Fun with anaerobes!

A "double zone" of hemolysis on blood agar plates is characteristic of: a) Clostridium perfringens b) Clostridium tetani c) Clostridium botulism d) all of the above

A

What are two kinds of commercial kits available for anaerobic identification?

1. API, 2. use of preformed enzymes

What are characteristics of the Bacteroides fragilis group?

1. Bile resistant; 2. more antibiotic resistance than other anaerobes; 3. non-sporeformer; 4. found in colon and female genital tract

What are methods used in the Identification of anaerobes?

1. Biochem - Bile, indole, catalase, 2. ID Discs - Kanamycin, Vancomycin, Colistin, 3. Commercial ID Systems, 4. Gas Liquid Chromatography (time consuming, impractical)

Besides wounds or puncture, what are additional modes of acquisition of Clostridium?

1. Ingestion of preformed toxins in foods - Botulism, C. perfringes, 2. GI colonization with toxin-producing organisms - infant botulism, 3. Person-to-person nosocomial spread of C. diff and bite wounds

What antibiotics are used in the identification of anaerobes?

1. Kanamycin, 2. Vancomycin, 3. Colistin

Why are swabs a poor alternative to the collection and transport of organisms? What is the best method of collection?

1. Swabs can lead to excessive drying, easier contamination, and retention of organisms in the fibers., 2. Tissue biopsies or aspiration using a needle and syringe.

What are the classical biochemicals used in the identification of anaerobic cultures?

1. growth in Bile, 2. spot indole, 3. catalase

All of the following are obligate anaerobes, EXCEPT: a) Citrobacter freundii b) Clostridium botulism c) Bacteroides fragilis d) Peptostreptococcus

A

What is a "holding jar" used for during anaerobic work ups?

A temporary holding container for anaerobic work ups, using N2 instead of CO2 that reacts with water to produce carbonic acid, thus promoting an anaerobic environment.

A gram stain from a swab of a hand wound reveals: moderate neutrophils, no squamous epithelial cells, moderate gram-positive cocci in chains, moderate large gram-negative bacilli. Select the appropriate media that will selectively isolate each organism. A. Columbia CNA, MacConkey; B. KV-Laked agar, Thayer Martin; C. Columbia CNA, chocolate; D. sheep blood, MacConkey

A. Columbia CNA, MacConkey CNA will select for gram positives and MacConkey will select for gram negs.

Which of the following is the most appropriate organism and media combination? A. Yersinia enterocolitica—cefsulodin-igrasin-novobiocin (CIN); B. Legionella species—Regan Lowe; C. Campylobacter species—charcoal yeast extcract; D. Clostridium difficile—phenylethyl alcohol (PEA)

A. Yersinia enterocolitica—cefsulodin-igrasin-novobiocin (CIN)

The most common cause for failure of a GasPak anaerobic jar to establish an adequate environment for anaerobic incubation is: A. catalyst that have become inactivated after repeated use B. the failure of the oxidation-reduction potential indicator system due to deterioration of methylene blue C. condensation of water on the inner surface off the jar D. the failure of the packet to generate adequate H2 and/or CO2

A. catalyst that have become inactivated after repeated use

An aspirate of a deep wound was plated on blood agar plates and incubated aerobically and anaerobically. At 24 hours there was growth on both plates. This indicates that the organism is a(n): A. facultative anaerobe B. nonfermenter C. aerobe D. obligate anaerobe

A. facultative anaerobe

Define obligate aerobe

An aerobic organism that requires O2 to grow; uses O2 to oxidize substances, like sugars or fats for energy.

Define aerobe

An organism that can survive & grow in an oxygenated environment

Define anaerobe

An organism that doesn't require O2 for growth

A tech sets up an anaerobic culture in a GAS PAK jar. Upon reexamination of the jar 2 hrs later, the tech notes that the methylene-blue indicator strip is blue in color. This would indicate: a) O2 is removed from the system b) anaerobic conditions have not been achieved c) palladium catalysts have been preheated & the GAS PAK properly activated

B

A wound culture swab was sent to the clinical lab & inoculated onto the appropriate media. After a 24-hr incubation, all plates incubated at 37C in ambient atmospheres had no growth. However, growth was demonstrated in the bottom of the fluid thio tube. In all likelihood, the causative organism is: a) aerobic b) anaerobic c) microaerophilic d) dead

B

An anaerobic, spore-forming, nonmotile, gram-positive bacillus isolated from a foot wound is most likely: a Actinomyces israelii; b Clostridium perfringens; c Bacillus subtilis; d Eubacterium lentum

B

What are the two most important species of Bacteroides?

