MIDTERM: Aerobic Gram Positive Bacilli Flashcards
Although several of these organisms are frequently isolated in the clinical laboratory, they are typically considered contaminants or commensals (e.g., Bacillus and Corynebacterium)
Aerobic Gram Positive Bacilli
Enumerate the spore formers bacteria
Bacillus anthracis
Bacillus cereus
Enumerate the non-spore formers bacteria
Corynebacterium
Listeria
Erysipelothrix
Gardnerella
Enumerate the Branching, non-spore-formers
Nocardia
Aerobic actenomycetes
This is also a part of the non spore formers bacteria and is formerly known as Corynebacterium equi which is an opportunistic pathogen that affects immunocompromised patients
Rhodococcus equi
Members of the genus Bacillus can be confused with
aerotolerant strains of the other primary endospore-forming genus, _______ as they are both gram positive
Clostridium
______ Is aerobic or facultative anaerobic while ______ is anaerobic
Bacillus spp., Clostridium, respectively
Enumerate at least two general characteristics of a Bacillus
- Gram positive
- Aerobic or facultative anaerobic bacilli
- Endospores
- Catalase positive
________ form endospores aerobically and anaerobically, whereas _________ form endospores anaerobically only
Bacillus
spp., Clostridium spp., respectively
What are the two groups of Bacillus spp.?
Bacillus anthracis &
Bacillus cereus
Bacillus spp. are divided into groups based on?
genetic identity and morphologic features
The causative agent of anthrax
Bacillus anthracis
Primarily a disease of wild and domestic animals including sheep, goats, horses, and cattle.
Anthrax
Humans acquire infections (anthrax) when inoculated with
endospores, either by traumatic introduction, injection, ingestion, or inhalation during exposure to contaminated animal products, such as hides
Enumerate the types of Anthrax
- Cutaneous Anthrax
- Gastrointestinal/Ingestion Anthrax
- Inhalation Anthrax (wool sorter’s disease)
- Injectional Anthrax
Accounts for most human infections and is associated with contact with infected animal products.
Cutaneous Anthrax
Infection results from close contact and inoculation of endospores through a break in the skin
Cutaneous Anthrax
This infection is typically effectively treated with antibiotics.
Cutaneous Anthrax
The typical presentation of the ulceration in cutaneous anthrax is a black, necrotic lesion known a
eschar
Incubation period of cutaneous anthrax
approximately 2 to 6 days
Mortality rate for untreated cutaneous anthrax
low, at approximately 1%
Fatalities may occur in cutaneous anthrax when the lesions form on
face or neck
These causes an obstructed airway after edema or progression to systemic disease
Face and neck lesions (Cutaneous anthrax)
Results from ingestion of endospores and presents in two forms: oral/oro-pharyngeal anthrax and gastrointestinal anthrax
Gastrointestinal/ Ingestion Anthrax
This anthrax causes lesion to appear in the buccal cavity or on the tongue, tonsils, or pharyngeal mucosa
oral or oro-pharyngeal anthrax (Gastrointestinal/ Ingestion Anthrax)
This anthrax causes lesion to develop in the mucosa of the terminal ileum or cecum
gastrointestinal anthrax (Gastrointestinal/ Ingestion Anthrax)
Its mortality rate is much higher than that of cutaneous anthrax and is usually attributed to toxemia and sepsis.
gastrointestinal anthrax
The most fatal type of anthrax
Inhalation Anthrax (wool sorter’s disease)
Inhalation Anthrax is previously referred to as
pulmonary anthrax, Wool sorters’ disease and ragpickers’ disease
In Inhalation Anthrax , the endospores are ingested by?
macrophages in the lungs
Terms used to describe respiratory infections that result from exposure to endospores during the handling of animal hides, hair, fibers, and other animal products.
Wool sorters’ disease and ragpickers’ disease
The initial, mild form of the Wool sorters’ disease or ragpickers’ disease lasts for?
2-3 days
The severe phase of this disease has a
high mortality rate
Wool sorters’ disease or ragpickers’ disease
The course of the severe phase (onset of respiratory symptoms to death) of Wool sorters’ disease or ragpickers’ disease may last only for?
24 hours
This has been considered a candidate for biological terrorism and warfare.
