Transcript Document 7497167

The Anaerobes
Clostridium
Bacteriodaceae
Anaerobes of Clinical
Importance
• Gram(+) Spore-Forming Bacilli
– Clostridium
• Gram(-) Bacilli: Bacteriodaceae:
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Bacteroides
Fusobacterium
Porphyromonas
Prevotella
Clostridium
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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)
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
Clostridium: Staining
• G(+) large bacilli
• All motile - except C.
perfringens
• Form endospore – oval,
subterminal
• C. tetani - terminal spore
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)
Clostridium: Lab Culture
• CCFA (Cycloserine-cefoxitinfructose 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
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
C. perfringens:
Infection 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)
C. perfringens:
Food 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
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
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
C. perfringens:
Treatment and Prevention
• Myonecrosis, tissue infection
– Require aggressive treatment
– Surgical debridement
– High dose penicillin
• Food poisoning
– Supportive treatment
– Antibiotics not necessary, intoxication
not infection
Clostridium botulinum:
• “sausage” – insufficiently smoked sausage
• Found in soil and water
• Botulinum exotoxin
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Most powerful biological poison known
Works at neuromuscular junction
Prevent release neurotransmitter acetylcholine
Stops signal for muscle stimulation
Leads to flaccid paralysis
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
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)
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
• Doesn’t occur in adults due to
competing NF of GI tract
C. botulinum:
Treatment 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 4°C)
– Destroy preformed toxin by adequate cooking
of food (20 minutes, 80°C)
– Infants (<1 year) not fed honey
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
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
C. tetani : Tetanus
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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
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
– >90% death of infants non-immune mothers (no
DTaP vaccine)
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
Clostridium difficile
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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
C. difficile:
Pseudomembranous Colitis
• Often after taking ampicillin,
clindamycin, cephalosporin
• Endogenous infection - C.
difficile NF in G.I. tract
• Exogenous infection - personto-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
C. difficile:
Treatment 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
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
Bacteriodaceae: Gram Stain
• G(-) straight, curved, helical rods
• Bacteroides – pleomorhpic
• Fusobacterium – long, slender,
pointed ends
• Porphyromonas – small, pigments
• Prevotella – small, pigments
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
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
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 byproducts, mixed acids
Bacteriodaceae:
Virulence Factors
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Capsule – adhesin, antiphagocytic
Fimbriae – adhesin
Endotoxin – LPS of gram(-) cell wall
Protease – degrade IgA
Enzymes - collagenase, phosphotase,
RNAse, DNAse
Bacteriodaceae:
Clinical 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
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
Bacteriodes: Treatment and
Prevention
• Manage infection – antibiotics + surgical
intervention (incision, drainage, aerate)
• Many isolates produce β-lactamases
• Antibiotics:
– Metronidazole (anaerobes incorporate drug
into DNA; making it unstable and disrupted)
– Carbapenems (imipenem)
– β-lactam + β-lactamase inhibitor
(piperacillin-tazabactam)
• Bacteroides NF, endogenous infection
difficult to prevent
• Prophylactic antibiotics - patients with
mucosa disrupted by diagnostic or
surgical procedure
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).
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.
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?
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