Screening and Confirmation of mecA producing Staphylococcus aureus
Purpose: Screening and confirmation of the mecA gene
are essential steps in the detection of methicillin-resistant Staphylococcus
aureus (MRSA), a bacterium that is resistant to many commonly used antibiotics,
including methicillin. The mecA gene is involved in resistance to
beta-lactamases by methicillin-resistance in Staphylococcus aureus.
Principle: The mecA gene is a gene found in bacterial cells. The most commonly
known carrier of the mecA gene is the bacterium known as MRSA. The mecA gene
allows a bacterium to be resistant to antibiotics such as Methicillin,
Penicillin and other penicillin-like antibiotics. The mecA gene does not allow
the ring like structure of penicillin-like antibiotics to attack the enzymes
that help form the cell wall of the bacterium (transpeptidases), and hence the
bacteria is allowed to replicate as normal.
The mecA gene encodes
the protein PBP2A (Penicillin binding protein 2A). PBP2A has a low affinity for
beta-lactams such as Methicillin, Penicillin and this enables transpeptidase
activity in the presence of beta-lactams to allow cell wall synthesis.
It is also found in
Streptococcus pneumoniae resistant to penicillins.
Performance characteristics: To identify Methicillin Resistant
Staphylococcus aureus.
Required
equipment and Reagents:
- Muller Hinton Agar
- Cefoxitin (30mcg)
- Incubator
Procedural
steps:
Sample
Collection: Start by collecting
a clinical sample, typically from a wound, nasal swab, blood, or other relevant
sources, where MRSA is suspected.
Culture and Isolation: The first step is to culture the collected
sample on a selective medium incubated at 37±2°C at ambient air in 18-24 hrs that
encourages the growth of Staphylococcus aureus. MRSA typically grows on
selective media like Mannitol Salt Agar or ChromAgar MRSA.
Gram Staining: Perform a Gram stain to confirm the presence
of Gram-positive cocci in clusters, which is characteristic of Staphylococcus
species.
Screening
for Methicillin Resistance:
Disk Diffusion Method: Use the Kirby-Bauer disk diffusion method
with a cefoxitin disk (rather than methicillin, which is not widely used
anymore).
Molecular Methods (PCR): This is the most specific and reliable
method. PCR (Polymerase Chain Reaction) can be used to detect the mecA gene
directly from the bacterial DNA. This confirms the presence of mecA and,
therefore, methicillin resistance. Alternatively, molecular methods like
real-time PCR can be used to detect MRSA-specific genes like mecA.
Confirmation
of mecA:
PCR Sequencing: For further confirmation, you can sequence
the PCR amplicon to identify the mecA gene specifically.
Other Molecular Tests: There are various other molecular methods
and techniques available for confirming mecA, such as PCR with specific primers
and probes.
Infection Control Measures: If MRSA is confirmed, implement infection
control measures to prevent further transmission within healthcare settings.
Quality
control procedures:
Each new batch of
medium should be tested with a control organism.
S. aureus ATCC 25923
Interferences
and cross reactions:
If the zone of
inhibition is small (usually ≤ 21 mm), it suggests methicillin resistance.
A zone mm of ≤ 21mm
is positive and ≥22 mm is negative.
Cefoxitin is used as
a surrogate for mecA mediated oxacillin resistance. Isolates that test as mecA positive are
reported as oxacillin resistant; other β-lactam agents should also be
considered resistance for clinical therapy.
.bmp) |
| MRSA SCREENING |
Laboratory clinical interpretation:
mecA negative- Report
as – Methicillin sensitive Staphylococcus aureus
mecA positive –
Report as – Methicillin Resistant Staphylococcus aureus.
Reference:
- Clinical and Laboratory Standards Institute (CLSI). Perormance Standards for Antimicrobial susceptibility testing; 12th Informational Supplement. CLSI document M100-S20, Vol 30, No.1.
- Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2010.
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