The Anaerobes
Bacteriodaceae, Clostridium, and the anaerobic cocci
Bacteriodaceae
Classification Bacteroidaceae family includes the following
genera Bacteroides Fusobacterium Leptotrichia (rare in human diseases) Prevotella Porphyromomas
B. fragilis is the most commonly isolated anaerobic G-B.
All are nonsporing, anaerobic, G-B
Bacteriodaceae
They may be NF of the oropharynx, urogenital tract, and colon and are considered opportunistic pathogens
Morphology/cultural characteristicsPleomorphic G-B showing irregular or
bipolar staining. Fusobacterium nucleatum characteristically is
long and slender with pointed ends
Bacteroides sp. Gram stain
Fusobacterium sp.
Fusobacterium nucleatum
Bacteriodaceae
To grow these organisms, nonselective anaerobic BA plates, selective anaerobic plates, and liquid should be used for primary isolation.
Nonselective anaerobic BA plates= CBA plates plus vitamin K1, hemin, yeast extract, and L-cystine (supplemented BA)
Selective media (all for Bacteroides species) Anaerobic PEA BA – suppresses aerobic G-B Kanamycin-Vancomycin BA – inhibits G+ and
facultatively anaerobic G -
Bacteriodaceae
Kanamycin-Vancomycin laked BA (KVLB) prepared by freezing and thawing whole blood
Bacteroides Bile Esculin (BBE) agar
Liquid media Thioglycollate Chopped meat glucose
Must incubate under strict anaerobic conditions
Incubation at 35-370 C for 48 hours before opening an anaerobic jar.
B. fragilis
Bacteriodaceae
Each colony type that grows should be Gram stained and subcultured to plates grown under both aerobic and anaerobic conditions to confirm that it is an anaerobe.
Biochemistry The Bacteroides group which now includes
Prevotella and Porphyromonas species are divided into groups based on bile tolerance, pigment production, and sensitivity to the antibiotics Vancomycin (V), Kanamycin (K), and Colistin (C)
Bacteriodaceae
V K C Bile pigmentBacteroides fragilis R R R R -Prevotella R R S S +/-Porphyromonas S R R S +
B. fragilis is catalase + GLC – used to differentiate Fusobacterium from
the others. The major by-product of Fusobacterium is butyric acid while the
others produce mixtures of acids. F. nucleatum and F. necrophorum (lipase+) are the major
pathogens
GLC
Bacteriodaceae
Virulence factorsFusobacterium – endotoxin; the endotoxin
of Bacteroides is not highly toxicB. fragilis – capsuleSome in the Bacteroides group produce IgA
protease,collagenase, phosphotase, RNAse, or DNAse
Clinical significance
Bacteriodaceae
Clinical significance These organisms are NF of the oropharynx,
urogenital tract, and colon and cause serious infections when they gain access to tissues and organs.
Most commonly they cause intra-abdominal infections
The infections are usually polymicrobial infections They can also be found causing:
Peridontal disease
Bacteriodaceae
Chronic otitis media Chronic sinusitis Wound infections Pneumonia Female genital tract infections Brain abscesses Bacterial endocarditis Bone infections
Antimicrobial susceptability/treatment Incision and drainage Chloramphenicol, clindamycin, cefoxitin, or
metronidazole
Clostridium
Classification – no family designation Most are strict anaerobes Are widely distributed in soil and water Some are NF in the GI tract of man and other
animals Morphology/cultural characteristics
Are endospore forming large G+B The site at which the endospore forms in the vegetative
cell is characteristic and helps in differentiating C. tetani (terminal) from the others (oval and subterminal)
C. perfringens Gram stain
Spore stain with time
C. tetani spores (terminal)
Clostridium
All except C. perfringens are motile Nonselective, selective, and liquid media should be
used for primary isolation Nonselective – supplemented anaerobic BA
C. perfringens produces a classic double zone of hemolysis Nonselective, differential – Egg yolk agar
Allows differentiation based on Lecithinase production (white precipitate) Lipase production (sheen around surface of colonies) Protease production(clearing)
C. perfringens double zone hemolysis
Lecithinase production
Lipase production
Clostridium
Selective – Cycloserine-cefoxitin-egg yolk-fructose agar(CCFA) is selective for C. difficile
Liquid Thioglycollate Chopped meat
Special isolation procedures – Clostridia usually occur in mixed cultures with G-B and nonsporing anaerobes – use heat or alcohol treatment to kill others before plating
C. perfringens grows rapidly at 450 C
CCFA
Clostridium
Biochemistry O2 tolerance – C. tertium and C. histolyticum are
aerotolerant, but catalase - Lipase vs lecithinase vs protease production on egg
yolk agar Naegler reaction - smear ½ of an egg yolk agar
plate with type A anti-toxin (anti-lecithinase), streak organism in a single line, and look for inhibition of lecithinase production
Sugar fermentations
Clostridium
Milk digestion Esculin hydrolysis Gelatin hydrolysis Reverse CAMP – is presumptive for C. perfringens
Mechanisms of Virulence – most Clostridia are not invasive, but many produce powerful toxins and enzymes C. perfringens produces a capsule C. botulinum – produces a potent exotoxin
The organisms are divided into 8 different types based on which of the 8 serotypes of exotoxin are produced.
