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Journal of Clinical Microbiology, June 2007, p. 1830-1837, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02402-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection{triangledown}

Barry D. Cookson,1 D. Ashley Robinson,2 Alastair B. Monk,3 Stephen Murchan,4 Ariane Deplano,5 Rafaël de Ryck,5 Marc J. Struelens,5 Christina Scheel,6 Vivian Fussing,6 Saara Salmenlinna,7 Jaana Vuopio-Varkila,7 Christina Cuny,8 Wolfgang Witte,8 Panayotis T. Tassios,9 Nikolas J. Legakis,9 Willem van Leeuwen,10 Alex van Belkum,10 Anna Vindel,11 Javier Garaizar,12 Sara Haeggman,13 Barbro Olsson-Liljequist,13 Ulrika Ransjo,14 Manica Muller-Premru,15 Waleria Hryniewicz,16 Angela Rossney,17 Brian O'Connell,17 Benjamin D. Short,18 Jonathan Thomas,18 Simon O'Hanlon,18 and Mark C. Enright18*

Laboratory of Hospital Infection, Centre for Infections, Health Protection Agency, London, United Kingdom,1 New York Medical School, New York, New York,2 Virginia Commonwealth University, Richmond, Virginia,3 Health Protection Surveillance Centre, Dublin, Ireland,4 Laboratoire de Microbiologie, Hopital Erasme, Bruxelles, Belgium,5 Statens Serum Institut, Copenhagen, Denmark,6 National Public Health Laboratory (KTL), Helsinki, Finland,7 Robert Koch-Institut, Wernigerode (Harz), Germany,8 Department of Microbiology, University of Athens, Athens, Greece,9 Erasmus MC Center, Rotterdam, The Netherlands,10 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain,11 Dpt. Immunol., Microbiol. y Parasitol., F. Farmacia, UPV/EHU, Vitoria-Gasteiz, Spain,12 Swedish Institute for Infectious Disease Control, Solna, Sweden,13 Swedish Institute for Infectious Disease Control, Solna, Sweden,14 Institute of Microbiology, University of Ljubljana, Ljubljana, Slovenia,15 Sera and Vaccines Central Research Laboratory, Warsaw, Poland,16 National MRSA Reference Laboratory, St James's Hospital, James's St., Dublin 8, Ireland,17 Department of Infectious Disease Epidemiology, Imperial College, London, United Kingdom,18

Received 29 November 2006/ Returned for modification 8 January 2007/ Accepted 1 March 2007


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ABSTRACT
 
We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.


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INTRODUCTION
 
Methicillin-resistant Staphylococcus aureus (MRSA) is among the most common nosocomial pathogens globally and is generally acknowledged as the most significant due to the burden of disease it causes and to the evolution and global spread of multidrug-resistant clones. MRSA isolation rates in the United States, parts of Europe, and Asia have been increasing for more than 4 decades (36), and recent figures show that in some areas >50% of S. aureus bacteremias are caused by MRSA (4, 5, 6). Emerging intermediate, and more recently high-level (vanA-encoded), vancomycin resistance (8, 22) and increasing numbers of multidrug-resistant MRSA emphasize the importance of effective antimicrobial prescribing and infection control measures that can be informed usefully by molecular-typing results.

Epidemic spread of S. aureus in hospitals and intercontinental spread of a particular clone was first demonstrated in the 1950s using bacteriophage typing (37). This method has largely been replaced in centers with low workloads as an epidemiological tool by genotyping approaches that index chromosomal variation and offer advantages in typeability, discrimination, and reproducibility. Many different genotyping methods are currently in use, but the most popular is pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA (27). Other techniques that have been explored include ribotyping (34) and PCR-based methods, such as repetitive-element PCR (13), amplified fragment length polymorphism typing (23), and, more recently, DNA-sequencing approaches (16, 35, 39, 40). Typing methods should have high and relevant discriminatory power and typeability, good reproducibility, applicability to all organisms of interest, ease of use, portability (that is, they should produce data that can easily be transferred between laboratories or presented in published work), and low cost (43).

PFGE is the most commonly used method for studying local or short-term S. aureus epidemiology. It has proven invaluable in investigations of nosocomial outbreaks, but difficulties in reproducibility and interlaboratory reliability have limited its application to relatively small studies (10). Multicenter studies using PFGE are now possible due to recent advances in the standardization of electrophoresis conditions (10, 32) and the development of normalization and analysis software (15). Interpretive criteria for use in comparing complex PFGE patterns in outbreaks have been applied to nonoutbreak situations to track the national and international dissemination of S. aureus clones (44). The use of PFGE typing with adjusted interpretation criteria for grouping patterns with <7 bands difference has been shown to correspond to clonal assignments made by other methods (12). The main criticisms of this technique for S. aureus are that PFGE may on occasion be too discriminatory for other than local or short-term epidemiological analyses, the arbitrary nature of the interpretive criteria used, and the occasional requirement for subjective analysis of complex band patterns (30).

