacterium necroforum is relatively common in wound anaerobic infections. The rate of its isolation increases in systemic bacterial infections such as endocarditis, bacteremia and sepsis.
- Overall, usually five and more of bacterial species can be isolated from inflammatory site, including facultatively anaerobic and anaerobic bacteria.
- Oral cavity harbors large amounts of porphyromonads (P. gingivalis, P. endodontalis and many others), prevotellas (P. intermedia, P. melaninogenica) and tannerellas (T. forsythia). In various combinations with other microbial pathogens they actively contribute to progression of periodontites
- Porphyromonads can be isolated also in suppurative bacterial infections of various localizations.
- Likewise, members of prevotella species can affect female genital tract, causing pelvic inflammatory disease and tubo-ovarian abscesses.
Laboratory Diagnosis of Anaerobic Infections
- The material is obtained in anaerobic conditions, e.g. by abscess puncture with syringe that contains appropriate medium for anaerobes (e.g., thioglycolic medium). The specimen should be transferred immediately into the sealed bottle with transport anaerobic medium and delivered to the laboratory within 1-1.5 h.
- Specimens, obtained from closed purulent foci, blood and cerebrospinal fluid are examined.
- Since various anaerobic species produce different spectra of short-chain fatty acids they can be identified by gas-liquid chromatography. This method may be used for rapid diagnosis of anaerobic infection.
- Also rapid identification of non-sporeforming anaerobic bacteria in clinical specimens is performed by serological (ELISA) and molecular genetic tests (PCR).
- Anaerobes are cultivated upon blood agar, trypticase soy agar, brain-heart infusion agar and other enriched media in anaerobic jars. The jars are usually supplied with disposable packets that produce hydrogen, and a catalyst that combines the hydrogen with any free oxygen to form water. Cultures are incubated at 35-37°C with addition of CO2.
- The microbial isolates are further identified by their morphology, cultural properties and biochemical activities.
Treatment and Prophylaxis of Anaerobic Infections
- Prophylaxis of anaerobic infections is non-specific and includes adequate surgical treatment followed by drainage that ensures sufficient oxygen access to affected site.
- The most active antibiotics for treatment of anaerobic infections are metronidazole and clindamycin. Most of non-sporeforming anaerobic bacterial strains are shown to develop no resistance to clindamycin and metronidazole. In severe cases of anaerobic infections carbapenems are successfully used. Sensitive strains are treated with other β-lactam antibiotics (e.g., penicillins and cephalosporins).