GNB NF, GPB, Mycobacterium, Anaerobes updated 4-25-21

What is the purpose of the digestion-decontamination process in the recovery of mycobacteria

1. to liquefy the sample though digestion 2. to allow the chemical decontamination agent to contact and kill non-mycobacterial organisms.

How long is the incubation for Mycoplasma pneumoniae

2 to 3 weeks headache, low-grade fever, malaise, anorexia, sore throat, dry cough, earaches

Where is Mycoplasma pneumoniae isolated

20% in general populations - school-age children and young adults 50% in confined settings - prisoners, college students, and military personnel

What enhances the growth of M. tuberculosis

5-10% CO2

M. tuberculosis is enhanced by an atmosphere of 5-10% CO2 and requires a pH of?

6.5 and 6.8

Cystine-tellurite agar

A modification of Tinsdale that contains sheep RBCs, bovine serum, cystine, and potassium tellurite selective and differential potassium tellurite inhibits noncoryneform bacteria corynebacteria form black or brownish colonies due to tellurite reduction Corynebacterium diphtheria

Corynebacterium pseudotuberculosis identification

Catalase positive Nitrate variable Urease positive <-- differentiates between C. diphtheriae

Corynebacterium ulcerans identification

Catalase positive Nitrate variable Urease positive <-- differentiates between C. diphtheriae

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.

Elek Test

Detected by Elek immunodiffusion test. Antitoxin diffuses away from the strip of filter paper and toxin produced by toxin-producing strains diffuse away from growth. At the zone of equivalence, a precipitin line is formed. a key test that is used to detect diphtheria toxin

Disease caused by C. diphtheriae is called

Diphtheria

C. diptheriae disease

Diphtheria (caused by diphtheria toxin) Pharyngitis Cutaneous lesions

Corynebacterium ulcerans

Diphtheria-like isolated from skin ulcers and exudative pharyngitis veterinary pathogen: cattle

Transmission of Mycoplasma

Direct sexual contact, during delivery, respiratory secretions, or fomites susceptible to heat and drying

Digestion and Decontamination Agents to help recover mycobacteria

Done to kill non-mycobacterial organisms Sodium Hydroxide concentration 2-4% is a digestant and decontamination agent N-acetyl-L-cysteine (NALC) liquefaction agent that allows the decontamination agent to come into contact with the bacteria

Mycobacteria characteristics

- Obligate/strict aerobes (like a lot of air that is why TB can grow in the lungs the best) but increase carbon dioxide can enhance growth - Slender rod-shaped organisms - non-motile - non-sporulating - ubiquitous microbes that can be found in animals and humans They are SLOW GROWING and fastidious

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.

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.

Culture Media that is enriched with protein

Lowensteiin-Jensen (LJ) media: Eggs, salts, glycerol, potato flour contains malachite green to supress growth of gram-positive bacteria Middlebrook media: contain many factors, olec acid (fats), serum

Lymph node involvement of TB is called _____. TB infection on the skin is called _____.

Lymphadenitis, Cutaneous tuberculosis

Not usually seen in United States

M. leprae​ Leprosy (Hansen's disease)​ If humoral immunity and not cell-mediated immunity​ M. africanum​ African tuberculosis​ M. microti​ Not associated with human disease

Mycobacterium bovis is closely related to what genus

M. tuberculosis

Main U.S. pathogens

M. tuberculosis​ M. avium-intracellulare complex​ M. bovis​ Usually animals​ Reactivation can occur: Advanced age​ Immunocompromised​ Malnutrition

The microscopic morphology of C. diphtheriae on Methylene blue stain

Metachromatic granules at ends

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.

Concentration procedures of

Mycobacteria has a low specific gravity therefore needs to be centrifuged at a high speed and treated with NALC to split mucoprotein.

