The AnaerobesThe AnaerobesThe AnaerobesThe Anaerobes

Clostridium Clostridium

BacteriodaceaeBacteriodaceae

Anaerobes of Clinical Importance

• Gram(+) Spore-Forming Bacilli– Clostridium

• Gram(-) Bacilli: Bacteriodaceae:– Bacteroides– Fusobacterium– Porphyromonas– Prevotella

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)

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-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

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

– 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• 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 • 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 - 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

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 by-products, mixed acids

Bacteriodaceae:Virulence Factors

• 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