Sunday, September 11, 2011

Anaerobic Infections

nullDefinition of anaerobe infection:
It is a microbe that can only grow under anaerobic conditions only and is sensitive to metronidazole on routine disc testing anaerobically.
To, prove that isolate is anaerobic bacteria
Subculture isolate both aerobically and anaerobically with CO2 enrichment for 5 days. Microaerophilic carbon dioxide dependent coccus appear on aerobic plate on fifth day, while strict anaerobe appear on 2nd day under anaerobic conditions

Spore Forming anaerobes.
Clostridium tetani causing tetanus.
-Clostridium perferingens, Clostridium sporogenes, Clostridium oedemetiens causing gas gangrene.
- Clostridium Botulinium causing food poisoning botulinism.
-Clostridium difficile causing Pseudomembranous colitis.
Predisposing Factors for anerobes infection:
Lower Oxygen tension and decrease oxidation-reduction potential.
1-Trauma with dead tissue in deep or extensive wound.
2-Impaired blood supply e.g. ischaemic arterial disease in foot or leg in diabetic patients.
4-Clostridium difficile and Pseudomembranous Colitis
Source - feces of nondiarrheic humans: 5-10% - hospital environment: up to 25% of patients - soil, marine sediments (mostly spores),dogs and cats: up to 35% Disruption of intestinal flora by antibiotics, chemotherapeutics - clindamycin: most cases per amount used - ampicillin, cephalosporins: most commonly associated (more widely used)
Onset 4 - 10 days after start of antibiotic, up to 2 weeks after termination

Transmission usually via spores, vegetative cells oxygen sensitive -> die rapidly - aerosol (diarrhea), lack of hygiene (fecal-oral) - pass through stomach - bile acids induce germination.
Antibiotic levels fall, C. difficile grows rapidly in unoccupied niches
Vegetative cells produce toxins.
1-Toxin A 308 kDa (largest known exotoxin, maybe largest known prokaryotic protein).
-Enterotoxic in vivo: fluid accumulation with tissue damage (blood and mucus), cells can no longer control water movement
-Causes diarrhea, intense inflammatory response when fed to hamsters
-Chemoattractant for neutrophils
-Must be internalized for toxic effect
C-terminal 1/3: - host cell binding (trisaccharide receptor) - five repetitive peptides - not toxic but required for toxic effect
N-terminal 1/3: - toxin domain - inactivation of Rho-protein by monoglucosylation (Rho-protein induces polymerization of actin)
Cytotoxic, cytotonic

2-Toxin B 269 kDa
-No enterotoxic activity in vivo
-Trace amounts of toxin A or mucosal damage necessary for toxic effect in rodent bowel
-cytotoxic in vitro(~1000-times more active than toxin A)
-63% amino acid homology with toxin A (gene duplication)
N-terminal domain: highly conserved, same activity as in toxin A
C-terminal domain: quite different, may recognize different receptor, also contains repetitive sequences
-Toxins act synergistically: toxin A damage to mucosal cells allows toxin B maximal effect
-Damage to colonic mucosa - accumulation of fibrin, mucin, dead host cells (yellowish layer on surface = pseudomembrane) - separate lesions coalesce
Symptoms: - severe abdominal pain - water, nonbloody diarrhea - high number of neutrophils in stool

Diagnosis: - detection of the organism:    * culture of feces for C. difficile(48-72h)    * immunological assay for somatic antigens
- detection of toxins:    * tissue culture
Fatality rate: 27-44% if untreated
Treatment - cessation of antibiotic, if possible - treatment with anti-C. difficile-drugs: vancomycin, metronidazole - extended course may be required to prevent recurrence - restoration of normal intestinal flora: fecal enema from family member
Prophylaxis - feeding of Saccharomyces boulardii (nonpathogenic yeast) - administration of toxin-neutralizing antibodies
1-Selection of specimens for anaerobic culture:
Anaerobic infection can occur in all body sites including CNS, head and neck, oral cavity, chest, abdomen, pelvic, urogenital, skin and soft tissues and blood.
So, the specimens that are suitable for isolation of anaerobes are
-----Blood samples
-----Aspirateed pus
-----Aspirated body fluids ----CSF

2- Samples Transport
Protection of anaerobic bacteria from oxygen exposure is critical step in the recovery of these organisms.
1-The anaport system: Consists of 2 tubes with rubber stopper one contains sterile swab in oxygen free CO2 or N2 atmosphere and other contain few ml of reduced salt solution.
2- Vacutainer transport: used for swabs, fluid and tissue specimens consists of outer glass tube and inner glass and fixed with rubber stopper

3) Biobag system: Clear gas impermeable bag commercially available.
4) Hungate tube, contains O2 free gas and agar indicator system
5) Use of transport media Staurt s media in which the sample is immersed and covered and transportes rapidly to the lab.

3-Laboratory Examination of anaerobic bacteria
1-Direct examination
2-Culture and biochemical identification of anaerobes.
3- Antibiotic susceptibility tests
4-Serological Identification
5-Mollecular typing
6-Gas Liquid Chromatography
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