Anaerobic Infections By Maysaa ElSayed Zaki Professor Of Clinical Pathology Definition of anaerobe: It is a microbe that can only grow under anaerobic conditions only and is sensitive to metronidazole on routine disc testing anaerobically. Anaerobes are the most primitive bacteria in terms of oxidation (the greater the oxidation, the more advanced evolutionarily). They use a relatively inefficient metabolic system: anaerobic fermentation provides energy at 10% the level of aerobic respiration 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. Classification of anaerobes: Non Spore anaerobes (NSA): Gram negative bacilli NSA -Bacteroides fragilis. -Prevotella melaninogemica -Prophyromonas Spp. -Leptotrichia buccalis(Vincentent s fusiform bacillus) Gram negative cocci NSA. -Fusiform species -Veillonella species Gram Positive Cocci NSA -Anaerobic cocci Peptococci Peptostreptococci Gram Positive Anaerobic Bacill NSA -Propionobacteria anaerobic diphteroids skin common contaminants ( a rare cause of infection after neurosurgery or heart valve operations) -Actinomyces israelli anaerobic Gram positive branching bacillus the cause of actinomycosis). Spore Forming Anaerobes: CLOSTRIDIUM Species -The anaerobic Clostridium species are large anaerobic, Gram-positive, motile rods. The Clostridium species are spore-formers, with spores generally wider than the diameter of the rod-shaped cells, and placement is central, subterminal, or terminal. Most of the species are motile and possess numerous flagella all over the cell (peritrichous flagella). -Many decompose proteins or form toxins, and some do both. The natural habitat of Clostridium species is soil or the intestinal tract of animals and humans, where they live as saprophytes. - Clostridia produce serious disease including botulism, tetanus, gas gangrene, pseudomembranous ulcerative colitis, and food poisoning. Clinically Important Colistridium Species: -Clostridium tetani causing tetanus. -Clostridium perferingens, Clostridium sporogenes, Clostridium oedemetiens causing gas gangrene. - Clostridium Botulinium causing food poisoning botulinism. -Clostridium difficile causing Pseudomembranous colitis. Effect of Disinfectant on anaerobe -Kill NSA, while spore forming anaerobes are resistant. 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. 3- Presence of other organisms- infection or colonization with other bacteria as E.coli in an abdominal wound may enhance growth of B.fragilis, or presence of synergistic infection as in Meleney's gangrene--- Staph aureus+ microaerophilic or anaerobic streptococci. 4- Presence of foreign bodies e.g clothes or soil inserted following car accident Autogonous Infection associated with NSA. -Mouth Vincent s infection, gingivitis -necrotic purulent tonsillitis Dental sepsis may lead to anaerobic endocarditis Leptotrichia, fusiforms and spirochetes Actinomyces israelli Anaerobic cocci and anerobic Gram negative bacilli -Ear, Sinus Chronic suppurative otitis media and sinusitis Bacteroides species in a minority of cases -Lower respiratory tract Aspiration pneumonia Lung absess Empyema Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis -Abdomen Wound infection Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Faecal anaerobic bacteria mainly Bacteroides fragilis -Skin, soft tissues Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Anaerobic cocci and anerobic gram negative bacilli -Female genital tract Post hysterectomy and occasionally post caesarean section wound infections Pelvic actinomycossis in association with intra-uterine device Pyosalpinx Septic abortion Bartholin s abscess Fusiform, Prevotella Melaninenica , Bacteroides fragilis Anaerobic cocci Actinomyces israelii Fusiform, Prevotella melaningenica Bacteroides fragilis Anaerobic cocci Spore Forming Anaerobes: 1-Colistridium tetani -Gram+ rod with terminal spore drum stick appearance. -Causes tetanus (lock jaw) in humans -Spores can be acquired from any types of skin trauma involving an infected device -If an anerobic environment is present, the spores will germinate and eventually form active C.tetani cells. -At the tissue level, the bacterium releases an exotoxin that causes nervous system irregularities -Toxin s effect includes constant skeletal muscle contraction -Due to a blockage of inhibitory interneurons that regulate muscle contraction. -Immunization prevents C.tetani infections in children and adults. -The first four shots are administrated within two years of birth, followed with periodic booster shots given every ten years. 