The Anaerobes

1
The Anaerobes

• Clostridium
• Bacteriodaceae

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Anaerobes of Clinical Importance

• Gram() Spore-Forming Bacilli
• Clostridium
• Gram(-) Bacilli Bacteriodaceae
• Bacteroides
• Fusobacterium
• Porphyromonas
• Prevotella

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Clostridium

• Strict anaerobes, some aerotolerant
• Widely distributed soil, water, sewage
• NF in GI tract animals, humans
• Most are saprophytes
• Disease-causing species
• Survive adverse conditions by spore formation
• Rapid growth in nutrition rich, decrease oxygen
  site
• Most not invasive but produce powerful exotoxins
  (cytotoxin, enterotoxin, neurotoxin)

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Clostridium Genera

• C. perfringens
• Food poisoning - intoxication
• Myonecrosis - gas gangarene
• Soft-tissue infection
• C. botulinum
• Botulism - food poisoning (intoxication,
  infection)
• C. tetani
• Tetanus - lockjaw
• C. difficile
• Pseudomembranous colitis - antibiotic-associated
  disease

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Clostridium Staining

• G() large bacilli
• All motile - except C. perfringens
• Form endospore oval, subterminal
• C. tetani - terminal spore

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Clostridium Lab Culture

• Blood Agar - Enriched
• Supplemented anaerobic BA
• C. perfringens produces classic double zone
  hemolysis
• Egg Yolk Agar - Differential
• Lecithinase production (white precipitate)
• Lipase production (sheen around surface of
  colonies)

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Clostridium Lab Culture

• CCFA (Cycloserine-cefoxitin-fructose agar)
• Selective by antibiotics
• Differential by fructose fermentation
• C. difficile (yellow, ground-glass colony)
• Thioglycollate broth
• Reducing agents eliminate oxygen
• Chopped meat for nutrients
• Special isolation procedures
• Usually mixed culture specimens
• Use heat or alcohol to kill NF before plating for
  Clostridium

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Clostridium perfringens Virulence Factors

• At least 12 exotoxins and enzymes
• Alpha Toxin - phosphopipase C)
• Vascular permeability
• Massive hemolysis bleeding, tissue destruction
  (myonecrosis)
• Hepatic toxicity, myocardial dysfunction
• Enzymes - gelatinase, collagenase, protease,
  hyaluronidase, DNase, neuraminidase
• Enterotoxin - food poisoning
• Meats, poultry, gravy
• Action resembles cholera toxin

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C. perfringensInfection and Disease

• Exogenous infection from external source (soil,
  food, trauma)
• Endogenous infection GI tract to sterile areas
  (tissues, blood)
• At risk
• Surgical patients
• Skin trauma with soil contamination
• Ingest contaminated meat products, without proper
  refrigeration or reheating (enterotoxin heat
  labile)

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C. perfringensFood Poisoning

• Relatively common
• Meat products infected large number MO multiply,
  produce enterotoxin
• Ingestion of toxin contaminated food
  Intoxication
• Short incubation, 8-24 hours before symptoms
• Abdominal cramps, watery diarrhea, nausea and
  vomiting no fever
• Short, self-limiting
• MO and toxin may be detected in feces but not
  usually tested

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C. perfringens Myonecrosis (Gas Gangrene)

• Life-threatening disease
• Virulence of cytotoxins
• Intense pain 1 week after introduction into
  tissue
• Severe systemic toxicity
• Painful, edematous wound, sweet or foul smelling
  discharge
• Muscle necrosis, shock, renal failure
• Untreated may result in death

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C. perfringens Soft Tissue Infection

• Simple contaminant of wound, heal normally with
  treatment
• Cellulitis - invasion necrotic wound
• Gas accumulation
• Discoloration of skin
• Malodorous brown, purulent discharge
• Fasciitis infection of muscle
• Possible rapid spread and death
• MO easily Gram-stained and cultured from infected
  tissue

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C. perfringensTreatment and Prevention

• Myonecrosis, tissue infection
• Require aggressive treatment
• Surgical debridement
• High dose penicillin
• Food poisoning
• Supportive treatment
• Antibiotics not necessary, intoxication not
  infection

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Clostridium botulinum

• sausage insufficiently smoked sausage
• Found in soil and water
• Botulinum exotoxin
• Most powerful biological poison known
• Works at neuromuscular junction
• Prevent release neurotransmitter acetylcholine
• Stops signal for muscle stimulation
• Leads to flaccid paralysis

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Food Botulism

• In U.S. uncommon disease usually occurs
  following ingestion of inadequately processed
  home-canned food
• Contaminated with C. botulinum spores
• Composition and nutritive properties allow
  germination and toxin production i.e. pH (7),
  warm temperature
• Ingest inadequately heated or processed food
  (toxin heat labile) Intoxication
• Food does not appear spoiled by smell or taste

