anaerobic bacteriology

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Anaerobic Bacteriology

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  • 1.ANAEROBICBACTERIOLOGY Dr.T.V.Rao MD Dr.T.V.Rao MD 1

2. What Are Anaerobic Microorganisms2 Anaerobicmicroorganismsare widespreadand veryimportant Do not requireoxygen for growth- often extremelytoxic Dr.T.V.Rao MD 3. The Requirements for Growth:Related to Oxygen Oxygen (O2)Dr.T.V.Rao MD 3Table 6.1 4. Anaerobes differ from AerobicBacteria Anaerobes generate energy by fermentation Lack the capacity to utilize O2 as a terminal hydrogenacceptor Some are sensitive to O2 concentration as low as 0.5%O2 Most can survive in 3%-5% O2 A few can grow poorly in the presence of air aerotolerant anaerobes Many are members of the normal flora created by presence of facultativeanaerobes Dr.T.V.Rao MD 4 5. DEFINITIONS OBLIGAETE ANAEROBE Lack superoxide dismutase and/or catalase toxic radicals formed by oxidative enzymes killorganisms AERO-TOLERANT ANAEROBES survive in presence of oxygen Do not use oxygen for energy requirements FACULTATIVE ANAEROBES Dr.T.V.Rao MD5 6. Anaerobes and Oxygen Anaerobes generate energy by fermentation Lack the capacity to utilize O2 as a terminal hydrogenacceptor Some are sensitive to O2 concentration as low as 0.5% O2 Most can survive in 3%-5% O2 A few can grow poorly in the presence of air aero tolerantanaerobes Many are members of the normal flora created by presence of facultativeanaerobesDr.T.V.Rao MD6 7. Anaerobic and Aerobic RespirationReaction name Reduc Oxid. Reaction kcal/ t.Stoichiometry molAerobic CHO O2C6H12O6 + 6O2 ==>686Respiration6CO2 + 6H2ONitrate CHO NO3- CHO + NO3- + H+ 649Reduction ==> CO2+ N2+ H2OSulfate CHO SO42- 2CHO + SO42-+2H+ 190Reduction=> 2CO2+ H+ 2H2OMethanogenesis CHOCO24H2 + CO2 ==> CH4 8.3or H2 + 2H2ODr.T.V.Rao MD7 8. FACTORS THAT INHIBIT THEGROWTH OF ANAEROBES BYOXYGEN1.Toxic compounds are produced e.g. H2O2 , Superoxides2. Absence of catalase & Superoxide dismutase3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them Dr.T.V.Rao MD8 9. Strict Anaerobic Bacteria9 Obligate (strict)anaerobes - oxygen istoxic to theseorganisms, do not useoxygen as terminalelectron acceptor. Archaea such asmethanogens andBacteria, e.g Clostridia,Bacteriodes etc. etc. Dr.T.V.Rao MD 10. ROS production during respiration O2 + e- => O2- superoxide anion O2- + e- + 2H+ => H2O2 hydrogen peroxide H2O2 + e- + H+ => H2O + OH. Hydroxyl radical OH. + e- + H+ => H2O water Dr.T.V.Rao MD10 11. Oxygen Toxicity Oxygen is used by aerobic and facultativelyanaerobic organisms as its strong oxidisingability makes it an excellent electronacceptor During the stepwise reduction ofoxygen, which takes place in respirationtoxic and highly reactive intermediatesare produced reactive oxygen species (ROS). Dr.T.V.Rao MD11 12. FACTORS THAT INHIBIT THEGROWTH OF ANAEROBES BYOXYGEN1.Toxic compounds are produced e.g. H2O2 , Superoxides2. Absence of catalase & Superoxide dismutase3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate themDr.T.V.Rao MD 12 13. Chemical Dynamics in AnaerobicBacteria Organisms that use O2 have developed defence mechanisms toprotect themselves from these toxic forms of oxygen -enzymes Catalase: H2O2 + H2O2 => 2H2O + O2 Peroxidase: H2O2 + NADH + H+ => 2H2O +NAD+ Superoxide dismutase: O2- + O2- + 2H+ => H2O2 + O2 Dr.T.V.Rao MD 13 14. Anaerobic environments exist inNature too Anaerobic environments (low reductionpotential) include: Sediments of lakes, rivers and oceans; bogs,marshes, flooded soils, intestinal tract ofanimals; oral cavity of animals, deepunderground areas, e.g. oil packets and someaquifers Anaerobes also important in some infections,e.g. C. tetanii and C. perfringens important indeep puncture wound infectionsDr.T.V.Rao MD 14 15. ANAEROBES OF CLINICAL IMPORTANCE CLOSTRIDIA C tetani; C perfringens; C difficile; C botulinum BACTEROIDES B fragilis; Prevotella Porphyromonas ACTINOMYCES FUSOBACTERIUM ANAEROBIC STREPTOCOCCIDr.T.V.Rao MD 15 16. FACTORS RESPONSIBLE FOR THEIR VIRULENCE1. Lipopolysaccharide- promotes abscess formation, enhanced coagulation2. Polysaccharide capsule - correlated with abscess production3. Enzymesa. Collagenaseb. Heparinize* develop thrombophlebitis & septic emboli4. Short chained fatty acidsa. Butyrate- seen in dental plaqueb. succinic acid reduces phagocytic killing Dr.T.V.Rao MD 16 17. Multiplication of the opportunistic pathogens is facilitated by:1. Inhibition of phagocytosis & intracellularkillingby PMN in the presence of Bacteroides by:a. competition of opsoninsb. inhibition by capsular materials2. Protection of antibiotic susceptibilitystrains in mixtures thru destruction by the -lactamases3. Utilization of O2 by facultative species thataids in producing a suitable environmentfor growth of anaerobe Dr.T.V.Rao MD 17 18. CLINICAL MANIFESTATIONClinical finding suggestive of Anaerobic infection 1. odor 2. tissue 3. location 