anaerobic bacteria
TRANSCRIPT
*Definition of terms: bacterial growth in relation with respiratory
processes (use of O2, CO2)
• Obligate aerobes need oxygen because they cannot ferment or respire anaerobically (e.g. Mycobacterium tuberculosis)
• Obligate anaerobes are poisoned by oxygen
• Facultative anaerobes can grow with or without oxygen (e.g. Staphylococcus, Streptococcus, E.coli)
• Microaerophiles need some amount of oxygen but are poisoned by high concentrations of oxygen (e.g. Campylobacter, Helicobacter, Neisseria gonorrhoeae)
(Obligate) Anaerobic bacteria- general aspects & definition of terms -
• Energy generated exclusively by anaerobic fermentation (does not generate superoxide radicals = O2 anions)
• Can only grow in the total absence of O2;
• WHY?: Anaerobic bacteria lack the enzymes:– Superoxide dismutase (SOD) – Catalase
(Obligate) Anaerobic bacteria- general aspects & definition of terms - continued
• Superoxide dismutases - antioxidant factors; enzymes which catalyze the dismutation (partitioning) of the superoxide radical (O2 anion) into:– Molecular O2 or – Hydrogen peroxide (H2O2)
• Superoxide = by-product of O2 metabolism; high cellular toxicity
• Hydrogen peroxide = less toxic; degraded by catalase
(Obligate) Anaerobic bacteria- general aspects & definition of terms - continued
CONCLUSIONS: • fermentation in the presence of O2 → superoxide
radicals – toxic if not dismuted (partitioned) by superoxid dismutase into O2 / H2O2;
• furthermore H2O2 – toxic if not decomposed by catalase
• Anaerobic bacteria lack both enzymes (superoxid dismutase and catalase) → mandatory absence of O2 in order for anaerobic bacteria to avoid toxic effects of superoxide radicals and/or H2O2
(Obligate) Anaerobic bacteria- general aspects & definition of terms -
”Friends or Foes?”
• Colonize the human body • involved in the balance of the normal microbial flora:
skin, oropharynx, gastro-intestinal tract, uro-genital tract (urethra, vagina)
• Cause severe infections (endogenous and exogenous)
Obligate Anaerobic Bacteria- Collection and transport of specimens -
• Inoculation asap (within 10 minutes) due to toxicity of atmospheric O2
• Transport: anaerobic tubes with transportation media e.g. modified Cary Blair, Stuart– minimal nutrients to increase survival of organisms without
multiplication – sodium thioglycollate - to provide low oxidation-reduction
potential – alkaline pH – to minimize bacterial destruction by acid
production– phenol red indicator (red at alkaline pH, yellow at acidic pH)– Redox indicator: resazurin – turns pink in the presence of O2
Obligate Anaerobic Bacteria- Collection and transport of specimens -
continued“Hungate tubes”:• Disposable/autoclavable
screw thread style tube designed to maintain anaerobic culture conditions
• butyl rubber stoppers,• screw cap 9 mm opening
Obligate Anaerobic Bacteria- Macroscopic and microscopic exam -
• Suggestive signs of anaerobic infection:– Fetid odour– Purulent aspect– Necrotic tissues
– Gas (e.g. ”gas gangrene” – wound infected by Clostridium perfringens)
• Microscopy: Gram stained smears (methanol fixation to preserve cellular elements)
Obligate Anaerobic Bacteria- Isolation -
• Nonselective media: blood agar
• Selective media requirements (CLSI: Clinical Laboratory Standards Institute): – 3 days of strict anaerobic
incubation → mandatory growth of:
– Bacterioides fragilis, Clostridium perfringens + Fusobacterium nucleatum, Peptostreptococcus anaerobius, Bacteroides levii
Obligate Anaerobic Bacteria- Identification -
• API 20 A Identification of anaerobes in 24-48 hours
• Fermentation tests, which are the reference tests for the identification of anaerobes
• Easy-to-use: suspension prepared directly in the API 20 A medium.
