Anaerobic Bacteriology

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Anaerobic Bacteriology

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<ul><li>1.ANAEROBICBACTERIOLOGY Dr.T.V.Rao MD Dr.T.V.Rao MD 1</li></ul><p>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 ==&gt;686Respiration6CO2 + 6H2ONitrate CHO NO3- CHO + NO3- + H+ 649Reduction ==&gt; CO2+ N2+ H2OSulfate CHO SO42- 2CHO + SO42-+2H+ 190Reduction=&gt; 2CO2+ H+ 2H2OMethanogenesis CHOCO24H2 + CO2 ==&gt; 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 &amp; 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- =&gt; O2- superoxide anion O2- + e- + 2H+ =&gt; H2O2 hydrogen peroxide H2O2 + e- + H+ =&gt; H2O + OH. Hydroxyl radical OH. + e- + H+ =&gt; 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 &amp; 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 =&gt; 2H2O + O2 Peroxidase: H2O2 + NADH + H+ =&gt; 2H2O +NAD+ Superoxide dismutase: O2- + O2- + 2H+ =&gt; 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 &amp; 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 &amp; 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 &amp; 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 &amp; 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 &amp; 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 MD34 35. Clostridium botulinum Fastidious spore forming anaerobic grampositive bacillus Produces 8 antigenically distinct toxins Human disease described with types A, B &amp; E Heavy chain binds to ganglioside receptor Toxin internalized and prevents release of acetylcholine from vesicles Clinical Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis) infant botulism (floppy baby) wound botulism Dr.T.V.Rao MD35 36. ANAEROBIC GRAM NEGATIVE BACILLI Bacteroides, Prevotolla, Porphyromonas andFusobacterium Present in GI tract -form large component ofnormal flora &gt;80% of human infections associated with Bfragilis virulence factors - capsule, LPS, agglutinins and enzymes Clinical - Endogenous infections Intra-abdominal pyogenic infections pleuro-pulmonary infctions genital infectionDr.T.V.Rao MD36 37. ACTINOMYCES Strict anaerobic Gram positive bacilli typically arranged in hyphae whichfragment into short bacilli Normal flora of upper respiratory tract, GI tract and female genital tract. Low virulence produce disease when mucosal barrier is breached (eg: following dentaltrauma or surgery) ENDOGENOUS Establishes chronic infection that spreads through normal anatomicalbarriers Clinical -cervicofacial, abdominal and thoracic Diagnosis: Gram stain of sulpher granules culture Treatment - surgery and long term penicillin Dr.T.V.Rao MD 37 38. LABORATORY DIAGNOSIS A. COLLECTIONAnaerobes are endogenous in natureI. Appropriate specimens for anaerobic culture :1. pus2. pleural fluid3. urine4. pulmonary secretions5. uterine secretions or sinus tract materialDr.T.V.Rao MD 38 39. Aspiration is ideal Avoid SwabsII. Collection by needleaspiration ispreferable than swabculture because ofa. better survival ofpathogenb. greater quantity ofspecimenc. less contaminationwith extraneousorganism are oftenachieved Dr.T.V.Rao MD 39 40. HANDLINGIf a swab must be used, a 2 tube system must be used 1st tube contains swab in O2 free CO2 2nd tube contains PRAS (pre-reducedanaerobically sterilized culture media)Specimen should be placed in anaerobictransport device with gas mixtureDr.T.V.Rao MD 40 41. IsolationGram stain should be done in thelaboratory : a. choice of appropriate media &amp;methods for culture b. quality control for the types ofbacteria that laboratory culturereveal Dr.T.V.Rao MD 41 42. A solid or liquid medium maybe used &amp; must provide an anaerobic environment Anaerobic CultureSystem A. ANAEROBIC JAR 1. Candle Jar - reduces O2 environment - only CO2 tension2. Gas Pak Jar a. Palladium aluminum coated pellets- catalyst- chemically reduces O2- reacts with residual O2 in the presence of H2 toform H2O Dr.T.V.Rao MD 42 43. b. Gas Pak envelope- generates CO2 &amp; H2 gases c. Methylene blue strip - indicator blue (+) O2 white (-) O2II. Anaerobic Glove Chamber- close system- used for premature babies- e.g. incubatorIII. Roll Tube- has a pedal gas ( CO2 &amp; H2 ) would come out- place test tube directly to the outletDr.T.V.Rao MD 43 44. IDENTIFICATIONPlates are checked at&gt; 18-24 hours for faster growing species likeCl. Perfringens &amp; B.fragilis &amp; daily thereafter up to&gt; 5-7 days for slowly growing species likeActinomyces, Eubacterium &amp; propionibacterium Genus is determined by - gram stain, cellular morphology, Gas-liquid chromotography Species determination is based on fermentation of sugars &amp; other biochemical determination Dr.T.V.Rao MD44 45. ANAEROBIC GRAM NEGATIVE BACILLI Bactericides, Prevotolla, Porphyromonas andFusobacterium Present in GI tract -form large component of normalflora &gt;80% of human infections associated withBfragilis virulence factors - capsule, LPS, agglutinins and enzymes Clinical - Endogenous infections Intra-abdominal pyogenic infections pleuro-pulmonary infections genital infectionDr.T.V.Rao MD45 46. ACTINOMYCES Strict anaerobic Gram positive bacilli typically arranged in hyphaewhich fragment into short bacilli Normal flora of upper respiratory tract, GI tract and female genitaltract. Low virulence produce disease when mucosal barrier is breached (eg: followingdental trauma or surgery) ENDOGENOUS Establishes chronic infection that spreads through normalanatomical barriers Clinical-cervicofacial, abdominal and thoracic Diagnosis: Gram stain of sulpher granules cultureDr.T.V.Rao MD46 47. Culturing of anaerobes need special skills Culture of anaerobes is extremelydifficult due to the need to excludeoxygen, slow growth and complexgrowth requirements Molecular methods based on DNAanalysis and direct microscopy haveshown that we are largely ignorant of themicrobial world and previously unknown diversity has been discoveredDr.T.V.Rao MD 47 48. 48 Culture methods Anaerobes differ in their sensitivity to oxygen and the culture methods employed reflect this - some are simple and suitable for less sensitive organisms, others more complex but necessary for fastidious anaerobes Vessels filled to the top with culture medium can be use...</p>