Harr 7.7 - Microbiology - Anaerobic Bacteria

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

Decreased O2

The anaerobic bacteria are subdivided according to their requirement for O2. Obligate anaerobes are killed by exposure to atmospheric O2 for 10 min or longer. Facultative anaerobes grow under aerobic or anaerobic conditions. Microaerophilic organisms do not grow in an aerobic incubator on solid media and only minimally under anaerobic conditions. However, they will grow in minimal oxygen (5% O2). Superoxide dismutase (SOD) is produced by many anaerobes, which catalyzes the conversion of superoxide radicals to less toxic H2O2 and molecular O2.

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

pH

The Eh is most affected by pH and is expressed at pH 7.0. In cultivating anaerobic bacteria, reducing agents such as thioglycollate and L-cysteine are added to anaerobic transport and culture media in order to maintain a low Eh. Certain anaerobes do not grow in the media above a specific critical Eh level.

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

Kanamycin-vancomycin (KV) agar

KV allows the growth of Bacteroides spp., Prevotella spp., and Fusobacterium spp. and inhibits most facultative anaerobic gram-negative rods and gram-positive bacteria (both aerobic and anaerobic). PEA inhibits facultative gram-negative bacteria but will support gram-positive aerobes and anaerobes and gram-negative obligate anaerobes. CCFA is selective for C. difficile from stool, while THIO broth supports gram-positive and gram-negative aerobes and anaerobes.

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

Urinary tract infections

The incidence of anaerobic bacteria recovered from the urine is approximately 1% of isolates. The other three types of infection are associated with a 60%-93% incidence of anaerobic recovery. Urine is not cultured routinely under anaerobic conditions unless obtained surgically (e.g., suprapubic aspiration).

Methods other than packaged microsystems used to identify anaerobes include:

Gas-liquid chromatography (GLC)

Anaerobic bacteria can be identified by analysis

of metabolic products using gas-liquid chromatography. Results are evaluated along

with Gram staining characteristics, spore formation, and cellular morphology in order to make the identification.

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

Prereduced peptone-yeast extract-glucose (PYG)

Peptone yeast and chopped meat with carbohydrates support the growth of anaerobic bacteria. The end products from the metabolism of the peptone and carbohydrates are volatile fatty acids that help to identify the bacteria. After incubation, the broth is centrifuged, and the supernatant injected into a gas-liquid chromatograph. Peaks for acetic, butyric, or formic acid, for example, can be identified by comparison to the elution time of volatile organic acid standards.

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

Clostridium perfringens

C. perfringens produces a double zone of β-hemolysis on blood agar, which makes identification relatively easy. The inner zone of complete hemolysis is caused by a θ-toxin and the outer zone of incomplete hemolysis is caused by an α-toxin (lecithinase activity). The Bacteroides spp. are gram-negative bacilli, and C. difficile is lecithinase negative and does not produce a double zone of β-hemolysis.

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

Lecithinase

Egg yolk agar (modified McClung's or neomycin egg yolk agar) is used to determine the presence of lecithinase activity, which causes an insoluble, opaque, whitish precipitate within the agar. Lipase activity is indicated by an iridescent sheen or pearly layer on the surface of the agar.

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

Clostridium botulinum, Clostridium sporogenes, Clostridium novyi (A)

Lipase is produced by some Clostridium spp. and is seen as an iridescent pearly layer on the surface of the colonies that extends onto the surface of the egg yolk agar medium surrounding them. C. perfringens, the most frequently isolated Clostridium species, is negative for lipase production.

Which spore type and location is found on Clostridium tetani?

Round, terminal spores

Spore appearance and location, along with Gram stain morphology, aids in distinguishing the Clostridium spp. Round, terminal spores (drumstick spores) are demonstrated when C. tetani is grown in chopped meat with glucose broth. Recognition of spores is particularly important because C. tetani sometimes appears as gram negative.

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

Spores seen = Neg

Motility = Neg

Hemolysis = + (double zone)

Lecithinase = +

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

What is the most likely identification?

Clostridium perfringens

Spores are generally not demonstrated from clinical specimens containing C. perfringens, which is the only species producing a double zone of hemolysis. The reactions in the chart above distinguish the four species listed.

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

Ingestion of spores in food or liquid

Infant botulism is the most frequent form occurring in the United States. Epidemiological studies have demonstrated that infant botulism results from the ingestion of spores via breastfeeding or exposure to honey. Preformed toxin has not been detected in food or liquids taken by the infants. C. botulinum multiplies in the gut of the infant and produces the neurotoxin in situ.

