J. Clin. Microbiol. 1998 Hookey 1083 9

8/11/2019 J. Clin. Microbiol. 1998 Hookey 1083 9

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1998, 36(4):1083.J. Clin. Microbiol.

John V. Hookey, Judith F. Richardson and Barry D. Cookson

the Coagulase GenePolymorphism and DNA Sequence Analysis ofBased on PCR Restriction Fragment Length

Staphylococcus aureusMolecular Typing of

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JOURNAL OFCLINICALMICROBIOLOGY,0095-1137/98/$04.000

Apr. 1998, p. 10831089 Vol. 36, No. 4

Copyright 1998, American Society for Microbiology

Molecular Typing of Staphylococcus aureus Based on PCRRestriction Fragment Length Polymorphism and DNA

Sequence Analysis of the Coagulase GeneJOHN V. HOOKEY,1* JUDITH F. RICHARDSON,2 ANDBARRY D. COOKSON3

Molecular Biology Unit, Virus Reference Division,1 Staphylococcal Reference Section,2 and Laboratory of HospitalInfection,3 Central Public Health Laboratory, Colindale, London NW9 5HT, United Kingdom

Received 23 December 1996/Returned for modification 21 April 1997/Accepted 17 December 1997

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of thecoagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Thosestrains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCRproducts were digested with AluI and CfoI, and the fragments were separated by gel electrophoresis. Tendistinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10propagating strains (PS) of methicillin-sensitiveS. aureus(MSSA) examined. RFLP patterns 1, 2, and 3 werespecific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen witha heterogeneous collection of strains, together with drug-resistant forms ofS. aureus isolated in Europe andfour propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolateand PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germanygave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains(RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively.The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for bothstrands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distancesbetween isolates.

Resistance to methicillin was first described for Staphylococ-cus aureus in 1960, shortly after the introduction of the druginto clinical practice (20). Since then, methicillin-resistant S.

aureus(MRSA) has become a widely recognized cause of mor-bidity and mortality throughout the world (16).

Accurate and rapid typing of S. aureus is crucial to thecontrol of infectious organisms (37), and numerous methodshave been described elsewhere (8, 19, 28). A bacteriophagetyping scheme forS. aureushas been agreed on internationallysince 1951, but although it remains a cost-effective approach totyping the large number of referred isolates, it has some lim-itations. The reagents are not commercially available, and insome instances and certain parts of the world, MRSA strainsare nontypeable with phages (5). Of the other methods, plas-mid analysis has drawbacks, since the plasmids may be absentfrom isolates, may vary in size, or may be readily lost (18), andantibiogram schemes are often uninformative, as many strainsare multiply drug resistant (6).

Recently, several investigators have described DNA-basedtechniques for typing strains (13, 17, 34, 40). Pulsed-field gelelectrophoresis (PFGE) is now recognized as being the most

discriminatory method for gene typing strains ofS. aureus, andit has been used to investigate nosocomial outbreaks (4, 39).However, PFGE is costly and technically complex and lacks anagreed criterion for the interpretation of banding patterns (4,9). Furthermore, for most national reference centers, it is not

practical to use PFGE to type the large numbers of referredisolates.

In the 1980s, epidemic methicillin-resistant S. aureus 1(EMRSA-1) was the principal MRSA strain identified byphage typing in England (27). By 1986, a further 13 EMRSA

strains were recognized (EMRSA-2 to EMRSA-14). Recently,EMRSA-15 and EMRSA-16 were described (12, 31). Cur-rently, the major United Kingdom EMRSA strains are 3, 15,and 16. In 1996, these comprised approximately 50% of theisolates referred for phage typing to our staphylococcus refer-ence service (1). However, some strains phage typed weakly ornot at all, even at a 100 routine test dilution (RTD). Toconfirm phage type and/or to answer particular epidemiologi-cal questions, PFGE has been used periodically, and yet analternative rapid and cost-effective confirmatory test would beof value in clinical and reference centers.

Coagulase is produced by all strains of S. aureus (24). Itsproduction is the principal criterion used in the clinical micro-biology laboratory for the identification ofS. aureusin humaninfections, and it is thought to be an important virulence factor.

