02 - Aerobic Gram-Positive Bacteria(Exam #2) Flashcards
Describe three types of hemolysis that can be observed on a 5% sheep blood agar plate.
- Alpha (α) = partial lysis of the RBCs has occurred. You see green to greenish-brown discoloration of the medium.
- Beta (β) = complete lysis of the RBCs has occurred. You see a clear, colorless zone around the colonies.
- Gamma (γ) = no hemolysis has occurred. The RBCs surrounding the colonies are intact.
Explain the catalase test including principle, procedure, and interpretation.
Principle
Detects the enzyme catalase which breaks down hydrogen peroxide into water and gas.
Procedure
Using a sterile loop, place a few colonies in the center of a glass slide
Add a drop of 3% hydrogen peroxide
Interpretation
* Positive reaction = immediate formation of bubbles
* Weak positive reaction = formation of 1 or 2 bubbles
* Negative reaction = no formation of bubbles or few bubbles after 20 seconds
Catalase can be used to quickly differentiate streptococci from staphylococci. What is the catalase result for each genus?
Streptococcus spp. (Negative)
VS.
Staphylococcus spp. (Positive)
Explain the PYR test including principle, procedure, and interpretation.
Principle:
The pyrrolidonyl-α-naphthylamide (PYR) hydrolysis test is a colorimetric test that detects pyrrolidonyl aminopeptidase.
Procedure:
Apply a few drops of reagent 1 (buffer) to the test card
Using a sterile loop grab several colonies from a fresh transfer and smear onto test card
Incubate at room temperature for 2 mins
Apply a few drops of reagent 2 (p-dimethylaminocinnamaldehyde)
Interpretation:
- Positive reaction = bright red or cherry color
- Negative reaction = no color change
A positive PYR test is commonly associated with what three gram positive cocci?
- Enterococci
- Streptococcus pyogenes (Group A streptococci)
- Staphylococcus lugdunensis
Which species of staphylococci is coagulase (staphaurex) positive?
Staphylococcus aureus
What are three methods for detecting coagulase?
- Slide coagulase
- Tube coagulase
- Staphaurex (latex agglutination)
Explain the principle, procedure, and purpose of the slide coagulase test.
Principle
Detects bound coagulase also known as clumping factor. Clumping factor converts fibrinogen into fibrin.
Procedure
Using a china marker draw two circles on a glass slide, one circle will be used as a negative saline control.
Place one drop of saline in the circle closest to the frosted edge of the glass slide
Place one drop of rabbit plasma in the circle furtherest from the frosted edge of the glass slide
Using a sterile loop pick up a few colonies and throughly mix into the circle with saline.
Using a new sterile loop pick up few colonies and throughly mix into the circle with rabbit plasma.
Interpretation
Auto saline control should have no clumps. If clumps are present the test is invalid.
Positive= clumps aka clots in rabbit plasma
Negative= no clumps in rabbit plasma
Which two common species of staphylococci are typically slide coagulase positive?
- Staphylcoccus aureus
- Staphylococcus lugdunensis
Explain the principle, procedure, and interpretation of the tube coagulase test
Principle
Detects free coagulase also known as staphylocoagulase which reacts with a thermostable, thrombin-like molecule called coagulase-reacting factor (CRF) to form coagulase-CRF complex. The coagulase-CRF complex resembles thrombin and indirectly converts fibrinogen to fibrin.
Procedure
Using a sterile pipette add, 0.5 mL of rabbit plasma to a sterile test tube
Using a sterile loop, pick up several colonies off a fresh transfer of the test isolate and emulsify into the rabbit plasma
Incubate in a 37 degree incubator
If negative at 6 hours, incubate for 24 hours
Interpretation
* Positive result = plasma will coagulate and form a gel
* Negative result = plasma will remain a liquid
Describe the colony morphology of Staphylococcus aureus.
White & hemolytic (older colonies may turn yellow-aureus is greek for golden)
What type of infection does Staphylococcus saprophyticus cause?
2nd most common cause of urinary tract infections (UTIs), in young women. “honeymoon cystitis”.
Define Enterotoxins
heat stable exotoxins that cause nausea vomiting, and diarrhea
Includes TSST-1 (Toxic Shock Syndrome Toxin-1)
Toxic Shock Syndrome Toxin-1
- A superantigen, produced by Staphylococcus aureus, that stimulates T-cell proliferation and the subsequent production of a large amount of cytokines that activate an aggressive, overreactive immune response.
- Previously referred to as enterotoxin F.
- Causes the majority of cases of menstruating-associated TSS
Exfoliative Toxin
- Toxin produced by Staphylococcus aureus
- Causes Scalded Skin Syndrome (the epidermal layer of the skin sloughs off)
- Most common in newborns and infants
Cytolytic Toxins
- Extracellular proteins produced by Staphylococcus aureus that affect red blood cells and leukocytes.