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

Preformed toxin in food

Foodborne botulism in adults and children is caused by ingestion of the preformed toxin (botulinum toxins A, B, E, and F) in food. The neurotoxins of C. botulinum are protoplastic proteins made during the growing phase and released during lysis of the organisms. Confirmation of botulism is made by demonstration of the toxin in serum, gastric, or stool specimens.

Which test is performed in order to confirm an infection with Clostridium botulinum?

Toxin neutralization

C. botulinum and C. sporogenes have similar characteristics biochemically (see the following chart), and definitive identification of C. botulinum is made by the toxin neutralization test for its neurotoxins in serum or feces. Specimens should be kept at 4°C and sent to the CDC for culture and toxin assays.

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

C. difficile

C. difficile is also implicated in hospital acquired diarrhea and colitis. Clinical testing for C. difficile includes culture and cytotoxin testing. Because culture takes 3 days and will detect nontoxigenic strains that do not cause diarrheal disease, immunoassays using antibodies against either the A toxin or both the A and B toxins are most frequently employed. Assays detecting both toxins are only slightly more sensitive, since infections producing only B toxin are infrequent. The cytotoxin assay requires that specimens be shipped to a reference laboratory on dry ice or kept at 4°C-6°C if done in-house.

Identification of Clostridium tetani is based upon:

Clinical findings

The culture and Gram stain of the puncture wound site usually does not produce any evidence of C. tetani. The diagnosis is usually based upon clinical findings, which are characterized by spastic muscle contractions, lockjaw, and backward arching of the back caused by muscle contraction.

Obligate anaerobic gram-negative bacilli that do not form spores grow well in 20% bile and are resistant to penicillin 2-unit disks are most likely:

Bacteroides spp.

The Bacteroides group grows well in 20% bile and is resistant to penicillin 2-unit disks with the exception of B. ureolyticus. Most Prevotella are also resistant to penicillin 2-unit disks, but most Fusobacterium and Porphyromonas are sensitive.

Which Bacteroides spp. is noted for "pitting" of the agar and is sensitive to penicillin 2-unit disks?

B. ureolyticus

B. ureolyticus is the only species listed that is susceptible to penicillin and produces urease. The other organisms listed are resistant to penicillin.

Which gram-negative bacilli produce black pigment and brick red fluorescence when exposed to an ultraviolet light source?

Porphyromonas spp. and Prevotella spp.

Pigmenting Porphyromonas spp. and Prevotella spp. also show hemolysis on sheep blood agar.

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

Gram stain: long, slender rods with pointed ends

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

Penicillin 2-unit disk test: Susceptible

Fusobacterium spp.

Fusobacterium spp. are usually spindle-shaped,

slim rods, whereas the other genera are small

rods (variable length for Bacteroides spp. and tiny coccoid rods for Prevotella and Porphyromonas spp.). Fusobacterium spp. and Porphyromonas spp. are susceptible to penicillin 2-unit disks, while most Bacteroides spp. and Prevotella spp. are resistant.

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

Veillonella spp.

Veillonella spp. are gram-negative cocci. All four genera are part of the normal human flora and are the anaerobic cocci most frequently isolated from blood cultures, abscesses, wounds, and body fluids. The Streptococcus spp. are facultative anaerobes, but only Streptococcus intermedius is classified as an obligate anaerobe.

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

Propionibacterium acnes

P. acnes is a nonspore former and is described as a diphtheroid-shaped rod. It is part of the normal skin, nasopharynx, genitourinary, and gastrointestinal tract flora but is implicated as an occasional cause of endocarditis.

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

C. perfringens

Wounds infected with clostridia are characterized by invasion and liquefactive necrosis of muscle tissue with gas formation. The most frequent isolate is C. perfringens followed by C. novyi and C. septicum.

Gram stain 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). Colonies on blood agar resembled "molar teeth" in formation. The most likely organism is:

Actinomyces israelii

A. israelii is part of the normal flora of the mouth

and tonsils but may cause upper or lower respiratory tract infections. The sulfur granules are granular microcolonies with a purulent exudate. Like Nocardia, Actinomyces produces unbranched mycelia and is sometimes (erroneously) considered a fungus. It has also been implicated in pelvic infection associated with intrauterine contraceptive devices (IUDs).

Antimicrobial susceptibility testing of anaerobes is done by which of the following methods?

Microtube broth dilution

The anaerobes are not suited for the broth disk elution or disk agar diffusion tests because of their slow rate of growth. Kirby-Bauer method reference charts are not designed to be used as a reference of susceptibility for anaerobes.