Clostridium
Serotypes are A, B, C1, C2, D, E, F, and G Serotype A is the most potent. Types A,B, E, F, and G can cause botulism in man Botulinal exotoxin is the most powerful exotoxin known. It works at the neuromuscular junction and in the
autonomic nervous system to prevent the release of the neurotransmitter acetylcholine.
This leads to flaccid paralysis. The toxin has two components, B for binding, and A for
the toxic activity. The toxic part cleaves proteins that mediate fusion of
synaptic vesicles with the cell membrane and subsequent release of acetylcholine.
The toxin is part of the bacteria and not released until the death of the bacteria.
Normal release of acetylcholine
Proteins involved in membrane fusion to release acetylcholine
C. botulinum toxin prevents acetylcholine release
Botox treatment – relaxes muscle spasms
Clostridium
C. tetani -produces two exotoxins A hemolysin Tetanospasmin – can travel to the CNS humorally through
blood and lymph, or neurally through tissue spaces of the peripheral nerves.
The toxin binds to sialic acid containing gangliosides of the CNS to prevent the release of the inhibitory neurotransmitters GABA and glycine from synapses (by cleaving VAMP) in the inhibitory nerve system of the spinal cord.
This is a system that prevents the contraction of a muscle when the muscle of the opposite action contracts.
This leads to both sets of muscles contracting at the same time and spastic paralysis.
Called lockjaw when the jaw is affected. Contractions can break the back.
Tetanospasmin activity
Spastic paralysis
Tetanospasmin and botulinism toxin action
Clostridium
C. perfringens – is divided into 5 types, A through E, based on the major lethal toxins produced.
All types produce the alpha toxin – is a lethal, necrotizing lecithinase which is responsible for the outer zone of hemolysis.
Other toxins that may be produced include beta, epsilon, or iota which are all lethal and necrotizing, delta and theta which are lethal and hemolytic and are responsible for the inner zone of hemolysis.
Clostridium
Enzymes produces may include gelatinase, collagenase, protease, hyaluronidase, DNAse, and neuraminidase
Types A and C produce an enterotoxin responsible for causing an intoxication type of food poisoning in meats, poultry, and gravy.
Its action resembles that of the cholera toxin.
Clostridium
C. difficile – produces two exotoxins both of which inactivate Rho proteins by adding a glucose.
Rho proteins function as molecular switches in cytoskeletal dynamics and many signal transduction pathways
Enterotoxin A stimulates fluid and electrolyte losses from the intestinal tract
Cytotoxin B kills mammalian cells
Clostridium
Clinical significance C. tetani – causes tetanus, a disease due to the
toxin tetanospasmin. Spores are found in feces, soil, and dust. Spores
enter the body through wounds where they germinate into vegetative cells and subsequently produce toxin when a sufficiently low O/R potential is established in the infected tissue (usually a deep wound).
Clostridium
The incubation is 1-54 days with an average of 6-15 days. The longer the incubation, the better the prognosis.
Symptoms begin with cramps and twitching of muscles around the wound.
Headache and neck stiffness also occur. These are followed by trismus (lockjaw) and more
generalized symptoms. Death, if it occurs, results from respiratory failure within 4
days. Neonatal tetanus is a consequence of infection of the
umbilicus through septic midwifery and it occurs in underdeveloped countries.
