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- 3. • Anaerobes Microorganisms that grow only in complete or nearly complete absence of molecular oxygen. Non-sporing Anaerobes – These do not form spores and are those which usually form the Normal flora Of Human beings and usually present in Skin, Oral cavity, GIT, Genitourinary tract and are opportunistic in nature. Introduction
- 4. HABITAT • Normal flora of skin, mucosal surfaces, mouth, respiratory tract, GIT, genital tract • Outnumber aerobes in many habitats – mouth and skin -10 to 30 times > aerobes – Intestines-1000 times >aerobes • Estimated no of anaerobes in: – Saliva – 108/ ml – Small intestine – 105/ ml – Colon – 1011/ gm 4
- 5. 5 Gram negative bacilli Gram positive bacilli Bacteroides Prevotella Porphyromonas Fusobacterium Leptotrichia Eubacterium Propionibacterium Lactobacillus Mobiluncus Bifidobacterium Actinomyces Cocci Peptococci Peptostreptococci Veillonella (gram negative)
- 6. ANAEROBIC INFECTION EXOGENOUS ORIGIN: • Superficial infection • Infection following animal or human infection • Infection of injection • Septic abortion ENDOGENOUS ORIGIN: • Actinomycosis • Aspiration pneumonia • Complications of appendicitis • Dental and duodenal infections • Septic arthritis • Subdural emphysema • Thoracic emphysema
- 8. VIRULENCE FACTORS Actinomyces species Including A.israeli, A.meyeri A.naeslundii A.odontolyticus Not well characerised. Infections usually require disruptions of protective mucosal surface of the oral cavity, respiratory tract, GIT Propionibacterium species Bifidobacterium species Eubacterium species Mobiluncus species No definitive virulence factors are known Bacteroides species including B.fragilis B.gracilis B.ureolyticus Provotella species Porphyromonas species Fusobacterium nucleatum These produce capsules and succinic acid, which inhibits phagocytosis and various enzymes that mediate cell damage
- 10. ANAEROBIC COCCI • Peptococcus & Peptostreptococcus – usually produce mixed infections along with Clostridia or anaerobic gram negative bacilli – Puerperal sepsis & other genital infections – Wound infections – Gangrenous appendicitis – UTI – Osteomyelitis – Abscesses in brain, lungs & other internal organs 10
- 12. GRAM POSITIVE BACILLI A,israelii,A.meyeri A.naeslundii A.odontolyticus species Usually involved in mixed oral or cervicofacial, thoracic,pelvic and abdominal infections caused by patients endogenous strains Propionibacterium species Usually involved in mixed oral or cervicofacial, thoracic, pelvic and abdominal infections caused by patients endogenous strains Bifidobacterium species Eubacterium species Usually encountered in mixed infections of pelvis or genito urinary tract Mobiluncus species Usually encountered in mixed infections of pelvis or genito urinary tract
- 14. PATHOGENICITY OF GRAM NEGATIVE ANAEROBIC BACILLI Bacteroides fragilis Brain abscess, intra abdominal abscess, infections of female genitalia, cellulitis, diabetic ulcer, septicaemia Prevotella melaninogenica Lung or liver abscess, empyema, pelvic infections in females, breast abscess, wound infections Porphyromonas Dental root canal infections, periodontal disease Fusobacterium necrophorum Fusobacterium nucleatum Aspiration pneumonia, lung/ liver abscess, oral infections, chronic sinusitis, abdominal infections
- 15. • Bacteroides fragilis Prevotella
- 17. PREDISPOSING FACTORS • Trauma, Tissue necrosis, Impaired circulation, hematoma formation or the presence of foreign bodies • Diabetes, Malnutrition, Malignancy or prolonged treatment with antibiotics. • They are usually polymicrobial in nature, more than one anaerobe can be responsible besides aerobic bacteria. • When the infection is usually localised, it gets generalised by hematogenous route causing Bacteremia.