2-Colistridium perfringens: -Can be contracted from dirt via large cuts or wounds -Release of their exotoxin which causes necrosis of the surrounding tissue, also produce gas which leads to a bubbly deformation of infected tissues(gas gangrene). -Can release an enterotoxin that may lead to severe diarrhea. 3-Colistridium botulinum: -produce one of the most potent toxins. -Causes the deadly botulism food poisoning. -May find their way into foods that were placed in anaerobic storage such as cans or jars. -Once the jars are sealed, the spores germinate and the bacteria release their potent toxin. -Patient experience muscular paralysis and blurred vision. -Immediate treatment with anti-toxin is required for the patient to survive. -Infantile botulism is much milder than the adult version. _Honey is the most coomon spores which germinate in the child s intestinal tract. -Symptoms last a few days and then subside without the use of an antitoxin. 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% - wide variety of other animals • Disruption of intestinal flora by antibiotics, chemotherapeutics - clindamycin: most cases per amount used - ampicillin, cephalosporins: most commonly associated (more widely used) • C. difficile often sensitive to antibiotics which initiate the episode • 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 * immunological assay for toxin antigens Treatment • 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 Microbiological diagnosis of the anaerobic Infections: Proper management of anaerobic infections depend on appropriate documentation of the bacteria causing the infection, as certain or all anaerobes may not recovered when the specimen is not placed under anaerobic condition. 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 -----Aspirated pus -----Aspirated body fluids ----CSF Pleural fluid Peritoneal fluid Synovial fluid Pericardial fluid -----Direct lung aspirates, trans- tracheal aspirate, thoracocentesis or BAL. ------In urinary tract infections the suitable samples are by cystescopy or nephrestomy, ureterostomy or suprapubic aspiration. -----Swabs are least advisable and less satisfactory than aspirates or pus samples. 2- Samples Transport; Protection of anaerobic bacteria from oxygen exposure is critical step in the recovery of these organisms. There are many of transport media: 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. It 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 or thioglycate broth in which the sample is immersed and covered and transported 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 1-Direct Examination: -Macroscopic -Microscopic -Macroscopic examination for foul smell, black colour, purulent appearance, necrotic tissues, gas or sulphur granules. -Microscopic examination: -Direct Gram Stain:---for the presence of cocci or bacilli in the sample -Dark field examination or phase contrast for motile organisms or refractile spores. 2- Inoculation of Appropriate anaerobic media: Non Selective media: -Scheadler blood agar -Brucella blood agar -Columbia blood agar -Brain heart infusion -Trypicase Soya agar -Egg Yolk agar Selective Media -Anaerobic Kanamycin-Vancomycin laked blood agar(AKVLB) -Bacteroide bile esculin (BBE). -Anaerobic Phenylethyel alcohol agar (PEA). -Cycloserine Cefoxitin fructose agar(CCFA). 3-Anaerobic Incubation System. The specimens are processed and incubated into appropriate media anaerobically at 37 C in any anaerobic systems The anaerobic systems are: 1-Anaerobic jar. 2- Anaerobic work station or anaerobic chambers. 3-Anaerobic gas bags (biobag system). Duration of anaerobic incubation: At least 48 hours before examination of plate -at least 72-96 hours for slowely growing suspected organisms. -at least 9 days before discarding as negative sample. Processing of colonies suspected to be anaerobes Examination of each colony type appear in plate. Colony morphology(shape,size,appearance,colou,etc…) Grome reaction Pigmentation Hemolysis Flourescence Biochemical Identification of the isolated colonies: A-Preliminary B-Definitive A-Preliminary: Colony morphology,Grom raction, pigments,hemolysis Flourescence character Specific disc—for peptostreptococus anaerobes Nitrate disc for----B.ureolyticus and Veillonella sp. Lipase test-----Clostridium spp, Bacteroides and Fusobacterium Lecithinase---as above Indole test for indole +ve give blue or green colour Catalase test----For Bacteroides and Peptococcus Bile test------ to differentiate bile resistant from bile tolerant Urease test---for Clostridium and Bacteroid urelyticus Ethanol spare test---- Clostidium species Naglar test-----For Clostridium perferingens. B) Definitive Identification of anaerobes: 1- Conventional tubed Biochemical Identification System; Biochemically based this envolves preparing inoculum directely from pure culture and exposued to different tests as indole, sucrose, hippurate, etc….. Commercially available systems include API20A, ATB32A, Minitik system. 