Gram Positive Cocci Flashcards
(39 cards)
String Test
Reagent : 3% KOH Gram+/- Positive reaction= String formation Positive = Gram - Negative = Gram +
Gram-Sure: Remel
Reagent : L-alanine-7-amido-4-
methylcoumarin
Gram -/+
LanaGram: Hardy Diagnostics
Reagent : L-alanine-4-nitroanilide
Gram -/+
APNA; Key Scientific
Reagent : L-alanine-p-nitroanilide
Gram -/+
STAPHYLOCOCCUS AUREUS Clinical Importance
-Different Virulence factors, such as adhesins, enzymes, and
toxins.
-Resistant strains, most common cause of nosocomial
pneumonia & skin infections.
-S.aureus is second after CoNS as a cause of primary
bacteremia in hospitals.
-Persistent carriers (10-35%)
-Intermittent carriers (20-75%)
-Noncarriers (5-50%)
-(MRSA, MRSE)
Catalase Test
- 3% Hydrogen Peroxide
- Catalase Positive V. Negative
- Creation of bubbles
- Staphylococcus V. Streptococcus
- Staph = Positive
- Strept = Negative
STAPHYLOCOCCUS:
General Characteristics
Gram-positive cocci in pairs, tetrads, and
clusters
-Catalase positive
- Most species are halotolerant: resistant to
10% NaCl
-Susceptible to lysostaphin, resistant to
lysozyme
STAPHYLOCOCCUS Aureus : Presumptive Positive
-Catalase positive
-Gram-positive cocci in clusters
-Tube or slide coagulase or latex
agglutination test positive
Slide Coagulase Test
Clumping Factor: Demonstrated by the ability of the organism to act directly on the fibrinogen in the plasma to clump it in a slide assay. -Rabbit Plasma EDTA- -S. Aureus= Coag + if Negative move to tube test
Tube Coagulase Test-Rabbit
Plasma Method
Tube Coagulase:
Coagulase is a thermostable thrombin-like
substance that activates fibrinogen to form fibrin,
resulting in a fibrin clot. This is demonstrated in
the test tube by the formation of clot when rabbit
plasma is inoculated with the Staphylococcus for 4
to 24 hrs.
The coagulase clot can be destroyed by S.aureus
fibrinolysin or staphylokinase, an enzyme which is
more active at 35°C.
-Rabbit Plasma EDTA-
-S. Aureus= Coag +
Coagulase Test-Protein A/
Clumping Factor Agglutination
Method
Staphylococcal protein A (SAG) or clumping
factor agglutination:
S. aureus produces another substance in its cell
wall, protein A, which binds to the FC part of
IgG. If latex coated with IgG and with human
fibrinogen, a Staphylococcus will agglutinate if
either clumping factor or protein A is present in
the bacterial cell wall.**
-S. Aureus= Coag +
Voges–Proskauer (VP Test)
-Voges–Proskauer: VP is a test used to
detect acetoin in a bacterial broth culture.
-The test is performed by adding alpha-
naphthol and 40% potassium hydroxide to culture if acetylmethyl carbinol(acetoin) is present then it turns red.
-Incubate 24hr take 2mls added reagents and mix to check for color change.
-Voges-Proskauer broth which has been
inoculated with bacteria. A cherry red color
indicates a positive result, while a yellow-
brown color indicates a negative result.
-All staphylococci except, S. intermedius
and S. hyicus, are positive for VP.
PYR Test
- L-pyroglutamic acid beta-
naphthylamide
-pyrolidonyl arylamidase
-PYR test is a rapid colorimetric method for
presumptive identification of certain groups of
bacteria based on the activity of the enzyme
pyrolidonyl arylamidase. L-pyroglutamic acid beta-
naphthylamide is impregnated into the test disk
and serves as the substrate for the detection of
pyrolidonyl arylamidase. Hydrolysis of the
substrate yields beta-naphthylamide which
combines with the PYR Reagent (p-dimethylamino-
cinnamaldehyde) to form a bright pink to cherry
red color within 2 minutes. - PYR activity is a key
test for differentiation of some species of
coagulase-negative Staphylococcus.
Ornithine Decarboxylase
- The purpose is to see if the microbe can use the amino acid ornithine as a source of carbon and energy for growth. Use of ornithine is accomplished by the enzyme ornithine decarboxylase.