- 18. Sequence of the events Trauma to the sites of protective barriers allow anaerobes of indigenous flora to gain access to deeper tissues. Vascular stasis Growth & multiplication of anaerobes Anaerobic infection
- 19. Brain abscesses, Chronic sinusitis, Periodontal infections caused by: • Peptostreptococcus anaerobius •Finegoldia magna •Bacteroides spp. •Fusobacterium nucleatum Bacteremia, Septicemia, Endocarditis, Lung abscess caused by: • Bacteroides fragillis • Clostridium perfringens • Fusobacterium spp. Hepatic, abdominal infections • Bacteroides fragilis • Clostridium spp. Pelvic abscesses in females • Bacteroides bivius •Bacteroides disiens •Peptostreptococcus Necrotizing fascitis • Peptostreptococcus spp. Gas gangrene • Clostridium septicum •Clostridium novyi
- 20. Polymicrobial nature of the anaerobic infection • Contamination of the tissue by the normal flora of the mucosa of the mouth, pharynx, GIT or genital tract. • Multiple species are present including other anerobes and facultative anaerobes. • Aerobic bacteria may also be present.
- 21. CHARACTERISTICS SUGGESTING ANAEROBIC INFECTIONS Foul smelling discharge Infection in close proximity to mucosal surface. Tendency to form closed space infections either as discrete abscess (lung, brain, pleura) or by burrowing through tissue layers Polymicrobial nature. Infection associated with necrotic tissue (poor blood supply).
- 22. Gas formation in tissues Failure to isolate organisms from pus (sterile pus) & negative aerobic cultures. Lack of response to usual antibiotic therapy. Infection of human or animal bite wounds. Detection of sulphur granules in pus . Gram negative bacteraemia is more common.
- 24. Acceptable specimens 1. Head and neck 2.Respiratory tract 3.Abdomen 4.Urinary tract 5.Bone and joint 6.Genital tract •Tissue fluid aspirate ,cerebrospinal fluid . •pleural fluid,broncho alveolar lavage fluid. •Peritoneal (ascitic fluid) abscess aspirate. •Suprapubic aspirate . •Bone marrow ,synovial fluid. •Endoscopy specimen,endometrial aspirate. Suitable specimens
- 25. 1. Head and neck 2. Respiratory tract 3. Abdomen 4.Urinary tract 5.Genital tract 6.Soft tissue Throat swab,nasopharayngeal swab. Expectorated sputum,nasal swab. Gastric lavage, rectal swab, colostomy drainage. Voided urine, catheterised urine, urethral swab. Vaginal swab, cervical swab. Superficial material swab U U n a n c s c u e i t p a t b a l b e l e s p s e p c e i c m i m e n e s n s
- 26. Collection and transport of specimen •Aspirate collection by syringe can be sent by plugging the needle with a cork. • Other specimen can be transported in robertson cookedmeat media or in anaerobic transport media
- 27. Transport of samples • Enriched thioglycollate broth • Oxygen free systems gassed with CO2 or N2 • Anaerobic pouches
- 28. Anaerobic specimen processing and identification SPECIMEN 1.Gross examination Purulence , necrosis, foul odour, sulphur granules, fluoresence . 2. Gram stain •Methanol fixed (30 sec ) smear. •Pale , pleomorphic filamentous Gram negative bacteria with or with out vacuoles , irregular staining bacteriodes species. •Pale gram negative cocco bacilli , pigmented prevotella species or porphyromonas species . 3. inoculation into culture media •Brucella blood agar with 5% sheep blood , neomycin , hemin and vitamin k. • Phenyl ethyl blood agar. •Robertson cooked meat media or enriched thioglycolate media with vitamin k and hemin.
- 29. •Thin pale gram negative bacteria with tapering ends(fusiform). Fusobacterium nucleatum. •Very small gram negative cocci Veillonella species. •Kanamycin-vancomycin laked blood agar if pale gram negative cocco bacilli found on gram stain. •Bacteroides bile esculin agar for bacteriodes fragils.
