This document discusses methods for detecting Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA is any strain of S. aureus that is resistant to beta-lactam antibiotics due to the mecA gene. Rapid detection of MRSA is important for optimal treatment and reducing costs. The document describes several screening methods, focusing on the oxacillin salt agar screening test which involves growing bacterial samples on agar containing oxacillin and 4% NaCl. Growth of more than one colony indicates oxacillin resistance and identifies the strain as MRSA.

1. MRSA Detection
Dr. Samira Fattah
PhD in Medical Bacteriology
College of Health Sciences-HMU

2. MRSA Detection
• Methicillin Resistant Staphylococcus aureus (MRSA) is any strain of S. aureus that has
developed a multiple drug resistance to beta-lactam antibiotics.
• Beta-lactam (β-lactam) antibiotics are a broad-spectrum group that include some penams
(penicillin derivatives such as methicillin and oxacillin) and cephems such as the
cephalosporins.
• Strains unable to resist these antibiotics are classified as methicillin-susceptible S. aureus,
or MSSA.
• Methicillin resistance is due to a resistance gene mecA, which stops β-lactam antibiotics
from inactivating the enzymes (transpeptidases) critical for cell wall synthesis.

3. MRSA Detection
• Methicillin-resistant S. aureus are associated with serious nosocomial
infections in hospitals which lead to higher costs, longer
hospitalizations, increased mortality and limits the treatment
possibilities .
• Rapid differentiation between methicillin-resistant and methicillin-
susceptible S. aureus species is necessary to optimize treatment and
minimize costs.
• Making the correct choice of antibiotics based on the antimicrobial
susceptibility of MRSA isolates may help to reduce the resistance
problem by reducing the over-prescription of ineffective antibiotics

4. Screening for MRSA
1. Disc diffusion
- Oxacillin disc diffusion tests
- Cefoxitin disc diffusion tests
2. Oxacillin Salt Agar screening method
3. Dilution methods
- Agar dilution
- Broth microdilution
4. E test method
5. Latex agglutination (detection of PBP2a )
6. Automated methods (Vitek2 system- Cefoxitin screen)
7. Molecular methods (detection of mecA gene)

5. Oxacillin Salt Agar Screening test
Materials:
1. Staphylococcus aureus bacteria
2. MHA with 4%NaCl
3. 6 µg/mL Oxacillin
4. Incubator

6. Oxacillin Salt Agar Screening test
Method:
1. Prepare 1 liter of MHA media
• Add 40 gm NaCl
• Autoclave
• After cooling to 45°C add 6mg Oxacillin
(6 µg/mL oxacillin ˟ 1000ml =6000 µg=6mg=0.006 gm )
2. Pour to plates
3. Colony suspension to obtain 0.5 McFarland turbidity.
4. Using a swab dipped in the suspension and the excess liquid expressed, spot an
area or streak an entire quadrant.
5. Incubate at 33 to 35°C for 24 hours (Testing at temperatures above 35°C may
not detect MRSA.)

7. Oxacillin Salt Agar Screening test
Result reading:
1. Examine carefully with transmitted light for > 1 colony or light film
of growth.
2. > 1 colony = oxacillin resistant
3. Oxacillin-resistant staphylococci are resistant to all β-lactam agents;
other β-lactam agents should be reported as resistant or should not
be reported.

8. Staphylococcus aureus strains sensitive to oxacillin (1)
and resistant to oxacillin (2 and 3) by the agar screening
method (Mueller-Hinton agar containing 6 μg/mL
oxacillin + 4% NaCl).
MRSA -
MRSA +

9. Oxacillin Agar Screening test
Limitation of the method
When performed properly, the oxacillin agar method will detect most
mecA-positive S. aureus strains. Occasionally a heteroresistant mecA-
positive strain is not detected and may be missed , in part due to a low
frequency of resistance expression and slow growth rate.