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Food Botulism

• Following ingestion, toxin absorbed from
  intestine, transported via blood and lymph to PNS
• Incubation - 8 hours to 8 days, 18-36 hours most
  common
• Symptoms - nausea, vomiting and diarrhea
  symmetric, descending paralysis (eyes, throat,
  neck, trunk, then limbs)
• Death by paralysis of respiratory muscles
• Lab diagnosis by detecting toxin in food and
  patient (serum, feces, gastric fluid)

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Infant Botulism

• Follows ingestion of spores which germinate in
  intestine Infection
• Illness may range from subclinical to sudden
  infant death syndrome
• Honey implicated as source of spores
• Doesnt occur in adults due to competing NF of
  GI tract

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C. botulinumTreatment and Prevention

• Respiratory, ventilatory support to patient
• Eliminate MO from GI tact gastric lavage,
  antibiotics (metronidazole, penicillin)
• Administer botulinum antitoxin antibody binds
  and neutralizes toxin circulating in blood
• Prevention
• Not practical to destroy spores in food
• Prevent spore germination (acid pH, high sugar
  content, store food at 4C)
• Destroy preformed toxin by adequate cooking of
  food (20 minutes, 80C)
• Infants (lt1 year) not fed honey

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Clostridium tetani

• Spores found in soil
• Transient NF GI tract of animals, humans
• In USA, exposure common, but disease uncommon due
  to DTaP vaccine
• Developing countries, poor access to vaccine,
  medical care
• 1 M cases/year
• 20-50 mortality
• Many neonatal infections
• Diagnosis by clinical disease presentation as lab
  tests (stain, culture) usually unsuccessful as MO
  extremely oxygen sensitive, low number tests for
  tetanus toxin insenstive

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C. tetani Exotoxins

• Tetanolysin hemolysin
• Tetanospasmin neurotoxin
• Travel to CNS through blood, lymph, tissue
  spaces, peripheral nerves
• Stops release inhibitory Glycine from synapse (no
  signal to stop muscle contraction)
• Continued excitement at synapse, spastic
  paralysis
• lockjaw - muscles of jaw affected
• May result in respiratory failure, death

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C. tetani Tetanus

• Due to tetanospasmin toxin
• Minor trauma, skin break (i.e. splinter)
• Infection requires relatively few MO
• Spores enter through wound, germinate into
  vegetative cells produce toxin when sufficiently
  low O/R infected tissue (usually deep wound)
• Incubation 1-54 days, average 6-15 days
• Longer incubation, better prognosis

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Tetanus

• Symptoms - cramps, twitching of muscles around
  wound headache, neck stiffness
• Followed by - trismus (lockjaw), generalized
  symptoms (drooling, sweating, irritability, back
  spasms)
• Severe disease involves CNS cardiac arrhythnia,
  fluctuation blood pressure, sweating,
  dehydration)
• Death, if occurs, from respiratory failure
• Neonatal tetanus
• Developing countries
• Umbilical stump infection by septic midwifery
• gt90 death of infants non-immune mothers (no DTaP
  vaccine)

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Tetanus Treatment and Prevention

• Debride wound, aerate well
• Maintain open airway
• Administer antitoxin human tetanus IgG
  neutralizes toxin (but not in CNS)
• Metronidazole - to kill vegetative cells
• If no serious CNS symptoms and toxic effects
  controlled, prognosis for recovery is good
• Prevent disease by vaccination with tetanus
  toxoid part of DTaP trivalent vaccine

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Clostridium difficile

• Part of GI tract NF (in small number)
• In past, rarely associated human disease
• Today, antibiotic-associated GI disease
• Produces two exotoxins
• Enterotoxin A - stimulates fluid and electrolyte
  losses, hemorrhagic necrosis
• Cytotoxin B depolymerize actin, loss of cell
  cytoskeleton, cell death
• Antibiotic therapy can result in diarrhea, permit
  overgrowth of resistant MO

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C. difficile Pseudomembranous Colitis

• Often after taking ampicillin, clindamycin,
  cephalosporin
• Endogenous infection - C. difficile NF in G.I.
  tract
• Exogenous infection - person-to-person in
  hospital
• Multiplies in colon, produces toxin
• Colonic plaques coalesce, form pseudomembrane
  mucin, fibrin, epithelial, inflammatory cells
• Complications - dehydration, electrolyte loss,
  colonic perforation
• Toxin detection in stool confirms diagnosis

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C. difficileTreatment and Prevention

• Mild disease allevate by discontinue
  antibiotics
• Serious disease require antibiotics
  (metronidazole, vancomycin)
• Relapse 20-30 patients due to resistant spores
  allow time for spores to germinate, retreat with
  same antibiotics
• Supportive give fluid and electrolyte
  replacement

