CHAPTER 42Overview of Anaerobic Organisms
EPIDEMIOLOGY • Most are part of the normal flora• Endogenous: developing from within the body• Others inhabit soil and environment• Mode of acquisition: • Nosocomial• Person to person• Normal flora gains access to a sterile site (disruption of
anatomic barrier)• Contamination of existing wound or puncture (ex. Clostridium)• Ingestion of preformed toxins in vegetable- or meat-based
foods • Colonization of GI tract
GRAM POSITIVE, SPORE FORMING BACILLI:
•Clostridia • Endospore-forming, obligate anaerobe (or
aerotolerant)• Catalase negative, gram positive bacilli• Pleomorphic and may be arranged in pairs or short
chains• C. perfringens may appear as weakly catalase
positive• Present in large numbers as normal flora in GI tract of
humans animals, female genital tract and the oral mucosa
GRAM POSITIVE, SPORE FORMING BACILLI:
•Clostridia • Contribute to virulence: collagenase, hyaluronidase,
lecithinase (cell destruction) and phospholipase• Histotoxic clostridia (myonecrosis): C. perfringens,
novyi, septicum, histolyticum, and bifermetans• C. septicum—is a marker for a malignancy in the GI
tract, “smooth swarming” growth on plated media• C. sordelii—is the only urease positive clostridia
GENERAL CHARACTERISTICS OF CLOSTRIDIA:
• Form endospores anaerobically• Motile with peritrichous flagella except C. perfringens, C.
ranosum, and C. innocum• Have swollen sporangia except C. perfringens and C.
bifermentans• Non-encapsulated except C. perfringens• Has a single hemolytic reaction except C. perfringens (double
hemolysis) • Carb fermenters except C. tetani and C. histolyticum• Some members are killed immediately (C. novyi) in the presence
of oxygen
CLOSTRIDIUM PERFRINGENS: GAS GANGRENE BACILLUS
• It is formerly known as Clostridium welchii• It is the commonly isolated member of Clostridia in blood cultures• Microscopy: “boxcar” shaped bacilli; spores are seldom seen , but it
is oval, central to subterminal • Culture: dome-shaped, gray to white colonies—BAP• Double zone of hemolysis (alpha and beta zone) • “Stormy fermentation of milk”—litmus milk medium (production
of acid and gas)
CLOSTRIDIUM PERFRINGENS: GAS GANGRENE BACILLUS
•Produces deoxyribonuclease—lowers the viscosity of exudates; for mobility of bacteria
• It lacks the ability to produce a number of essential amino acids—it causes outbreak after ingestion of contaminated milk and gravy
CLOSTRIDIUM PERFRINGENS: GAS GANGRENE BACILLUS
• Biochemical tests: very fermentative, nonmotile• Lecithinase positive—detected using egg yolk agar
(EYA)• Nagler test positive—lecithovitalin reaction (lecithinase
C) on EYA• Reverse CAMP test positive—arrowhead zone of
hemolysis• Virulence factors: a-toxin and enterotoxin• Alpha toxin—principal virulence of myonecrosis;
lecithinase (phospholipase C), it lyses RBC, WBC
• Nagler
CLOSTRIDIUM PERFRINGENS: GAS GANGRENE BACILLUSClinical infections:
1. Gas gangrene/ Myonecrosis (“Eating sore”)• A life threatening destruction of
muscle and other tissues; necrotizing infections of skeletal muscles• It is caused by a-toxin, a
lecithinase (phospholipase C)• Organisms contaminate wounds
either thru trauma, frostbite or surgery• It is accompanied by bullae
(fluid-filled blisters), pain, swelling serous discharge, discoloration and tissue necrosis
CLOSTRIDIUM PERFRINGENS: GAS GANGRENE BACILLUS
Clinical infections:2. Food poisoning/ enteritis necrotans (“pig-bel”)
• Characterized by ingestion of enterotoxin in contaminated food • Improperly stored food allows germination of the spores and growth of
vegetative bacteria• Symptoms: diarrhea (foul-smelling stool) and crampy abdominal feeling• 2 types of Food poisoning:
A. Type A food poisoning: mild and self-limiting GI illness; diarrhea and cramping abdominal pain
B. Type C food poisoning: serious but rarely encountered in humans; bloody diarrhea and vomiting
CLOSTRIDIUM TETANI (TACK-HEAD BACILLUS)
•Soil and environmental inhabitants •Endospores are found in hospital environments, in soil and dust and in the feces of many farm animals•Microscopy: with terminal spore and swollen sporanigia—“drumstick/lollipop/tennis racket” appearance
CLOSTRIDIUM TETANI (TACK-HEAD BACILLUS)
• Culture: heavy, “smooth swarming” anaerobically but grow slowly• Colonies are with matte surface, narrow zone of B-hemolysis—BAP
• Biochemical test: motile; gelatinase and indole positive; lecithinase and lipase negative
CLOSTRIDIUM TETANI (TACK-HEAD BACILLUS)