4. necrotic tissue 5. endocarditis with (-) blood culture 6. infection associated with malignancy 7. black discoloration 8. blood containing exudates 9. associated with sulfur granules 10. Bacteremic feature with jaundice 11. human bitesDr.T.V.Rao MD18 19. Dr.T.V.Rao MD 19 20. Dr.T.V.Rao MD 20 21. Anaerobic Bacteria of Medical Interest MORPHOLOGY GRAM STAIN GENUS Spore forming (+) ClostridiumNon-spore forming bacilli Actinomycetes,Bifidobacterium,Eubacte- (+)rium,Propionibacerium,Mobilncus,Lactobacillus(-) Bacteroides,FusobacteriumPrevotella,PorphyromonasNon-sporefoming cocci Peptococcus, (+)Pepto-streptococcusStreptococcus(-) VeilonellaDr.T.V.Rao MD 21 22. Pathogenesis of anaerobic infections Contamination of site with spores Factors which promote anaerobiasis crush injuries with interruption of blood supply,contamination with foreign bodies (dirt), tissuedamage Germination of spores Toxin release Binding of toxin to receptor Resulting effect produces symptom(s) ofdiseaseDr.T.V.Rao MD22 23. Gram-positive anaerobes Actinomyces (head, neck, pelvic infections;aspiration pneumonia) Bifid bacterium (ear infections, abdominalinfections) Clostridium (gas, gangrene, food poisoning,tetanus, pseudomembranous colitis) Peptostreptococcus (oral, respiratory, andintra-abdominal infections) Propionibacterium (shunt infections) Dr.T.V.Rao MD23 24. Gram-negative anaerobes Bactericides (the most commonly foundanaerobes in cultures; intra-abdominal infections,rectal abscesses, soft tissue infections, liverinfection) Fusobacterium (abscesses, wound infections,pulmonary and intracranial infections) Porphyromonas (aspiration pneumonia,periodontitis) Prevotella (intra-abdominal infections, soft tissueinfections) Dr.T.V.Rao MD24 25. FACTORS RESPONSIBLE FORTHEIR VIRULENCE1. Lipopolysaccharide- promotes abscess formation, enhancedcoagulation2. Polysaccharide capsule - correlated with abscess production3. Enzymesa. Collagenaseb. Heparinase* develop thrombophlebitis & septic emboli4. Short chained fatty acidsa. Butyrate- seen in dental plaqueb. succinic acid reduces phagocytic killing Dr.T.V.Rao MD25 26. Common HumanAnaerobic InfectionsDr.T.V.Rao MD 26 27. 27CLOSTRIDIA Gram positive spore forming bacilli ubiquitous intestines of man and animals animal and human faeces contaminated soil and water Several species associated with human diseaseDr.T.V.Rao MD 28. Clostridium perfringens Large rectangular Gram positive bacillus Spores seldom seen in vivo or in vitro non motile Produces several toxins alpha (lecithinase), beta, epsilon ...... enterotoxin Causes a spectrum of human diseases Bacteraemia Myonecrosis food poisoning enteritis necrotica (pig bel)Dr.T.V.Rao MD 28 29. Clostridium tetani Small motile spore forming gram positive bacillus withround terminal spores Causes tetanus Pathogenesis: produces tetanospasmin during stationary phase which is released when cell lysis occurs heavy chain binds to ganglioside on neuronal membranes toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport crosses synapse and localized within vesicles acts by blocking release of inhibitory neurotransmittors (eg GABA) Dr.T.V.Rao MD29 30. Clostridium tetani Small motile spore forming gram positive bacilluswith round terminal spores Causes tetanus Pathogenesis: produces tetanospasmin during stationary phase which is released when cell lysis occurs heavy chain binds to ganglioside on neuronal membranes toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport crosses synapse and localized within vesicles acts by blocking release of inhibitory neurotransmitters (eg GABA) Dr.T.V.Rao MD 30 31. TETANUS Clinical syndromes dueto unregulatedexcitatory synapticactivity resulting inspastic paralysis Generalised tetanus Neonatal tetanus localized tetanus Dr.T.V.Rao MD 31 32. Prevention and treatment Active immunization with tetanus toxoid Wound toilet and active/passive immunizationof risk injuries management of wound tetanus toxoid Anti-tetanus serum (ARS -horse serum) or HumanTetanus ImmunoGlobulin (HTIG) Penicillin or Metronidazole Management of patient with tetanus reduce stimuli respiratory and CVS supportDr.T.V.Rao MD32 33. Clostridium difficile Associated with human disease in mid-1970s Found in human GIT in small numbers With antibiotic use, increase in number in GIT Clindamycin, ampicillin, cephalosporins ....... Produces 2 entero toxins Toxin A -enterotoxin & Toxin B -cytotoxin Diagnosis Detection of toxins in stools, culture of organism Clinical - AAC Pseudomembranous colitis Treatment omit antibiotic if possible oral vancomycin (125mg qds or metronidazole Dr.T.V.Rao MD33 34. Clostridium difficle Associated with human disease in mid-1970s Found in human GIT in small numbers With antibiotic use, increase in number in GIT Clindamycin, ampicillin, cephalosporins ....... Produces 2 entero toxins Toxin A -enterotoxin & Toxin B -cytotoxin Diagnosis Detection of toxins in stools, culture of organ