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Journal of Clinical Microbiology, January 2006, p. 271-273, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.271-273.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Use of Variations in Staphylococcal Interspersed Repeat Units for Molecular Typing of Methicillin-Resistant Staphylococcus aureus Strains

Katherine J. Hardy,1,2* Beryl A. Oppenheim,1 Savita Gossain,1 Fang Gao,3 and Peter M. Hawkey1,2

West Midlands Public Health Laboratory, Health Protection Agency, Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS, United Kingdom,1 Division of Infection and Immunity, University of Birmingham, Birmingham B15 2TT, United Kingdom,2 Intensive Care Unit, Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, United Kingdom3

Received 29 July 2005/ Returned for modification 10 September 2005/ Accepted 9 October 2005


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ABSTRACT
 
Staphylococcal interspersed repeat unit typing has previously been shown to have the ability to discriminate between epidemic methicillin-resistant Staphylococcus aureus strains in the United Kingdom. The current study illustrates its ability to distinguish between strains within an endemic setting thereby providing a rapid transportable typing method for the identification of transmission events.


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TEXT
 
Methicillin-resistant Staphylococcus aureus (MRSA) causes a significant degree of morbidity and mortality. An understanding of the genetic relatedness and clonal spread of strains is necessary to facilitate the control of outbreaks. At present, there are numerous typing methods available but no consensus regarding which method is the best (13). Pulsed-field gel electrophoresis (PFGE) is a highly discriminatory typing method which is best suited to investigating microevolution and recent transmission events within a hospital (1). However, it has several limitations, including the ease of reproducibility and interpretation of the banding patterns, even when using a standardized method (7, 8). Studies of the evolution of MRSA have been facilitated by multilocus sequence typing (MLST), which examines the slowly evolving genomic core (2). MLST is less discriminatory than PFGE, so although it is useful for defining evolutionary events, it does not provide a good marker for microevolution events within the hospital. Variable-number tandem repeats (VNTRs) have been used as markers for strain typing of various bacteria (5, 9, 10). Determination of the numbers of repeats at each locus produces a digital profile providing a highly portable typing method allowing comparison between laboratories. We have previously described the presence of VNTRs in staphylococci, termed staphylococcal interspersed repeat units (SIRUs), in all seven sequenced S. aureus genomes and their ability to discriminate between the main epidemic MRSA (EMRSA) strains within the United Kingdom (4).

The aim of the present study was to determine the ability of SIRUs to type MRSA strains within an endemic setting in an intensive care unit (ICU). During two 8-month study periods, 215 and 197 patients admitted to a nine-bed general ICU were screened three times a week for MRSA. All swabs were placed into brain heart infusion broth and then subcultured on oxacillin-resistant screening agar (Oxoid Unipath, Hants, Basingstoke, United Kingdom) and confirmed as MRSA by using the previously described multiplex PCR for the detection of the coagulase gene (coa) and mecA gene (6). Totals of 56/215 (26%) and 61/197 (30.9%) of patients were colonized with MRSA, of which 32 and 29 patients acquired MRSA in the ICU. Isolates from all patients were typed using both SIRUs and PFGE as has been previously described (4, 8). For SIRU typing, each locus was amplified using the oligonucleotide sequences detailed in Table 1, the size of each amplicon determined and the number of repeats calculated (Table 1), and PFGE was carried out using SmaI restriction digestion. SIRU and PFGE results were entered into Bionumerics (Applied Maths, St.-Martin-Latem, Belgium) and analyzed using the Dice coefficient for PFGE and the Euclidian coefficient for SIRU analysis and displayed using unweighted pair group method using arithmetic averages. All isolates were typeable by both SIRU typing and PFGE and were considered unique if they differed by ≥1 band by PFGE or if they differed by ≥1 repeat in any locus by SIRU typing. A total of 18 different SIRU profiles and 21 different PFGE profiles were identified. For SIRU typing, all loci added to the discriminatory power of the technique with variation in repeat number occurring at all loci (Fig. 1). SIRU 21 had the shortest repeat unit with, in this study, the repeat number being defined as 24 bp and not 48 bp as previously described (4). The greatest variation in repeat number was observed in this locus, and although the factors responsible for the difference in mutation rates are not clear, short repeat units have been cited as one of the possible factors responsible for higher mutation rates in tandem repeat sequences (11). Cluster analysis of both SIRU typing and PFGE revealed that 16 SIRU profiles and 16 PFGE profiles were variants of EMRSA-15, and this accounted for isolates from 48/56 and 49/61 of the patients colonized with MRSA in each study period (Fig. 2). Of the remaining patients, 6 and 12 patients in the first and second study periods were colonized with EMRSA-16, and 2 patients in the first study period were colonized with strains which did not have a high degree of similarity to either EMRSA-15 or EMRSA-16. With greater than 80% of patients colonized with EMRSA-15, it is important in aiding the study of transmission events to have an epidemiological typing method that has the ability to distinguish between these isolates. Although the clustering of epidemic strains was the same by SIRU typing and PFGE, subtyping of EMRSA-15 and EMRSA-16 isolates differed between the two. With EMRSA-15, SIRU typing was able to distinguish between isolates with the same PFGE profile and vice versa. This has been described previously for bacteria other than S. aureus, with Noller and colleagues demonstrating the ability of VNTRs to distinguish between isolates with the same PFGE profile during an investigation of an Escherichia coli O157:H7 outbreak. Isolates previously thought to be part of an outbreak according to PFGE were redefined as being sporadic isolates when investigated using VNTRs (9). Within EMRSA-16, PFGE had a greater discriminatory power than SIRU typing. However, the number of patients colonized with EMRSA-16 was significantly smaller than with EMRSA-15, and additionally, 16 of the 20 patients had indistinguishable PFGE profiles.


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  		TABLE 1. Repeat length and oligonucleotide sequences for each of the loci


Figure 1
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  		FIG. 1. Overview of the different numbers of repeat units identified in the loci. Letters (a through h) are used to represent loci that have more than nine repeats.


Figure 2
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  		FIG. 2. Clustering of patient isolates from both study periods by both SIRU typing and PFGE. The boxes contain the numbers of patients with each of the SIRU and PFGE profiles. Light gray boxes represent clustering of EMRSA-15 isolates; dark gray boxes represent clustering of EMRSA-16 isolates.

A greater understanding of the transmission of MRSA is necessary in order for us to be able to control the spread. SIRU typing has several advantages over PFGE both in terms of the transportability of the results and the ease of the technique which can be automated using a denaturing high-performance liquid chromatography analyzer or a DNA sequencer (3, 12). It provides a typing technique that is reproducible and is able to distinguish between MRSA strains within an endemic setting.


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ACKNOWLEDGMENTS
 
We thank Wyeth for the support of K. J. Hardy.


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FOOTNOTES
 
* Corresponding author. Mailing address: West Midlands Public Health Laboratory, Health Protection Agency, Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, United Kingdom. Phone: 44 (0) 121 4240250. Fax: 44 (0) 121 772622. E-mail: Katie.Hardy{at}heartofengland. nhs.uk. Back


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Journal of Clinical Microbiology, January 2006, p. 271-273, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.271-273.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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