• Polyvalent system for all anaerobes, both Gram (+) and Gram (-)
Obligate Anaerobic Bacteria- Human infections -
Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:
– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may
cause both exogenous and endogenous infections)
Endogenous nonsporulating• Gram negative bacilli:
– Family Bacteroidaceae:• Bacteroides, Prevotella,
Prophyromonas, Fusobacterium
• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii
• Gram positive cocci:– Peptococcus,
Peptostreptococcus• Gram negative cocci:
– Veillonella
Bacterial survival outside host
Spores: reproductive structures adapted
for longtime survival in unfavourable
conditions
(etymology: ancient Greek spora = seed)
Bacterial spores - outer layer of keratin resistant to chemicals, staining and heat → bacterium able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients
Spore forming bacteria:
• Genus Clostridium;
• Bacillus spp (B. anthracis).
Genus Clostridium- Clinical significance -
• Exogenous infections: – Gas gangrene – Tetanus– Botulism– Food poisoning
• Endogenous infections: • Clostridium difficile
Definition of terms: Gangrene
• tisular death (necrosis) caused by lack of blood supply (= absence of O2 and nutrients in the respective area of the
organism)• potentially life-threatening condition • may occur by:
– injuries, trauma (compression of blood vessels)– frostbite (freezing of exposed extremities)– infection– chronic diseases affecting blood circulation e.g. diabetes
Gas gangrene clostridia
• Gas gangrene – severe invasive infection starting from infected wounds, rapid systemic invasion
• Caused by: Clostridium perfringens, + Cl. oedematiens, Cl. histolyticum – found in water, soil air + intestinal comensals
• Severity augmented by toxin production
• Collection of specimens: profound wound secretion, tissue fragments
Gas gangrene: Clostridium perfringens
• Oedema, necrosis, large blisters, crepitation
• Lower image: large incision for oxygen exposure of infected tissues
Gas gangrene clostridia- Microscopic examination -
Gram stained smear: • total absence of cells (no
PMNs, no epithelial cells, etc)
• short, thick, Gram positive bacilli
• no spores (no spore forming in vivo)
Gas gangrene clostridia: Cl. perfringens- Isolation and identification -
Blood agar: double hemolysis
Gas gangrene clostridia: Cl. perfringens- Isolation and identification - continued
Egg yolk agar (EYA): enriched
medium for presumptive id of
anaerobes e.g. Clostridium • Egg yolk suspension: detection
of enzymes:– lipase (iridescent sheen on
colony surface) and – lecitinase (opaque precipitate
around colonies)
Gas gangrene clostridia: Cl. perfringens- Isolation and identification - continued
• Reverse CAMP test Principle: synergistic effect between Streptococcus agalactiae (group B) and hemolytic Clostridium perfringens
• Initially intended as an improvement of CAMP test i.e. replacement of S.aureus by Clostridium perfringens for the identification of Streptococcus agalactiae (Group B)
• Then the idea comes up to use Streptococcus agalactiae (group B) to identify Clostridium perfringens (= the reverse CAMP test)
The CAMP test: id of Streptococcus agalactiae
(A) Streptococcus (group B) positive test (enhanced hemolysis)
(B) Streptococcus pyogenes (group A) negative test
(C) Staphylococcus aureus – replaced by Clostridium perfringens in reverse CAMP test (see next slide)
The reverse CAMP test
Identificationof Clostridiumperfringens: (A) Reverse CAMP-
positive Clostridium perfringens (”bow tie”)
(B) reverse CAMP-negative Clostridium septicum streaked at right angles to
(C) Streptococcus agalactiae (group B)