- Referred to as Hemolysins (alpha, beta, gamma and delta)
Panton-Valentine leukocidin (PVL).
- γ-cytolytic toxin produced by Staphylococcus aureus
- Exotoxin lethal to polymorphonuclear leukocytes.
Describe Staphylococcus saprophyticus AST testing
- CLSI guidelines do not recommend antimicrobial susceptibility testing of isolates from urine because they are typically sensitive to agents commonly used to treat UTIs (nitrofurantoin)
Staphylococcus lugdunensis has the same AST breakpoints as
Staphylococcus aureus
Explain the principle, procedure, and interpretation of the staphaurex test.
Principle
A latex agglutination test containing latex beads covered in fibrinogen and IgG. Beads will bind to bacteria that produce both clumping factor (bound coagulase) and protein A
Procedure
Mix latex reagent
Dispense one drop onto a latex card
using a sterile loop, pick up several colonies and emulsify into latex reagent
Spread mixture over half the are of the circle
Rotate card for up to 20 seconds
Interpretation
Positive= clumping
Negative= no clumping
What does MRSA stand for?
Methicillin Resistant Staphylococcus aureus
What does MSSA stand for?
Methicillin Susceptible Staphylococcus aureus
What does CA-MRSA stand for?
Community-associated methicillin-resistant Staphylococcus aureus
What does HA-MRSA stand for?
Hospital-associated methicillin-resistant Staphylococcus aureus
What two drugs are used to detect methicillin resistance staphylococci?
- Oxacillin
- Cefoxitin (gold standard)
Which gene is responsible for methicillin (oxacillin) resistance?
mecA gene
What does the mecA gene code for?
- An Altered Penicillin-Binding Protein (PBP) = PBP2a
- The altered PBP does not bind oxacillin, rendering all beta-lactam drugs ineffective
Why are 90% of staphylococci resistant to pencillin?
Acquired penicillinases (a type of beta-lactamase) which breaks down the β-lactam ring of many penicillins rendering them ineffective.
What AST results would trigger a D-ZONE Test?
When testing staphylococci, large colony beta-hemolytic streptococci, or Streptococcus pneumoniae and observe a resistant erythromycin and susceptible clindamycin AST result.
If the D-Zone test is positive, clindamycin should be reported as
Resistant
If the D-Zone test is negative, clindamycin should be reported as
Susceptible
What gene would cause a positive D-Zone test?
The erm gene (erythromycin ribosome methylase)
What is the drug of choice for MSSA?
Oxacillin
What is the drug of choice for MRSA?
Vancomycin
What cell wall component can be used to serogroup most streptococci?
- C carbohydrate
- AKA Lancefield grouping (A,B,C,D,F,G)
List infections caused by Streptococcus pyogenes (Group A Strep).
- Bacterial pharyngitis (Strep throat)
- Scarlet Fever
- Nonbullous impetigo
- Erysipelas
- Cellulitis
- Necrotizing fascitis
- Toxic Shock Syndrome
What protein helps Streptococcus pyogenes resist phagocytosis and adhere to mucosal cells?
M Protein
Streptolysin O
Oxygen labile hemolysins (O) produced by Streptococcus pyogenes
Streptolysin S
Oxygen stable hemolysins (S) produced by streptococci
Bacterial pharyngitis
- Strep throat is most commonly caused by Streptococcus pyogenes and is often seen in children between 5 and 15 years of age.
Describe
Scarlet Fever
- Infection with strains of S. pyogenes that produce streptococcal pyrogenic exotoxins
- Characterized by a diffuse red rash that appears on the upper chest and spreads to the trunk and extremities.
Necrotizing fasciitis
- An invasive infection characterized by rapidly progressing inflammation and necrosis of the skin, subcutaneous fat, and fascia.
- Is a life-threatening infection.
- “flesh eating disease”
- associated with GAS
Streptococcal Toxic Shock Syndrome (TSS)
- A condition in which the entire organ system collapses, leading to death.
- TSS produce a streptococcal pyrogenic exotoxin, notably SpeA.
- Patients are often bacteremic and have NF.
Poststreptococcal sequelae
Two serious complications, or sequelae, of GAS disease are rheumatic fever and acute glomerulonephritis.
GAS and GBS are instrinsically susceptible to what drug?
Penicillin
If patient is pen allergic, what is the drug of choice for treating GAS?
Erythromycin
When identifying GAS, bacitracin is _______ and PYR is _____________
- Susceptible
- PYR Positive
For Large Beta Hemolytic Streps (other than group A), the bacitracin disc is _________ and the PYR is ___________
- Resistant
- PYR Negative
Streptococcus agalactiae (GBS) is important because it ….
is a leading cause of meningitis in newborns. Because of this, clinical laboratories screen pregnant women to see if they are carriers.