B. fragilis and B. thetaiomicron

Anaerobic susceptibility testing is helpful in the management of patients with: A. pilonidal sinuses; B. synovial infections; C. streptococcal pharyngitis; D. rectal abscesses

B. synovial infections Normally sterile site.

How are all anaerobic cultures plated (at RGH)?

BAP, anaerobic Columbia, and Laked Kanamycin/Vancomycin Blood Agar.

What anaerobe is recovered from most intra -abdominal abscesses, and is the dominant normal colon flora?

Bacteroides fragilis

What is the most commonly encountered anaerobe in infections?

Bacteroides fragilis

What susceptibility testing is performed on anaerobes?

Beta-lactamase, E-test, Agar dilution MIC

How can anaerobic bacteria be differentiated from facultative anaerobes?

By anaerobes inability to grow in the presence of oxygen and their susceptibility to matronizadole

An anaerobic GPR w/ a terminal spore is: a)Veillonella b) Bacteroides sporogenes c) Clostridium tetani d) Propionibacterium acnes

C

The optimal wound specimen for culture of anaerobic organisms should be: A. a syringe filled with pus, obtained after administration of antibiotics; B. a swab of lesion obtained before administration of antibiotics; C. a syringe filled with pus, obtained before administration of antibiotics; D. a swab of lesion obtained after administration of antibiotics

C. a syringe filled with pus, obtained before administration of antibiotics

Which of the following groups of specimens would be acceptable for anaerobic culture? A. urine, sputum; B. vaginal, eye; C. pleural fluid, brain abscess; D. ear, leg tissue

C. pleural fluid, brain abscess

Define facultative anaerobe

Can grow w/out O2 but can utilize O2 if it's present

Define obligate anaerobe

Can't use O2 for growth & are even harmed by it

Define aerotolerant

Can't use O2 for growth, but isn't harmed by it

Besides O2, what are anaerobes sensitive to?

Carbonic acid

What is the general treatment for anaerobic infections?

Clindamycin, metranidazole, and 3rd generation cephalosporins

What are examples of common exogenous pathogens and what do they cause?

Clostridium tetani - tetanus, Clostridium perfringes - gas gangrene, Clostridium botulinum - botulism

What characteristics are observed during examination of cultures?

Colony morphology, fluorescence, pigmentation, gram stain, and aerotolerance

What non-selective media is used in the detection of anaerobes?

Columbia agar. Used for anaerobes and facultative.

What two mediums used to culture anaerobes are incubated in anaerobe jars or pouches?

Columbia and LKV

Anaerobic organisms should be incubated: a) at room temp b) in 5-10% CO2 c) in the dark d) 48 hrs before compromising (entering) the anaerobic conditions

D

When a Brucella species is suspected in a blood culture, the bottle should be held for a minimum of: a) 5 days b) 7 days c) 14 days d) 21 days

D

A 21-year-old patient presents with pharyngitis. A throat swab is collected and submitted for anaerobic culture. This specimen should be: A. sent to a reference laboratory; B. set up immediately; C. inoculated into thioglycolate broth; D. rejected as unacceptable

D. rejected as unacceptable Anaerobes do not cause pharyngitis. Most common cause is strep.

What medium and conditions are used for aerotolerance testing?

Each colony type is subbed to a Choc in CO2 and BAP incubated anaerobically.

What type of organism is characterized by growing equally well with or without oxygen, representing most pathogens?

Facultative anaerobe

True or False. Most clinically relevant anaerobes are not susceptible to first-line antimicrobials.

False.

What is the morphology and gram stain of the most commonly encountered anaerobic bacteria in clinical infections?

Gram negative rods

How many plates can anaerobic jars and pouches hold?

Jars = 15, pouches = 5

What selective media is used in the detection of anaerobes?

LKV. Selective for Prevotella and Bacteroides

What is Laked Blood?

Laked blood media is hemolyzed blood.

Where in the body are gram negative anaerobic bacteria considered normal flora?

Mouth, upper respiratory tract, intestinal tract, and urogenital tract.

When is susceptibility testing warranted for anaerobes?

When an infection is caused by a single anaerobe. However if multiple organisms are identified, testing will not be performed.

When is an endogenous strain (normal flora) pathogenic?

When they gain access to normally sterile sites.

What type of organism requires O2 as its final electron acceptor?

Obligate aerobe

What type of organism toxically affected by O2?

Obligate anaerobe

When is beta-lactamase testing on anaerobes accurate?

