8.7 - Anaerobic Bacteria

Obligate anaerobes, facultative anaerobes, and microaerophiles are terms referring to bacteria that require:

A. Increased nitrogen

B. Decreased CO2

C. Increased O2

D. Decreased O2

D. Decreased O2

Oxygen requirement terminology
• Obligate anaerobes – grow only in the absence of oxygen (O₂ is toxic)
• Facultative anaerobes – grow with or without O₂, prefer O₂ if available
• Microaerophiles – require reduced O₂ levels (5–10%), higher O₂ inhibits growth
• Reflects organisms’ ability to detoxify reactive oxygen species (superoxide, peroxide)

Quick differential:
• Aerobes: require full atmospheric O₂ (e.g., Pseudomonas)
• Capnophiles: need increased CO₂, not decreased O₂

Summary: These bacteria thrive in low-oxygen environments, not high-oxygen ones.

Which of the following most affects the oxidation-reduction potential (Eh, or redox potential) of media for anaerobic bacteria?

A. Oxygen

B. Nitrogen

C. pH

D. Glucose

C. pH

Redox potential (Eh) background
• pH directly affects the oxidation-reduction balance of culture media
• As pH decreases (more acidic), the Eh also decreases, creating more reducing conditions favorable for anaerobes
• Conversely, higher pH values increase Eh and inhibit anaerobic growth
• Oxygen exposure still influences Eh but pH exerts the most significant chemical control on reduction potential within the medium

Quick differential:
• Oxygen: external oxidizer, but not intrinsic to Eh chemistry
• Nitrogen: inert gas, no effect on redox balance
• Glucose: energy source, negligible direct effect on Eh

Summary: pH has the greatest chemical impact on a medium’s redox potential, influencing anaerobe growth conditions.

Which of the following is the medium of choice for the selective recovery of gram-negative anaerobes?

A. Kanamycin-vancomycin-anaerobe (KVA) agar

B. Phenylethyl alcohol (PEA) agar

C. Cycloserine cefoxitin fructose agar (CCFA)

D. THIO broth

A. Kanamycin-vancomycin-anaerobe (KVA) agar

KVA agar background/use
• Selective medium designed for the recovery of gram-negative anaerobes, particularly Bacteroides and Prevotella spp
• Contains kanamycin and vancomycin to inhibit most gram-positive and facultative organisms
• Supports anaerobic growth when incubated in an oxygen-free environment

Quick differential:
• PEA agar: inhibits gram-negative facultatives, enhances gram-positive anaerobes (Clostridium, Peptostreptococcus)
• CCFA agar: selective for Clostridioides difficile (fructose-fermenter)
• THIO broth: enrichment broth for both aerobes and anaerobes; non-selective

Summary: KVA agar is the selective medium of choice for isolating gram-negative anaerobes such as Bacteroides.

Anaerobic bacteria are routinely isolated from all of the following types of infections except:

A. Lung abscesses

B. Brain abscesses

C. Dental infections

D. Urinary tract infections

D. Urinary tract infections

Anaerobic infection background
• Anaerobes thrive in low-oxygen, necrotic tissues such as abscesses
• Common anaerobic infection sites:
– Lung abscesses (aspiration, mixed flora)
– Brain abscesses (extension from ear/sinus infections)
– Dental infections (gingival crevices, periodontal abscesses)
• Urinary tract infections are rarely anaerobic because urine and the urinary tract are well oxygenated and flushed regularly

Quick differential:
• Anaerobic UTI may occur only in severe mixed infections (e.g., obstruction, trauma, instrumentation)
• Most UTIs caused by Enterobacterales and other facultative GNRs

Summary: Anaerobes rarely cause UTIs due to the oxygenated, self-cleansing nature of the urinary tract.