Streptococcus agalactiae (GBS) is positive for what 2 tests?
- CAMP test
- Hippurate Hydrolysis
Explain the principle, procedure, and interpretation of the CAMP test.
Principle
Detects ability of a bacteria to produce enhanced hemolysis in the presence of a beta-lysin producing strain of staphylococcus.
Procedure
Using a sterile loop, streak a line of Staphylococcus aureus (ATCC 25923) down the center of the plate
Using a second sterile loop, pick up several 18-24 hours colonies of the test isolate and create a perpendicular streak from the edge of the plate up to the Staphylococcus aureus streak.
Incubate in an 37 degree celsius O2 incubator for 18-24 hours
Interpretation (standard method)
- Positive result = enhanced hemolysis, “arrowhead”, at junction of Staphylococcus and test microorganism
- Negative results = no enhanced hemolysis at junction of Staphylococcus and test microorganism
Streptococcus pneumoniae causes
- pneumonia, sinusitis, otitis media, bacteremia, and meningitis.
- #1 cause of bacterial pneumonia.
- Pneumococcal pneumonia is characterized by sudden onset of chills, dyspnea, and cough.
What is the main virulence factor of Streptococcus pnemoniae?
Capsule
Streptococcus pneumoniae
4 identifying characteristics
- Gram positive cocci in pairs (diplococci) and LANCET shaped
- Alpha hemolytic colonies that can be mucoid or umbilicate
- Susceptible to optochin disk
- Bile solubility positive (activates ability to autolyse)
Describe the princple, procedure, and interpretation of the optochin test.
Principle
The Optochin test is used to differentiate Streptococcus pneumoniae (susceptible) from other alpha-hemolytic streptococci (resistant).
Procedure
Using the general purpose isolation streak, transfer a 18-24 hour test isolate to a 5% sheep blood plate.
Using sterile forceps, place a p disk where the first and second quadrant overlap.
Incubate in a 37 degree celsius CO2 incubator for 18-24 hours
Interpretation
* Susceptible to Optochin = zone of inhibition ≥14mm around the disk
* Resistant = zone of inhibition <14mm around the disk
Describe the princple, procedure, and interpretation of the bile solubility test.
Principle
This test is useful to differentiate Streptococcus pneumoniae (positive) from alpha-hemolytic Streptococcus spp. (negative).The bile solubility test is based on the observation that pneumococcal cells lyse in the presence of bile salts (sodium desoxycholate).
Tube Test:
Procedure
1. Prepare a saline suspension of the test isolate from an 18-24 hour , pure culture
2. Adjust turbidity to that of a 0.5 to 1.0 McFarland standard or equivalent.
3. Aliquot 0.5 mL of the suspension into a steile test tube
4. Add 0.5 mL of 10% sodium desoxycholate
5. Incubate tubes at 37 degrees C and examine peroidicallly for up to 3 hours.
Interpretation
Positive- clearing of the test suspension within 3 hours
Negative- remains turbid for 3 hours.
Spot Test:
1.Add 1 drop of 10% sodium desoxycholate to a well isolated, 18-24 hour colony of the test isolate growing on sheep blood agar.
2.Incubate the plate aerobically in an upright position at 35 degree C. and examine periodically for up to 30 minutes. Leave lid slightly ajar to enhance evaporation of the reagent.
Interpretation
* Positive: colony disintegration within 30 minutes
* Negative = Colony remains intact within 30 mintues.
What is the drug of choice for treating Streptococcus pneumoniae infections?
- Penicillin
- (if pen is resistant, than ceftriaxone)
Viridans Streptococci
- Normal microbiota of the upper respiratory tract
- Opportunistic pathogens
- Are the most common cause of subacute bacterial endocarditis
- Divided into 5 groups
1. Streptococcus mitis group
2. Streptococcus mutans group
3. Streptococcus salivarius group
4. Streptococcus bovis group (group D)
5. Streptococcus anginosis
S. anginosus group
- Pinpoint alpha, gamma, or beta colonies with a sweet “butterscotch” odor
S. anginosus group is positive for what 2 tests?
- VP (Vouges-Proskauer)
- Arginine Hydrolysis
S. bovis group
- Often encountered in blood cultures of patients with gastrointestinal carcinoma.
S. bovis group is:
Bile Esculin (BEM) ________
PYR_______
Grow in 6.5% NaCL (salt)?
- Bile esculin (BEM) Positive
- PYR Negative
- No growth in salt
Describe the principle, procedure, and interpretation of the Bile Esculin Test.