Clostridium perfringens- Antimicrobial susceptibility -
• Sensitivity to: penicillin G, erythromycin, ampicillin, metronidazole
• Natural resistance to tetracyclines
Obligate Anaerobic Bacteria- Human infections -
Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:
– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may
cause both exogenous and endogenous infections)
Endogenous nonsporulating• Gram negative bacilli:
– Family Bacteroidaceae:• Bacteroides, Prevotella,
Prophyromonas, Fusobacterium
• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii
• Gram positive cocci:– Peptococcus,
Peptostreptococcus• Gram negative cocci:
– Veillonella
Clostridium tetani
• Habitat: intestinal tract of animals (sheep, cattle); vegetative bacteria eliminated with faceces; contamination of soil (spore formation)
• Infection occurs via:– Skin lesions contaminated with spores e.g. wound highly
contaminated with dirt, dust; extensive wounds with crushed tissues and foreign bodies (accidents);
– spores germinate into vegetative bacteria which multiply at the entry and produce tetanic toxin (disseminated) – 2 components:
• Tetanospasmin – muscle spasms• Tetanolysin – cardiotoxic
Clostridium tetani
Clinical significance:• Tetanus = Generalized tetanus (most comon form):
– onset with trismus (spasms of the face and chewing muscles popularly called “lockjaw”→ characteristic facial expression risus sardonicus or sardonic grin);
– further evolution: swallowing becomes increasingly difficult; severe spastic hyperextension of head, neck and spine (opisthotonos)
(effects of the tetanus exotoxin: tetanospasmin)
• Lethal outcome ~ 1 in 10 cases - spastic paralysis of respiratory muscles
Left: Risus sardonicus (rigid facial grin)Right: Opisthotonos (spastic contraction with
hyperextension of head, neck and spine)
Tetanus
• Vaccine preventable disease: several vaccines used to prevent tetanus among children, adolescents, and adults; e.g. combined vaccines against diphteria, pertusis and tetanus (e.g. DTaP) or tetanus and diphteria (e.g.TD) – Immunization schedules
• In Romania: tetanus containing vaccines given at the ages of:– 2, 4, 6, 13 months, 4 years (DTP) + 14 years (dT) +– (recommended) dT every 10 years
Definition of terms: Immunization schedule
• series of vaccinations, including the timing of all doses, which may be either recommended or compulsory,
depending on the country of residence • Examples:• http://www.cdc.gov/vaccines/schedules/hcp/child-adolescent.html#printable
• http://www.nhs.uk/Conditions/vaccinations/Pages/vaccination-schedule-age-checklist.aspx
• http://en.pediatricblog.info/2011/02/romanian-mandatory-vaccination-schedule.html
Tetanus prophylaxix in routine wound management
1st step: Assess wound - Clean, minor wound: • Q1: Has patient completed a primary tetanus-diphteria
series? (= minimum 3 doses of tetanus- and diphteria containing vaccine: e.g. at 2, 4 and 6 months of age)– NO/Unknown: Administer vaccine today (i.e. complete series per
age-appropriate vaccine schedule)– YES→Q2: Was the most recent dose within the past 10 years?
• NO: Administer vaccine today (next dose per age-appropriate schedule)
• YES: vaccine not needed today; next dose will be given at 10 years after the last dose
Tetanus prophylaxix in routine wound management - continued
1st step: Assess wound: “tetanigenic potential”:contaminated with dirt, faeces, saliva, soil; puncture wounds (lack of O2 in profound layers of wound); animal bites, burns, frostbite
• Q1: Has patient completed a primary tetanus-diphteria series?– NO/Unknown: vaccine + tetanus immune globulin (TIG) today– YES→Q2: Was the most recent dose within the past 5 years?