The sizes and DNA restriction endonuclease site polymor-phisms at the 3 coding region of the coagulase gene have beenutilized in PCR-based restriction fragment length polymor-phism (RFLP) analysis ofS. aureus (15, 25, 26, 29, 38, 39).

We describe here a coagulase gene-based PCR RFLP tech-nique that differentiated among the major current UnitedKingdom EMRSA strains, i.e., EMRSA-3, EMRSA-15, andEMRSA-16, as well as minor epidemic strains. The PCR prim-ers were designed to encompass the entire 3 repeat elements,thereby avoiding the variable regions within the coagulasegene. Comparisons between DNA sequence data from the 3

variable region of the coagulase gene then allowed phyloge-

* Corresponding author. Mailing address: Molecular Biology Unit,Virus Reference Division, Central Public Health Laboratory, Colin-dale, London NW9 5HT, United Kingdom. Phone: (44) 181 200 4400.Fax: (44) 181 200 1569. E-mail: [email protected].

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netic groups to be identified and permitted inferences to bedrawn about some of the lineages ofS. aureus.

MATERIALS AND METHODS

Bacterial strains. Bacterial strains were examined under a code that wasbroken upon completion of the analysis of the results. Eighty-five S. aureusstrains representing EMRSA-1 to -16, including the original Jevons strain

(NCTC 10442) and two duplicates, together with 10 methicillin-sensitive S.aureus propagating strains (PS), were studied (Table 1) (2, 12, 31, 33, 34, 41).Negative controls comprising three coagulase-negative staphylococcal species, S.

epidermidis(NCTC 11047), S. haemolyticus(NCTC 11042), and S. saprophyticus(NCTC 7292), were also included. Bacteria were grown overnight on blood agarplates at 37C, in an aerobic atmosphere. Stock clinical cultures were maintainedin blood glycerol (16% [vol/vol]) broth on Preserver Beads (Technical ServiceConsultants, Heywood, Lancashire, United Kingdom) at 70C.

Bacteriophage typing. This was done by the method described by Blair andWilliams (5) at the RTD and a 100 RTD with the current set of internationalphages (3) and supplementary phages (32).

Enterotoxin production.Isolates were examined for the production of entero-toxins A, B, and C and toxic shock syndrome toxin 1 (TSST-1) by reverse passivelatex agglutination according to the manufacturers instructions (OxoidUnipath).

Protein A production. A rapid, semiquantitative dot blot analysis was em-ployed (33).

Urease production.Conventional urea slopes were inoculated with 100 to 200l of an overnight broth culture with a Pasteur pipette to ensure that the slope

was i noculated evenly. Slopes were incubated at 37C for up to 7 days (11).DNA preparation.Two methods were used to prepare DNA from strains ofS.

aureus.(i) Lysostaphin-sodium chloride-cetyltrimethylammonium bromide. Chromo-

somal DNA was isolated as described by Jones (21), with modifications. Thebacteria were harvested from one-half the area of a blood agar plate, suspendedin 1 ml of TE-glucose (25 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0], 1.0%[wt/vol] D-glucose), and centrifuged at 7,500 g for 5 min. The cells wereresuspended in 100 l of lysostaphin (1 mg/ml in TE-glucose; Sigma)50l oflysozyme (50 mg/ml in TE-glucose; Sigma) and incubated at 37C for 1 h. Eightymicroliters of NaCl-cetyltrimethylammonium bromide solution (0.7 M NaCl,10% [wt/vol] cetyltrimethylammonium bromide; Sigma) was added with mixingand incubated at 65C for 10 min. Sodium chloride (100 l of a 5 M stocksolution), sodium dodecyl sulfate (30 l of 10% [wt/vol] sodium dodecyl sulfate;Sigma), and proteinase K (4 mg of proteinase K; Sigma) were added with mixingand incubated at 55C for 30 min. The lysate was extracted with equal volumesof phenol-chloroform, and the DNA was precipitated from the aqueous phase

with 1 volume of isopropanol and resuspended in 100 l of sterile distilledPCR-quality water (Sigma). The DNA concentration was determined by UV