Tetanus
Clostridium
C. botulinum – causes botulismFood botulism – in the U.S. this usually occurs
following ingestion of inadequately processed home-canned food. For this to occur you need:
Food must be contaminated with C. botulinum spores Food must possess composition and nutritive
properties that allow germination and toxin production Food must have suitable pH and temperature Food must have been inadequately heated or
processed (toxin is heat labile)
Clostridium
Following ingestion, toxin is absorbed from the intestine and transported via blood and lymph to the PNS.
The incubation is 8 hours to 8 days with 18-36 hours most common
The first symptoms include nausea, vomiting, and diarrhea followed by symmetric, descending paralysis (eyes, throat, neck, trunk, and then the limbs)
Paralysis of respiratory muscles results in death
Infant botulism – follows ingestion of spores which germinate in the intestine.
Clostridium
Illness may range from subclinical to sudden infant death syndrome
Honey has been implicated as a source of spores. This doesn’t occur in adults because of competing NF
Wound botulism – can follow C. botulinum toxin production in a traumatic wound.
Clostridial wound infections – most species of Clostridium are saprophytic bacteria living in soil and water.
C. perfringens and other are found in the intestines of man and other animals.
Clostridium Most clostridial wound infections occur as simple
contaminants of a fresh wound and most heal normally with simple therapy
Anaerobic cellulitis follows invasion of necrotic wound tissue by proteolytic bacteria and is characterized by gas accumulation, discoloration of the skin, and a malodorous brown, purulent discharge.
Clostridial myonecrosis or gas gangrene – involves invasion of normal, healthy muscle tissue surrounding the wound site.
C. perfringens is most commonly isolated. Need a lowered O/R potential in the wound which causes reduction of pyruvate to lactate and a decreased pH which activates the proteolytic enzymes.
Clinical features include severe systemic toxicity, a painful, edematous wound with a sweet or foul smelling discharge.
Untreated cases may result in death.
Gas gangrene
Clostridium
C. difficile – Antibiotic therapy of any kind can result in diarrhea.
The severity may range from simple diarrhea to severe antibiotic associated pseudomembranous colitis.
This is characterized by colonic plaques that coalesce to form a pseudomembrane of mucin, fibrin, sloughed off epithelial cells, and acute inflammatory cells.
Complications include dehydration, electrolyte imbalance and colonic perforation.
Most often occurs following therapy with ampicillin, clindamycin, or cephalosporins and is usually due to C. difficile which may be NF in the G.I. tract.
Pseudomembranous colitis plaques
Pseudomembranous colitis plaques
Clostridium
Antimicrobial therapy/treatment C. tetani - use antitoxin, however, toxin already in
the CNS is not neutralized; maintain open airway; remove infected tissue.vaccination - part of DPT – need booster every 10 years
C. botulinum – administer antitoxin before it is translocated inside tissues: aggressive respiratory support
Wound infections – debridement and antibiotics; hyperbaric chamber
Clostridium
C. difficile – metronidazole and vancomycin; fluid and electrolyte replacement
Anaerobic cocci
Classification Peptococcus Peptostreptococcus – anaerobic coccus most often
associated with human disease Veillonella All can be found as NF of oral cavity, Genital and
urinary tracts, G.I. tract , and skin Morphology and general characteristics
Peptococcus – G+C; 1 species, P. niger, produces black colonies
Peptostreptococcus G+C; 9 species. P. anaerobius is the most commonly isolated species
Peptostreptococcus Gram stain
Anaerobic cocci
Veillonella – tiny G-C; V. parvula is the major isolate of clinical importance.
All are slow growing and may require prolonged incubation
Biochemisrty I.D. by GLCP. anaerobius is presumptively identified
based on sensitivity to polyanethol sulfonate (SPS)
Anaerobic cocci
Clinical significanceUsually in polymicrobial infections with
aerobic organisms and Caused from spread from a site they
normally colonize to an adjacent sterile siteCause pleuropulmonary infections, sinusitis,
brain abscesses, intra-abdominal infections, pelvic infections, endocarditis, and osteomyelitis
Anaerobic cocci
Antimicrobial therapyCephalosporins, clindamycin, and
chloramphenicol
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