• Virulence factor: tetanospasmin (neurotoxin) • Tetanospasmin: • is an endopeptidase that selectively cleaves the synaptobrevin; • causes tension or cramping and twisting in skeletal muscles surrounding the wound and tightness of the jaw muscles
CLOSTRIDIUM TETANI (TACK-HEAD BACILLUS)
Clinical infection/ disease:1. Tetanus:• It is characterized by “trismus” (lockjaw) and “risus sardonicus” (distorted grin)• It occurs when the organism (spore) enters an open wound and elaborates the potent toxin that mediates generalized muscle spasms
CONT.Clinical infection/ disease:1. Tetanus: • Symptoms: muscular rigidity (jaws, neck and lumbar
region), difficulty in swallowing, rigidity of the abdomen, chest, back and limbs• Incubation period: 3 to 21 days (the long incubation
period is related to the distance from the injury to the CNS)• Tetanus neonatorum—contaminated instruments used
for newborns
CLOSTRIDIUM BOTULINUM (CANNED GOOD BACILLUS)
• It is found in soil and aquatic sediments• Potential agent of bioterrorism• Characterized by the presence of
subterminal spore; B-hemolytic on BAP• There are seven types of botulism
toxin (A-G)
CLOSTRIDIUM BOTULINUM (CANNED GOOD BACILLUS)
• Virulence factor: • botulism toxin—neurotoxin (most potent toxin known to
man)• It takes only a small amount of this toxin to produce
paralysis and death• Botulinum toxin is the most lethal of all biologic
compounds• 7 antigenically different botulism toxins (A to G) – only
types A, B. and E are associated with human infection
CLOSTRIDIUM BOTULINUM (CANNED GOOD BACILLUS)
• Toxin type A—used medically to treat strabismus (wandering eye) and as a beauty enhancer by temporarily improving frown lines• Botulism toxin—selectively
cleaves the synaptic vesicle membrane protein synaptobrevin, thus preventing exocytosis and release of the neurotransmitter acetylcholine
• Microscopy:• Gram positive straight
rods with blunt ends• Swollen terminal spores• Tennis racket appearance
CLOSTRIDIUM BOTULINUM (CANNED GOOD BACILLUS)
Clinical infection: • Botulism• Main features: double or blurred
vision, impaired speech, difficulty in swallowing, weakness and paralysis
1. Food botulism2. Infant botulism3. Wound botulism
1. Foodborne botulism• It results from ingestion of
preformed toxin in nonacidic vegetable, preserved food, meat-based food or mushroom foodstuffs (botulism toxin A-most common cause)
• Absorption of the toxin leads to nearly complete flaccid (rag doll) paralysis of respiratory and other essential muscle groups.
2. Infant botulism:• Is an actual infection caused by
ingesting the organism from honey or via breast feeding
• Occurs when the organism elaborates the toxin after it has colonized the gastrointestinal tract of infants
3. Wound botulism: • Occurs when C. botulinum
produces the toxin from an infected wound site
CLOSTRIDIUM DIFFICILE•Most common cause of antibiotic-associated diarrhea and pseudomembranous colitis Acquired in the hospitals by individuals receiving antibiotics • An infection control dilemma among hospitalized patients• Found as part of the GI biota in about 5% of individuals • Present occasionally on the hands of hospitalized personnel
CLOSTRIDIUM DIFFICILE• Microscopy: chains up to 6 cells aligned end to end ;
endospores may be oval and subterminal• Culture: yellow, ground glass colonies—CCFA • “horse stable” or “barnyard” odor—BAP• Fluoresce chartreuse under long wave UV• Nonhemolytic—BAP • Ferments fructose: CCFA—orginal pink colored medium
turns yellow• Virulence factors: toxin A (enterotoxin) and toxin B
(cytotoxin)
IDENTIFICATION OF CLOSTRIDIUM SPECIES
Swarming
Double Zone of
β-hemolysis
Chartreuse Fluorescence
Lecithinase Lipase Spore
Position
C. perfringens - + - + -ST
C. botulinum - - - - + ST
C. tetani + - - - - TC. difficile - - + - - STC. septicum + - - - - ST
IDENTIFICATION OF CLOSTRIDIUM SPECIES
Swarming
Double Zone of
β-hemolysis
Chartreuse Fluorescence
Lecithi-nase Lipase Spore
Position
C. perfringens - + - + - ST
C. botulinum - - - - + ST
C. tetani + - - - - TC. difficile - - + - - ST
ANAEROBIC NONSPOREFORMING BACILLI AND COCCI
Organism Gram stain reaction Distinguishing characteristics
Others
Actinomyces spp. Gram (+), straight or slightly curved filamentous rods; may be beaded or banded
Young colonies—”spider-like” or woolyOld colonies—”molar tooth” (A. israelii)
“sulfur granules” on direct exam of purulent exudate
Bacteroides fragilis Gram (-), pale staining, pleomorphic rods; resembles “safety pin”
Gray-white, circular, smooth, nonhemolytic
Isolated from the mucosal surfaces of the human oral cavity and GI tract
Bacteroides ureolyticus Gram (-), pale staining, thin rods; some curved