- A medium containing ornithine and a pH indicator is used. When ornithine is used, the pH of the medium rises and the indicator changes color.
- The medium used is ornithine decarboxylase broth. The medium is a nutrient broth to which 0.5% ornithine is added. An important component of the medium is a modest amount of glucose, necessary for the process to proceed. The pH indicator brom cresol purple is purple at neutral or alkaline/basic pH but turns yellow at pH <5.2.
- An inoculum from a pure culture is transferred aseptically to a sterile tube of ornithine decarboxylase broth. The inoculated tube is incubated at 35-37 C for 24 hours and the preliminary results are determined. The microbe must first use the glucose present to cause the pH to drop. This is indicated by a change from purple to yellow. Once the medium has been acidified, the enzyme ornithine decarboxylase is activated. The culture is incubated an additional 24 hours at 35-37 C to allow the microbe to now use the ornithine. The final results are then obtained by observing the tube at 48 hours. Change back to purple from yellow indicates a positive test for ornithine decarboxylase. Failure to turn yellow at 24 hours or to revert back to purple at 48 hours indicates a negative result.
- Differentiation of S. Lugdensis
- Arginine decarboxylase is useful in the identification of Enterococcus to the species level; Enterococcus faecalis and Enterococcus faecium are arginine positive but, Enterococcus avium is arginine negative.
Staphylococcus saprophyticus
Catalase positive Coagulase negative Non hemolytic Novobiocin resistant (<16mm) UTI -Polymyxin B sensitive Other novobiocin-resistant species: S.cohnii S.kloosii S.xylosus
Staphylococcus epidermidis
Catalase positive Coagulase negative Non hemolytic Novobiocin sensitive Most common cause of nosocomial infectious associated with indwelling or implanted foreign polymer bodies
Staphylococcus intermedius &
lugdunensis
Staphylococcus intermedius:
Catalase positive
Coagulase positive
VP negative to differentiate from S.aureus
Veterinary pathogen; dog bites
Implicated in food poisoning
Staphylococcus lugdunensis: (may be confused with
S.aureus)
Beta hemolytic
Slide coagulase +/-
Ornithine positive to differentiate from S. aureus
Antimicrobial Susceptibility Test
Prepare a bacterial suspension equal to 0.5 MacFarland in normal saline Use a swab to spread the suspension softly on the Mueller Hinton Agar (MHA) Let it sit 5-15 minutes to absorb Apply antimicrobial disks with sterile forceps with enough distance from each other and the edge of the plate Incubate for 18-24hrs @ 35°C
Pyogenic Group Beta hem(large colony)
- S.pyogenes
- S.agalactiae
-S.dysagalaciae subsp.equisimilis
….Veterinary Pathogenes
Non-Pyogenic Group
Alpha, Gama, or Beta
- S.mitis group
- S.anginosus group
- S.mutans group
- S.salivarius group
- S.bovis group
Streptococcus pyogenes
CLINICAL SIGNIFICANCE
- Infection-Septic sore throat, tonsillitis, scarlet fever
- Skin-STSS, necrotizing faciitis, wounds, burns, impetigo, erysipelas, cellulitis
- Acute hemorrhagic glomerulonephritis (GN)- linked to skin infections
- Female genital tract- puerperal sepsis (child-bed fever)
- Rheumatic fever
Streptococcus agalactiae
CLINICAL SIGNIFICANCE
-Early-onset neonatal disease:
sepsis & pneumoniae between first 7 days
-Late-onset neonatal disease:
meningitis and sepsis between day 7 and 3 months of age
- Found in 10-30% of pregnant women
- *prophylaxis 4 hours before delivery
Streptococcus dysagalactiae
CLINICAL SIGNIFICANCE
-Large colony forming beta hemolytic
(Lancefield Group C or G), BGUR positive
- Resembles infections caused by Group A strep
- Infections- necrotizing fasciitis, bacteremia, and endocarditis
Streptococcus anginosus Group
CLINICAL SIGNIFICANCE
The small-colony-forming species S. anginosus, S. constelaltus, and S. intermedius belong to the S. anginosus group. They can be non-, alpha-, or beta-hemolytic and often harmless commensals of oropharayngeal, urogenital and gastrointestinal microbiota.
Strongly associated with abscess of brain, oropharynx or peritoneal cavity.
It is important to reliably distinguish them from large colony-forming (>0.5mm) β-hem strep of pyogenic group. (S. anginosus group is VP positive).