Anthrax
Patients can develop meningitis within 6 days after exposure to this
Anthrax
Virulence in B. anthracis is attributed to the production of?
plasmid-borne anthrax toxins: lethal toxin (LT), and edema toxin (ET)
What are the two proteins that consist Lethal toxin and edema toxin?
protective antigen (PA) and the functional
enzyme, lethal factor (LF) and edema factor (EF)
Considered opportunistic pathogens and often associated with food borne illness
B. cereus group
These are often associated with infections in immunocompromised patients who have debilitating
disease such as cancer or diabetes
B. cereus group
Associated with the ingestion of a wide variety of foods including meats, vegetables, desserts, sauces, and milk
B. cereus group “food poisoning”
A higher incidence of B. cereus is seen after the ingestion of?
Rice dishes
One of the two types of symptoms patient present after ingestion of foods infected with B. cereus
diarrhea and emetic (vomiting)
The most common type of non gastrointestinal infection caused by B. cereus
eye infections (Endophthalmitis)
Enumerate the virulence factors of B. cereus
- hemolysin BL (HBL)
- Nonhemolytic enterotoxin (Nhe)
- cytotoxin K (Cytk) (hemolysin IV)
- Cereulide
These three toxins of B. cereus are believed to act synergistically
- hemolysin BL (HBL)
- Nonhemolytic enterotoxin (Nhe)
- cytotoxin K (Cytk) (hemolysin IV)
Toxin responsible for the major symptoms in the diarrheal presentation of B. cereus infection
Nonhemolytic enterotoxin (Nhe)
The _________ of illness is associated with a heat-stable, proteolysis, and acid-resistant toxin
emetic form
Heat-stable, proteolysis, and acid-resistant toxin which is produced in food
cereulide
Specimen processing:
Heat
62°C to 65°C for ____
70°C for ____
80°C for _____
- 15-20 minutes (B. anthracis only)
- 30 minutes
- 10 minutes
This removes contaminating organisms, and only the spore-forming bacilli survive
pretreatment (alcohol shock)
This technique is considered an enrichment and selection procedure designed to increase the chance for laboratory isolation of the organisms.
Alcohol shock
The only clinically relevant aerobic organisms capable of producing endospores in the presence of oxygen
Bacillus spp.
Sporulation is inhibited by high concentrations of?
CO2 (carbon dioxide)
The production of spores may be induced by growth in
triple sugar iron (TSI), urea, or nutrient agar containing 5mg/L manganese sulfate
What is the special stain required to visualize endospores?
malachite green
What counterstain follows the primary stain in gram staining endospores?
safranin
The _____ stain green, and the ______ will appear pink from the secondary stain, safranin
endospores and vegetative cells, respectively
This is attributed by the fact that the endospores may appear as intracellular or extracellular clear oval structures upon Gram staining.
Box car or bamboo rod appearance
All Bacillus and related genera grow well on
- 5% sheep blood agar
- chocolate agar
- routine blood culture media
- nutrient broths
Isolates susceptible to nalidixic acid will not grow on
Columbia agar with nalidixic acid and colistin (CNA)
An additional selective agar for gram-positive organisms. Also useful for the removal of contaminating organisms and the isolation of Bacillus spp.
Phenylethyl alcohol agar (PEA)
This can be used for selection and isolation from contaminated specimens (fecal)
Polymyxin-lysozyme-EDTA-thallous acetate (PLET)
Used to induce B. anthracis capsule formation, providing a means for presumptive morphologic identification
Bicarbonate agar
The current CDC level A testing protocol for the presumptive
identification of anthrax recommends using _____ for
stools suspected to contain B. anthracis, in addition to SBA
and other commonly used media.
PEA agar
Because B. anthracis would typically be isolated from normally sterile sites, such as
blood, lung tissue, and CSF
This should be incubated
in increase carbon dioxide environment
Bicarbonate agar
TRUE OR FALSE:
Increase CO2 promotes spore formation.
FALSE. It does not promote spore formation
On sheep blood agar (SBA), colonies of B. anthracis appear?
nonhemolytic,
large (2 to 5 mm), gray, and flat with an irregular margin
Term used to describe the colony morphology of B. anthracis.
Medusa head
These have a tenacious consistency, holding tightly to the agar surface, and when the edges are lifted with a loop, they stand upright without support.