Multilocus sequence typing (MLST) (26) has had a large impact on the field of bacterial typing, and it has been used as an investigatory tool in many studies of S. aureus evolution and epidemiology (2, 11, 16, 17, 28). MLST has a major advantage over PFGE as a reference method due to the unambiguous nature of DNA sequences, which can be stored easily along with corresponding clinical information on each isolate in Internet-linked databases. The S. aureus MLST website (http://www.mlst.net) currently contains information on >1,500 isolates from humans and animals from 40 different countries and represents a useful global resource for the study of the epidemiology of this species and the surveillance of hypervirulent and/or antibiotic-resistant clones.

In order to define MRSA clones, MLST has been used in conjunction with PCR analysis of the element conferring methicillin resistance—SCCmec (for staphylococcal chromosomal cassette mec) (17, 25). An international nomenclature for MRSA clones using the MLST and SCCmec designations has been accepted by the International Union of Microbiological Societies subcommittee on S. aureus typing. This unambiguous nomenclature characterizes clones based on their MLST sequence type (ST), SCCmec type, and phenotype. For example, members of the United Kingdom Epidemic MRSA –15 clone would be referred to as ST22-MRSA-IV—MLST ST22, MRSA, SCCmec type IV. Vancomycin intermediately susceptible descendants would be referred to as ST22-VISA-IV. This scheme allows the efficient worldwide tracking and surveillance of MRSA clones.

MLST/SCCmec typing is widely regarded as the reference method for defining MRSA clones. However, it is not as widely applicable as other methods for high-volume analysis of MRSA due to the cost of DNA sequencing (MLST requires seven PCRs plus 14 DNA-sequencing reactions per isolate). In addition, SCCmec typing requires an ever-increasing number of primers as new alleles are found (9). spa typing has been proposed as a rapid sequence-based approach to characterize MRSA. It has high portability, discrimination, and ease of use (39). An automatically curated, Internet-accessible spa sequence database is now available (1). Several recent epidemiological studies have used PFGE and/or spa typing to determine the genetic relatedness of large numbers of isolates and MLST/SCCmec typing for further characterization of representative isolates of each genotype found (3, 12, 18).

The EU-funded HARMONY project (29) standardized PFGE in eight European centers to facilitate the transfer of molecular typing data between laboratories (further details of HARMONY can be obtained from barry.cookson{at}hpa.org.uk). It now has the support of the International Union of Microbiological Societies subcommittee on S. aureus typing with the longer-term goal of allowing the development of networks of reference laboratories and research centers to provide epidemiologically useful information about the emergence and spread of MRSA clones. The potential benefits of such networks would be most obvious in the early detection of MRSA with new or multiple antibiotic resistance or pathogenic potential, determining whether such strains had emerged from parallel evolution, and monitoring their intra- or intercountry spread.

In this study, we analyzed a broadly representative collection of MRSA isolates from 11 European countries. Isolates were assigned to MRSA clones on the basis of MLST/SCCmec typing, and the congruence between these groupings and those made using PFGE and spa typing was assessed.


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MATERIALS AND METHODS
 
Bacterial isolates. The HARMONY collection of MRSA was used in this study. It consists of 98 clinical isolates collected from 11 European countries between 1981 and 1998 as part of the HARMONY project (contract number BMH4-CT96) (29). The countries that submitted isolates were Belgium, Finland, France, Germany, Greece, Ireland, Poland, Slovenia, Spain, Sweden, and the United Kingdom. Details on each isolate are available from the HARMONY website (http://www.hpa.org.uk/cfi/bioinformatics/harmony/harmonydb.htm). The isolates were chosen by each contributing center to represent currently circulating epidemic or otherwise important nosocomial clones from the mid-1980s to 1998. These isolates still represent the major clones currently causing hospital-acquired MRSA outbreaks in Europe (17). Countries were invited where possible to submit isolates that represented closely related PFGE types and important variants of these representative clones.

All isolates were stored in 30% glycerol-nutrient broth at –80°C and were grown on blood agar plates at 37°C overnight.