Loeffler's agar slant is a special culture medium used to recover which organism? a. Streptococcus pyogenes b. Corynebacterium diphtheriae c. Bordetella pertussis d. Neisseria meningitidis

b. Corynebacterium diphtheriae

Corynebacterium pseudotuberculosis growth

black on cystine tellurite brown halo on Tinsdale agar

Hematogenous spread

cancerous cells invade the circulatory system and spread via blood vessels

Mycoplasma are sometimes referred to as____because it is deficient in this

cell wall

What affects the recovery of Mycobacterium spp.

digstion-decontamination concentration length of specimen exposure to the agent centrifugation speed

Corynebacterium jeikeium

gram positive bacilli that is nonhemolytic, urease negative, and nitrate reduction negative. Resistant to antimicrobials including penicillins, cephalosporins, aminoglycosides drug for treating vancomycin

Corynebacterium pseudotuberculosis microscopic morphology

gram-positive small pleomorphic bacilli

Corynebacterium ulcerans Microscopic morphology

gram-positive small pleomorphic bacilli

Tinsdale agar

growth media used to culture C. diphtheria

Mycoplasma hominis

infection of the lower urogenital tract that is found in 50% of healthy patients. Infections of the upper urinary tract in sexually active people

Mycoplasma tuberculosis treatment

lacks of cell wall makes it resistant to Beta-lactams-penicillins and cephalosporins Antibiotics Sulfonamides, trimethoprim, rifampin Tetracycline is more adults only due to side effects

Ureaplasma urealyticum normal flora of the?

lower urinary tract of women can cause upper urinary tract infections in newborns Significant due to infection of fetus Chorioamnionitis (infection of placental membrane) Congenital pneumonia Chronic lung disease in premature infants Meningitis of newborns with negative cultures

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.

Corynebacterium ulcerans diease

pharyngitis

Mycobacterium bovis

produces Tuberculosis in cattle, rodents, other farm animals, along with dogs, and cats. related to M. tuberculosis and is thus treated similarly

Multidrug-Resistant Mycobacterium tuberculosis

resistant to isoniazid and rifampin

How is Mycoplasma pneumoniae transmitted?

respiratory droplets or aerosol

Extensively resistant TB (XDR-TB) results in strains resistant to what antibiotics

rifampin and isoniazid any fluoroquinolone at one of three injectable second-line anti-TB drugs: amikacin, kanamycin, or capreomycin

Corynebacteria gram stain

slightly curved gram-positive rods with nonparallel sides and slightly wider ends "club shaped"

Mycobacteria growth rate

slow grower 2 to 6 weeks

Identification of Mycobacterium tuberculosis

slow-growing species that is raised with a dry rough appearance non-pigmented colonies cauliflower looking positive for niacin accumulation reduces nitrate to nitrite

M. bovis growth

slowly on egg-based media produces small, granular, rounded, nonpigmented colonies Niacin negative ---M. tuberculosis positive does not reduce nitrate --M. tuberculosis does does not grow in the presence of T2H

Corynebacterium pseudotuberculosis disease

the book says granulomatous lymphadenitis in humans lab 17 says pharyngitis

Miliary tuberculosis

the development of active tubercles throughout the body

Mycobacteria take up dye with increased staining time or application of heat but resist decolorization with acid-ethanol why?

this is because of the mycolic acid

Catalase Test

used to identify organisms that produce the enzyme catalase To differentiate catalase-positive organisms like micrococci and staphylococci from catalase-negative organisms like streptococci.

Corynebacterium pseudotuberculosis

veterinary pathogen associated with sheep contact

What causes meningitis by Mycobacterium tuberculosis

when a tubercle erupts in the subarachnoid space more so than though hematogenous spread

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.

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.

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).

The extrapulmonary complications of Mycoplasma pneumoniae

Cardiovascular, central nervous system, dermatologic and gastrointestinal problems.

C. diptheriae identification

Catalase positive Nitrate positive Urase negative Elektoxin test positive

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.

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.

