ANAEROBIC BACTERIOLOGY

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

2. What Are Anaerobic Microorganisms • Anaerobic microorganisms are widespread and very important • Do not require oxygen for growth - often extremely toxic Dr.T.V.Rao MD

3. The Requirements for Growth: Related to Oxygen • Oxygen (O2) Dr.T.V.Rao MD Table 6.1

4. Anaerobes differ from Aerobic Bacteria • Anaerobes generate energy by fermentation • Lack the capacity to utilize O2 as a terminal hydrogen acceptor • 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 tolerant anaerobes • Many are members of the normal flora  created by presence of facultative anaerobes Dr.T.V.Rao MD

5. DEFINITIONS • OBLIGAETE ANAEROBE • Lack superoxide dismutase and/or catalase • toxic radicals formed by oxidative enzymes kill organisms • AERO-TOLERANT ANAEROBES • survive in presence of oxygen • Do not use oxygen for energy requirements • FACULTATIVE ANAEROBES Dr.T.V.Rao MD

6. Anaerobes and Oxygen • Anaerobes generate energy by fermentation • Lack the capacity to utilize O2 as a terminal hydrogen acceptor • 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 tolerant anaerobes • Many are members of the normal flora  created by presence of facultative anaerobes Dr.T.V.Rao MD

7. Anaerobic and Aerobic Respiration Dr.T.V.Rao MD

8. FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN 1. Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them Dr.T.V.Rao MD

9. Strict Anaerobic Bacteria • Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor. • Archaea such as methanogens and Bacteria, 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 MD

11. Oxygen Toxicity • Oxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidising ability makes it an excellent electron acceptor • During the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS). Dr.T.V.Rao MD

12. FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN 1. Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them Dr.T.V.Rao MD

13. Chemical Dynamics in Anaerobic Bacteria • Organisms that use O2 have developed defence mechanisms to protect 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

14. Anaerobic environments exist in Nature too • Anaerobic environments (low reduction potential) include: • Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils, intestinal tract of animals; oral cavity of animals, deep underground areas, e.g. oil packets and some aquifers • Anaerobes also important in some infections, e.g. C. tetanii and C. perfringens important in deep puncture wound infections Dr.T.V.Rao MD

15. ANAEROBES OF CLINICAL IMPORTANCE • CLOSTRIDIA • C tetani; C perfringens; C difficile; C botulinum • BACTEROIDES • B fragilis; • Prevotella • Porphyromonas • ACTINOMYCES • FUSOBACTERIUM • ANAEROBIC STREPTOCOCCI Dr.T.V.Rao MD

16. FACTORS RESPONSIBLE FOR THEIR VIRULENCE 1. Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinize * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid – reduces phagocytic killing Dr.T.V.Rao MD

17. Multiplication of the opportunistic pathogens is facilitated by: 1. Inhibition of phagocytosis & intracellular killing by PMN in the presence of Bacteroides by: a. competition of opsonins b. inhibition by capsular materials 2. Protection of antibiotic susceptibility strains in mixtures thru destruction by the ß-lactamases 3. Utilization of O2 by facultative species that aids in producing a suitable environment for growth of anaerobe Dr.T.V.Rao MD

18. CLINICAL MANIFESTATION Clinical 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 bites Dr.T.V.Rao MD

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21. Anaerobic Bacteria of Medical Interest Dr.T.V.Rao MD

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), tissue damage • Germination of spores • Toxin release • Binding of toxin to receptor • Resulting effect produces symptom(s) of disease Dr.T.V.Rao MD

23. Gram-positive anaerobes • Actinomyces (head, neck, pelvic infections; aspiration pneumonia) • Bifid bacterium (ear infections, abdominal infections) • Clostridium (gas, gangrene, food poisoning, tetanus, pseudomembranous colitis) • Peptostreptococcus (oral, respiratory, and intra-abdominal infections) • Propionibacterium (shunt infections) Dr.T.V.Rao MD

24. Gram-negative anaerobes • Bactericides (the most commonly found anaerobes in cultures; intra-abdominal infections, rectal abscesses, soft tissue infections, liver infection) • Fusobacterium (abscesses, wound infections, pulmonary and intracranial infections) • Porphyromonas (aspiration pneumonia, periodontitis) • Prevotella (intra-abdominal infections, soft tissue infections) Dr.T.V.Rao MD

25. FACTORS RESPONSIBLE FOR THEIR VIRULENCE 1. Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinase * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid – reduces phagocytic killing Dr.T.V.Rao MD

26. Common Human Anaerobic Infections Dr.T.V.Rao MD

27. CLOSTRIDIA • Gram positive spore forming bacilli • ubiquitous • intestines of man and animals • animal and human faeces contaminated soil and water • Several species associated with human disease Dr.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

29. Clostridium tetani • Small motile spore forming gram positive bacillus with 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 neurotransmittors (eg GABA) Dr.T.V.Rao MD

30. Clostridium tetani • Small motile spore forming gram positive bacillus with 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

31. TETANUS • Clinical syndromes due to unregulated excitatory synaptic activity resulting in spastic paralysis • Generalised tetanus • Neonatal tetanus • localized tetanus Dr.T.V.Rao MD

32. Prevention and treatment • Active immunization with tetanus toxoid • Wound toilet and active/passive immunization of ‘risk’ injuries • management of wound • tetanus toxoid • Anti-tetanus serum (ARS -horse serum) or Human Tetanus ImmunoGlobulin (HTIG) • Penicillin or Metronidazole • Management of patient with tetanus • reduce stimuli • respiratory and CVS support Dr.T.V.Rao MD