TABLE 1. Strains ofS. aureusstudied and their phenotypic features

Strain (isolate [no. of isolates])e Phage typ e(s) Su pplemen tary phage(s) Toxin

productiona Ureaseb Protein Ac

EMRSA-1 [4] 85/88A/932 616/617/620/622/626/630 A EMRSA-1 (CRF 616 [1]) 85/88A/932 620/622/617/626/630 A EMRSA-1 (QC 21 [1]) 85 620/622/617/626/630 A EMRSA-2 [6] 80/85/90/932 616/617/622/626/630 A EMRSA-2 (QC 15 [1]) 80/85/90/932 616/617/622/626/630 A EMRSA-3 [10] 75/83A/932 618/620/623/629 -ve EMRSA-4 [4] 85/90/932 623 A EMRSA-5 [3] 77/84 618/620 A, B, C EMRSA-6 [4] 90/932 NTd A EMRSA-7 [2] 85inh NT A, C EMRSA-8 [3] 83A/83C/932 620/617/622/630 -ve EMRSA-9 [2] 77/84/932 620/622/617/626/630 -ve EMRSA-10 [2] 77/83A/29/75/85 626/617/618/622/630 A, B EMRSA-11 [2] 84 617/618/620/622 A EMRSA-12 [3] 75/83A/83C/932 617/622/629 -ve EMRSA-12 (QC 03 [1]) 75/83A/54/75 617/622/629 -ve EMRSA-13 [2] 29/83C/932 620/629/630 -ve EMRSA-14 [2] 29/6/47/54/90/932 629/630 -ve EMRSA-15 [11] 75 NT C EMRSA-15 (91/11046 [1]) 75 NT C EMRSA-16 [10] 29inh/52inh/75/77/83A 618 A, TSST-1 EMRSA-16 (K 06 [1]) 29inh/52inh/75/77/83A 618 A, TSST-1 EMRSA-16 (QC 01 France [1]) 77/84 Not tested Not tested Not tested Not testedEMRSA-16 (QC 07 France [1]) 77/84 Not tested Not tested Not tested Not testedEMRSA-16 (211 Spain [1]) 29/77/84/932 618 A EMRSA-16 (212 Spain [1]) 29/77/84/932 NT A EMRSA-16 (QC 04 Germany [1]) 84 618/620 A EMRSA-16 (94/14103 Germany [1]) 54 625 Not tested Not tested Not testedNCTC 10442T [1] 47/53/54/75/77/84/85 616/617/618/622/623/625/626/629/630 B Not tested PS 6, III, NCTC 8509 [1] 6/47/53/54/75/83A 616/617/620/622/623/626 -ve Not testedPS 29, I, NCTC 8331 [1] 29 NT C, TSST-1 Not testedPS 42E, III, NCTC 8357 [1] 42E NT -ve Not testedPS 52, I, NCTC 8507 [1] 52 NT -ve Not testedPS 53, III, NCTC 8511 [1] 53/54/75/77/84/85 617/622 A, B Not testedPS 71, II, NCTC 9315 [1] 3C/55/71 NT TSST-1 Not testedPS 75, III, NCTC 8354 [1] 53/75/77/84/85 618/626 B Not testedPS 94, V, NCTC 10970 [1] 94/96 NT B Not testedPS 95, misc, NCTC 10971 [1] 95 NT B Not testedPS 96, V, NCTC 10972 [1] 94/96 NT B Not tested

a Toxins A, B, and C and/or TSST-1; -ve, no toxin produced.b , negative; , positive; , strongly positive.c , weakly positive; , positive; , strongly positive.d NT, non-phage-typeable.e PS, methicillin-sensitiveS. aureuspropagating strain for the international set; I, II, III, and V, phage groups; misc, miscellaneous phage group; T, type strain (20)

obtained from the National Collection of Type Cultures, Public Health Laboratory Service, London, United Kingdom.

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spectrophotometry at A260, and the extract was stored at 4C. Extraction timewas 1 to 2 days. Approximately 50 to 100 ng of DNA was taken for PCRamplification.

(ii) Chelex extraction.A half-loopful (approximately 25 l) of bacterial growthwas removed from a blood agar plate, suspended in 1 ml of TE-glucose, andcentrifuged at 7,500 g for 5 min. The cells were resuspended in 100 l oflysostaphin solution plus 50 l of lysozyme and incubated at 37C for 1 h. Onehundred microliters of a 5 M NaCl solution and 30 l of proteinase K wereadded, and the lysate was incubated at 55C for 30 min. Five microliters of thelysate was diluted in 45 l of PCR-quality water. Ten microliters of Chelex 100

resin (sodium form; 100/200 mesh size; final concentration, 5% [wt/vol]; pH 7.0;Sigma)Nonidet P-40 (Sigma; final concentration, 0.4% [vol/vol]) solution wasadded and incubated at 55C for 30 min. The lysate was then overlayered with 2drops of mineral oil (Sigma) and heated at 99C for 20 min to denature theproteinase K. One microliter of lysate was taken for PCR amplification.