Methods other than packaged Microsystems used to identify anaerobes include:

A. Antimicrobial susceptibility testing

B. Gas-liquid chromatography (GLC)

C. Special staining

D. Enzyme immunoassay

B. Gas-liquid chromatography (GLC)

Anaerobe identification methods
• Gas-liquid chromatography (GLC) analyzes volatile fatty acids (VFAs) produced by anaerobes during metabolism
• Each anaerobic genus/species yields a distinct chromatographic pattern, aiding identification
• GLC is a biochemical-based method, not dependent on commercial test panels
• Still useful for Bacteroides, Clostridium, and Fusobacterium differentiation

Quick differential:
• Antimicrobial susceptibility: determines resistance, not identification
• Special staining: aids visualization (e.g., spore, Gram) but not species ID
• Enzyme immunoassay: detects antigens/toxins, not used for routine anaerobe ID

Summary: Gas-liquid chromatography is the traditional non–Microsystem method for identifying anaerobes by their metabolic end products.

Which broth is used for the cultivation of anaerobic bacteria to detect volatile fatty acids as an aid to identification?

A. Prereduced peptone-yeast extract-glucose (PYG)

B. THIO broth

C. Gram-negative (GN) broth

D. Selenite (SEL) broth

A. Prereduced peptone-yeast extract-glucose (PYG)

PYG broth background/use
• Enrichment broth formulated for anaerobic cultivation
• Supports growth of a wide variety of anaerobes and production of volatile fatty acids (VFAs)
• VFAs detected via gas-liquid chromatography (GLC) for organism identification
• Prereduced to eliminate oxygen and maintain low redox potential

Quick differential:
• THIO broth: general enrichment for both aerobes and anaerobes; not ideal for VFA detection
• GN broth: supports facultative gram-negative rods (Enterobacterales), not anaerobes
• SEL broth: selective enrichment for Salmonella spp, not anaerobes

Summary: PYG broth is the medium of choice for anaerobic cultivation when detecting VFAs for identification.

A gram-positive spore-forming bacillus growing on sheep blood agar anaerobically produces a double zone of beta-hemolysis and is positive for lecithinase. What is the presumptive identification?

A. Bacteroides ureolyticus

B. Bacteroides fragilis

C. Clostridium perfringens

D. Clostridium difficile

C. Clostridium perfringens

Clostridium perfringens background/morphology
• Large, boxcar-shaped, gram-positive, spore-forming bacillus
• Anaerobic growth on SBA with double zone of beta-hemolysis (inner complete, outer partial)
• Lecithinase positive on egg yolk agar (opalescent zone)
• Causes gas gangrene (myonecrosis), food poisoning, and necrotizing enteritis
• Produces alpha toxin (lecithinase) and other exotoxins

Quick differential:
• Bacteroides spp: gram-negative, non–spore-forming rods
• C. difficile: chartreuse fluorescence, toxin A/B detection, not lecithinase positive
• C. septicum: beta-hemolytic but lecithinase negative

Summary: Anaerobic, lecithinase-positive bacillus with double hemolysis = Clostridium perfringens.

Egg yolk agar is used to detect which enzyme produced by several Clostridium species?

A. Lecithinase

B. B-Lactamase

C. Catalase

D. Oxidase

A. Lecithinase

Egg yolk agar background/use
• Detects lecithinase activity, common in Clostridium spp, especially C. perfringens
• Positive reaction produces an opaque, opalescent zone around colonies
• Reaction occurs due to hydrolysis of lecithin in the medium
• Helps differentiate C. perfringens from other Clostridia

Quick differential:
• β-lactamase: antimicrobial resistance mechanism, not detected on EYA
• Catalase: aerobic bacteria; most Clostridia are catalase negative
• Oxidase: not used for anaerobic GPR identification

Summary: Egg yolk agar detects lecithinase, a hallmark enzyme of Clostridium perfringens.

Which of the following organisms will display lipase activity on egg yolk agar?

A. Clostridium botulinum

B. Clostridium sporogenes

C. Clostridium novyi

D. All of these options

D. All of these options

Lipase on egg yolk agar background
• Lipase breaks down triglycerides in egg yolk
• Produces an iridescent/oily sheen on agar surface
• Several Clostridium spp show lipase activity, including:
– C. botulinum
– C. sporogenes
– C. novyi

Quick differential:
• Lecithinase activity instead produces opaque precipitation (C. perfringens hallmark)
• Lipase vs lecithinase reactions help distinguish Clostridium species in mixed anaerobic cultures

Summary: Multiple Clostridium species demonstrate lipase-positive reactions on egg yolk agar.