Only when done anaerobically.

Define microaerophilic

Requires O2 to survive, but requires environments containing lower levels of O2 than are present in the atmospher; can be cultivated in a candle jar

What condition must be met if anaerobic specimens are to be processed on open bench-tops?

Specimens must be incubated immediately thereafter, in anaerobic jars, pouches, or performed in an anaerobic chamber.

How do endogenous organisms (normal flora) gain access to normally sterile sites?

Surgery or trauma, or if normal defense mechanisms are compromised via malignancy, immunosuppressive therapy, or diabetes.

Define facultative aerobe

They grow & survive in an oxygenated environment; makes ATP by aerobic respiration if O2 is present but is capable of switching to fermentation

How do exogenous pathogens gain access to the body?

Through existing wounds or by puncture with objects contaminated with toxigenic Clostridium species.

What is the biggest factor when dealing with anaerobic infections?

Time. After culture, have to prove organism is an anaerobe and do testing. By then, the patient would have already been treated.

True or False. Both anaerobic jar and pouches use a hydrogen and CO2 generating system.

True

True or False. Most anaerobic gram negative rods are positive during beta-lacatamse testing.

True

How is an agar dilution MIC performed?

Use pre-reduced anaerobically sterilized media and run multiple organisms at once.

A strict anaerobe that produces terminal spores is: a Clostridium tetani; b Corynebacte rium diphtheria; c Bacillus anthracis; d Propionibacterium acnes

a

Acceptable specimens for culture of anaerobic bacteria that cause disease include: a abscesses; b gingival swabs; c skin swabs

a

The Gram stain of drainage from a pulmonary sinus tract shows many WBCs and 3+ branching gram-positive bacilli. Colonies grow only on anaerobic media after 3 days incubation. They are yellow-tan and have a molar tooth appearance. The most likely genus is: a Actinomyces; b Bacteroides; c Fusobacterium; d Nocardia

a

The most meaningful laboratory procedure in confirming the diagnosis of clinical botulism is: a demonstration of toxin in the patient's serum; b recovery of Clostridium botulinum from suspected food; c recovery of Clostridium botulinum from the patient's stool; d Gram stain of suspected food for gram-positive, sporulating bacilli

a

Which of the following pairs of organisms usually grow on kanamycin, vancomycin, laked blood agar? a Bacteroides and Prevotella; b Mobiluncus and Gardnerella; c Porphyromonas and Enterococcus; d Veillonella and Capnocytophaga

a

Which one of the following anaerobes would be negative for indole? a Bacteroides fragilis; b Fusobacterium nucleatum; c Porphyromonas asaccharolytica; d Proteus mirabilis

a

Which organism is the most common anaerobic bacteria isolated from infectious processes of soft tissue and anaerobic bacteremia? a Bacteroides fragilis; b Fusobacterium nucleatum; c Porphyromonas asaccharolytica; d Clostridium perfringens

a

What type of organism grows poorly in O2?

aerotolerant

A control strain of Clostridium should be used in an anaerobe jar to assure: a that plate media is working; b that an anaerobic environment is achieved; c that the jar is filled with a sufficient number of plates; d that the indicator strip is checked

b

A patient has a suspected diagnosis of subacute bacterial endocarditis. His blood cultures grow non-spore-forming pleomorphic gram-positive bacilli only in the anaerobic bottle. What test(s) will give a presumptive identification of this microorganism? a beta-hemolysis and oxidase; b catalase and spot indole; c esculin hydrolysis; d hydrolysis of gelatin

b

An aspirate of a deep wound was plated on blood agar plates aerobically and anaerobically. At 24 hours there was growth on the anaerobic plate only. The next step in the evaluation of this culture is to: a reincubate for another 24 hours; b begin organism identification; c issue the final report; d set up a Bauer-Kirby sensitivity

b

An organism from a peritoneal abscess is isolated on kanamycin-vancomycin laked blood agar and grows black colonies on BBE agar. It is nonpigmented, catalase positive, and indole negative. The genus of this organism is: a Acidominococcus; b Bacteroides; c Porphyromonas

b

The presence of 20% bile in agar will allow growth of: a Fusobacterium necrophorum; b Bacteroides ovatus; c Prevotella melaninogenica; d Porphyromonas gingivalis

b

Gram stain of a thigh wound showed many gram-positive spore-forming bacilli. The specimen was placed on brain heart infusion blood agar and incubated aerobically at 35"C for 3 days. At the end of that time, the plates showed no growth. The most likely explanation is that some of the specimen should have been incubated: a on chocolate agar; b for 5 days; c under 5% C02; d anaerobically

