Recognition of SCCmec Types According to Typing Pattern Determined by Multienzyme Multiplex PCR Amplified Fragment Length Polymorphism Analysis of Methicillin Resistant Staphylococcus aureus

0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.12.6042–6047.2005

Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Recognition of SCC

mec

Types According to Typing Pattern

Determined by Multienzyme Multiplex PCR-Amplified

Fragment Length Polymorphism Analysis of

Methicillin-Resistant

Staphylococcus aureus

Anneke van der Zee,

* Max Heck,

Marcel Sterks,

Airien Harpal,

Emile Spalburg,

Leonard Kazobagora,

and Wim Wannet

Laboratory of Medical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands,1_{and Diagnostic Laboratory for Infectious}

Diseases and Perinatal Screening, National Institute of Public Health and the Environment, Bilthoven, The Netherlands2

Received 29 July 2005/Accepted 10 September 2005

Multienzyme multiplex PCR-amplified fragment length polymorphism (ME-AFLP) typing is a reliable and simple method for typing of bacterial species. In this study we analyzed two well-documented strain collections

ofStaphylococcus aureusand compared ME-AFLP typing results with results of various other typing methods.

The discriminatory power of ME-AFLP was found comparable to pulsed-field gel electrophoresis, and typing results were highly concordant. ME-AFLP typing presents a suitable method for prescreening of large strain collections. Furthermore, the obtained typing patterns were found to cluster according to the staphylococcal cassette chromosome mec types of the strains.

Staphylococcus aureus is the most significant pathogen in-volved in nosocomial infections. A rapid epidemiological typ-ing system is needed for the identification of outbreaks and to monitor spread. Some methicillin-resistantS. aureus(MRSA) strains are known to spread more easily and are designated epidemic MRSA, while other strains lack this capacity and are usually associated with sporadic cases. In The Netherlands strict rules for containment of MRSA-infected patients are followed. Due to the policy of “search and destroy,” MRSA is not endemic in The Netherlands (24). Pulsed-field gel electro-phoresis (PFGE) is regarded as the gold standard for epide-miological typing of MRSA, and a new harmonized protocol for PFGE has been developed enabling the establishment of a European web-based database for comparison of intercenter MRSA PFGE patterns (10). However, PFGE is a time-con-suming and tedious method and therefore is not best suited for use in the hospital clinical microbiology laboratory and in large-scale studies of outbreaks. Several alternative typing methods have been published recently in order to replace PFGE. These include multilocus sequence typing (12, 22), MecA-associated variable element genotyping (4, 15), fluores-cent amplified fragment length polymorphism (5), binary typ-ing (23), variable number of tandem repeat analysis (14), and others (1, 2, 13). In our laboratory we developed multienzyme amplified fragment length polymorphism (ME-AFLP) for typ-ingKlebsiellaspp. (20) and various other gram-negative bacte-ria. This method relies on the amplification of selections of restriction fragments which represent a total chromosomal im-age. Since this broadly applicable method would also be very convenient for typing of MRSA we investigated the

perfor-mance of ME-AFLP typing of MRSA in comparison with PFGE. Our aim was to be able to use ME-AFLP typing for prescreening MRSA strains and thus limit the number of strains to be subjected to PFGE. We used the two well-docu-mented strain collections (17, 18, 19) that were described pre-viously with various different other pheno- and genotyping methods.

MATERIALS AND METHODS

Bacterial strains.The present study includes the use of two well-defined MRSA strain collections. The first set consists of 47 isolates which have been described in great detail (17–19). Strains that could not be revitalized and were not included in the present study were SA5, -8, -10, and -16; SB3, -5, -15, and -18; and SC3, -5, -7, -13, and -17. Outbreak related isolates were SA1, -2, -3, -9, -13, -14, -15, -17, and -19 (outbreak NH1/2) and SB2, -4, -6, -11 (outbreak II), -10, -12, -19, and -20 (outbreak I) (19). SC isolates were all outbreak related (III and IV), except SC8. The second strain collection consisted of 32 epidemiologically un-related MRSA isolates from a Dutch surveillance study, 19 of which were clas-sified as epidemic MRSA (2). In addition, 14 well-documented epidemic MRSA strains from the United Kingdom were included (2, 19).

