Staphylococcus Species Flashcards
Give the preliminary ID Tests for staphylococci
(3)
Gram positive
Cocci
Catalase +
How do you speciate staphylococci?
(3)
Coagulase test
+ = S. aureus
- = S. epidermidis or saprophyticus
What are the three clinically significant species of staphylococci
S.aureus
S. epidermidis
S. saprophyticus
What is a gram positive cocci but catalase negative species
Micrococcaceae
How many genera of staphyloccaceae are there
6
What is the most clinically significant micrococcaceae
Micrococcus
How are staphylococci arranged?
Grape like clusters
How many species of staphylococci are there?
40+
Why are there only a few species of staphylococci that are considered clinically significant
Most species only exist as commensals
Name four species of commensal staphylococci that have some varying pathogenic potential
S. aureus
S. epidermidis
S. saprophyticus
S. haemolyticus
What clinical specimens can staphylococcus be found in?
(6)
Skin wab
Pus specimen
Wound swab
Blood - blood cultures for BSI
Sputum
Urine
What must be done with specimens potentially containing staph
(2)
MRSA surveillance to detect carriage
Screening swabs can be taken from the nasal, axilla groin or carriage
In general what are the growth characteristics of staph?
Facultative aerobe
Halophilic
What does halophilic mean?
Can grow in NaCl as high as 15%
What is query staph put up on on day 1 of investigation
(4)
General purpose: Blood agar
Selective agar
- Mannitol salt agar
- Chromogenic media e.g. S. aureus ID or CHROMagar MRSA
How should you incubate staphylococcus on blood agar?
Incubate at 37 degrees for 24 hours
How should you incubate the selective and differential medias for Staphylococci?
Incubate for 24-48 hours at 37 degrees Celsius
What are the selective agents of Mannitol Salt agar and how do they work?
7.5% salt
High salt favours growth of salt-tolerant Staphylococcus species
What are the differential agents of mannitol salt agar and how do they work?
(3)
Mannitol is the differential agent
S. aureus can ferment mannitol producing yellow colonies
S. epidermidis cannot ferment mannitol and will grow as pink or white colonies
List the Chromogenic Mediums that can be used for Staphylococci
(3)
Oxoid Chromogenic MRSA Agar
BioMérieux Chrome ID
ChromID MRSA
How does Oxoid Chromogenic MRSA Agar work?
(3)
Chromogens detect phosphatase activity in S. aureus -> this will produce denim-blue colonies
Cefoxitin (methicillin) will inhibit MSSA colonies
This allows for the accurate detection of MRSA
How does the BioMérieux- Chrome ID work?
(3)
SAID -> S. aureus ID
Chromogens detect alpha glucuronidase in S. aureus
This forms green colonies i.e. S, aureus = green colonies
How does the ChromID MRSA agar work?
MRSA stains indicated by green colonies
Results from alpha-glucuronidase producing colonies in the presence of cefoxitin
How does the ChromID MRSA agar work?
(2)
MRSA stains indicated by green colonies
Results from alpha-glucuronidase producing colonies in the presence of cefoxitin
What tests are carried out on Day 2 to identify Staphylococcus?
(9)
Colonial morphology
Gram stain -> GPC, grape-like clusters
Catalase positive
Coagulase
Protein A
DN’ase
Biochemical profile - Vitek/Phoenix
Protein profile - MALDI
Molecular ID systems - Real Time PCR
What are the four classifications of tests carried out on day 2 for the identification of an organism
Basic characterisation tests
Manual Conventional confirmatory tests
Automated conventional confirmatory tests
Molecular confirmatory tests
What manual conventional confirmatory tests are carried out for Staphylococcus
(3)
Coagulase
Protein A
DN’ase
What automated conventional confirmatory tests are carried out for staphylococcus?
Biochemical profile on the Vitek/Phoenix
Protein profile on the MALDI
What molecular confirmatory tests are carried out for Staphylococcus?
Molecular ID systems - Real Time PCR
What is the definitive test to identify S. aureus?