Principle
The bile esculin test identifies if a microorganism can hydrolyze esculin in the presence of bile to esculetin. Esculetin reacts with ferric ammonium citrate to produce brown-black colonies.
Procedure
Using a sterile loop, streak the slant.
Incubate at 37 degrees C for 18-24 hours.
Interpretation
- Positive = blackening of the media
- Negative = no color change of the media
For what two catalase-negative gram-positive cocci would bile esculin agar be positive?
- Group D streptococci
- Enterococci
Describe
Enterococcus
- Natural inhabitants of the intestinal tracts
- Enterococci are frequent causes of nosocomial infections.
- Of these, UTIs are the most common (usually due to indwelling catheters), followed by bacteremia.
4 Identification characteristics of Enterococcus
- PYR positive
- Bile esculin positive
- Ability to grow in 6.5% NaCL (salt)
- Lancefield Group D
The 2 most clinically significant species of Enterococcus are
- E. faecium
- E. faecalis
What does VRE stand for?
Vancomycin Resistant Enterococcus
Which Enterococcus species is most commonly associated with aquired VRE?
E. faecium
What genes carry vancomycin resistance?
- VanA
- VanB
- VanC
Describe the principle, procedure, and interpretation of vancyomycin agar screen?
Principle
Screen for vancomycin resistant enterococci
Procedure
Using a 18-24 hour test isolate, prepare a 0.5 McFarland.
Using a sterile swab, dip in 0.5 McFarland and apply to the brain-heart infusion plate containing 6 ug of vancomycin
Incubate at 37 degrees celsius in O2 fir 20-24 hours
Interpretation
Resistant: growth
Susceptible: no growth
CLSI recommends screening of enterococci isolated from blood cultures or specimens such as heart valve tissue for high-level aminoglycoside resistance with both
Gentamicin and Streptomycin.
E. faecium is usually _____ to ampicillin.
Resistant
E. faecalis is usually _____ to ampicillin.
Susceptible
Streptococcus-Like Organisms
- The genera Aerococcus, Gemella, Leuconostoc, Abiotrophia, Granullicatella, and Pediococcus consist of organisms that resemble viridans streptococci macroscopically.
- Present as contaminants or as opportunistic pathogens
- Low virulence and pathogenicity
- Can cause endocarditis
Abiotrophia and Granulicatella
- Formerly known as the nutritionally variant streptococci
- Grows on chocolate agar
- Requires 0.01% pyridoxal hydrochloride (Vitamin B6) overlaid on TSA sheep blood agar to grow
- Vancomycin susceptible
- Satellites around the vicinity of the Staphylococcus hemolysis due to the diffusion of metabolites in agar (Staphylococcal Cross Streak)
Leucostonoc spp. and Pediococcus spp. are both intrinsically resistant to what drug?
Vancomycin
Aerobic, Spore-Forming, Nonbranching, Catalase-Positive Bacilli are likely to be
Bacillus spp.
Many Bacillus spp. are commonly isolated and often regarded as laboratory contaminants. Which 2 species are associated with human infections?
B. anthracis and B. cereus
B. anthracis is the causative agent of what disease?
- Anthrax (Cutaneous, GI, Inhalational, or Injectional).
- Because B. anthracis is considered to be a potential bioterrorism agent, it is important for clinical laboratories to rule out this organism when Bacillus spp. are isolated
B. anthracis colonies are commonly described as…..
- Tenacious
- “Medusa’s head”
B. anthracis is both…….
- Non-hemolytic
- Non-motile
Describe the princple, procedure, and interpretaion of the motility test.
Princple
The tube motility method uses a semi-solid agar designed to demonstrate motility by diffusion. Sometimes the motility test medium contains an indicator dye for added visual interpretation.
Procedure
Clear semisolid media is used.
Using a sterile needle, pick up a few colonies of the test isolate.
Stab the medium.
Incubate for 18-24 hours at 37 degrees celcius. Add a second if enhance motility is supected at room temperature.
Interpretation
Positive- growth (tubidity) in the medium outiside of the stab line
Negative- growth only in stab line
Wet Mount Motility
The wet mount motility method is the direct observation of a microorganism suspension on a microscope slide under a microscope.
Be careful not to confuse Brownian motion with motility. Brownian motion is the continuous, normal drifting of particles in a solution. Motile bacteria will be seen moving at different angles of direction in the solution.
Interpretation
- Positive or motile = directional or tumbling movement of cells
- Negative or non-motile = cells stay in same relative position
B. cereus is a relatively common cause of
Food poisoning (fried rice)
Listeria monocytogenes
- Large, multistate outbreaks of illness caused by L. monocytogenes have occurred several times, usually associated with foods, such as various types of cheese or deli meats.
- Uncommon but serious infection primarily of neonates, pregnant women.
