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 Arylamidase (PYR) test is a colorimetric test that detects pyrrolidonyl aminopeptidase by the hydrolysis of the substrate, L-Pyrrolidonyl-Beta-Naphthylamide.
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 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 drug 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?
staphylococci, large colony beta-hemolytic streptococci, and Streptococcus pneumoniae with 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 in the first quadrant
Incubate in a 37 degree celsius CO2 incubator for 18-24 hours
Interpretation
* Susceptible to Optochin = zone of inhibition ≥14mm (15 - 30mm) 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 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
Aerobic, Spore-Forming, Nonbranching, Catalase-Positive Bacilli are likely to be
Bacillus spp.
B. anthracis colonies are commonly described as…..
- Tenacious
- “Medusa’s head”
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
B. cereus is a relatively common cause of
Food poisoning (fried rice)
- 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
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
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
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)
Lactobacillus (gram positive non-spore forming rods)
- medium, straight, uniform Gram positive rods
- may form chains
- Normal vaginal flora
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
22.3.2. Recall additional organisms that are associated with BV.
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