Why is Mycobacterium tuberculosis difficult to stain?

Because it lacks a cell wall

other means of digesting and decontaminating mycobacterium

Benzalkonium chloride Oxalic acid​-to decontaminate P. aeruginosa from CF patients​

Corynebacterium ulcerans growth characteristics

Black on cystine tellurite Brown halo on Tinsdale agar

Growth characteristics of C. diphtheriae

Black on cystine tellurite agar Brown halo on Tinsdale agar

Mycoplasma pneumoniae diseases

Bronchitis Pharyngitis Walking pneumonia Mostly asymptomatic (90%)

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.

____is a facultative anaerobe that grows best under aerobic conditions with an optimal growth temp of 37C

C. diphtheriae

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.

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.

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.

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.

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.

Mycoplasma pneumoniae treatment

Erythromycin​ Tetracycline

Corynebacterium ulcerans natural habitat

Humans and animals (cattle and other domesticated animals)

C. diptheriae natural habitat

In humans only throats of carriers diphtheritic skin ulcers

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.

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.

Skeletal TB of the spine is called?

Pott disease

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 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.

Mycobacterium tuberculosis

Primary tuberculosis​ Transmitted by airborne droplet nuclei​ Infect the deep lung in the alveoli​ Bacteria get phagocytosed and continue to multiply​ 4-6 weeks later T cells sensitize and allow the destruction of intracellular bacteria​ Regression and healing of the primary lesion​ Systemic, "Consumption"

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.

Nitrate test

Purpose: differentiate Enterobacteria from other Gram-negative rods that can perform a one-step reduction of Nitrate to nitrite. Procedure: Inoculate with loop to nitrate broth at 35C for 24 to 48 hours Medium: nitrate broth substrate:Nitrate Enzyme: nitrate reductase NO3 to NO2 end product:Nitrite pH indicator: n/a reagent: Nitrate A and Nitrate B, if not color change add zinc + result: if gas produced or if red (1st) or when added zinc no color change (+) - result: colorless (1st), when zinc is added it turned red (-)

Ureaplasma urealyticum treatment

Resistant to clindamycin or lincomycin; use erythromycin

Mycoplasma hominis treatment

Resistant to erythromycin; use clindamycin or lincomycin

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.

Mycoplasma hominis dieases

Salpingitis: inflammation of the fallopian tubes​ Pyelonephritis: infection of kidney and ducts​ Pelvic inflammatory disease (PID)​ Postpartum fevers

Middlebrook agar

Selective Mycobacterium tuberculosis Malachite green inhibits other organisms clear media

The microscopic morphology of C. diphtheriae on Loeffler's agar

Small, pleomorphic, beaded, palisading, clubbed

General characteristics of Mycobacteria

Strictly aerobic Nonmotile, slender, slightly curved, rod-shaped bacilli; non-sporeforming The cell wall has high lipid content (mycolic acid) acid-fast requires complex media

Urease Test

Test for microorganisms that possess the enzyme urease that hydrolyzes urea to form ammonia. pH indicator: phenol red When the pH increase (alkaline) the medium turns from yellow to red red = positive for urease C. diphtheriae is negative C. ulcerans and C. pseudotuberculosis are positive

Why must specimens be processes for the recovery of mycobacteria?

The specimens may contain mucin, or other organisms that are nonmycobacterial that can quickly over grow and take over the slow growing mycobateria

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.

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).

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.

Staining for Acid-fast bacilli

Ziehl-Neelsen​: Carbolfuchsin (primary stain) while heating​ Acid alcohol (decolorizer)​ Methylene blue (counterstain)​ Kinyoun ​ Similar except no heating​

acid fast stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol

Diphtheritic membrane

a thin coating on the surface of an epithelial lined organ (e.g. intestine) that is composed of necrotic cellular debris, inflammatory cells and fibrin.

Corynebacterium pseudotuberculosis natural habitat

associated with sheep contact and rare