LAB - Staphylococci & MRSA Flashcards
Staph family
Micrococcaceae
Major staph species associated with human infection
S. aureus
S. epidermidis
S. saprophyticus
S. aureus
- skin infections
- wound infections
- enterotoxin-associated food poisoning
- endocarditis
- TSS
- scalded skin syndrome
S. epidermidis
- normal flora
- hospital-acquired in immunosuppressed patients as a result of catheterization or valve replacement
S. saprophyticus
- normal flora
- UTI in young females
S. aureus is carried in the nose of ____% of the general population
20-40%
carriage is higher in hospital personnel
Micrococcus colonies
- slower growing than staphylococci
- most produce yellow-orange pigment
- non-hemolytic on BAP
- larger cocci, often in tetrads
Catalase test
- enzyme that converts hydrogen peroxide into water and oxygen = visible bubbles
a hemoprotein similar in structure to Hb
catalase
- present in most cytochrome-containing aerobic and facultatively anaerobic bacteria
pseudoperoxidase
RBCs produce a weakly + catalase so sample from tops of colonies!!!
some bacteria in this family produce pseudocatalases which are capable of decomposing H2O2 and producing weak bubbles with some delay
Enterococci
- avoid by reading test within ten seconds
T or F. Coagulase should be performed using selective media
F! use nonselective (BAP)
bc colonies isolated on selective media tend to agglutinate in saline, resulting in false positives
Slide coagulase test
- bound coagulase or clumping factor (surface of cell wall of most strains of S. aureus); acts directly upon fibrinogen
- should be read within ten seconds
- screening procedure
- isolates positive for this are reported as presumptive S. aureus
- MRSA could be neg for this but pos for tube coag
Tube coag test
- detects coagulase secreted extracellularly
- reacts with a substance in plasma called coag-reacting factor (CRF) = thrombin-like complex that converts fibrinogen to fibrin causing clot formation in plasma
- test read by gently tilting tube
- false pos or nef can occur with non-sterile plasma
- read within 4 hours but if neg, held overnight to inspect for delayed clot formation
Latex agglutination
- replaces slide and tube coag
- detects bound coag and a S. aureus cell wall antigen = protein A
- late particles coated with fibrinogen and IgG
DNAse test
- heat stable DNAse, thermonuclease test
- S. aureus produces heat-tolerant DNAse; different from other Micrococcus sp.
- medium containing DNA with methyl green or toluidine blue O as an indicator
- zone of clearing if DNAse is present for methyl green
- toluidine = positive will show pink around colonies; neg = remain blue
Mannitol Salt Agar Test
- selective/differential
- high salt (7.5%) inhibit most bacteria except Staph species
- S. aureus ferments mannitol
- acid produced lowers pH; indicator phenol red changes to yellow
- other Staph don’t ferment mannitol so red colonies with red/purple zone
Resistance to novobiocin is used as a presumptive test for recognition of this organism
S. saprophyticus
- zone of inhibition less than or equal to 16 mm are resistant
Oxidase test with 6% TMPD for Micrococci vs. Staphylococci
cytochrome C in Micro but not in Staph
both are oxidase neg by traditional test
Bacitracin for Micrococci vs. Staphylococci
micro is inhibited but staph shows no zone of inhibition
Furazolidine
growth of Staph inhibited but Micro shows no zone of inhibition
OF test
majority of Staph produce acid from glucose anaerobically (ferment), Micro are oxidative glucose utilizers
This Staph enterotoxin is associated with majority of food outbreaks
Staphylococcus Enterotoxin A (SEA)
- intoxication
- detected in filtrates prepared from vomitus or food filtrates (not stool bc absorbed in gut)
iatrogenic infection
physician transmitted
These two methods are used to separate strains into specific groups or types (Staph strains)
- phage typing: based on susceptibility of Staph to certain bacterial phages or viruses
- DNA analysis
Direct immunofluorescence antibody (DFA; 1 step)
- used to ID unknown bacterial antigen
- antiserum conjugated with fluorescein isothiocyanate is prepared + join with patient sample
- smear viewed under UV microscope; if fluorescence = bacteria is present
- faster = one incubation period
- more specific method for detecting antigens
IFA (2 steps)
- detection of either Ab or Ag
- if detecting Abs in patient serum = prepare known Ags + patient sample = Ag-specific Abs will bind; wash away excess; THEN labelled antihuman globulin (AHG) applied = microscopy
- more sensitive method for detecting antigens
MRSA identification strategies
- Oxacillin Agar Screen
- Oxacillin Disc Screen
- MRSA Chromagar
- PCR = gold standard (identifying methicillin resistance mecA gene)
Oxacillin Agar Screen
- Mueller-Hinton agar medium = 6 micrograms of oxacillin/ml and 4% NaCl
- after incubation, any growth = resistance