PFGE. PFGE following SmaI digestion was performed and analyzed according to the HARMONY protocol (29). Comparisons and groupings of PFGE patterns by Dice coefficients (14) and unweighted-pair group method using average linkages (41), respectively, were performed using GelCompar and BioNumerics software (Applied Maths, Belgium). The results were represented both as distinct PFGE patterns (indistinguishable band patterns were assigned the same PFGE pattern number) and as PFGE types (each type included closely related PFGE patterns that differed by ≤3 bands). By using this definition of PFGE types, isolates assumed to be closely related according to the guidelines proposed by Tenover et al. (44) for short-term outbreak-associated isolates were grouped.

MLST. MLST was performed as described previously (16). Briefly, seven housekeeping gene fragments (~500 bp) were sequenced and compared to known alleles at each locus on the MLST website (http://www.mlst.net). Allelic profiles, each consisting of seven allele numbers, defined STs. STs sharing 100% genetic identity in at least five of seven MLST loci were grouped into a clonal complex (CC) named after its presumed ancestral genotype, as described previously (19).

SCCmec typing. The four main SCCmec structures were differentiated by PCR detection of the SCC type (I, II, III, or IV) and the class of the mec region (A or B) using conventional (25, 31) and multiplex (33) PCRs.

spa sequence typing. Sequencing of the short sequence repeat region of the spa gene was performed as described previously (39). The sequences obtained were compared to those held on the SpaServer (http://www.ridom.de/spaserver/query) (21).

DI. The discriminatory power of each typing method was represented by an index of diversity (DI) (24), which represented the probability that two isolates selected from the sample at random would have different types. Ninety-five percent confidence intervals (CIs) were calculated as described by Grundmann et al. (20).


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RESULTS
 
PFGE. PFGE following SmaI digestion resolved 81 PFGE patterns whose profiles differed by at least one band (Table 1). PFGE patterns 8 and 13 were the most common, shared by four isolates each. PFGE pattern 8 isolates were from four different countries, Finland, Germany, Sweden, and Spain, and those having PFGE pattern 13 were from France and Belgium only. Three PFGE patterns (16, 18, and 70) were shared by three isolates each, and five patterns (1, 7, 37, 40, and 50) by two isolates each. The majority of study isolates (71/98, or 72.4%) had unique PFGE patterns.


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  		TABLE 1. Characteristics of study isolates

The number of PFGE types (PFGE patterns that differed at ≤3 band positions) was 34. They were numbered (1 to 34) arbitrarily. Nineteen of these types comprised only one PFGE pattern each. Twenty-four isolates from seven countries belonged to PFGE type 3, which contained all of the isolates with PFGE patterns 8 and 13 detailed above. Eight isolates (from Germany and Slovenia) were of type 13 (the southern German clone), seven isolates (Sweden, Ireland, and United Kingdom) were of type 25, six isolates (France) were of type 1, five isolates (Greece and France) were of type 7, and five isolates (Germany) were of type 24 (the Berlin clone). Four isolates (from Belgium, Sweden, and Finland) belonged to type 22, and four isolates (Finland and United Kingdom) were of type 27 (UK EMRSA-16). Three isolates each were of types 5 (Finland and Poland) and 8 (Germany), and two isolates each were of types 4 (Finland and Slovenia), 9 (Finland and Slovenia), 14 (Germany), 15 (Slovenia), and 19 (Belgium).

spa typing. Thirty-one spa sequence types were resolved and identified by interrogating the RIDOM SpaServer. Eighteen spa types were unique to one isolate. spa type t051 was found in 18 isolates; t001 and t037 in 13 isolates each; t008 in 8; t004 in 5; t018 and t030 in 4 each; t009 and t022 in 3 each; and t015, t032, t038, and t045 in 2 isolates each.

MLST/SCCmec typing. Seventeen MLST STs were resolved, the most common of which was ST247, shared by 23 isolates (Table 1). Six isolates were not typeable using published primers recommended for analysis of SCCmec (25, 31) and may therefore represent novel types of this element. Combined MLST and SCCmec typing resolved 24 MRSA clones, which included five STs with >1 type of SCCmec element (Table 1). The common clone names that have been used in other published studies of MRSA epidemiology are also shown in Table 1.

DI. Table 2 details the discriminatory power of PFGE, MLST, MLST/SCCmec, and spa typing for the 98 MRSA isolates of the HARMONY collection. PFGE pattern analysis gave a DI of 99.5% (95% CI, 99.1 to 99.9), indicating that when two isolates were chosen at random they would have different patterns 99.5% of the time. The four other methods gave lower levels of discrimination of between 88.7% and 92.1%, with overlapping 95% CIs. spa typing was the second most discriminatory method, followed by PFGE type using the ≤3-band difference criterion, and then MLST/SCCmec typing, followed by MLST alone.


