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Genotyping of epidemic methicillin-resistant Staphylococcus aureus phage type 15 isolates by fluorescent
1Molecular Biology Unit, Central Public Health Laboratory, London NW9 5HT, United Kingdom. rgrady@phls.nhs.uk
Abstract:
Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plus TaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.
Insights
Fluorescent amplified-fragment length polymorphism (FAFLP) effectively subtypes methicillin-resistant Staphylococcus aureus (MRSA), offering a robust and reproducible method for hospital infection control. This technique distinguishes strains better than PCR-RFLP and PFGE.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat in healthcare settings.
- Accurate subtyping of MRSA strains is crucial for effective infection control and epidemiological tracking.
- Epidemic MRSA strain 15 (EMRSA-15) is a prevalent and concerning MRSA phage type.
Purpose of the Study:
- To evaluate the efficacy of Fluorescent Amplified-Fragment Length Polymorphism (FAFLP) for identifying and subtyping MRSA isolates, specifically EMRSA-15.
- To compare FAFLP with other molecular typing methods like PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE).
Main Methods:
- FAFLP analysis involved double DNA digestion, adaptor ligation, and PCR amplification with adaptor-specific primers.
- Amplified fragments were analyzed using an automated sequencer and specialized software to generate FAFLP profiles.
- Comparative analysis included PCR-RFLP of the coagulase gene and PFGE.
Main Results:
- FAFLP successfully differentiated EMRSA-15 from other MRSA isolates and revealed heterogeneity within the EMRSA-15 phage type.
- The discriminatory power of FAFLP was high, dividing 24 isolates into 11 profiles when combining enzyme-primer data.
- PCR-RFLP failed to discriminate among EMRSA-15 isolates, while PFGE showed good discrimination but was less reproducible than FAFLP.
Conclusions:
- FAFLP is a robust, reproducible, and highly discriminatory method for subtyping MRSA, including EMRSA-15.
- FAFLP offers advantages over PCR-RFLP and PFGE in terms of ease of performance and reproducibility.
- FAFLP has significant potential for use in hospital infection control for MRSA surveillance and outbreak investigation.