2- Enzyme based systems : That based on the presence of performed enz. Consists of small plastic cards or panels that inoculated and require no anaerobic conditions. Most of them generate code number and referred to manufactures supplied code book. Example---------ANI card of Vitek ----------- ANIDENT, rapid ------------Ana 11, Microscan walkaway ------------API Zym system 3-Antimicrobial Susceptibility of Anaerobes 1-Selection of an appropriate antimicrobial agents as: - Metronidazole -Clindamycine and Lincomycin - Penicillin G -Vancomycin for G+ve -Macrolides –Erythromycine - Zithromycin -Carbapenem -Cephalosporin (variable effect) Method: 1-Conventional Methods -Dilution method to asses MIC Agar dilution Method Broth Macrodilution method Broth microdilution method 2-E test 3-Automated method as Vitek, sensititre and Walkaway N.B Disc diffusion method is not suitable for anaerobes 4- Serological Identification For Cl. Difficile and Cl. Botulinium toxins . Flourescent antibodies for bacteroid spp. As fragilis, melaninog. 5-Molecular Identification: To assess the pathogen genotype We use----Restriction endonuclease analysis (REA) ----REA with pulsed gel electrophoresis ------Polyacrimide gel electrophoresis -----------Restriction fragment length polymorphism (RFLP) ----------Hybridization methods Ribotyping. 6-Gas Liquid Chromatography To detect volatile free fatty acids of anaerobes (valeric acid,isovaleric acid, butyric acid and isobutyric acids-they are responsible for foul odour of anaerobeic infection) usefull for Bacteroides, Colistridium, and Fusebacterium spp.
Anaerobic Infections
Anaerobic Infections By Maysaa ElSayed Zaki Professor Of Clinical Pathology Definition of anaerobe: It is a microbe that can only grow under anaerobic conditions only and is sensitive to metronidazole on routine disc testing anaerobically. Anaerobes are the most primitive bacteria in terms of oxidation (the greater the oxidation, the more advanced evolutionarily). They use a relatively inefficient metabolic system: anaerobic fermentation provides energy at 10% the level of aerobic respiration 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. Classification of anaerobes: Non Spore anaerobes (NSA): Gram negative bacilli NSA -Bacteroides fragilis. -Prevotella melaninogemica -Prophyromonas Spp. -Leptotrichia buccalis(Vincentent s fusiform bacillus) Gram negative cocci NSA. -Fusiform species -Veillonella species Gram Positive Cocci NSA -Anaerobic cocci Peptococci Peptostreptococci Gram Positive Anaerobic Bacill NSA -Propionobacteria anaerobic diphteroids skin common contaminants ( a rare cause of infection after neurosurgery or heart valve operations) -Actinomyces israelli anaerobic Gram positive branching bacillus the cause of actinomycosis). Spore Forming Anaerobes: CLOSTRIDIUM Species -The anaerobic Clostridium species are large anaerobic, Gram-positive, motile rods. The Clostridium species are spore-formers, with spores generally wider than the diameter of the rod-shaped cells, and placement is central, subterminal, or terminal. Most of the species are motile and possess numerous flagella all over the cell (peritrichous flagella). -Many decompose proteins or form toxins, and some do both. The natural habitat of Clostridium species is soil or the intestinal tract of animals and humans, where they live as saprophytes. - Clostridia produce serious disease including botulism, tetanus, gas gangrene, pseudomembranous ulcerative colitis, and food poisoning. Clinically Important Colistridium Species: -Clostridium tetani causing tetanus. -Clostridium perferingens, Clostridium sporogenes, Clostridium oedemetiens causing gas gangrene. - Clostridium Botulinium causing food poisoning botulinism. -Clostridium difficile causing Pseudomembranous colitis. Effect of Disinfectant on anaerobe -Kill NSA, while spore forming anaerobes are resistant. 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. 