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  		TABLE 2. Resolution of typing methods for MRSA

Comparison of clonal assignments. MLST/SCCmec typing identified 10 clones containing at least two isolates. They represent the majority of pandemic nosocomial MRSA clones described in the scientific literature (17) (Tables 1 and 3). ST247-MRSA-I, commonly referred to as the Iberian clone (38), was the most frequently represented clone in this study, with isolates coming from seven countries (Table 1). Twenty-one of 23 isolates belonging to the ST247-MRSA-I clone were of PFGE type 3, but PFGE type 3 isolates were not exclusive to this clone, as three PFGE type 3 isolates were ST8-MRSA-IV. However, ST247 and ST8 are closely related, both belonging to CC8. Two isolates of ST247-MRSA-I were PFGE type 4. Of the 21 ST247-MRSA-I isolates, 18 had the same spa type (t051) and three were unique (t008, t052, and t200).


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  		TABLE 3. PFGE and spa sequence typing subdivisions of MLST/SCCmec clones

PFGE was the most discriminatory technique, followed by spa typing and then PFGE using ≤3 bands difference, although the last two methods differed by very little in their discriminatory powers (Table 2). MLST and MLST augmented with SCCmec typing were also very similar in their discriminatory powers due to the fact that only ST5 isolates were found with different SCCmec elements (disregarding SCCmec nontypeables). However, study isolates were not randomly selected, as some participating centers submitted several PFGE variants of their main types. There was perfect concordance between MLST CC and PFGE analysis at the 65% level (data not shown), and this might be explored as a way of designing a nomenclature (it has been adopted by some member of the HARMONY group).

spa sequence types were not shared between isolates from different CCs (Table 1). The same spa type, however, was shared by different clones in two cases: t008 was present in ST247-MRSA-I and ST8-MRSA-IV (both CC8), and t001 was found in both ST228-MRSA-I and ST5-MRSA-I (both CC5).


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DISCUSSION
 
The choice of typing method used to investigate the epidemiology of MRSA should primarily be dictated by the environment and/or timescale being examined and the aims of the investigation. For a single MRSA outbreak in one hospital, for example, a highly discriminatory typing method that could differentiate between index case-associated isolates and isolates that were not associated with the index case would be required. As a short timescale is being examined, little genomic variation would be expected to occur in index case-associated isolates during the course of the outbreak, and minimal data interpretation would be required. PFGE typing has been the method applied most commonly in such scenarios. Interpretation of the banding patterns generated by PFGE was refined by the suggested guidelines of Tenover et al. (44), which allow variant PFGE patterns to be included in outbreak assignments. These criteria are biologically based and validated in outbreak investigations (7, 42) and have been used in numerous studies of MRSA epidemiology. However, they should only be used for short-term outbreak investigation and need validation outside of such scenarios (42).

An approach recommended for more widespread epidemiological typing is to initially use PFGE and then select isolates for clonal confirmation by SCCmec analysis and MLST. spa typing could be used instead of PFGE if sequencing capacity and expense are not issues. spa typing provides data that are as concordant with the MLST designation as PFGE, but the use of spa typing does not obviate the need for SCCmec analysis to determine the clonal designation of an isolate.

A potential problem with spa typing is that it involves sequencing of only one small region of the chromosome, which is subject to recombination between unrelated clones. This could result in isolates exhibiting the same spa type when they are shown to be unrelated by other methods. PFGE and MLST both investigate multiple locations around the chromosome. However, spa typing and MLST are portable and can be compared directly using global databases.

Potential difficulties may arise when using PFGE to analyze epidemiological scenarios involving large numbers of study centers or long timescales. PFGE has been used in many studies to show the international spread of MRSA, and frequently the criteria used for microepidemiological analyses have been employed. SmaI PFGE patterns of MRSA clones are known to change considerably during transmission of some strains over the course of years. In a number of cases, isolates assigned to different clones by PFGE have been shown to be genetically highly related using MLST. These limitations should not hinder the continued development of networks of centers using methods such as PFGE and spa typing, as long as examples of each genotype are assigned to MRSA clones using MLST and SCCmec typing.


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ACKNOWLEDGMENTS
 
This paper is dedicated to the memory of Névine El Solh.

M.C.E. is a Royal Society University Research Fellow. This work was funded by the Wellcome Trust (grant GR073363 to M.C.E.) and a EUDGXII grant (contract no. BMH4-CT96b) to B.D.C. (project leader).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Disease Epidemiology, Imperial College, London, United Kingdom. Phone: 44 207 5943417. Fax: 44 207 594 3693. E-mail: m.c.enright{at}imperial.ac.uk Back

{triangledown} Published ahead of print on 11 April 2007. Back


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Journal of Clinical Microbiology, June 2007, p. 1830-1837, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02402-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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