B. anthracis colonies
This has been described as having the appearance or characteristic of beaten egg whites.
B. anthracis colonies
This organism ferments glucose but does not ferment mannitol, arabinose or xylose
B. anthracis
This produces lecithinase, grows in 7% NaCl and pH < 6 and is susceptible to penicillin (10 U/mL)
B. anthracis
B. anthracis grow on?
Egg Yolk Agar
Opaque zone around colonies
Lecithinase
In Gelatin hydrolysis test, the hydrolyzed tube remain liquid even after freezing at?
4°C
Oily sheen
Lipase
This can be tested by either wet mount preparation or inoculation into motility test medium
Motility
Capsule production by B. anthracis can be detected by ____________ on blood or CSF specimens or on cells isolated in media supplemented with ____________
India ink staining and sodium bicarbonate, respectively
Identify:
- Susceptible to penicillin
- Positive for Lecithinase
- Negative for motility
- No hemolysis on BAP
- Negative for Gelatin hydrolysis
- Negative for growth in PEA
B. anthracis
Serodiagnosis of B. anthracis is typically available for the detection of
PA antigen or toxin protein, LF, and EF
Immunochromatographic test that presumptively identifies B. anthracis from blood agar within 15 minutes
Red Line Alert Test
Identify:
- Resistant to penicillin
- Positive for Lecithinase
- Positive for motility
- Shows beta hemolysis on BAP
- Positive for Gelatin hydrolysis
- Positive for growth in PEA
B. cereus
This test is a presumptive test and should not be used independently for the diagnosis of anthrax.
Red Line Alert Test
Most rapid detection method and differentiation of B. anthracis
PCR
Give the expected results in Penicillin susceptibility for:
- B. anthracis
- B. cereus
B. anthracis- susceptible
B. cereus- resistant
Prevention and treatment of Anthrax
- Vaccine
- Ciprofloxacin (or doxycycline)
Give the expected results in Growth in Phenylethyl Alc. agar for:
- B. anthracis
- B. cereus
B. anthracis- negative
B. cereus- positive
Give the expected results in Lecithinase for:
- B. anthracis
- B. cereus
B. anthracis- positive
B. cereus- positive
Cell-free inactivated vaccine are given in what doses and how many boosters?
Three primary doses followed by two boosters thereafter (0 weeks, 1month, 6 months, 12 months, and 18 months)
Give the expected results for Fermentation of salicin:
- B. anthracis
- B. cereus
B. anthracis- negative
B. cereus- positive/ negative
Recommended after aerosol exposure to B. anthracis, such as in a bioterrorist event.
Chemoprophylaxis with ciprofloxacin (or doxycycline)
These are available for immunizing high-risk adults (i.e., public health laboratory workers, workers handling potentially contaminated industrial raw materials, and military personnel) against anthrax
Cell-free inactivated vaccine (BioThrax, Emergent Biode fense Operations, Lansing, MI)
This organism is gram positive “club-shape” coryneform /
diptheroid, nonlipophilic, lipophilic, catalase +, and nonmotile
Corynebacterium (non-spore formers)
On Gram stain, these are slightly curved, gram- positive rods with nonparallel sides and slightly wider ends, producing the described “club shape”.
corynebacteria
A form of Diptheria that is often considered fastidious and grow slowly on standard culture
media
Lipophilic corynebacteria
Disease caused by C. diphtheriae is referred to as
diphtheria
The bacteria are carried in the upper respiratory tract and spread by droplet or hand-to-mouth contact.
Respiratory Diptheria
The most common site of infection in Respiratory Diptheria
tonsils or the pharynx
Some C. diptheriae cannot cause diptheria but only those who that have been infected by ________
bacteriophage
Cause tissue necrosis and exudate formation triggering an inflammatory reaction
Diptheria
Combination of cell necrosis and exudate forms a tough gray-to white ___________, which attaches to the tissue
pseudomembrane
A form of Diptheria that consists of nonhealing ulcers with a dirty gray membrane
Cutaneous Diptheria
This toxin is produced by strains of C. diphtheriae infected with a lysogenic β-phage (carrier of gene tox)
Diphtheria toxin
The major virulence factor associated with C. diphtheriae
Diphtheria toxin
One of the Corynebacterium species most frequently recovered from human specimens
C. Amycolatum
Often been misidentified by clinical laboratories as C. striatum, C. xerosis, and C. minutissimum
C. Amycolatum
Named after Johnson and Kaye. Most common cause of Corynebacterium- associated prosthetic valve endocarditis in
adults
C. Jeikeium
Infections are typically limited to patients who are immunocompromised, have undergone invasive procedures, or have central line catheters or prosthetic devices
C. Jeikeium
Primarily a veterinary pathogen. Human infections typically have been associated with contact with sheep and are rare
C. pseudotuberculosis
The organism produces a dermonecrotic toxin that causes death of various cell types, and it can produce diphtheria toxin.