3- Presence of other organisms- infection or colonization with other bacteria as E.coli in an abdominal wound may enhance growth of B.fragilis, or presence of synergistic infection as in Meleney's gangrene--- Staph aureus+ microaerophilic or anaerobic streptococci. 4- Presence of foreign bodies e.g clothes or soil inserted following car accident Autogonous Infection associated with NSA. -Mouth Vincent s infection, gingivitis -necrotic purulent tonsillitis Dental sepsis may lead to anaerobic endocarditis Leptotrichia, fusiforms and spirochetes Actinomyces israelli Anaerobic cocci and anerobic Gram negative bacilli -Ear, Sinus Chronic suppurative otitis media and sinusitis Bacteroides species in a minority of cases -Lower respiratory tract Aspiration pneumonia Lung absess Empyema Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis -Abdomen Wound infection Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Faecal anaerobic bacteria mainly Bacteroides fragilis -Skin, soft tissues Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Anaerobic cocci and anerobic gram negative bacilli -Female genital tract Post hysterectomy and occasionally post caesarean section wound infections Pelvic actinomycossis in association with intra-uterine device Pyosalpinx Septic abortion Bartholin s abscess Fusiform, Prevotella Melaninenica , Bacteroides fragilis Anaerobic cocci Actinomyces israelii Fusiform, Prevotella melaningenica Bacteroides fragilis Anaerobic cocci Spore Forming Anaerobes: 1-Colistridium tetani -Gram+ rod with terminal spore drum stick appearance. -Causes tetanus (lock jaw) in humans -Spores can be acquired from any types of skin trauma involving an infected device -If an anerobic environment is present, the spores will germinate and eventually form active C.tetani cells. -At the tissue level, the bacterium releases an exotoxin that causes nervous system irregularities -Toxin s effect includes constant skeletal muscle contraction -Due to a blockage of inhibitory interneurons that regulate muscle contraction. -Immunization prevents C.tetani infections in children and adults. -The first four shots are administrated within two years of birth, followed with periodic booster shots given every ten years. 2-Colistridium perfringens: -Can be contracted from dirt via large cuts or wounds -Release of their exotoxin which causes necrosis of the surrounding tissue, also produce gas which leads to a bubbly deformation of infected tissues(gas gangrene). -Can release an enterotoxin that may lead to severe diarrhea. 3-Colistridium botulinum: -produce one of the most potent toxins. -Causes the deadly botulism food poisoning. -May find their way into foods that were placed in anaerobic storage such as cans or jars. -Once the jars are sealed, the spores germinate and the bacteria release their potent toxin. -Patient experience muscular paralysis and blurred vision. -Immediate treatment with anti-toxin is required for the patient to survive. -Infantile botulism is much milder than the adult version. _Honey is the most coomon spores which germinate in the child s intestinal tract. -Symptoms last a few days and then subside without the use of an antitoxin. 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% - wide variety of other animals • Disruption of intestinal flora by antibiotics, chemotherapeutics - clindamycin: most cases per amount used - ampicillin, cephalosporins: most commonly associated (more widely used) • C. difficile often sensitive to antibiotics which initiate the episode • 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 * immunological assay for toxin antigens Treatment • 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 Microbiological diagnosis of the anaerobic Infections: Proper management of anaerobic infections depend on appropriate documentation of the bacteria causing the infection, as certain or all anaerobes may not recovered when the specimen is not placed under anaerobic condition. 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 -----Aspirated pus -----Aspirated body fluids ----CSF Pleural fluid Peritoneal fluid Synovial fluid Pericardial fluid -----Direct lung aspirates, trans- tracheal aspirate, thoracocentesis or BAL. ------In urinary tract infections the suitable samples are by cystescopy or nephrestomy, ureterostomy or suprapubic aspiration. -----Swabs are least advisable and less satisfactory than aspirates or pus samples. 