• NO: Administer vaccine today (next dose per age-appropriate schedule)
• YES: Vaccine not needed today (next dose at 10 years from last dose)
Clostridium tetani
• Laboratory diagnosis only required in suspicion of iatrogenic infections e.g. infection of umbilical cord stump, post-partum infections, etc
• In most cases diagnosis relies on clinical aspect and history (tetanigenic circumstances e.g. wounds contaminated with dirt, faeces, saliva, soil; puncture wounds; animal bites, burns, frostbite)
• IMPORTANT FACTS:– no human to human transmission– Vaccine preventable
Obligate Anaerobic Bacteria- Human infections -
Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:
– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may
cause both exogenous and endogenous infections)
Endogenous nonsporulating• Gram negative bacilli:
– Family Bacteroidaceae:• Bacteroides, Prevotella,
Prophyromonas, Fusobacterium
• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii
• Gram positive cocci:– Peptococcus,
Peptostreptococcus• Gram negative cocci:
– Veillonella
Clostridium botulinum - Clinical significance -
Foodborne Botulism: • Ingestion of botulinic toxin
contained in canned vegetables, fish, meat (toxin production generated gas → can lid appears bulging/swollen)
• Toxin resists gastric acidity, enters the intestine → lymph vessels → blood stream→ acts on nervous system: progressive flaccid paralysis
Wound botulism: i.v. / intradermic drug users
• Possible use as biological weapon
Clostridium botulinum - Clinical significance - continued
• Onset symptoms: double vision, blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness
• if untreated: descending flaccid paralysis; death by paralysis of respiratory muscles
• Treatment: botulinum antitoxin – asap!!
Clostridium botulinum- Laboratory diagnosis -
• Performed in reference laboratories
• Specimens: blood for serology, vomit, faeces, suspected food
• Identification based on:– Gram stain: Gram positive
bacilli + spores; – anaerobic growth; – neurotoxin detection:
experimental disease in mice; immunoassay; molecular techniques
Obligate Anaerobic Bacteria- Human infections -
Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:
– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may
cause both exogenous and endogenous infections)
Endogenous nonsporulating• Gram negative bacilli:
– Family Bacteroidaceae:• Bacteroides, Prevotella,
Prophyromonas, Fusobacterium
• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii
• Gram positive cocci:– Peptococcus,
Peptostreptococcus• Gram negative cocci:
– Veillonella
Clostridium difficile- Clinical significance -
• Pseudomembranous colitis: bloating and severe diarrhoea
• Endogenous: bacteria replaces normal intestinal flora that has been compromised, usually following antibiotic treatment for an unrelated infection; C. difficile gains a growth advantage (positive selection) and overruns the intestinal microbiome; “antibiotic-associated diarrhoea”
• Exogenous: accidental ingestion of spores e.g. incomplete/incorrect hospital management of infected patient (isolation, disinfection, etc) leads to spore contamination of objects → spore ingested by another patient (when prevention guidelines are not strictly followed e.g. hand washing, cleaning, PPE, disinfection)
Clostridium difficile- Prevention guidelines in clinical settings -
Examples:• http
://www.documents.hps.scot.nhs.uk/about-hps/hpn/clostridium-difficile-infection-guidelines.pdf
• http://www.idsociety.org/uploadedFiles/IDSA/Guidelines-Patient_Care/PDF_Library/cdiff2010a.pdf
• http://d2j7fjepcxuj0a.cloudfront.net/wp-content/uploads/2013/04/ACG_Guideline_Cdifficile_April_2013.pdf
Obligate Anaerobic Bacteria- Human infections -
Exogenous sporulating• Gram positive bacilli:• Genus Clostridium:
– Gas gangrene clostridia– Clostridium tetani– Clostridium botulinum– Clostridium difficile* (may
cause both exogenous and endogenous infections)
Endogenous nonsporulating• Gram negative bacilli:
– Family Bacteroidaceae:• Bacteroides, Prevotella,
Prophyromonas, Fusobacterium
• Gram positive bacilli: – Propionibacterium acnes– Actinomyces israelii
• Gram positive cocci:– Peptococcus,
Peptostreptococcus• Gram negative cocci:
– Veillonella
Endogenous nonsporulating bacilli
Gram positive • Propionibacterium acnes:
involved in juvenile acne, blepharitis together with staphylococci, corynebacteria; morphology similar to corynebacteria
• Actinomyces israelii: comensal flora of the oral cavity; involved in periodontal disease, abscesses (in immune compromised patients)
Gram negativeBacteroides, Prevotella,Prophyromonas,
Fusobacterium- Normal flora- Isolation in naturally
sterile sites – always pathological