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Journal of Clinical Microbiology, October 1999, p. 3198-3203, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genotyping of Epidemic Methicillin-Resistant Staphylococcus aureus Phage Type 15 Isolates by Fluorescent Amplified-Fragment Length Polymorphism Analysis

Ruth Grady,1 Meeta Desai,1 Gael O'Neill,2 Barry Cookson,2 and John Stanley1,*

Molecular Biology Unit1 and Laboratory of Hospital Infection,2 Central Public Health Laboratory, London NW9 5HT, United Kingdom

Received 22 February 1999/Returned for modification 1 June 1999/Accepted 29 June 1999

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.

* Corresponding author. Mailing address: Molecular Biology Unit, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: (44) 181 200 4400, ext. 3090. Fax: (44) 181 200 1569. E-mail: rgrady{at}phls.nhs.uk.

Journal of Clinical Microbiology, October 1999, p. 3198-3203, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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