ME-AFLP procedure.Chromosomal DNA was isolated fromS. aureusstrains essentially as described elsewhere (1). After addition of 5␮l of RNase (1 U; Sigma), the DNA concentrations were measured by using the GeneQuant spec-trophotometer (260 nm; GeneQuant; Pharmacia Biotech, United Kingdom). The restriction-ligation reaction was performed with 500 ng of DNA in a final reaction volume of 30␮l. It contained restriction enzymes EcoRI, PstI, XbaI, and NheI (20 U each; Roche Molecular Biochemicals), 3␮l of T4 ligase buffer, 1 U of T4 ligase (Roche Molecular Biochemicals), and 20 pmol of each adaptor oligonucleotide (Table 1). The restriction-ligation mixture was incubated for 3 h. at 37°C. Subsequently, the DNA was precipitated with 2.5 M ammonium acetate in 100␮l of absolute ethanol (⫺20°C). After the DNA was washed with 70% ethanol, it was resuspended in 100␮l of Tris-EDTA buffer and stored at 4°C.

PCR’s were performed with Ready-to-Go beads (Amersham Pharmacia, Little Chalfont, United Kingdom) after the addition of 1␮l of MgCl2(25 mM), 10 pmol of each primer (Table 1), and 2␮l of template DNA (ca. 100 ng) in a final volume of 25␮l. Three PCRs were performed with different primer sets: Pxn/ Pst-ag, Pn-g/Pst-ag/Pst-at, and Px/Pst-ag (Table 1). The reaction mixtures were preheated for 4 min at 94°C in a DNA thermocycler (Perkin-Elmer GeneAmp PCR System 2400). Thirty-three amplification cycles of 1 min at 94°C, 1 min at 60°C, and 2.5 min at 72°C were performed.

* Corresponding author. Mailing address: Laboratory of Medical Microbiology, St. Elisabeth Hospital, PO Box 747, 5000AS Tilburg, The Netherlands. Phone: 0031-13-5392653. Fax: 0031-13-5441264. E-mail: [email protected].

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Analysis of banding patterns.Analysis of 10␮l of each PCR product was performed by agarose gel electrophoresis on a 1.5% agarose gel (MP Agarose; Roche Molecular Biochemicals). Marker DNA (100-bp ladder; Gibco-BRL/Life Technologies, Paisley, Scotland) was loaded after every four samples. Analysis of banding patterns on gel images was performed with the software program Gelcompar II included in Bionumerics (Applied Maths, Kortrijk, Belgium). The gel images were imported in the gel analysis software Bionumerics. The images were fitted in the framework bordered by the upper 2,000-bp and lower 100-bp marker bands. Cluster analysis of the fingerprints was performed with the un-weighted pair group using arithmetic averages, and similarities between ME-AFLP patterns were calculated with the Pearson product-moment correlation coefficient.

PCR analysis of SCCmec types.PCR was performed with the following primer pairs: CIF2 (50 pmol), KDP, MECI, DCS, RIF4, RIF5, IS431/pUB110, IS431/ pT181 (80 pmol each), and 20 pmol of MECA primers as described previously (11). PCR was performed with HotstarTaq polymerase (QIAGEN, Hilden, Ger-many). The reaction mixtures were preheated for 15 min at 95°C in a DNA thermocycler (Perkin-Elmer GeneAmp PCR System 2400). Thirty-five amplifi-cation cycles of 30 s at 95°C, 30 s at 53°C, and 1 min at 72°C were performed. The PCR was completed with a final round of 7 min at 72°C. PCR products were analyzed on a 3% agarose gel.