Which spore type and location is found on Clostridium tetani?

A. Round, terminal spores

B. Round, subterminal spores

C. Ovoid, subterminal spores

D. Ovoid, terminal spores

A. Round, terminal spores

Clostridium tetani background/morphology
• Gram-positive spore-forming anaerobic bacillus
• Produces round terminal spores giving a “drumstick” or tennis racket appearance
• Spores resistant to heat and disinfectants
• Produces tetanus neurotoxin (blocks inhibitory neurotransmitters → spastic paralysis)

Quick primer: spore shape + position
• Shape
– Round: perfectly circular
– Ovoid: oval/egg-shaped
• Location
– Terminal: at the very end of the bacillus
– Subterminal: slightly inside from the end

Quick differential:
• C. botulinum: oval, subterminal spores
• C. perfringens: rarely visible, central to subterminal when present
• C. sporogenes: oval, subterminal

Summary: C. tetani has round, terminal spores, creating the classic drumstick look.

Gram-positive bacilli recovered from two blood cultures from a 60-year-old patient with diabetes gave the following results:

Spores seen: neg

Motility: neg

Volatile acids by GLC (PYG): acetic acid (A) and butyric acid (B)

Hemolysis: + (double zone)

Lecithinase +

What is the most likely identification?

A. Clostridium tetani

B. Clostridium perfringens

C. Clostridium novyi

D. Clostridium sporogenes

B. Clostridium perfringens

Clostridium perfringens background/morphology
• Large “boxcar” GPR, nonmotile
• Lecithinase positive on egg yolk agar
• Double zone of beta-hemolysis on SBA (inner complete, outer partial)
• Spores often not seen in clinical material
• VFAs from PYG–GLC commonly include acetic (A) and butyric (B) acids
• Causes bacteremia, gas gangrene, and foodborne illness

Quick differential:
• C. tetani: motile, terminal spores, not lecithinase positive
• C. novyi: typically motile, lecithinase variable, lipase positive
• C. sporogenes: motile, lipase positive, not classically lecithinase positive

Summary: Nonmotile, lecithinase-positive GPR with double hemolysis fits C. perfringens.

Which mechanism is responsible for botulism in infants caused by C. botulinum?

A. Ingestion of spores in food or liquid

B. Ingestion of preformed toxin in food

C. Virulence of the organism

D. Lipase activity of the organism

A. Ingestion of spores in food or liquid

Infant botulism background/mechanism
• Infants ingest C. botulinum spores (commonly honey or environmental dust)
• Spores germinate in the immature gut, producing botulinum neurotoxin in vivo
• Toxin blocks acetylcholine release at neuromuscular junction → flaccid paralysis
• Not due to preformed toxin ingestion as in foodborne botulism in adults

Quick differential:
• Adults: typically ingest preformed toxin in contaminated foods
• Wound botulism: spores contaminate wounds → local toxin production
• Lipase: helpful in Clostridial ID, not a disease mechanism

Summary: Infants get botulism from ingesting spores, which then produce toxin inside the gut.

The classic form of foodborne botulism is characterized by the ingestion of:

A. Spores in food

B. Preformed toxin in food

C. Toxin H

D. All of these options

B. Preformed toxin in food

Foodborne botulism background/mechanism
• Caused by ingestion of preformed botulinum neurotoxin in improperly canned or stored foods
• Toxin blocks acetylcholine release → flaccid paralysis
• Symptoms include descending paralysis, blurred vision, dysphagia, and respiratory failure
• Spores are not the primary concern in foodborne disease (that’s infant botulism)

Quick differential:
• Infant botulism: spores ingested, toxin made in gut
• Wound botulism: spores in wound, toxin produced locally
• Toxin H is a subtype, not the transmission mechanism

Summary: Classic foodborne botulism results from consuming preformed toxin in contaminated food.

Which test is performed to confirm an infection with C. botulinum?

