1 ANAEROBES and Pseudomonas Lecture 39 ANAEROBES and Pseudomonas Lecture 39 Faculty: Dr. Alvin Fox

2 MAJOR POINTS MAJOR POINTS Overview of anaerobic bacteriology Anaerobic non-spore formers Anaerobic spore formers (clostridia) Pseudomonas (a strict aerobe)

3 KEY WORDS Obligate anaerobesObligate anaerobes Strict anaerobesStrict anaerobes Polymicrobic (mixed) infectionPolymicrobic (mixed) infection Spore formersSpore formers Non-spore formersNon-spore formers BacteroidesBacteroides B. fragilisB. fragilis Clostridium tetaniClostridium tetani - tetanospasmin - tetanospasmin C. perfringensC. perfringens - toxin (lecithinase phospholipase) - α toxin (lecithinase phospholipase) C. perfringensC. perfringens - enterotoxin - enterotoxin C. botulinumC. botulinum - botulinum toxin - botulinum toxin C. difficileC. difficile - exotoxin A (cytotoxin) - exotoxin A (cytotoxin) Pseudomonas aeruginosaPseudomonas aeruginosa - pyocyanin - pyroverdin - exotoxin A - exotoxin A

4 no oxidative phosphorylationno oxidative phosphorylation fermentationfermentation killed by oxygenkilled by oxygen lack certain enzymes:lack certain enzymes: superoxide dismutase O H + H 2 O 2 catalase H 2 O 2 H O 2 peroxidase H 2 O 2 + NADH + H + 2H NAD Obligate anaerobes

5 Polymicrobic anaerobic infection opportunistic growth –injured tissue *limited blood/O 2 no growth no growth – healthy tissues *high O 2 content Many species in human flora Many species in human flora Many grow simultaneously - opportunistic Many grow simultaneously - opportunistic conditions conditions

6 Simultaneous infection with facultative anaerobe –diminishes O 2 supply further – aids growth of obligate anaerobes Polymicrobic anaerobic infection

7 Endogenous versus exogenous infection Two sourcesTwo sources –normal human flora endogenousendogenous –environment (e.g. soil) exogenousexogenous

8 Source of spore-formers and non-spore formers Spore-formers (clostridia)Spore-formers (clostridia) –exotoxins –common in the environment (e.g. soil) –found in normal flora Non - spore-formersNon - spore-formers –no exotoxins –mostly normal flora

9 Sites of anaerobes in normal flora intestine intestine – major site – 95-99% total bacterial mass mouth mouth genitourinary tract genitourinary tract

10 Bacteroides fragilis minor component of gut flora most common (strict) anaerobic infection after abdominal surgery

11 Enterobacteriaceae (facultative anaerobes) – commonly cause disease – low numbers gut flora Strict anaerobes Strict anaerobes – much less commonly cause disease – high numbers gut flora.

12 Strict anaerobe infectious disease Sites throughout bodySites throughout body Muscle, cutaneous/sub-cutaneous necrosisMuscle, cutaneous/sub-cutaneous necrosis abscessesabscesses

13 Problems in identification of anaerobic infections air in sample (sampling, transportation)air in sample (sampling, transportation) –no growth identification takes several days or longeridentification takes several days or longer –limiting usefulness often derived from normal floraoften derived from normal flora –sample contamination can confuse

14 LABORATORY IDENTIFICATION BIOCHEMICAL KITSBIOCHEMICAL KITS - e.g. substrate utilization GAS CHROMATOGRAPHYGAS CHROMATOGRAPHY –volatile fermentation products

15 ANAEROBIC NON-SPORE-FORMERS OF CLINICAL IMPORTANCE Gram-positive cocci: Gram-negative cocci: Veillonella Bacteroides e.g. B. fragilis Fusobacterium, Porphyromonas, Prevotella Gram-positive rods: Actinomyces, Bifidobacterium, Eubacterium Lactobacillus, Mobiluncus, Propionibacterium Peptostreptococcus and Peptococcus Gram-negative rods:

16 Bacteroides fragilis Major disease causing strict anaerobicMajor disease causing strict anaerobic non-spore-former non-spore-former Prominent capsule Prominent capsule – anti-phagocytic – abscess formation Endotoxin Endotoxin – low toxicity – structure different than other lipolysaccharide – beta lactamase

