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 - - ± - + +