A. Toxin neutralization test

B. Spore-forming test

C. Lipase test

D. Gelatin hydrolysis test

A. Toxin neutralization test

Clostridium botulinum confirmation
• Detection of botulinum neurotoxin is required for definitive diagnosis
• Toxin neutralization assay (traditionally mouse bioassay) confirms specific toxin type
• Culture alone insufficient because toxigenicity must be proven
• Newer validated molecular and immunoassays may be used, but toxin detection remains gold standard

Quick differential:
• Spore-forming test: shows sporulation, not diagnostic
• Lipase: useful for ID on egg yolk agar but not confirmatory
• Gelatin hydrolysis: nonspecific, used for biochemical differentiation

Summary: Confirm C. botulinum infection by demonstrating toxin with a toxin neutralization test.

Which Clostridium spp causes pseudomembranous colitis or antibiotic-associated colitis?

A. Clostridium ramosum

B. Clostridium difficile

C. Clostridium perfringens

D. Clostridium sporogenes

B. Clostridium difficile

Clostridium difficile background/morphology
• Gram-positive, spore-forming anaerobic bacillus
• Produces Toxin A (enterotoxin) and Toxin B (cytotoxin)
• Major cause of pseudomembranous colitis and antibiotic-associated diarrhea
• Spores persist in hospital environments; spread via fecal–oral route
• Diagnosis via toxin detection (EIA/NAAT) or GDH screen + reflex toxin testing

Quick differential:
• C. perfringens: food poisoning, gas gangrene
• C. sporogenes: rarely pathogenic, lipase positive
• C. ramosum: opportunistic infections, no pseudomembranes

Summary: C. difficile causes antibiotic-associated colitis with toxin-mediated pseudomembranes.

Identification of C. tetani is usually based on:

A. Gram staining of the wound site

B. Anaerobic culture of the wound site

C. Blood culture results

D. Clinical findings

D. Clinical findings

Clostridium tetani diagnosis background
• Clinical diagnosis because the organism is difficult to isolate and recovery is not required to confirm disease
• Classic symptoms: lockjaw (trismus), risus sardonicus, spastic paralysis due to tetanus neurotoxin
• Spores may persist in wounds but culture is often negative
• Lab confirmation provides no added value and must not delay treatment

Quick differential:
• Botulism: flaccid paralysis, descending
• Tetanus: spastic paralysis, ascending
• C. perfringens: gas gangrene, food poisoning

Summary: Tetanus is diagnosed based on clinical presentation, not culture.

Obligate anaerobic gram-negative bacilli that do not form spores, grow well in 20% bile (Bacteroides bile esculin [BBE] agar), and are resistant to penicillin two-unit disks are most likely:

A. Porphyromonas spp

B. Bacteroides spp

C. Fusobacterium spp

D. Prevotella spp

B. Bacteroides spp

Bacteroides spp background/morphology
• Obligate anaerobic, gram-negative bacilli, non–spore-forming
• Grow on BBE agar with 20% bile tolerance
• Hydrolyze esculin → black colonies on BBE (especially B. fragilis group)
• Resistant to penicillin (β-lactamase production common)
• Normal GI flora; major cause of intra-abdominal abscesses and bacteremia

Quick differential:
• Porphyromonas: bile sensitive, black pigment colonies, often vancomycin susceptible
• Fusobacterium: bile sensitive, produce butyric acid, tapered ends
• Prevotella: bile sensitive, some pigmented, often indole positive

Summary: Bile-resistant, penicillin-resistant anaerobic GNR on BBE = Bacteroides fragilis group.

Which Bacteroides spp is noted for "pitting" of the agar and is sensitive to penicillin two-unit disks?

A. Bacteroides vulgatus

B. Bacteroides ovatus

C. Bacteroides thetaiotaomicron

D. Bacteroides ureolyticus

D. Bacteroides ureolyticus

Bacteroides ureolyticus background/morphology
• Obligate anaerobic, gram-negative rod
• Known for “pitting” or etching of the agar surface
• Sensitive to penicillin 2-unit disks
• Typically urease positive
• Isolated in genital tract infections and periodontal disease

Quick differential:
• B. fragilis group: bile resistant, penicillin resistant, no pitting
• B. vulgatus/ovatus/thetaiotaomicron: major GI flora, no agar pitting

Summary: The only pitting, penicillin-susceptible Bacteroides is Bacteroides ureolyticus.

Which gram-negative bacilli produce brown to black pigment on KVA agar and brick red fluorescence when exposed to an ultraviolet (UV) light source?