RESULTS

ME-AFLP typing of MRSA strains. The strain collection SA1 to -20, SB1 to -20, and SC1 to -20 comprised of sporadic isolates and epidemiologically related isolates that have been genotyped by using various methods (17–19). ME-AFLP typ-ing of these strains was carried out with three combinations of primers. The results were compared to those of PFGE typing (Fig. 1). Outbreak related strains were found clustered into four distinct groups—A, B, C, and D—of isolates (Fig. 1). Cluster analysis of single-set primer generated patterns showed the same clusters (not shown) as were obtained when cluster-ing was based on three fcluster-ingerprint types. Strains involved in outbreaks NH1/NH2, I/II, III, and IV, are clustered in groups A to D, respectively. Cluster C comprises mainly PFGE type A, while in cluster D PFGE type B is found. Strain SC6 (cluster C), although designated to PFGE type B, most likely is of type A as was also found with rep-PCR typing (19). PFGE type A is found in several clusters of strains as shown before by using other typing methods (17). Minor band differences within PFGE patterns as reflected in the type designations B/B1 and K2/3 are also found in corresponding ME-AFLP types of SC18/SC8, and SA9/SA19, respectively, but only when primers

Png/Pst-ag/Pst-at were used. For example, strains SC8 and SC18 showed an identical pattern when analyzed with primers Px⫹Pst-ag but clearly differed when the other two primer sets were used.

All distinctive ME-AFLP types were selected among the strain collection SA/B/C1 to -20 and clustered (Fig. 2). De-pending on the cutoff of 90 or 95% 13 or 17 distinctive ME-AFLP types, respectively, were found. The total number of different PFGE types among the same strain collection was 11 (17). Overall, there is good concordance between the results obtained with PFGE and ME-AFLP.

Comparison of ME-AFLP typing results with REP typing (19) showed that the results are highly consistent. For example, cluster D of strains in Fig. 1 contains isolates that were not involved in outbreaks but had the same Rep-PCR type as the strains that were; isolates SB8 and SC8 had the same Rep-PCR types of strains involved in outbreak IV. The ME-AFLP types are fully supportive of this finding.

ME-AFLP typing of epidemic and nonepidemic MRSA strains.Typing of epidemic and nonepidemic MRSA isolates revealed two main clusters of isolates: E and F (Fig. 3). The majority of epidemic strains were found to belong to the larger cluster. Strains within this cluster differed from the other clus-ter mainly by the presence of a 800-bp band. In order to find out whether this was a specific virulence trait, we isolated this band from the gel for sequence analysis. The DNA fragment was found to represent the PstI-NheI part of theorfXgene described by T. Ito et al. (NCBI AB014440) (6). Alignments with otherS. aureussequences revealed mutations in the NheI site resulting in the absence of this fragment size among the other MRSA genotypes. Although this sequence is located just downstream of the staphylococcal chromosomal cassette (SCCmec), it may reflect a particular SCCmectype.

SCCmectyping.In order to investigate whether ME-AFLP types might represent certain SCCmectypes, we subjected the SCCmec type strains I to IV (kindly provided by T. Ito) to ME-AFLP analysis. Four clinical isolates of MSSA were also typed by using ME-AFLP. Cluster analysis (Fig. 3) showed four groups—E, g, h, and i—and each group (except i) showed a high degree of similarity to one of each SCCmectype strains (except SCCmectype IVstrain). To confirm whether the

SC-TABLE 1. Restriction enzymes, adaptors, and primersb

Restriction enzyme Recognition site Adaptor oligonucleotidesa Primer

Sequence Name

XbaI 5⬘-T2CTAG A A GATC2T

5⬘-CTAGTACTGGCAGACTCT

3⬘-ATGACCG

5⬘-AGAGTCTGCCAGTACTAGA

5⬘-AGAGTCTGCCAGTACTAG

Px Pxn 5⬘-AGAGTCTGCCAGTACTAGCG Pn-g

NheI 5⬘-G2CTAG C C GATC2G

5⬘-CTAGTACTGGCAGACTCT

3⬘-ATGACCG

5⬘-AGAGTCTGCCAGTACTAGA

5⬘-AGAGTCTGCCAGTACTAG

Px

5⬘-AGAGTCTGCCAGTACTAGCG Pn-g

PstI 5⬘-C TGCA2G G2ACGT C

5⬘-CTCGT AGACTGCGTACATGCA

3⬘-CATCTGACGCATGT

5⬘-GACTGCGTACATGCAGAG 5⬘-GACTGCGTACATGCAGAT

Pst-ag Pst-at

EcoRI 5⬘-G2AATT C C TTAA2G

5⬘-AATTGGT ACGCAGTCTAC

3⬘-CCATGCGT CAGATGCTC

a_{One of the oligonucleotides of each adaptor was phosphorylated at its appropriate 5⬘site.} b_{Selective bases are indicated in boldface.}