33. Clostridium difficile • Associated with human disease in mid-1970’s • 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 MD

34. Clostridium difficle • Associated with human disease in mid-1970’s • 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 MD

35. Clostridium botulinum • Fastidious spore forming anaerobic gram positive bacillus • Produces 8 antigenically distinct toxins • Human disease described with types A, B & E • Heavy chain binds to ganglioside receptor • Toxin internalized and prevents release of acetyl choline from vesicles • Clinical • Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis) • infant botulism (floppy baby) • wound botulism Dr.T.V.Rao MD

36. ANAEROBIC GRAM NEGATIVE BACILLI • Bacteroides, Prevotolla, Porphyromonas and Fusobacterium • Present in GI tract -form large component of normal flora • >80% of human infections associated with B fragilis • virulence factors - capsule, LPS, agglutinins and enzymes • Clinical - Endogenous infections • Intra-abdominal pyogenic infections • pleuro-pulmonary infctions • genital infection Dr.T.V.Rao MD

37. ACTINOMYCES • Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli • Normal flora of upper respiratory tract, GI tract and female genital tract. • Low virulence • produce disease when mucosal barrier isbreached (eg: following dental trauma or surgery) ENDOGENOUS • Establishes chronic infection that spreads through normal anatomical barriers • Clinical -cervicofacial, abdominal and thoracic • Diagnosis: • Gram stain of ‘sulpher’ granules • culture • Treatment - surgery and long term penicillin Dr.T.V.Rao MD

38. LABORATORY DIAGNOSIS A. COLLECTION Anaerobes are endogenous in nature I. Appropriate specimens for anaerobic culture : 1. pus 2. pleural fluid 3. urine 4. pulmonary secretions 5. uterine secretions or sinus tract material Dr.T.V.Rao MD

39. Aspiration is ideal Avoid Swabs II. Collection by needle aspiration is preferable than swab culture because of a. better survival of pathogen b. greater quantity of specimen c. less contamination with extraneous organism are often achieved Dr.T.V.Rao MD

40. HANDLING If 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-reduced anaerobically sterilized culture media) Specimen should be placed in anaerobic transport device with gas mixture Dr.T.V.Rao MD

41. Isolation Gram stain should be done in the laboratory : a. choice of appropriate media & methods for culture b. quality control for the types of bacteria that laboratory culture reveal Dr.T.V.Rao MD

42. A solid or liquid medium maybe used & must provide ananaerobic environment Anaerobic Culture System • ANAEROBIC JAR 1. Candle Jar - reduces O2 environment - only ↑ CO2 tension 2. Gas Pak Jar a. Palladium aluminum coated pellets - catalyst - chemically reduces O2 - reacts with residual O2 in the presence of H2 to form H2O Dr.T.V.Rao MD

43. b. Gas Pak envelope - generates CO2 & H2 gases c. Methylene blue strip - indicator blue → (+) O2 white → (-) O2 II. Anaerobic Glove Chamber - close system - used for premature babies - e.g. incubator III. Roll Tube - has a pedal  gas ( CO2 & H2 ) would come out - place test tube directly to the outlet Dr.T.V.Rao MD

44. IDENTIFICATION Plates are checked at > 18-24 hours for faster growing species like Cl. Perfringens & B.fragilis & daily thereafter up to > 5-7 days for slowly growing species like Actinomyces, Eubacterium & propionibacterium Genus is determined by - gram stain, cellular morphology, Gas-liquid chromotography Species determination is based on fermentation of sugars & other biochemical determination Dr.T.V.Rao MD

45. ANAEROBIC GRAM NEGATIVE BACILLI • Bactericides, Prevotolla, Porphyromonas and Fusobacterium • Present in GI tract -form large component of normal flora • >80% of human infections associated with B fragilis • virulence factors - capsule, LPS, agglutinins and enzymes • Clinical - Endogenous infections • Intra-abdominal pyogenic infections • pleuro-pulmonary infections • genital infection Dr.T.V.Rao MD

46. ACTINOMYCES • Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli • Normal flora of upper respiratory tract, GI tract and female genital tract. • Low virulence • produce disease when mucosal barrier isbreached (eg: following dental trauma or surgery) ENDOGENOUS • Establishes chronic infection that spreads through normal anatomical barriers • Clinical -cervicofacial, abdominal and thoracic • Diagnosis: • Gram stain of ‘sulpher’ granules • culture Dr.T.V.Rao MD

47. Culturing of anaerobes need special skills • Culture of anaerobes is extremely difficult due to the need to exclude oxygen, slow growth and complex growth requirements • Molecular methods based on DNA analysis and direct microscopy have shown that we are largely ignorant of the microbial world and previously unknown diversity has been discovered Dr.T.V.Rao MD

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 used for organisms not too sensitive Dr.T.V.Rao MD

49. Appropriate Specimens for Anaerobic Cultures • The Microbiologists understanding of basic anaerobic bacteriology is critical in the interpretation of an anaerobic culture result for the diagnosis and treatment of anaerobic infection. Since anaerobes from part of the normal bacterial flora of the skin and mucous membrane, proper selection and collection of clinical specimens for the laboratory diagnosis of an anaerobic infection critical factors that will determine the clinical significance of the culture results Dr.T.V.Rao MD

50. Acceptable Specimens • Specimens for anaerobic cultures are ideally biopsy samples of needle aspirates. • Anaerobic swabs are discouraged but sometimes cannot be avoided. Swabs are the least desirable because of the small amount of the specimen and effect of drying. There is a greater chance of contamination with normal micro flora Dr.T.V.Rao MD