A. Porphyromonas spp and Prevotella spp

B. Fusobacterium spp and Actinomyces spp

C. Bacteroides spp and Fusobacterium spp

D. All of these options

A. Porphyromonas spp and Prevotella spp

Pigmented anaerobe background
• Porphyromonas and Prevotella produce brown to black pigment on KVA agar
• Show brick-red fluorescence under UV light (long-wave, 365 nm)
• Common in oral/dental and respiratory infections

KVA agar (Kanamycin-Vancomycin-Anaerobe agar)
• Selective medium for gram-negative anaerobes
• Kanamycin + vancomycin suppress gram-positive and facultative organisms
• Used to recover Bacteroides, Prevotella, Porphyromonas, etc.

Quick differential:
• Fusobacterium: nonpigmented, no brick-red fluorescence
• Bacteroides fragilis group: bile-resistant, nonpigmented
• Actinomyces spp: gram-positive, not fluorescent

Summary: Pigmented, UV-fluorescent anaerobic GNRs indicate Porphyromonas or Prevotella.

The following characteristics of an obligate anaerobic gram-negative bacilli best describe which of the listed genera?

Gram staining: long, slender rods w/ pointed ends

Colonial appearance: dry bread crumbs or "fried-egg" appearance

Penicillin two-unit disks test: susceptible

A. Bacteroides spp

B. Fusobacterium spp

C. Prevotella spp

D. Porphyromonas spp

B. Fusobacterium spp

Fusobacterium background/morphology
• Obligate anaerobic gram-negative bacilli
• Long, slender rods with tapered/pointed ends (“spindle-shaped”)
• Colony appearance described as dry bread crumbs or fried-egg
• Often susceptible to penicillin two-unit disk
• Known for producing butyric acid and foul odor
• Associated with Lemierre’s syndrome, periodontal and respiratory infections

Quick differential:
• Bacteroides fragilis group: bile resistant, no pointed ends
• Prevotella: pigmented, fluoresce under UV
• Porphyromonas: bile sensitive, pigmented

Summary: Pointed gram-negative anaerobic rods with dry colonies indicate Fusobacterium.

All of the following genera are anaerobic cocci that stain gram positive except:

A. Peptococcus spp

B. Peptostreptococcus spp

C. Streptococcus spp

D. Veillonella spp

D. Veillonella spp

Anaerobic cocci background
• Peptococcus and Peptostreptococcus are gram-positive anaerobic cocci
• Streptococcus are gram-positive cocci (mostly facultative, not strict anaerobes)
• Veillonella are the classic gram-negative anaerobic cocci

Quick differential:
• Peptostreptococcus: common in abscesses, brain/dental infections
• Peptococcus: less clinically significant, similar habitats
• Veillonella: from oral and GI flora, sometimes dental/resp infections

Summary: Veillonella stands out as the only gram-negative anaerobic coccus in the group.

The gram-positive non-spore-forming, anaerobic, short, pleomorphic rods most frequently recovered from blood cultures as a contaminant are:

A. Propionibacterium acnes

B. Clostridium perfringens

C. Staphylococcus intermedius

D. Veillonella parvula

A. Propionibacterium acnes

Propionibacterium acnes background/morphology
• Gram-positive, non–spore-forming, anaerobic, pleomorphic rods
• Normal skin flora; frequently a blood culture contaminant
• Slow-growing; forms small, white, opaque colonies
• Can cause true infections in prosthetic devices, shunts, and endocarditis

Quick differential:
• Clostridium perfringens: spore-forming, double-zone beta hemolysis
• Staphylococcus intermedius: gram-positive cocci, not anaerobic rod
• Veillonella parvula: gram-negative anaerobic coccus

Summary: Propionibacterium acnes is the classic skin contaminant in anaerobic blood cultures.

Which Clostridium species is most often recovered from a wound infection with gas gangrene?