2- Samples Transport; Protection of anaerobic bacteria from oxygen exposure is critical step in the recovery of these organisms. There are many of transport media: 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. It 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 or thioglycate broth in which the sample is immersed and covered and transported 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 1-Direct Examination: -Macroscopic -Microscopic -Macroscopic examination for foul smell, black colour, purulent appearance, necrotic tissues, gas or sulphur granules. -Microscopic examination: -Direct Gram Stain:---for the presence of cocci or bacilli in the sample -Dark field examination or phase contrast for motile organisms or refractile spores. 2- Inoculation of Appropriate anaerobic media: Non Selective media: -Scheadler blood agar -Brucella blood agar -Columbia blood agar -Brain heart infusion -Trypicase Soya agar -Egg Yolk agar Selective Media -Anaerobic Kanamycin-Vancomycin laked blood agar(AKVLB) -Bacteroide bile esculin (BBE). -Anaerobic Phenylethyel alcohol agar (PEA). -Cycloserine Cefoxitin fructose agar(CCFA). 3-Anaerobic Incubation System. The specimens are processed and incubated into appropriate media anaerobically at 37 C in any anaerobic systems The anaerobic systems are: 1-Anaerobic jar. 2- Anaerobic work station or anaerobic chambers. 3-Anaerobic gas bags (biobag system). Duration of anaerobic incubation: At least 48 hours before examination of plate -at least 72-96 hours for slowely growing suspected organisms. -at least 9 days before discarding as negative sample. Processing of colonies suspected to be anaerobes Examination of each colony type appear in plate. Colony morphology(shape,size,appearance,colou,etc…) Grome reaction Pigmentation Hemolysis Flourescence Biochemical Identification of the isolated colonies: A-Preliminary B-Definitive A-Preliminary: Colony morphology,Grom raction, pigments,hemolysis Flourescence character Specific disc—for peptostreptococus anaerobes Nitrate disc for----B.ureolyticus and Veillonella sp. Lipase test-----Clostridium spp, Bacteroides and Fusobacterium Lecithinase---as above Indole test for indole +ve give blue or green colour Catalase test----For Bacteroides and Peptococcus Bile test------ to differentiate bile resistant from bile tolerant Urease test---for Clostridium and Bacteroid urelyticus Ethanol spare test---- Clostidium species Naglar test-----For Clostridium perferingens. B) Definitive Identification of anaerobes: 1- Conventional tubed Biochemical Identification System; Biochemically based this envolves preparing inoculum directely from pure culture and exposued to different tests as indole, sucrose, hippurate, etc….. Commercially available systems include API20A, ATB32A, Minitik system. 2- Enzyme based systems : That based on the presence of performed enz. Consists of small plastic cards or panels that inoculated and require no anaerobic conditions. Most of them generate code number and referred to manufactures supplied code book. Example---------ANI card of Vitek ----------- ANIDENT, rapid ------------Ana 11, Microscan walkaway ------------API Zym system 3-Antimicrobial Susceptibility of Anaerobes 1-Selection of an appropriate antimicrobial agents as: - Metronidazole -Clindamycine and Lincomycin - Penicillin G -Vancomycin for G+ve -Macrolides –Erythromycine - Zithromycin -Carbapenem -Cephalosporin (variable effect) Method: 1-Conventional Methods -Dilution method to asses MIC Agar dilution Method Broth Macrodilution method Broth microdilution method 2-E test 3-Automated method as Vitek, sensititre and Walkaway N.B Disc diffusion method is not suitable for anaerobes 4- Serological Identification For Cl. Difficile and Cl. Botulinium toxins . Flourescent antibodies for bacteroid spp. As fragilis, melaninog. 5-Molecular Identification: To assess the pathogen genotype We use----Restriction endonuclease analysis (REA) ----REA with pulsed gel electrophoresis ------Polyacrimide gel electrophoresis -----------Restriction fragment length polymorphism (RFLP) ----------Hybridization methods Ribotyping. 6-Gas Liquid Chromatography To detect volatile free fatty acids of anaerobes (valeric acid,isovaleric acid, butyric acid and isobutyric acids-they are responsible for foul odour of anaerobeic infection) usefull for Bacteroides, Colistridium, and Fusebacterium spp.
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More about antibiotics uses, resistance will be found at my book
http://www.barnesandnoble.com/w/antibiotics-toward-gram-positive-cocci-maysaa-el-sayed-zaki/1116741402?ean=9783656484530
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