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Bacteriodaceae

• NF of oropharynx, urogenital tract, colon
• Anaerobes predominant over aerobes (10-1,000x) in
  colon
• Few cause infection, opportunistic pathogen
• Bacteroides fragilis - most commonly isolated
  anaerobe pathogen

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Bacteriodaceae Gram Stain

• G(-) straight, curved, helical rods
• Bacteroides pleomorhpic
• Fusobacterium long, slender,
• pointed ends
• Porphyromonas small, pigments
• Prevotella small, pigments

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Bacteriodaceae Lab Culture

• Nonselective media
• CBA plates plus vitamin K1, hemin, yeast extract,
  L-cystine
• Selective media
• KVLB (Kanamycin-Vancomycin Laked BA) - freezing,
  thawing whole blood
• BBE (Bacteroides Bile Esculin agar) selective,
  differential
• PEA (phenylethyl alcohol agar) growth all
  obligate anaerobes
• Incubate strict anaerobic conditions
• At 35-370C, 48 hours before opening anaerobic jar

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Bacteriodaceae Lab Culture

• Thioglycollate broth
• Liquid media
• Enriched chopped meat, glucose
• Thioglycolic acid (reducing agent) remove oxygen,
  anaerobic atmosphere deeper in tube
• Resazurin - reduction indicator presence of O2
  pink

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Bacteriodaceae Lab ID

• Each colony - Gram stain, subculture to plates
  (aerobic, anaerobic) to confirm anaerobe
• Species ID - bile tolerance, pigment production,
  sensitivity to antibiotics (vancomycin,
  kanamycin, colistin)
• Gas Liquid Chromatography (GLC) used to
  differentiate anaerobes by major by-products,
  mixed acids

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BacteriodaceaeVirulence Factors

• Capsule adhesin, antiphagocytic
• Fimbriae adhesin
• Endotoxin LPS of gram(-) cell wall
• Protease degrade IgA
• Enzymes - collagenase, phosphotase, RNAse, DNAse

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BacteriodaceaeClinical Significance

• As human NF cause serious infections when gain
  access to normally sterile tissue, organ, fluid
• At risk
• Surgical, trauma patient
• Disrupt patient normal mucosa
• Patient aspirate oral secretions (with NF) into RT

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Infection Mixed Culture Gram(-) Anaerobes

• Respiratory tract causes 50 chronic infection
  of sinus, ear may spread to blood, CNS (brain
  abscess)
• Peridontal - involved in all infections
• Intraabdominal anaerobes recovered
• Gynecological PID, abscess, endometritis,
  surgical wound infection
• Skin and soft tissue colonize wound, progress
  to disease

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Bacteriodes Treatment and Prevention

• Manage infection antibiotics surgical
  intervention (incision, drainage, aerate)
• Many isolates produce ß-lactamases
• Antibiotics
• Metronidazole (anaerobes incorporate drug into
  DNA making it unstable and disrupted)
• Carbapenems (imipenem)
• ß-lactam ß-lactamase inhibitor
  (piperacillin-tazabactam)
• Bacteroides NF, endogenous infection difficult to
  prevent
• Prophylactic antibiotics - patients with mucosa
  disrupted by diagnostic or surgical procedure

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Case Study 8 - Clostridium

• A 61-year-old woman with left-sided face pain
  came to the emergency department of a local
  hospital.
• She was unable to open her mouth because of
  facial muscle spasms and had been unable to eat
  for 4 days because of severe pain in her jaw.
• Her attending physician had noted trismus (motor
  disturbance of trigeminal nerve, spasm of
  masticatory muscles, difficulty in opening the
  mouth) and risus sardonicus (spasmodic grin).

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Case Study 8 - Clostridium

• The patient reported that 1 week before
  presentation, she had incurred a puncture wound
  to her toe while walking in her garden.
• She had cleaned the wound and removed small
  pieces of wood from it, but she had not sought
  medical attention.
• Although she had received tetanus immunizations
  as a child, she had not had a booster vaccination
  since she was 15 years old.
• The presumptive diagnosis was made.

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Case Study 8 - Questions

• 1. How should this diagnosis be confirmed?
• 2. What is the recommended procedure for treating
  this patient? Should management wait until the
  laboratory results are available? What is the
  long-term prognosis for this patient?
• 3. Compare the mode of action of the toxins
  produced by C. tetani and C. botulinum.
• 4. C. difficile causes what diseases? Why is it
  difficult to manage infections caused by this
  organism?

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Class Assignment

• Textbook Reading
• Chapter 22 Anaerobes of Clinical Significance
• Important Concepts In Anaerobic Bacteriology
• Frequently Encountered Anaerobes and Their
  Associated Diseases
• Omit Remaining last three Sections of reading
• Omit Key Terms
• Omit Learning Assessment Questions