Definition 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
Classification of anaerobes:
Non Spore anaerobes (NSA).
According to Gram stain and morphology if bacilli or Cocci.
Spore Forming anaerobes
Clostridium species
Gram negative bacilli NSA
Bacteroides fragilis.
Prevotella melaninogemica
Prophyromonas Spp.
Leptotrichia buccalis(Vincentent s fusiform bacillus)
Gram negative cocci.
-Fusiform species
-Veillonella species
Gram Positive Cocci NSA
-Anaerobic cocci
Peptococci
Peptostreptococci
Anaerobic Gram positive bacilli
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 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.
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
Autogenous Infection associated with NSA.
Leptotrichia, fusiforms and spirochetes
Actinomyces israelli
Anaerobic cocci and anerobic Gram negative b Vincent s infection, gingivitis
-necrotic purulent
tonsillitis
Dental sepsis may lead to anaerobic endocarditis Mouth
Bacteroides species in a minority of cases Chronic suppurative otitis media and sinusitis Ear, Sinus
Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis Aspiration pneumonia
Lung absess
Empyema Lower respiratory tract
Faecal anaerobic bacteria mainly Bacteroides fragilis Wound infection
Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Abdomen
Anaerobic cocci and anerobic gram negative bacilli Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Skin, soft tissues
Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis Aspiration pneumonia
Lung absess
Empyema Lower respiratory tract
Faecal anaerobic bacteria mainly Bacteroides fragilis Wound infection
Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Abdomen
Anaerobic cocci and anerobic gram negative bacilli Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Skin, soft tissues
Fusiform, Prevotella Melaninenica , Bacteroides fragilis
Anaerobic cocci
Actinomyces israelii
Fusiform, Prevotella melaningenica
Bacteroides fragilis
Anaerobic cocci Post hysterectomy and occasionally post caesarean section wound infections
Pelvic actinomycossis in association with intra-uterine device
Pyosalpinx
Septic abortion
Bartholin s abscess Female genital tract
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
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
-----Aspirateed pus
-----Aspirated body fluids ----CSF
Pleural Fluid
Peritoneal Fluid
Synovial Fluid
Pericardial fluid
Direct lung aspirates,tran tracheal, thoracocentesis or BAL.
In UTI the suitable samples are by cystescopy or nephrestomy, ureterostomy or suprapubic aspiration.
-----Swabs are least advisable and less satisfactory than aspirates or pus samples.
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
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:
a)Anaerobic jar.
b)Anaerobic work station or anaerobic chambers.
c) 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.
4-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
5- 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 tubes Biochemical Identification System.This involves preparing inoculums directly from pure culture and exposed 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-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.
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
Classification of anaerobes:
Non Spore anaerobes (NSA).
According to Gram stain and morphology if bacilli or Cocci.
Spore Forming anaerobes
Clostridium species
Gram negative bacilli NSA
Bacteroides fragilis.
Prevotella melaninogemica
Prophyromonas Spp.
Leptotrichia buccalis(Vincentent s fusiform bacillus)
Gram negative cocci.
-Fusiform species
-Veillonella species
Gram Positive Cocci NSA
-Anaerobic cocci
Peptococci
Peptostreptococci
Anaerobic Gram positive bacilli
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 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.
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
Autogenous Infection associated with NSA.
Leptotrichia, fusiforms and spirochetes
Actinomyces israelli
Anaerobic cocci and anerobic Gram negative b Vincent s infection, gingivitis
-necrotic purulent
tonsillitis
Dental sepsis may lead to anaerobic endocarditis Mouth
Bacteroides species in a minority of cases Chronic suppurative otitis media and sinusitis Ear, Sinus
Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis Aspiration pneumonia
Lung absess
Empyema Lower respiratory tract
Faecal anaerobic bacteria mainly Bacteroides fragilis Wound infection
Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Abdomen
Anaerobic cocci and anerobic gram negative bacilli Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Skin, soft tissues
Anaerobes from the mouth including Fusiform and anaerobic cocci occasionally Bacteroides fragilis Aspiration pneumonia
Lung absess
Empyema Lower respiratory tract
Faecal anaerobic bacteria mainly Bacteroides fragilis Wound infection
Abscesses or perotinitis associated with bowel cancer, appendicitis , diverticulitis and bowel surgery---bacteremia as complication Abdomen
Anaerobic cocci and anerobic gram negative bacilli Infected diabetic ulcers, deep pressure sores, axillary abscesses, infected sebaceous cyst, Meleney s synergestic gangrene Skin, soft tissues
Fusiform, Prevotella Melaninenica , Bacteroides fragilis
Anaerobic cocci
Actinomyces israelii
Fusiform, Prevotella melaningenica
Bacteroides fragilis
Anaerobic cocci Post hysterectomy and occasionally post caesarean section wound infections
Pelvic actinomycossis in association with intra-uterine device
Pyosalpinx
Septic abortion
Bartholin s abscess Female genital tract
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
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
-----Aspirateed pus
-----Aspirated body fluids ----CSF
Pleural Fluid
Peritoneal Fluid
Synovial Fluid
Pericardial fluid
Direct lung aspirates,tran tracheal, thoracocentesis or BAL.
In UTI the suitable samples are by cystescopy or nephrestomy, ureterostomy or suprapubic aspiration.
-----Swabs are least advisable and less satisfactory than aspirates or pus samples.
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
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:
a)Anaerobic jar.
b)Anaerobic work station or anaerobic chambers.
c) 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.
4-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
5- 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 tubes Biochemical Identification System.This involves preparing inoculums directly from pure culture and exposed 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-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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