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FIG. 1. Dendrogram of genotyping results. Clusters with homology⬎90% are designated A to D. Primer pairs used are indicated above typing patterns.

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Cmectype could be inferred from the ME-AFLP typing pat-tern, we selected two to four representative isolates from each cluster and performed the multiplex PCR for the SCCmec

typing method described by Oliveira et al. (11). The results (not shown) confirmed that the clustering of ME-AFLP types was according to the SCCmectypes of isolates.

DISCUSSION

PFGE is the gold standard in typing of S. aureus, and a web-based database has been recently set up to compare in-tercenter typing patterns that allow tracing of isolates (10). However, for typing of large strain collections this method is less suitable since it is rather time-consuming and tedious. In recent years many reports have focused on other typing meth-ods (13, 14, 16, 22). Theoretically, methmeth-ods based on sequence analysis that produce unambiguous numerical data are the most suitable for interlaboratory exchange of typing results. However, these methods are very expensive and consequently not applicable for the clinical laboratory.

ME-AFLP is very easy to perform and is used for typing of various bacterial species in our laboratory.

The reproducibility of ME-AFLP typing of MRSA was 100%. Restriction-ligation is controlled by inclusion of a con-trol strain, and negative concon-trols are included for concon-trol of contamination by previously amplified fragments. The method is very suitable to be used for prescreening of MRSA suspected outbreak strains in the clinical microbiology laboratory. Since ME-AFLP is more discriminative than PFGE, identical results

from ME-AFLP prescreening are very likely to represent iden-tical PFGE types. In contrast to REP-PCR typing (19), which generates DNA fragments with a single primer, ME-AFLP typing offers the possibility for direct sequencing of discrimi-native bands between strains. In the present study we se-quenced the discriminant band of cluster E in Fig. 3, which was found to present part of theorfXgene. This band seems to be a marker for SCCmectype I isolates. SCCmecsequences are composed of various genes and mobile genetic elements as insertion sequence elements and transposons (7, 8). Depend-ing on the composition and organization, five discrete SCCmec

types have been described (7, 9). Most epidemic strains (20 of 29 [69%]) analyzed in the present study were found among SCCmec type I isolates, whereas all of the SCCmec type II isolates except one were nonepidemic (13% epidemic), and SCCmec type III isolates comprised 38% of the epidemic strains. Type IV SCCmec, although it clusters with type II isolates, probably is not present among this strain collection, since all strains were hospital isolates. SCCmec type IV is predominantly found among patients with community-ac-quired MRSA (9).

Recently, a PCR-based detection method of MRSA in clin-ical material was described (3). This method uses various re-versed primers based on SCCmecsequences inserted into the bacterial chromosomal attachment siteattBand forward prim-ers located in theorfXgene.

Further study is needed with typing of strains and sequenc-ing of discriminant bands in typsequenc-ing patterns of strains havsequenc-ing variant SCCmectypes. A genotyping method that may support

FIG. 2. Dendrogram of 13 or 17 discriminate genotypes, respectively, depending on homology cutoffs of 90% or 95%, as indicated. Primer pairs used are indicated above typing patterns.

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FIG. 3. Clustering of genotypes of epidemic (e) and nonepidemic MRSA. Clusters E, g, h, and i are indicated and comprise, respectively, strains with SCCmectype I, SCCmectype II, SCCmectype III, and MSSA strains. The primer pair used is indicated above the typing pattern.

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the identification of novel SCCmec types may be helpful for inclusion of all SCCmecvariants in future PCR-based detec-tion of MRSA.

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