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Micro 443 Mark S. Chandler, Ph.D. Identification of Anaerobes Obligate anaerobic bacteria are those that grow in the absence of free oxygen but fail to grow in the presence of oxygen Growth Response at 48 hours Atmosphere Aerobesa Facultative anaerobesb Anaerobesc,d Air + + __ Anaerobic __ + + a Neisseria, Pseudomonas (obligate respirers) Staphylococcus, Corynebacterium, enteric GNRs, etc., (Streptococcus, aerotolerant anaerobe/obligate fermenter) c Bacteroides, Fusobacterium, Clostridium, Prevotella, Porphyromonas, Peptostreptococcus, Viellonella, and many others b d For good recovery of anaerobes, specimens are inoculated onto blood agar supplemented with vitamin K and hemin (reducing agents such as thioglycolate and L-cysteine may be added to anaerobic transport media and certain culture media to help maintain reduced conditions) Anaerobes vary considerably in their tolerance to atmospheric oxygen. Some of the proteins present in bacteria react with O2 to form oxygen molecules that are toxic to cells. Toxic forms of oxygen include singlet oxygen (O2*), superoxide anion (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH). Enzymes that destroy these toxic oxygen products include catalase, peroxidase, and superoxide dismutase. Low levels or complete absence of superoxide dismutase is likely a major reason why oxygen is toxic to anaerobes. Therefore rapid achievement and maintenance of low oxygen tension is essential for cultivation of anaerobes – use anaerobe jars and glove boxes in clinical labs. Micro 443 Mark S. Chandler, Ph.D. Common Medically Important Anaerobic Bacteria GNR Bacteroides fragilis group – these are the most common anaerobes recovered from clinical specimens. They are more resistant to penicillin and cephalosporins than other anaerobes (resistance in other groups is increasing). The bile-resistance of the B. fragilis group can be used as a simple overnight test to screen for them. (See separate description of presumptive identification). Prevotella group – includes the black pigmented Pr. melaninogenica Porphyromonas Fusobacterium spp. Others -- ≥20 genera GPR with spores Clostridium perfringens – does not produce spores in clinical specimens or on clinical media Clostridium difficile Clostridium tetani Clostridium botulinum, plus more than eight other species GPR no spores Actinomyces spp. Propionibacterium spp. Several other genera GPC Peptostreptococcus spp. GNC Veillonella spp. Anaerobic infections can involve virtually any organ when conditions are suitable. Trauma, poor circulation, and tissue necrosis provide favorable conditions for anaerobes to multiply. Exogenous sources Endogenous sources Micro 443 Mark S. Chandler, Ph.D. Important to isolate and identify the organism or group of organism as most anaerobic infections are associated with high morbidity and mortality (presumptive ID OK). Treatment involves antibiotics; prompt surgical intervention including debridement of necrotic tissue and/or amputation may be importantLaboratory isolation: Initial Work-up for Anaerobe Subculture Subculture each colony type Incubate 48h Air anaerobe jar Conclusion #2 and # 4 are anaerobes Micro 443 Mark S. Chandler, Ph.D. Identification (ID) of Anaerobic GNRs Physician must request species ID otherwise presumptive ID is done as species ID is costly and takes several days to a week or more and treatment is usually necessary immediately. Preliminary examination of isolates should include; Gram stain, colony morphology, pigment, fluorescence (long wave UV), hemolysis, antibiotic susceptibility, and biochemical screening. Most of the clinically important GNRs can be placed into broad groups on the basis of these relatively few tests. For Presumptive ID Antibiotic screening: Kanamycin, Vancomycin, Colistin (in lab we will use Penicillin) Biochemical screening: Bile (growth in 20 % bile), spot indole, catalase, red fluorescence, and black pigment. Bacteroides fragilis group is resistant to all the above antibiotics and to bile. Catalase + Prevotella are sensitive to bile, resistant to vancomycin and kanamycin, variable to colistin and may produce black colonies. Porphyromonas are susceptible to vancomycin and bile and resistant to colistin and may fluoresce red. Thus using this small battery of tests many anaerobes can be presumptively identified. For species ID Biochemical tests for definitive species identification. Overnight biochemical tests that require growth; API strip Also strips that test for a panel of preformed enzymes in ~4 hr; AnIdent Old method;Gas liquid chromotography (GLC) of fatty acids from glucose metabolism Presumtive ID of Clostridium perfringens Gram stain, hemolysis, lecithinase on Nagler’s egg yolk agar Presumtive ID of Clostridium difficile C. difficile is naturally resistant to many antibiotics Selective and differential media available for isolation of C. difficile CCFA medium has peptones and the antibiotics cycloserine and cefoxitin with fructose and a pH indicator. C. dif grows giving yellow colonies with yellow fluorescence Direct detection of toxins in stool is often used in place of culture for diagnosis