- Listeria monocytogenes*
- Identification*
- Gram-positive bacillus
- Beta hemolytic
- Cat positive
- “tumbling” and “umbrella” motility
- Grows at 4 ° C
- CAMP positive “matchstick” hemolysis
- Hippurate positive
Listeria resembles S. agalactiae (GBS). Name at least 2 tests to distinguish the difference
- Gram stain. GBS is GPC and Listeria are GPB
- Listeria is CAT positive. GBS in NEG
- CAMP both are POS but Listeria produces a block hemolysis while GBS is an arrow head type
Listeria is most motile at what temperature?
Room temperature (22 degrees C)
2 ways to describe Listeria monocytogenes motility
- Tumbling motility (on wet prep)
- Umbrella motility (tube motility at room temp 22deg C)
Corynebacterium
- Slightly curved, gram-positive rods with nonparallel sides and slightly wider ends, producing the described “club shape” or coryneform on gram stain.
- Catalase positive
- More than 100 species in the genus, and at least 50 are thought to be clinically significant.
- Most of the species are found as normal biota on the skins and mucous membranes
Corynebacterium diphtheriae
- Most significant pathogen of the group
- Cause of the disease “diphtheria” 2 forms: Respiratory and cutaneous
- Must produce toxin to cause disease
Diphtheria toxin
- Produced by strains of C. diphtheriae infected with a lysogenic β-phage
- Carries the gene tox
- Toxicity is caused by the ability of diphtheria toxin to block protein synthesis in eukaryotic cells.
- Only toxin-producing C. diphtheriae causes diphtheria
Diphtheria (the disease)
- Respiratory disease is characterized by low-grade fever, malaise, and a mild sore throat.
- The most common site of infection is the tonsils or the pharynx.
- The toxigenic strains of C. diphtheriae produce toxin locally, causing tissue necrosis and exudate formation triggering an inflammatory reaction.
- This combination of cell necrosis and exudate forms a tough gray-to-white pseudomembrane, which attaches to the tissues.
- It may appear on the tonsils and then spread downward into the larynx and the trachea. There is the potential for suffocation if the membrane blocks the air passage or if it is dislodged, perhaps as the result of sampling for a throat culture.
- Cutaneous diphtheria consists of nonhealing ulcers with a dirty gray membrane.
- There is an effective vaccine against the disease (TDAP)
The Elek test is used to detect
Toxin-producing stains of Corynebacterium diphtheriae.
Cystine-tellurite blood agar (CTBA) or Tinsdale agar is used to detect what organism and how does it appear?
C. diphtheriae form black colonies surrounded by brown halos from the reduction of tellurite.
Describe an alternate stain for detecting granules in the cells of C. diphtheria
Methlyene blue stain done on colonies growing on loefflers medium.
The metachromatic areas of the cell, which stain more intensely than other parts
showing Babès-Ernst granules.
Erysipelothrix rhusiopathiae
- Non-spore forming, nonbranching, CAT neg, GPB (long filaments)
- Usual route of infection is through cuts or scratches on skin and exposure to birds, pigs and fishes.
- Erysipeloid, the most common infection caused by E. rhusiopathiae in humans, is a localized skin infection
Erysipelothrix rhusiopathiae is H2S____
- H2S Positive on TSI slant
Hydrogen Sulfide Production Test
The hydrogen sulfide production test determines whether a microorganism can reduce sulfur containing compounds to hydrogen sulfide (H2S). There are several media that include sulfide indicators and can be used to detect H2S production.
Detection of H2S production can be used to separate Lactobacillus (negative) from Erysipelothrix (positive).
Interpretation of tube media
- Positive reaction = blackening of the media
- Negative reaction = no blackening in the media
Arcanobacterium haemolyticum is positive for what test?
Reverse CAMP. (compare to CAMP on picture)
Arcanobacterium haemolyticum can cause
Pharyngitis
Lactobacillus (gram positive non-spore forming rods)
- medium, straight, uniform Gram positive rods
- may form chains
- Normal vaginal flora
Gardnerella vaginalis is primarily known for its association with….
Bacterial vaginosis (BV)
Explain the Nugent scoring system for diagnosing BV
BV generally results from a reduction in the Lactobacillus population in the vagina, followed by an increase in vaginal pH; this results in overgrowth by G. vaginalis and other BV-associated organisms.
The Nugent scoring system quantitates the amount of lactobacillus (good bacteria) compared to BV morphotypes (bad bacteria) seen in a vaginal gram stain.
The score is added up to make a diagnosis
Good Vaginal Bacteria
Lactobacillus
Bad Vaginal Bacteria
Gardnerella, Bacteroides, Mobiluncus
Clue Cells
Large squamous epithelial cells with gram-positive and gram-variable bacilli and coccobacilli clustered on the edges, aids the diagnosis of BV
List the three common species of streptococci that produce colonies >0.5 mm in diameter and are beta-hemolytic on 5% sheep blood agar plates.