- 31. • Microscopy–Gramstain: different micro-organisms • Culturemedia – Brucellabloodagar (BRBA) – Phenyl ethyl alcohol agar (PEA) – Kanamycin-vancomycin-laked bloodagar(KVLBA) – Bacteroides bileesculin agar(BEA) – Neomycinbloodagar(NBA) • Culturemethods–Anaerobic jar, Gaspak 31
- 32. FUSOBACTERIUM NUCLEATUM ACTINOMYCES VELLIONELLA GRAM STAINING MORPHOLOGY BACTEROIDES FRAGILIS
- 33. Processing of samples Gross examination • Blood • Purulence • Necrotic tissue • Foul odor • Sulphur granules
- 34. Anaerobic Jar • Tightly sealed container in which oxygen is completely eliminated by hydrogen and a catalyst. • palladium coated aluminum pellets acts as a catalyst. • Methylene blue anaerobic indicator. • Anaerobic charge activated with H2SO4.
- 35. • Colony morphology • Agar pitting • Black or tan pigmentation • Brick red fluorescence • Fried egg Identification Bacteroides ureolyticus Porphyromonas Prevotella Fusobacterium necrophorum • Greening of medium • Molar tooth • Speckled or bread-crumb Fusobacterium varium Actinomyces Fusobacterium nucleatum
- 36. Pigment production • Porphyromonas,prevote lla on anaerobic media • Dark brown or black pigment.
- 37. Fluorescence • Porphyromonas species Prevotella species, brick red fluorescence. • Detected using 366nm UV light
- 38. Bile Resistance • Bacteroides resistance to bile on bacteroides bile esculin agar. • Confirmatory test for bacteroides.
- 39. Spot Indole test • Growth obtained from a single,pure culture on a blood agar plate is smeared on filter paper that has been saturated with 1% paradimethylaminocinnamaldehyde in 10% concentrated HCl. • Immediate formation of a blue color indicates positive reaction.
- 40. Gas Liquid Chromatography Analysis of metabolites • Acetic acid- eg:bifidobacterium • Propionic acid- eg:propionibacterium • Isobutyric acid- eg:porphyromonas • Valeric acid- eg:fusobacterium • Isocaproic acid- eg:peptostreptococcus
- 41. Identification by means of special-potency Antimicrobial agent disk Brucella agar plate with kanamycin, vancomycin, colistin disks
- 42. organisms Kanamycin 1000ug vancomycin 5ug Colistin 10ug Bacteroides fragilis R R R Bacteroides ureolyticus S R S Fusobacterium S R R Prevotella R R V veillonella S R S 3. Clinical Microbiology Proceedings handbook. 2nd Edition. Henry D. Isenberg.
- 43. Antibiotic resistance • Most of bacteroides fragilis,prevotella and porphyromonas species are resistant to penicillins and to many cephalosporins due to production of beta-lactamase. • The resistance is overcome by treating with high concentration of piperacillin,imipenem along with beta-lactamase inhibitors. • Bacteroides show plasmid mediated resistance to clindamycin.
- 44. Treatment • Surgical – – Drainage of pusfromabscess – W ounddebridement – Curettage &removalof necrotictissues • Antibiotics – – Metronidazole – Penicillin – Clindamycin – Cephalosporins – chloramphenicol 44
- 45. SUMMARY CNS Cerebral,epidural,subdural abscess. SSI W ounds, ulcers, abscess. RT Pneumonia, empyema,lung abscess. GIT Abdominal, liver abscess, appendicitis. FGT Bacterial vaginosis, abscess, septic abortion. Fusobacterium, Peptostreptococcus, Propionibacterium. Peptostreptococcus, Propionibacterium. Bacteroides,Actinomyces, Prevotella,Eubacterium. Fusobacterium, Eubacterium, Propionibacterium Bacteriodes,Mobiluncus, Veillonella 45
- 46. References 1. Konemans Color Atlas and Textbook of Diagnostic Microbiology. 6th Edition. Winn, Alen, Jenha. LWW 2. Bailey and Scotts Diagnostic Microbiology 12th Edition. Forbes, Sahn, Weissel. Mosby 3. Clinical Microbiology Proceedings handbook. 2nd Edition. Henry D. Isenberg. 4. Anaerobic bacteriology: Clinical and Laboratory Practice, 3rd Edition, A. Trevor Willis. 1979 Butterworths. 5. Mackie and McCartney practical medical microbiology. 14th Edition, J.G.Collee,A.G.Frases,B.P.Marmion,A.Simmons.
- 47. THANK YOU