C. pseudotuberculosis
Most commonly associated with UTIs, Up and incoming cause of cystitis
C.urealyticum
Has been isolated from humans with diphtheria-like illness, and a significant number of isolates produce the diphtheria toxin
C. ulcerans
As a veterinary pathogen, it causes mastitis in cattle and
other domestic and wild animals. Human infections are
usually acquired through contact with animals or by
ingestion of unpasteurized dairy products
C. ulcerans
These are highly pleomorphic (many shapes) gram- positive bacillus that appears in palisades (cells lie in parallel rows, picket-fence, chine letter arrangement) or as
individual cells lying at sharp angles to another in “V” and “L” formations
C. diphtheriae
These organisms often stain irregularly, especially when stained with methylene blue, giving them a beaded appearance.
C. diphtheriae
These produce much larger colonies when cultured on 5%sheepbloodagarsupplemented with 1% Tween 80
lipophilic (lipid loving) species (e.g.,C. jeikeium, C. urealyticum, C. afermentans subsp. lipophi lum, C. accolens, and C. macginleyi)
The metachromatic areas of the cell, which stainmore intensely than other parts, are called
Babès-Ernst granules or Volutin granules
They represent accumulation of polymerized polyphosphates. Their presence indicates the accumulation of nutrient reserves and differs with the type of medium and the metabolic state of the individual cells
Babès-Ernst granules
Demonstrate better growth in broths supplemented with rabbit serum (Corynebacterium are facultatively anarobe)
Lipophilic coryneform bacteria
Can promote production of metachromatic granules because it contains egg yolk
Loeffler medium
Produces brown-black colonies with a gray-brown halo
Tinsdale medium
A modification of Tinsdale medium, contains
sheep red blood cells, bovine serum, cystine, and
potassium tellurite. This is also both a selective medium and a differential medium
Cystine-tellurite blood agar (CTBA)
Useful for differentiating
corynebacteria because only C. diphtheriae, C. ulcerans, and C. pseudotuberculosis form a brown halo as a result of cystinase activity.
Cystine-tellurite blood agar (CTBA)
All medically important corynebacteria are
catalase positive and nonmotile
Inhibits many noncoryneform bacteria. When grown on CTBA
potassium tellurite
Due to reduction of tellurite, corynebacteria form?
black or brownish colonies
Indicators of clinical relevance include:
(1) isolation from normally sterile sites or multiple blood
culture bottles;
(2) isolation in pure culture or as the predominant organism from symptomatic patients who have not yielded any other known etiologic agent; and
(3) isolation from urine if present as a pure culture at
greater than 10,000 colony-forming units per milliliter
(CFU/mL) or the predominant organism at greater than 100,000 CFU/mL.
Distinguished from the other two species by
its lack of urease production. It ferments
glucose and maltose, producing acid, but not gas, and reduces nitrate to nitrite.
C. diptheriae
Colonies of this bacteria are flat and dry, have a matte or waxy appearance, and are nonlipophilic. It does not produce any halo in Tinsdale medium; negative for urease; is only able to produce acid from glucose
C. amycolatum
This organism is lipophilic and a strict aerobe that is nonhemolytic, does not produce urease, and is nitrate reduction negative
C. jeikeium
Produces urease and on SBA, forms small and yellowish-white colonies.