A. Clostridium sporogenes

B. Clostridium sordellii

C. Clostridium novyi

D. Clostridium perfringens

D. Clostridium perfringens

Clostridium perfringens background/morphology
• Gram-positive, spore-forming, nonmotile anaerobic bacillus
• Most common cause of gas gangrene (clostridial myonecrosis)
• Produces alpha toxin (lecithinase) causing tissue necrosis
• Double zone of beta-hemolysis on SBA
• Rapid disease progression with gas production and crepitus

Quick differential:
• C. sporogenes: motile, less virulent
• C. sordellii: associated with toxic shock–like syndromes
• C. novyi: gas gangrene possible but less frequent

Summary: C. perfringens is the primary Clostridium species behind gas gangrene.

Gram staining of a smear taken from the periodontal pockets of a 30-year-old man with poor dental hygiene showed sulfur granules containing gram-positive rods (short diphtheroids and some unbranched filaments) and were non-acid fast. Colonies on blood agar resembled "molar teeth" in formation. The most likely organism is:

A. Actinomyces israelii

B. Propionibacterium acnes

C. Staphylococcus intermedius

D. Peptostreptococcus anaerobius

A. Actinomyces israelii

Actinomyces israelii background/morphology
• Gram-positive, non–acid-fast, filamentous rods
• Found in oral cavity; causes cervicofacial actinomycosis (“lumpy jaw”)
• Sulfur granules in pus: yellow, gritty colonies of intertwined filaments
• Colonies on SBA show “molar tooth” morphology
• Associated with periodontal disease, poor dental hygiene, trauma

Quick differential:
• Propionibacterium acnes: pleomorphic rods, skin contaminant, no sulfur granules
• Staphylococcus intermedius: cocci, not filamentous
• Peptostreptococcus: gram-positive cocci, not rods/filaments

Summary: Non–acid-fast filaments with sulfur granules and molar-tooth colonies = Actinomyces israelii.

Antimicrobial susceptibility testing of Bacteroides and Clostridium spp anaerobes are done by which of the following methods?

A. Broth microdilution

B. Agar dilution

C. E-Test

D. All of the above

D. All of the above

Anaerobe susceptibility testing
• Routine for serious infections due to resistance concerns in Bacteroides and select Clostridium

Brief method descriptions
• Broth microdilution
– Organism inoculated into wells of broth with antimicrobial dilutions
– MIC determined as lowest concentration inhibiting visible growth

• Agar dilution (CLSI reference method)
– Organism spotted onto agar plates containing specific antimicrobial concentrations
– MIC = lowest concentration plate showing no growth

• E-test (gradient diffusion)
– Plastic strip with antimicrobial concentration gradient placed on agar
– MIC read where ellipse of inhibition intersects the strip

Quick differential application
• Bacteroides fragilis group: high beta-lactamase rates, test frequently when isolated from sterile sites
• Clostridium perfringens: typically predictable, testing based on clinical need
• C. difficile: treatment guided by toxin presence, not susceptibility testing

Summary: Broth microdilution, agar dilution, and E-test are all valid methods for anaerobic susceptibility testing.

An abdominal postoperative wound specimen grew catalase-positive, indole-negative, nonpigmented, nonfluorescent, gram-negative, pleomorphic bacilli (safety pin-shaped) on 20% bile media. The colonies also grew on KVA agar (resistant to kanamycin, vancomycin, and colistin). What is the most likely identification?

A. Fusobacterium nucleatum

B. Bacteroides fragilis

C. Prevotella spp

D. Porphyromonas spp

B. Bacteroides fragilis

Bacteroides fragilis background/morphology
• Gram-negative, pleomorphic bacilli; often “safety pin” appearance
• Catalase positive, indole negative
• Nonpigmented, nonfluorescent
• Bile tolerant: grows on 20% bile media (BBE); typically esculin hydrolysis → black colonies
• Grows on KVA agar and is resistant to kanamycin, vancomycin, and colistin (B. fragilis group profile)
• Major cause of intra-abdominal/postoperative infections

Quick differential:
• Fusobacterium nucleatum: bile sensitive, often indole positive, tapered ends
• Prevotella spp: many pigmented and brick-red fluorescence; generally kanamycin susceptible
• Porphyromonas spp: pigmented, vancomycin susceptible, bile sensitive

Summary: Bile-tolerant, K-V-C resistant, nonpigmented anaerobic GNR from an abdominal wound fits B. fragilis group.