- Streptococcus pyogenes
Streptococcus agalactiae
Streptococcus dysgalactiae
Which large colony beta hemolytic streptococci typically have small colonies with a large zone of beta hemolysis?
- *Streptococcus pyogenes
- Streptococcus dysgalactiae*
Which species of large colony beta hemolytic streptococci typically has a larger colony with a smaller zone of beta hemolysis?
- Streptococcus agalactiae
Describe the Gram stain of streptococci including gram reaction, cellular morphology, and cellular arrangment.
Gram-positive cocci in pairs and chains
List four common coagulase-negative staphylococci species.
- Staphylococcus epidermidis
- Staphylococcus saprophyticus
- Staphylococcus lugdenensis
- Staphylococcus haemolyticus
Describe the colony morphology of micrococci.
Most species produce yellow pigmented colonies that are nonhemolytic.
Describe the Gram stain including gram reaction, cellular morphology, and cellular arrangment of staphylococci.
Gram-positive cocci in clusters (may see some singularly, in pairs, or tetrads)
Describe the Gram stain including the gram reaction, cellular morhophology, and cellular arrangment of micrococci.
Gram-positive cocci in clusters (may see a large amount of tetrads)
Explain the principle, procedure, and interpretation of the bacitracin susceptibility test.
Principle
Used for the identification of bacteria known to be intrinsically susceptible to 0.04 units of bacitractin (A disk).
Procedure
Allow bacitracin disks to warm to room temperature.
Using a disposable loop transfer test isolate to a fresh 5% sheep blood agar plate using the general purpose isolation streak.
Using sterile forceps, grab an A disk and place where the 1st and 2nd quadrant overlap.
Incubate in a CO2 incubator for 18-24 hours.
Interpretation
Susceptible= zone of inhibition
Resistant= no zone of inhibition
Which species of large colony beta-hemolytic streptococci is susceptible to 0.04 units of bacitracin?
Streptococcus pyogenes
Describe the principle, procedure, and interpretation of the lysostaphin test.
Principle
The peptidoglycan layer of staphylococci is held together by peptide bridges rich in glycine. Lysostapin is a peptidase which lyses glycine bridges. Rendering the cell susceptible to osmotic rupture.
Procedure
Prepare a suspension of the test isolate from a pure, 18-24 hour culutre in 0.2 mL of sterile saline.
Add 0.2 mL of lysostaphin.
Incubate tubes at 37 degrees celcius for 2 hours
Interpretation
Positive test- solution clears, no turbidity remains
Negative test - solution remains turbid, does not clear
Describe the principle, procedure, and interpretation of the modified oxidase test (microdase test).
Principle
Detects cytochrome c using the compound p-aminodimethlaniline oxalate
Procedure
Using sterile forceps, place the disk on a clean glass slide.
Using a sterile loop, rub a small amount of several colonies of an 18- to 24-hour pure culture grown on blood agar onto a small area of the microdase disk.
Incubate at room temperature for 2 minutes.
Interpretation
Positive= purple
Negative= anything else
Describe the principle, procedure, and interpretation of the novobicin suceptibility test.
Principle
Used for the identification of bacteria known to be intrinsically resistant to 6 ug of novobicin (NB).
Procedure
Using a sterile swab and a pure 18-24 hour old transfer of the test isolate, create a 0.5 McFarland in saline.
Using a second swab, dip into the 0.5 McFarland standard and ring out excess fluid.
Create a bacterial lawn on a Mueller-Hinton plate.
Using sterile forceps, place a novobicin disk in the center of the plate.
Incubate in an O2 incubator for 18-24 hours
Interpretation
Susceptible= >16mm diameter of the zone of inhibition
Resistant= </= 16mm diameter of the zone of inhibition
Which coagulase negative staphylococcus species is resistant to 6 ug of novobicin?
Staphylococcus saprophyticus
Explain the principle, procedure, and interpretation of the ornithine decarboxylase (ODC) test.
Principle
Broth contains glucose and ornithine (amino acid) as well as the pH indicators bromcresol purple and cresol red. If bacteria can break down both glucose and ornithine, they will break down glucose first causing the pH to drop. After all the glucose is gone, the bacteria use ornithine decarboxylase to break down ornithine causing the pH to increase
Procedure
Using a sterile swab, select a few colonies from a fresh culture of the test isolate and emulsify in the ODC broth.
Overlay broth with mineral oil
Tighten cap and incubate at 37 degrees celsius
Examine at 24,48,72,96 hrs
Intrepretation
Negative = No color change
Positive = Purple
Which common coagulase negative staphylococci is positive for ornithine decarboxylase at 24 hours?