PCR amplification of the coagulase (coa) gene.An oligonucleotide primer pairwas designed by usin g the p rogram Primer (C. W. Dieffenbach, Department ofSurgery and Pathology, Uniformed Services University of the Health Sciences,Bethesda, Md.). To encompass the entire 3 repeat elements and avoid the

variable regions within the coagulase gene primer sequences, 5ATA GAG ATGCTG GTA CAG G3 (1513 to 1531; nucleotide numbering according to the workof Kaida et al. [23]; MRSA 213, accession no. X16457) and 5 GCT TCC GATTGT TCG ATG C3 (2188 to 2168) were chosen. Each amplification in sterilethin-walled glass capillaries (Idaho Technologies, Idaho Falls, Idaho) comprisedDNA template, 75 pmol of each primer, 50 M (each) deoxynucleoside triphos-phates (dATP, dCTP, dGTP, and dTTP), 1 buffer (Gibco BRL), 3.0 mMMgCl2, 1 bovine serum albumin (250 g/ml; BioGene Limited, Kimbolton,Bedfordshire, United Kingdom), and 12.5 U ofTaq DNA polymerase (GibcoBRL). Filter-sterilized (0.22-m pore size) PCR-quality water (Sigma) was

added to a final volume of 50 l. Thermal cycling took place on a hot-airRapidcycler (Idaho Technologies) following an initial denaturation at 94C for45 s. The cycling proceeded for 30 cycles of 94C for 20 s, 57C for 15 s, and 70Cfor 15 s with a final step at 72C for 2 min. The size of the PCR product (5- laliquot) was determined by comparison to the X174 DNA/HaeIII markers(Bio-Rad Laboratories) by electrophoresis on 1.0% (wt/vol) agarose gels.

DNA restriction endonuclease analysis of the PCR-amplified coagulase gene.Approximately 500 ng (7 to 10 l) of PCR product was digested with 2 U ofrestriction endonuclease (AluI,CfoI,HinfI, andSacI; Boehringer Mannheim) at37C for 1 h 30 min. Twenty microliters of digested PCR product was analyzedby electrophoresis on 2.75% (wt/vol) agarose gels (FMC BioProducts).

DNA sequencing of the PCR-amplified coagulase gene. The 875- to 550-bpPCR-amplified fragments were purified according to the method of Zhen andSwank (42). PCR products were directly sequenced on both overlapping strands

with DyeDeoxy Terminator kits (Applied Biosystems-Perkin-Elmer) accordingto the manufacturers protocol with a 377 DNA sequencer. The primers used

were those for PCR amplification.Data analysis. Sequences were aligned against S. aureus 213 (accession no.

X16457 [23]) and 8325-2 (accession no. Z33404 [30]) by using the program

Multalin (10) (Cherwell Scientific Publishing Limited, Oxford, United King-dom). Those base positions that could not be aligned unambiguously wereremoved. A total of 530 nucleotide bases for 28 strains comprised the finalalignment; this is available from us on request. Evolutionary analyses werecarried out with PHYLIP (J. Felsenstein, University of Washington, Seattle).The reliability of tree nodes was assessed by analyzing 1,000 data sets. Pairwisedistances between sequences were inferred under the Jukes and Cantor (22)one-parameter model. Trees were constructed by using neighbor joining(NEIGHBOR [35]) and the algorithm of Fitch and Margoliash (FITCH [14]). Amajority rule consensus tree was computed with the CONSENSE program. Thebootstrap percentages quoted in the legend to Fig. 3 are the percentages of timesthat a taxon to the right of that node occurred, and they provide some indicationof the stability of the branching order and the phylogenetic groupings.