C. pseudotuberculosis
Produce acid from starch and is gelatin hydrolysis positive. It does not reduce nitrate, differentiating it from C. diphtheriae, and it is urease positive
C. ulcerans
Identify:
Pinpoint, nonhemolytic, white colonies have characteristic coryneform microscopic morphology. Is nitrate negative, catalase positive, and urease positive within minutes after inoculation on a Christensen urea slant
C. urealyticum
For toxigenicity test, a patient may be infected with several strains at once, so testing is performed using a pooled inoculum of at least ________ is necessary
10 colonies
Used to ascertain whether diphtheria antitoxin neutralizes the lethal effect of a cell-free suspension of the suspect organism
Guinea pig lethality test
Utilizes the principle of immunodiffusion. A filter
paper strip impregnated with diphtheria antitoxin is buried just beneath the surface of a special agar plate before the agar hardens
ELEK test
In ELEK test, after 24 hours of incubation at 37°C, the plates are examined with transmitted light for the
presence of ________________ at a 45-degree angle to the streak
fine precipitin lines
A preparation of antibodies capable of toxin neutralization before its entry into the patient’s cells.
diphtheria antitoxin (DAT)
Why is it critical that DAT be administered as soon as a presumptive clinical diagnosis is completed?
Because of hypersensitivity reactions to antibodies (proteins) produced in horses
This is the only bacteria considered as an important human pathogen
Listeria monocytogenes
Clinically significant in specific populations of immunocompromised patients as well as being associated with foodborne infections in healthy individuals.
Listeria monocytogenes
Identify:
- facultative anaerobe
- catalase-positive
- nonbranching,
- oxidase-negative
- gram-positive rod
Listeria
Enumerate the toxin detection methods available
- Guinea pig lethality test
- Immunodiffusion (ELEK TEST
- Tissue culture cell test
- ΕΙΑ
- Detection of tox gene (PCR)
In ELEK test, the precipitin lines indicates that the
strain produced toxin that reacted with the homologous
antitoxin. With that, state the uses of Lines 1, 2, 3, and 4
Line 1: the negative control
Line 2: the positive control
Line 3: an unknown organism that is a nontoxigenic strain
Line 4: an unknown organism that is a toxigenic strain
The only effective control of diphtheria is through
Immunization with a multidose diphtheria toxoid prepared by inactivation of the toxin with formaldehyde
The colonies are small, round, smooth, and translucent. They
are surrounded by a narrow zone of β-hemolysis, which may be visualized only if the colony is removed. The colonies and hemolysis resemble those seen with S. agalactiae
L. monocytogenes colonies
Short, gram-positive rod that may occur singly or in
short chains, resembling streptococci
Listeria
The optimal growth temperature for L. monocytogenes is ___ to ___ but growth occurs over a wide range (0.5°to45° C).
30° and 35° C, respectively
Because of this, L. monocytogenes may be used to isolate from polymicrobial clinical specimens
cold enrichment technique (4° C)
In CAMP test, L.monocytogenes exhibit a
narrow zone formation but more of a block-type of hemolysis
Identify what listeria:
- Motile
- Ferments salicin
- Hydrolyzes esculin
L. monocytogenes
This organism is also called a refrigerator bacilli
L. monocytogenes
In wet mount, it exhibits tumbling motility (end-over-end motility)
L. monocytogenes
“umbrella” pattern is seen when the organism is incubated at room temperature(22° to25°C)but not at 35° C
L. monocytogenes
Widely distributed in nature and occasionally colonizes the human gastrointestinal tract. Many foods are contaminated with this, including milk, raw vegetables, cheese, and meats.
L. monocytogenes
Outbreaks have occurred as a result of eating contaminated cheese, coleslaw, and chicken. Contaminated ice cream, hotdogs, and luncheon meats have served as vehicles for this food borne disease as well.
Listeriosis
Major virulence factor. It is a pore forming toxin that reduces T-cell responsiveness.
Listeriolysin O
Organism produces a bacterial surface protein, _________, that induces host cell actin polymerization
Act A
Are found worldwide in a variety of vertebrate and invertebrate animals, including mammals, birds, and fish
Erysipelothrix rhusiopathiae
Identify:
- gram-positive
- catalase-negative
- non–spore-forming, pleomorphic rod that has
a tendency to form long filaments
Erysipelothrix rhusiopathiae
Associated with individuals
employed in occupations such as fish handlers, farmers, slaughterhouse workers, food preparation workers, and
veterinarians
Erysipelothrix infections
Specimen used for Erysipelothrix
Tissue biopsy of the skin lesion
The most common infection caused by E. rhusiopathiae in humans
Erysipeloid