Staphylococcus lugdunensis
Describe the colony morphology of Staphylococcus saprophyticus.
Bright white and nonhemolytic
Describe the colony morphology of Staphylococcus lugdunensis
White and slightly hemolytic
4.Differentiate the staphylococci based on coagulase, hemolysis, and other techniques.
Describe the colony morphology of Staphylococcus epidermidis.
White and nonhemolytic
Describe the colony morphology of Staphylococcus haemolyticus.
White and slight heme
Define the term superantigen
Antigen that interact with T cells triggering an overactive response from the immune system.
What are some examples of superantigens?
- Enterotoxins including toxic shock syndrome toxin 1
- Streptococcal pyrogenic exotoxins
What toxins can Staphylococcus aureus produce?
1-Enterotoxins
2-Exfoliative toxin (epidermolytic toxin)
3- Cytolytic toxins
5.Recall and describe the virulence factors and how they play in infections caused by Staphylococcus aureus.
What enymes do Staphylococcus aureus produce?
1- staphylocoagulase
2-protease
3- hyaluronidase
4-lipase
Explain the function of Protein A.
Protein, produced by Staphylococcus aureus, that binds to the Fc portion of IgG and blocks phagocytosis from the immune system.
5.Recall and describe the virulence factors and how they play in infections caused by Staphylococcus aureus.
30% of the population are carriers of Staphylococcus aureus. List some body sites that can become colonized with S. aureus.
- anterior nares
- umbilicus
- axilla
- perianal/rectal
- inguinal folds
List skin and wound infections are commonly caused Staphylococcus aureus.
Folliculitis
Furuncles
Bullous impetigo
Carbuncles
Cellulitis
List infections caused by toxins produced by Staphylococcus aureus.
- Food poisoning
- Scalded skin syndrome
- Toxic shock syndrome
What food is staphylococcal food poisoning associated with?
- Mayonniase based salads
- Eggs
- Cream filing
What type of bone infections can staphylococcus aureus cause?
- Osteomyelitis
- Septic arthritis
- Prosthetic joint infections
Staphylococcus lugdunensis causes many of the same infections Staphylococcus aureus; however, it is particularly associated with
catheter-related bacteremia and endocarditis.
Describe what type of infections Staphylococcus epidermidis causes.
Nosocomial infections - infections that result after placement of foreign devices such as cathers, shunts, and prosthetic devices. Often, a contaminant in improperly collected blood cutlures.
What type of microbiota are Micrococcus species typically apart of?
Skin microbiota
Mannitol Salts Agar (MSA)
A selective and differential medium for the isolation of Staphylococcus aureus from respiratory sites. Contains 7.5% NaCl, mannitol, and phenol red. Staphylococci can grow in high salt and Staphylococcus aureus can utilize mannitol changing the medium from pink to yellow.
*of note Staphylococcus saprophyticus can also utilize mannitol, but is not typically found in the respiratory tract.
What does the erm gene code for?
A methylase which alters the ribosomal binding site for macrolides, lincosamides, and stretogramin antibiotics, making antibiotics in these classes useless.
Describe the procedure and interpretation for the D-Zone test when performed on staphylococci.
Procedure
Make a 0.5 McFarland using saline and an 18-24 hr old test isolate.
Make a bacterial lawn on Mueller Hinton agar
Place a 15 ug erythromycin 15-26 mm from a 2 ug clindamycin disk
Incubate in 35 degree Celsuis O2 incubator for 16-18 hours
Interpretation
Positive = flattening of clindamycin zone of inhibition adjacent to the erythromycin disk (giving it a D appearance).
Negative= no flattening of clindamycin zone of inhibition
8.2.Describe the D Zone test for detecting macrolide resistance.
Describe the procedure and interpretation for the D-Zone test when performed on large colony beta-hemoltyic streptococci and Streptococcus pneumoniae.
Procedure
Make a 0.5 McFarland using saline and an 18-24 hr old test isolate.
Make a bacterial lawn on Mueller Hinton agar with 5% sheep blood
Place a 15 ug erythromycin 12mm from a 2 ug clindamycin disk
Incubate in 35 degree Celsuis CO2 incubator for 20-24 hours
Interpretation
Positive = flattening of clindamycin zone of inhibition adjacent to the erythromycin disk (giving it a D appearance).
Negative= no flattening of clindamycin zone of inhibition
8.2.Describe the D Zone test for detecting macrolide resistance.
How many ug/mL of oxacillin are in the oxacillin screen plate?
The oxacillin screen plate contains Mueller-Hinton supplemented with 4 % NacL and 6 ug/mL of oxacillin.
8.3.Describe the methods for methicillin resistance detection.