17 ANAEROBIC SPORE-FORMERS (CLOSTRIDIA) Gram-positive rods – human intestine – soil

18 Clostridium tetani spore vegetative

19 Clostridium tetani Non-invasiveNon-invasive

20 Tetanospasmin disseminates systemicallydisseminates systemically binds to ganglioside receptorsbinds to ganglioside receptors – inhibitory neurones in CNS, signal stopped muscles keep on workingmuscles keep on working spastic (rigid) paralysisspastic (rigid) paralysis glycine, neurotransmitterglycine, neurotransmitter

21 A severe case of tetanus. muscles, back and legs are rigid muscle spasms can break bones can be fatal(e.g respiratory failure) can be fatal (e.g respiratory failure)

22 infant infant DPT (diptheria, pertussis, tetanus) DPT (diptheria, pertussis, tetanus) tetanus extremely uncommon in US tetanus extremely uncommon in US tetanus toxoid tetanus toxoid – antigenic – no exotoxic activity Vaccination

23 C. perfringens soil, fecal contaminationsoil, fecal contamination especially in warespecially in war gas gangrenegas gangrene - swelling of tissues - gas release (fermentation products) - tissue destruction (see next slide)

24 tissue degrading enzymes tissue degrading enzymes – α toxin (lecithinase, phospholipase) destruction of blood vessels destruction of blood vessels myonecrosis myonecrosis anaerobic environment created anaerobic environment created organism spreads organism spreads α toxin

25 Without treatment death occurs within 2 days  effective antibiotic therapy  debridement  anti-toxin  amputation & death is rare

26 lecithinase production lecithinase production Laboratory identification

27 Food poisoning enterotoxin producing strainsenterotoxin producing strains

28 C. botulinum

29 Botulism food poisoningfood poisoning –rare –fatal germination of sporegermination of spore inadequately sterilized canned foodinadequately sterilized canned food –home not an infectionnot an infection

30 Botulinum toxin binds peripheral nerve receptorsbinds peripheral nerve receptors –acetylcholine neurotransmitter inhibits nerve impulsesinhibits nerve impulses flaccid paralysisflaccid paralysis deathdeath –respiratory –cardiac failure

31 Infection with C. botulinum Neonatal botulismNeonatal botulism –uncommon –the predominant form of botulism –colonization occurs limited normal flora to competelimited normal flora to compete unlike adultunlike adult

32 Wounds –extremely rare –an infection

33 Botulinum toxin BioterrorismBioterrorism –not an infection –resembles a chemical attack

34 anti-toxin anti-toxin antibiotic therapy (if infection) antibiotic therapy (if infection) Treatment

35 C. difficile pseudomembanous colitispseudomembanous colitis after antibiotic useafter antibiotic use intestinal normal flora intestinal normal flora –greatly decreased colonization occurscolonization occurs exotoxin A (enterotoxin, cytoxin)exotoxin A (enterotoxin, cytoxin) - most potent exotoxin Bexotoxin B - secondary (also a cytoxin)

36 Therapy discontinuation of initial antibiotic (e.g. ampicillin)discontinuation of initial antibiotic (e.g. ampicillin) specific antibiotic therapy (e.g. vancomycin)specific antibiotic therapy (e.g. vancomycin)

37 PSEUDOMONAS AERUGINOSA PSEUDOMONAS AERUGINOSA Gram negative rod

38 Pseudomonas AerobicAerobic Gram-negative rodGram-negative rod majority of human infectionsmajority of human infections –P. aeruginosa

39 Common in the environment water air soil

40 Burns and wounds Burns and wounds – destruction of blood vessels – phagocyte access limited cancercancer – cytotoxic drugs *destroy the immune system cystic fibrosiscystic fibrosis – altered respiratory epithelium – pneumonia. P. aeruginosa and compromised host

41 Identification Pigments Pigments – pyocyanin (blue-green) – pyoverdin (green-yellow, fluorescent) biochemical reactions biochemical reactions cultures have fruity smell cultures have fruity smell

42 Slime layer is anti-phagocytic Slime layer is anti-phagocytic Toxin A - ADP ribosylates EF2 Toxin A - ADP ribosylates EF2 – similar to diphtheria toxin Pathogenesis