Bacteriodaceae, Clostridium, and the anaerobic cocci 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 Morphology/cultural characteristics They may be NF of the oropharynx, urogenital tract, and colon and are considered opportunistic pathogens Morphology/cultural characteristics Pleomorphic 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 Must incubate under strict anaerobic conditions 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 Biochemistry 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 pigment Bacteroides 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 factors Clinical significance Fusobacterium – endotoxin; the endotoxin of Bacteroides is not highly toxic B. fragilis – capsule Some 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 Antimicrobial susceptability/treatment 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 botulism Food 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 Morphology and general characteristics 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 Biochemisrty 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 GLC P. anaerobius is presumptively identified based on sensitivity to polyanethol sulfonate (SPS)

Anaerobic cocci Clinical significance Usually in polymicrobial infections with aerobic organisms and Caused from spread from a site they normally colonize to an adjacent sterile site Cause pleuropulmonary infections, sinusitis, brain abscesses, intra-abdominal infections, pelvic infections, endocarditis, and osteomyelitis

Anaerobic cocci Antimicrobial therapy Cephalosporins, clindamycin, and chloramphenicol