Colonies corrode (pit) the agar
requires formate and fumarate for growth in broth culture
Bifidobacterium spp. Gram (+) diptheroid; coccoid or pointed shape; bifurcated (forked) end, resemble “dog bones”
Small, white, shiny, convex colonies
Eubacterium spp. Gram (+) pleomorphic , has seagull wing shape
Poorly characterized
a.Israelii—causes abdomen and chest infections abd pelvic actinomycosis in women with intrauterine devices,a.Most common pathogen
ANAEROBIC NONSPOREFORMING BACILLI AND COCCI
Organism Gram stain reaction Distinguishing characteristics
Others
Fusobacterium Gram (-) spindle shaped rod; resembles Capnocytophaga
Most spp. fluoresce chartreuse“breadcrumb like colonies”
Typically isolated from the oral cavity as integral components of dental biofilms
Lactobacilli spp. Gram variable rods, short coccobacilli, resembles streptococci
produces lactic acid from glucose fermentation
Leptotrichia spp. Gram (-) large fusiform rods
“raspberry-like” colonies Most often isolated from the oral cavity or urogenital tract
Peptococcus niger Gram (+) cocci, singly, pairs or tetrads
Black to olive green colonies
Porphyromonas spp. Gram (-) coccobacilli Brick red fluorescence
ANAEROBIC NONSPOREFORMING BACILLI AND COCCI
Organism Gram stain reaction Distinguishing characteristics
Others
Prevotella spp. Gram (-) rods Brick red fluorescence
Propionibacterium spp. Gram (+) palisade arrangement
produces propionic acid from glucose
Veillonella parvula Gram (-) tiny diplococci Red fluorescence ubiquitous as part of the normal flora of the human oral cavity and the genitourinary, respiratory and GI tracts
BACTEROIDES FRAGILIS• Most commonly isolated anaerobes
from blood cultures• Significant cause of intraabdominal
abscesses• Biochemical tests: • catalase (+); • growth (+) in 20% bile; • beta lactamase producers; • saccharolytic; • nonmotile
PREVOTELLA SPP.
Colony morphology• Fluoresces
brick red on blood agar
• Black pigment in KVLB agar.
KVLB
Ana BAP
PREVOTELLA SPP.Other Lab Characteristics• Ferments glucose
and other carbohydrates (saccharolytic)
• Inhibited by 20% bile
• Susceptible to Colistin
LACTOBACILLUS ACIDOPHILUS• Part of the normal flora of the mouth, GIT, and vaginal canal•Has an important role in the health of female genital tract• For protection
• Produces lactic acid from glycogen—lower the vaginal pH and suppresses the overgrowth of other bacteria
LACTOBACILLUS ACIDOPHILUS• It can grow at pH 3-4 (Tomato juice agar)
•Associated infection: • Bacterial vaginosis
•Biochemical test: (-) catalase, H2S, and esculin hydrolysis
LABORATORY DIAGNOSIS
AEROTOLERANCE TEST• Aerotolerance Test• The colony is subcultured:
i. Aerobic agar plateii. Anaerobic blood agar plate
Colony Type Aerobe Capnophilic
AerobeFacultative Anaerobe
Obligate Anaerobe
Non-CO2 + - + -CO2 + + + -Anaerobic - - + +
NAGLER TEST• This test is performed
to differentiate Clostridium perfringens from other Clostridium spp. • based on the
neutralization of lecithinase C activity by the presence of antitoxin.
LAKED KV BLOOD AGAR• For isolation of gram negative
anaerobes (Bacteroides, Prevotella and some Fusobacterium)• Kanamycin inhibits facultative gram (-) bacilli• Vancomycin inhibits facultative and obligate gram (+) bacteria
ANTIBIOTIC DISK TEST• To determine for characteristic
susceptability pattern to: • Colistin (10 µg),
Vancomycin (5 µg) and Kanamycin (1 mg)
• Resistant: Zone of inhibition of 10 mm or less• Susceptible: Zone greater
than 10 mm
Ana BAP
ADDITIONAL NOTES:• Bile disk test—utilized to identify B. fragilis (bile-tolerant)
• SPS disk—utilized for rapid identification of Peptostreptococcus anaerobius (sensitive) against Peptostreptococcus assacharolyticus (resistant)
ADDITIONAL NOTES:• Propionibacterium acnes—associated with endophthalmitis after cataract surgery
• Bacteroides ureolyticus—requires formate and fumarate for growth in broth culture
• Kanamycin, colistin, and vancomycin—preliminary antibiotics for testing
IDENTIFICATION OF GRAM POSITIVE SPECIES
48-hr colony <1mm
Rough colony
Branched Bacilli
Catalase/ Indole Comments
A. israelii + + + -Molar Tooth
Colony
Bifidobacterium - - - -Rods w/ forked ends
P. acnes - - - +
IDENTIFICATION OF ANAEROBIC GRAM NEGATIVE BACILLI
V K CoBile
Resistance
Esculin Hydrol
ysis
Growth on
KVLB
Brick Red
Fluorescence
Chartreuse
fluorescence
Iridescent
sheen
B. fragilis R R R + + + - - -
Prevotella R R V - - + + - -
Porphyromonas S R R - - - + - -
Fusobacterium R S S - - ± - + +
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