RESULTS

Size variation in the 3 region of the coagulase gene. Withthe exception of the coagulase-negative strains,S. epidermidis

NCTC 11047, S. haemolyticus NCTC 11042, and S. saprophy-ticus NCTC 7292, all strains examined produced a PCR am-plicon. The four PCR products obtained were either 875 (10bp,n 2), 660 (20 bp,n 10), 603 (20 bp,n 10), or 547(15 bp, n 10) bp. All EMRSA-16 isolates gave a 547-bpproduct (Fig. 1A).

PCR RFLP patterns of the coagulase gene. PCR productswere digested with AluI or CfoI, and the resulting fragmentswere separated (Fig. 1 and 2). No changes were observed in thesizes of the coagulase gene PCR products after repeated strainsubcultivation (seven times) and DNA extraction. The mean

values (standard errors of the means) from within-gel errors

(n 3) for duplicated strains were 10 bp for those fragmentsformed on AluI or CfoI digestion (Fig. 2).

Ten distinct RFLP patterns were observed among the 95strains examined on AluI (Fig. 2A) and CfoI (Fig. 2B) diges-tion. Other enzymes specific for AT-rich DNA, such as HinfIandSacI, were less discriminatory (data not shown). The num-ber of fragments produced upon AluI digestion varied fromone (RFLP pattern 6) to four, and their sizes varied from 80 to

660 bp (Fig. 2A). Isolates Airedale 16 and PS 95 were notdigested withAluI (RFLP pattern 6 [Fig. 2A]). The number ofCfoI fragments varied from two (a doublet appears for RFLPpattern 5) to five, and their sizes varied from 60 to 400 bp (Fig.1 and 2B). The assignments of isolates to one of the 10 AluIandCfoI RFLP patterns were similar, except for five isolates ofEMRSA-2 (cf. Fig. 2A and B). These five isolates were char-acterized as belonging to RFLP pattern 5 on AluI digestion,and yet they fell into RFLP pattern 8 when digested with CfoI(Fig. 2B). The German isolate 94/14013 was also found inRFLP pattern 8 (cf. Fig. 2A and B). RFLP patterns 1, 2, and3 corresponded to strains of EMRSA-3, EMRSA-15, andEMRSA-16, respectively (Fig. 2). RFLP patterns 4 and 5 en-compassed a collection of isolates belonging to heterogeneousEMRSA strains (Table 1), and they accounted for approxi-

mately 50% of the isolates examined. They included five epi-demic isolates from France, Spain, and Germany and fourpropagating strains (PS) used in the international phage set.RFLP pattern 4 was given by EMRSA-1, -4, -7, -9, and -11; theGerman isolate QC04; and PS 52. RFLP pattern 5 was char-acteristic of the type strain NCTC 10442; EMRSA-2, -5, -6, -8,-10, -12, -13, and -14; the French and Spanish isolates; and PS6, PS 53, and PS 75 (Fig. 2). RFLP pattern 6 was given by theisolate Airedale 16 and PS 95. RFLP pattern 7 encompassedEMRSA-2 isolate 331, PS 95, and PS 96. Eight methicillin-sensitive propagating strains (PS) shared five patterns (RFLPpatterns 3, 4, 5, 6, and 7) with methicillin-resistant strains.Representatives of lytic groups III (34) (PS 42E) and II (34)(PS 71) gave unique RFLP patterns 9 and 10, respectively.

Comparison of coagulase gene sequences. The 3 variableregions of the coagulase gene were sequenced for 26 isolatesrepresenting the 16 United Kingdom EMRSA strains and eachof the 10 PCR RFLP patterns. Sequences were aligned, andpairwise distance measurements, based on 530 nucleotides foreach strain, were used in the construction of a consensus phy-logenetic tree (Fig. 3).

The same sequence similarities (percent S value) were ob-tained for two pairs of strains, EMRSA-16 isolates 6 and 27and EMRSA-6 isolate 486 and EMRSA-12 isolate 607. Twomajor phylogenetic clusters were found. An outlier group wasformed by two isolates (11 and 203) of EMRSA-9. The isolates11 and 203 had 99.3% sequence similarity and were distantlyrelated (nucleotide substitution rate of 1.1421 [Knuc]) to otherisolates examined (Fig. 3). On this analysis, a larger clustercomprising 26 isolates was defined at and above 76.7% S and