What does the msrA gene encode for?
a macrolide efflux pump in staphyloccoci
List important biochemical results for Staphylococcus lugdunensis.
Catalase (+)
Staphaurex (-)
Bacitracin (R)
Novobicin (S)
PYR (+)
Ornithine decarboxylase (ODC) (+)
What does VISA stand for?
Vancomycin Intermediate Staphylcococcus aureus
What does VRSA stand for?
Vancomycin Resistant Staphylcoccus aureus
- Define the following terms.
7.5. VRSA
Explain heteroresistance (heterogeneity) to oxacilin in Staphylococcus aureus
In heteroresistant isolates, all cells in the population have the genetic element (mecA gene) for oxacillin resistance, but not all the cells express this resistance by virtue of PBP2a production. Consequently, in the oxacillin susceptibility test, some cells may appear resistant, and some can appear susceptible. If too few cells appear resistant, an oxacillin-resistant strain might not be detected.
Ways to reduce heteroresistance during susceptibility testing and interpretation of Staphylcoccus aureus include:
*Incubation of tests at temperatures no higher than 35° C
*Making final test readings after a full 24hours of incubation *Supplementation of Mueller-Hinton broth or agar with 2% NaCl for dilution tests
*For oxacillin disk diffusion tests, zones of inhibition must be examined by using transmitted light and any growth is considered significant.
Describe the Gram stain including gram reaction, cellular morphology, and cellular arrangment of most streptococci.
Gram-positive cocci in pairs in chains
Describe the Gram stain including gram reaction, cellular morphology, and cellular arrangment of enterococci
Gram positive cocci in pairs and chains
9.Differentiate Streptococcus species, Enterococcus species and other Strep-like species utilizing microscopic features, colony morphology, hemolysis and biochemicals.
Describe the Gram stain including gram reaction, cellular morphology, and cellular arrangment of the following genra:
Leuconostoc
Abiotrophia
Granulicatella
Gram-positive cocci in pairs and chains
Describe the Gram stain including gram reaction, cellular morphology, and cellular arrangment of the following genera:
Pediococcus
Aerococcus
Gram-positive cocci in clusters
What C type of carbohydrate does Streptococcus pyogenes have?
A. Sometimes Streptococcus pyogenes is refered to as Group A streptococci (GAS)
What type of C carbohydrate does Streptococcus agalactiae have?
B. Sometimes, Streptococcus agalactiae is referred to as Group B streptococci (GBS)
What type of C carbohydrate does Streptococcus dysgalactiae have?
Some strains are C others are G.
- Associate the serologic grouping schemes with the corresponding genus and species.
What type of C carbohydrate does Streptococcus pnemoniae have?
None. It is nontypeable.
- Associate the serologic grouping schemes with the corresponding genus and species.
What does the Van a and Van b gene code for?
Alteration in the peptiglycan structure. Instead of the amino acid chains ending in D-Ala they end in D-Lac. Making vancomycin useless.
What does the Van c for?
Alteration in the peptiglycan structure. Instead of the amino acid chains ending in D-Ala they end in D-Ser. Making vancomycin useless.
What two species of enterococci intrinsically carry the Van c gene?
- Enterococcus casselifavus
- Enterococcus gallinarum
How can Enterococcus faecium be differentiated from Enterococcus faecalis?
- Hemolysis
- Arabinose (sugar)
E. faecalis= gamma hemolytic and cannot utilize arabinose
E.facium= alpha hemolytic and can utilize arbinose.
9.Differentiate Streptococcus species, Enterococcus species and other Strep-like species utilizing microscopic features, colony morphology, hemolysis and biochemicals.
What does VSE stand for?
Vancomycin susceptible enterococcus
Describe the principle, procedure, and interpretation of the Leucine aminopetidase (LAP) test.
Principle
The RemelTM LAP Test Kit is a rapid, colorimetric test for use in qualitative procedures for the detection of leucine aminopeptidase (LAP) activity in isolates of streptococci and related genera.
Procedure
Using forceps, place the disk on a clean microscope slide or in the lid of an agar plate free from excess moisture.
2. Moisten the disk slightly with 5-10 μl of demineralized water using an adjustable micropipette or a 10μl inoculating loop. DO NOT OVERSATURATE. Alternatively, the disk may be placed on the agar surface of the plated medium for rehydration.
3. Pick up a visible “paste” from an 18-72-hour culture using a sterile loop or wooden applicator stick.
4. Rub the inoculum gently into a small area of the disk.
5. Incubate at room temperature for 5 minutes.
6. Add one (1) drop of LAP Color Developer to the disk.
7. Allow up to one minute for a color change.
Interpretation
Positive Test -
Pink to red color development within one minute of applying LAP Color Developer
Negative Test -
No color change or a slight yellow color development within one minute of applying LAP Color Developer