Coagulase test
Test for the detection of coagulase enzyme
What are the two ways of carrying out coagulase test?
Slide coagulase/clumping factor test
Tube method
What is the slide coagulase/clumping factor test and how do you carry it out?
Detects bound coagulase only
Mix bacterial saline suspension with rabbit plasma (source of fibrinogen), observe agglutination
What is the slide coagulase/clumping factor test and how do you carry it out?
Detects bound coagulase only
Mix bacterial saline suspension with rabbit plasma (source of fibrinogen), observe agglutination
What is the tube method of coagulase test and how is it carried out?
Detects free and bound coagulase
Mix 9 drops of 3hr incubated test suspension with 1 drop rabbit plasma -> incubate over night -> observe clot
What does a positive coagulase test mean?
This is sufficient to identify S. aureus
What does a negative coagulase mean?
Coagulase negative staphylococcus species
These are collectively called CNS
What is the most common way of detecting coagulase
Staphaurex Plus
What is Staphaurex Plus
Rapid latex agglutination test for the identification of S. aureus
Why should Staphaurex be used instead of methods of testing for coagulase?
(3)
In addition to coagulase, protein A is also found on the surface of 95% of human strains of S. aureus
Certain MRSA possess a capsule that masks both protein A and the coagulase
With Staphaurex, rapid agglutination will occur using any three of these components
What will cause rapid agglutination in the Staphaurex Plus test?
(3)
Reaction between fibrinogen and coagulase
Binding between Protein A and the Fc portion of IgG
Binding between MRSA capsular polysaccharide and specific IgG
Explain in your own words how the Staphaurex Plus test works
(4)
Test latex beads are coated in porcine fibrinogen which will detect coagulase
Latex bead also coated with IgG antibodies
IgG anti capsular polysaccharide to detect MRSA capsule
Fc portion of IgG will detect Protein A
What is the DN’ase test?
Deoxyribonuclease Agar
Nutrient agar that has 0.2% DNA added
How is DN’ase agar used?
(5)
Spot inoculate DNase agar and incubate overnight
Flood with 1 molar HCL
HCL precipitates DNA which turns the medium cloudy
DNase-positive cultures -> DNA is unavailable for precipitation so there will be a clear zone around the inoculum e.g. S. aureus
DNase negative will have no zone of clearing e.g. CNS
What automation is used in the identification of Staphylococcus
(5)
VITEK GP Card - biochemical reactions
MALDI-TOF -> protein profile
These are used to ID S. aureus and speciate Coagulase negative staff if it’s required
Molecular ID systems- real time PCR detection systems such as the Xpert MRSA/SA assay can be used
- these target protein A (SA) or mecA (meth resistance) DNA sequences
- mostly used in MRSA surveillance for direct screening or confirmation of MRSA from nasal swabs
How does the Xpert MRSA/SA assay work?
(2)
Molecular ID system which uses real-time PCR
It detects targets such as Protein A (found in S. aureus) or mecA (found in methicilin resistant strains) DNA sequences
When is the Gene Xpert used for Staphylococci
Used in MRSA surveillance -> for direct screening or confirmation of MRSA from nasal swabs
What test do you carry out on Coagulase negative staph to speciate them?
Automated VITEK/MALDTOF
Novobiocin resistance
How do you carry out the novobiocin test, what are the results
(4)
Nutrient agar with novobiocin discs
Lawn inoculum
S. aureus/CNS are novobiocin susceptible
S. saprophyticus are novobiocin resistant
How is methicillin susceptibility performed?
Using cefoxitin
What are the three classifications of S. aureus infection
Superficial infection
Serious infection with S. aureus
Serious infection with MRSA
How is a superficial staph infection treated?
Topical = mupirocin
Oral = augmentin
How is a serious infection with S. aureus treated?
Requires aggressive treatment including incision and drainage of lesions
Systemic antibiotic needed = Methicillin
How is a serious infection with MRSA treated?
Aggressive treatment including incision and drainage of lesions
Systemic antibiotic = Vancomycin