was bounded by EMRSA-15 isolate 558 and EMRSA-2 isolate

277. This cluster was subdivided into six groups (A, B, C, D, F,and G [Fig. 3]). Phylogenetic group B representing RFLPpatterns 2 and 5, composed of EMRSA-15 isolates 558 and 28and EMRSA-13 isolate 275, had sequence similarities betweenthe isolates of 95.3% S. Strain PS 71 (lytic group II) (34) andthe Airedale 16 isolate were distinct from each other (88.2%S)and from the isolate of group D (Fig. 3). Five representativesof RFLP pattern 4 formed a closely related group (D) at 98.9%S. PS 42E and the German isolate 94/14013 were separatedfrom each other and from neighboring group A strains. Isolate213, EMRSA-3 isolate 12, and EMRSA-14 isolate 587 hadsequence similarities of 98.2% S and 88.6% S, respectively.

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FIG. 1. Agarose gel electrophoresis of PCR-amplified coagulase genes from representatives ofS. aureus. (A) Uncut PCR-amplified coagulase gene. Lanes 1 and6, X174 restriction fragment DNA/HaeIII marker; lane 2, PS 71; lane 3, EMRSA-15 isolate 2; lane 4, NCTC 10442T (20); lane 5, EMRSA-16 isolate 6. (B)PCR-amplified coagulase gene digested with the DNA restriction endonuclease AluI. Lanes 1, 6, 11, and 14, X174 restriction fragment DNA/HaeIII marker; lanes2, 3, 4, and 5, RFLP patterns 1, 2, 3, and 4, respectively; lanes 7, 8, 9, and 10, RFLP patterns 5, 6, 7, and 8, respectively; lanes 12 and 13, RFLP patterns 9 and 10 (Fig.2B). (C) PCR-amplified coagulase gene digested withCfoI. Lanes 1, 6, 11, and 14, X174 restriction fragment DNA/HaeIII marker; lanes 2, 3, 4, and 5, RFLP patterns1, 2, 3, and 4, respectively; lanes 7, 8, 9, and 10, RFLP patterns 5, 6, 7, and 8, respectively; lanes 12 and 13, RFLP patterns 9 and 10, respectively (Fig. 2B).

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Most isolates comprising RFLP pattern 5 formed a group, G,that was related at and above the 97.5% S level. EMRSA-8isolate 279 had sequence similarity identical to that of NCTC10442. EMRSA-16 isolates 6 and 27 (RFLP pattern 3) andEMRSA-2 isolates 331 and 277 were contained within groupsC and F, respectively (Fig. 3). There was good congruencebetween the coagulase RFLP patterns and phylogenetic group-ings (Table 2).

DISCUSSION

The object of this study was to determine whether PCRRFLP patterns of the coagulase gene could be used to differ-entiate the major epidemic United Kingdom strains of MRSA.The coagulase genes from 95 isolates, representing predomi-nant United Kingdom EMRSA strains, the Jevons MRSAstrain, and propagating strains (PS), were amplified by PCR,and the products were digested with both AluI andCfoI. In thisstudy, the parallel use of two DNA restriction endonucleasesto digest the coagulase gene was beneficial in confirming the 10distinct RFLP patterns among S. aureus strains. In addition,PCRCfoI RFLP pattern analysis allowed the differentiation of

five EMRSA-2 isolates (Fig. 2B, RFLP pattern 8); this was notpossible with AluI.

Other authors have used PCR RFLP pattern analysis tostudyS. aureus, but only Tenover et al. (39) phage typed any ofthe strains. Furthermore, reference strains were not used, anddiffering PCR primers were employed (15, 25, 26, 29, 38, 39).It is therefore not possible to compare the results of this study

with those of previous coagulase PCR RFLP pattern analyses.The RFLP patterns 1, 2, and 3 were simple (three to four

bands) and unique and allowed the typing of the important

United Kingdom epidemic strains, EMRSA-3, EMRSA-15,and EMRSA-16 (Fig. 2). These EMRSA strains also clustered

within the phylogenetically distinct groups A, B, and C, respec-tively (Table 2) (Fig. 3). In the light of coagulase PCR RFLPand sequence comparisons, most isolates with RFLP patterns 4and 5 gave distinct groups D and G, respectively (Table 2) (Fig.2 and 3). Isolates of EMRSA-2 were exceptional, in that theyformed a single phylogenetic group (F) and yet gave two RFLPpatterns, 5 and 8 (Table 2).