d

Propionibacterium acnes is most often associated with: a normal oral flora; b post-antibiotic diarrhea; c tooth decay; d blood culture contamination

d

A 1-2 mm translucent, nonpigmented colony, isolated from an anaerobic culture of a lung abscess after 72 hours, was found to fluoresce brick-red under ultraviolet light. A Gram stain of the organism revealed a coccobacillus that had the following characteristics: growth in bile: inhibited; vancomycin: resistant; kanamycin: resistant; colistin: susceptible; catalase: negative; esculin hydrolysis: negative; indole: negative. The identification of this isolate is: a Bacteroides ovatus; b Prevotella oralis; c Prevotella melaninogenica; d Porphyromonas asaccharolytica

c

A Gram stain of a necrotic wound specimen showed large gram-positive bacilli. There was 3+ growth on anaerobic media only, with colonies producing a double zone of hemolysis. To identify the organism, the microbiologist should: a determine if the organism ferments glucose; b perform the oxidase test; c set up egg yolk agar plate; d test for bile tolerance

c

A stool sample is sent to the laboratory for culture to rule out Clostridium difftcile. What media should the microbiologist use and what is the appearance of the organisms on this media? a BBE: colonies turn black; b Brucella agar: red pigmented colonies; c CCFA: yellow, ground glass colonies; d CNA: double zone hemolytic colonies

c

A thin, gram-negative bacillus with tapered ends isolated from an empyema specimen grew only on anaerobic sheep blood agar. It was found to be indole positive, lipase negative, and was inhibited by 20% bile. The most probable identification of this isolate would be: a Bacteroides distasonis; b Prevotella melaninogenica; c Fusobacterium nucleatum; d Clostridium septicum

c

Anaerobic infections differ from aerobic infections in which of the following? a they usually respond favorably with aminoglycoside therapy; b they usually arise from exogenous sources; c they are usually polymicrobic; d Gram stains of specimens are less helpful in diagnosis

c

Fluid from a cutaneous black lesion was submitted for routine bacterial culture. After 18 hours of incubation at 35·c there was no growth on MacConkey agar, but 3+ growth on sheep blood agar. The colonies were nonhemolytic, nonmotile, 4-5 mm in diameter and off-white with a ground glass appearance. Each colony had an irregular edge with comma-shaped outgrowths that stood up like "beaten egg whites" when gently lifted with an inoculating needle. A Gram stain of a typical colony showed large, gram-positive rectangular bacilli. The organism is most likely: a Clostridium perfringens; b Aeromonas hydrophila; c Bacillus anthracis; d Mycobacterium marinum

c

The following growth results were observed on media inoculated with a foot abscess aspirate and incubated in 3%-5% C02. SBA: 2+ large gray colonies; PEA: no growth; chocolate: 3+ large gray colonies; MacConkey: 3+ lactose fermenters; trypticase soy broth: gram-negative bacilli and gram-positive bacilli. Biochemicals were set up on the colonies from the MacConkey agar plate. What should the microbiologist do next? a set up biochemicals on the colonies from SBA; b send out final report to the physician after biochemicals are interpreted; c subculture TSB to SBA aerobic and SBA anaerobic; d test colonies on chocolate agar with hemin and NAD

c

Which of the following sets of organisms may exhibit a brick red fluorescence? a Porphyromonas asaccharolytica and Clostridium ramosum; b Clostridium difficile and Fusobacterium sp; c Veillonella parvula and Prevotella melaninogenica; d Fusobacterium sp and Veillonella parvula

c

Which one of the following organisms could be used as the positive quality control test for lecithinase on egg yolk agar? a Bacteroides fragilis; b Fusobacterium necrophorum; c Clostridium perfringens; d Clostridium sporogenes

c

The characteristic that is most commonly associated with the presence of strict anaerobic bacteria and can be taken as presumptive evidence of their presence in a clinical specimen is the: a presence of a single bacterial species; b production of gas in a thioglycollate broth culture; c growth on a blood agar plate incubated in an anaerobic jar; d presence of a foul, putrid odor from tissue specimens and cultures

d

The etiologic agent of botulism is: a highly motile; b non-spore-forming; c Clostridium perfringens; d an exotoxin producer

d

Which of the following genera include anaerobic gram-negative nonsporulating bacilli? a Brucella; b Pasteurella; c Actinomyces; d Bacteroides

d

What type of organism requires decreased O2 tension?

microaerophilic