Isolates of MRSA from France (QC01 and QC07) and Spain(211 and 212) have given similar patterns on ribotyping, PFGE(2, 34), and coagulase typing (this study) and are thought to be

FIG. 2. Schematic representations of PCR-amplified coagulase gene fromS. aureus. (A) PCR-amplified coagulase gene digested with AluI. a , PCR RFLP patternnumber; b , duplicate strains prepared on different occasions; , methicillin-sensitiveS. aureuspropagating strains (PS) used for the international set of phages; T, typestrain (20) obtained from the National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. The darker, thicker bands represent

doublets. The schematic was prepared with Adobe Photoshop. (B) PCR-amplified coagulase gene digested with CfoI.a

, PCR RFLP pattern number;b

, duplicate strainsprepared on different occasions; , methicillin-sensitiveS. aureus propagating (PS) strains used for the international set of phages; T , type strain (20) obtained fromthe National Collection of Type Cultures. The darker, thicker bands represent doublets.

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representative of an epidemic clone circulating within Europe.In contrast, the German isolate 94/14013 was distinct andclearly separable from the other European isolates studied (2,41) (Fig. 2 and 3).

The propagating strains, PS 42E and PS 71, represent someof the diverse types of isolates from human clinical material.They can be differentiated by ribotype (34) and by their unique

coagulase RFLP patterns, 9 and 10, respectively (Fig. 2).Isolate Airedale 16 has phage type EMRSA-15, and yet

unlike other EMRSA-15 strains, it does not produce entero-toxin C, has a unique PCR RFLP pattern, and is atypical onPFGE (31). It is a sporadic outbreak strain which may haveoriginated from horizontal genetic transfer of resistance genesfrom MRSA to methicillin-sensitive S. aureus (cf. reference36). It is evident from the populations studied so far thatantibiotic-sensitive strains exhibited greater genetic diversitythan did resistant strains (reference 7 and this study).

This study demonstrates the value of PCR RFLP (AluI andCfoI) pattern analysis of the coagulase gene for the rapid initial

genotyping ofS. aureus, particularly of the major United King-dom epidemic strains, EMRSA-3, EMRSA-15, and EMRSA-

16. The RFLP patterns observed in this study were substanti-ated by the analysis of sequence data in that the patterns gaverise to parallel phylogenetic groups.

ACKNOWLEDGMENTS

We are grateful to Philip P. Mortimer, Jonathan P. Clewley, andJohn Stanley for critical reading of the manuscript and to Jon Whitefor artwork.

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1. Anonymous. 1996. Epidemic methicillin resistant Staphylococcus aureus.Commun. Dis. Rep. 6:1.

2. Aparicio, P., J. Richardson, S. Martin, A. Vindel, R. R. Marples, and B. D.Cookson. 1992. An epidemic methicillin-resistant strain ofStaphylococcus

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14. Fitch, W. M., and E. Margoliash. 1967. Construction of phylogenetic trees.

FIG. 3. Unrooted consensus Jukes and Cantor (22) one-parameter and Fitchand Margoliash (14) distance tree showing the lineages of strains ofS. aureus. a ,

unless otherwise stated, the RFLP pattern numbers were the same for both AluIandCfoI; b, GenBank accession numbers: the accession number forS. aureus213was X16457 (23) and that for S. aureus 8325-4 was X17679 (30); c, assigned tophylogenetic group; , methicillin-sensitive S. aureus propagating strains (PS)used for international phage set; T , type strain (20) obtained from the NationalCollection of Type Cultures. The bootstrap values are the percentages of timesthat a taxon at that node occurred. The scale bar represents 0.1 substitution persequence position (Knuc).

TABLE 2. RFLP pattern and phylogenetic group of strainsof EMRSA

RFLP patterna Phylogenetic groupb Strain(s) of EMRSA

1 A 32 B 153 C 16

4 D 1, 4, 7, 114 E 9

5 A 145 B 135 F 28c F 25 G 5, 6, 8, 10

a Unless otherwise stated, PCR RFLP (AluI and CfoI).b Phylogenetic group assigned on the basis of coagulase gene sequence com-

parisons.c Pattern formed on CfoI.

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