MICROBIAL DRUG RESISTANCEVolume 4, Number 2, 1998Mary Ann Liebert, Inc.

Spread of the Multiresistant Iberian Clone ofMethicillin-Resistant Staphylococcus aureus (MRSA)

to Italy and Scotland

R. MATO,1 I. SANTOS SANCHES,1,2 M. VENDITTI,3 DJ. PLATT,4 A. BROWN,4 M. CHUNG,5and H. de LENCASTRE1,2,5

ABSTRACT

The mult ¡drug-resist ant "Iberian" clone of methicillin-resistant Staphylococcus aureus (MRSA) was first iden-tified on the basis of its unique DNA fingerprints as the strain responsible for the massive 1989 outbreak ofMRSA disease in the hospital Princeps d'Espanya, Barcelona, Spain. Most Iberian MRSA carry a constitu-tive /?-lactamase. They are resistant to most /3-lactam antibiotics, macrolides, aminoglycosides, tetracycline,rifampin and ciprofloxacin and are susceptible to fosfomycin, fusidic acid, mupirocin, sulfa-methoxazole/trimethoprim and vancomycin. The characteristic DNA fingerprints of the clone include the mecApolymorph I, Tn554 pattern E (or its variants), a chromosomal macrorestriction pattern (pulsed-field gel elec-trophoretic type) A (or its subtype variants), the lack of the mecí regulatory gene and a homogeneous, highlevel of expression of melhicillin resistance. Molecular surveillance studies have documented the extensivespread of this clone to many Portuguese hospitals during the 1990s. In this article, we describe the spread ofthe Iberian MRSA to hospitals in Rome, Italy, and Scotland.

INTRODUCTION tributor to this increase in the frequency of multidrug-resistantMRSA, involve the spread of unique genetic lineages of these

Since the mid-1980s and throughout the 1990s, the incidence bacteria over very large geographic distances.3,11,16,18,19 In thisof staphylococcal infections and the proportion of methi- study, we used DNA fingerprinting techniques to document the

cillin-resistant Staphylococcus aureus (MRSA) strains has risen appearance of one of these clones of MRSA, the Iberian MRSA,worldwide among nosocomial pathogens. This shift in etiology, already widely spread in Spain and Portugal, in hospitals inincrease in antibiotic resistance and frequency of hospital-borne Rome, Italy, and Scotland,staphylococcal infections must reflect, in part, changes in thepattern of antibiotic use and also the increased interhospitaltransfer of patients, as well as the greatly increased mobility of MATERIALS AND METHODShealthy carriers colonized by staphylococci. Of particular con-

cern is the rise in frequency of MRSA that are also resistant to We studied 53 Italian MRSA isolates collected between No-antibiotics other than members of the /3-lactam family. Mul- vember, 1993 and May, 1995 in Rome: 40 isolates were re-

tiresistant MRSA strains, against which the glycopeptide an- covered in eight different hospitals and 13 isolates came fromtibiotics (vancomycin and teicoplanin) have remained the only four private or ambulatory clinical laboratories. An additionalchemotherapeutically useful agents, have now been described. 13 MRSA isolates recovered between October, 1990 and De-Introduction of molecular fingerprinting techniques for the cember, 1993 were obtained from ten different hospitals locatedcharacterization of MRSA isolates has led to the surprising find- in the west of Scotland (see Table 1). As control strains for theing that, at least in some parts of the world, a significant con- comparison of molecular fingerprints, the following represen-

'Unidade de Genética Molecular, Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa (ITQB/UNL), Oeiras,Portugal.

2Faculdade de Ciencias e Tecnologia (FCT), Universidade Nova de Lisboa (UNL), Monte da Caparica, Portugal.3Universidade "La Sapienza," Rome, Italy.4University Department of Bacteriology, Royal Infirmary, Glasgow, Scotland.'Laboratory of Microbiology, The Rockefeller University, New York, NY 10021.

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MRSA IBERIAN CLONE SPREAD 109

tatives of the Iberian MRSA were used: strain S. aureus PER34,isolated at the end of 1989 from the hospital Princeps d'Es-panya, Barcelona, Spain;5 strain HPV107 recovered from thehospital Pulido Valente in Lisbon, Portugal;16 and strainHDG89 from the Hospital de Sousa Martins in Guarda, Portu-gal.15 Reference strains for the mecA and Tn5J4 fingerprints ofchromosomal Clal digests and pulsed-field gel electrophoretic(PFGE) types were obtained from The Rockefeller Universityand from the strain collection at the Instituto de TecnologíaQuímica e Biológica da Universidade Nova de Lisboa (ITQB/UNL). Strain NCTC8325 was used as molecular weight stan-dard in PFGE gels.12 The DNA probes used were a 1.196 kbPstl-Xbal internal fragment of the mecA gene, cloned inpMF13;2,9 a 5.5 kb EcoRV fragment from the plasmid pBlue-script II that contained a probe specific for the transposonTn554,6,8 and a 2.2 kb Hindlll fragment of the mecI-mecRI re-

gion cloned in E. coli WT183 from S. epidermidis? The en-

zymes Pstl, Xbal, EcoRV, and Hindlll were obtained fromStratagene, La Jolla, California.

Methods used for the phenotypic and genotypic characteri-zation were as described before.1,2 Antimicrobial susceptibili-ties were determined by disc diffusion methods10 followingbreakpoint definitions, as recommended by the NCCLS. Themethicillin-resistance phenotype was determined using discscontaining 1 mg of methicillin.4 Methods for the preparation ofchromosomal DNA, endonuclease restriction, running condi-tions for both conventional and PFGE were carried out, as de-scribed.2,16 DNA fixation to the membranes was done by UVcross-linking.14 Probe labeling and hybridization were done byusing the ECL-random prime labeling and detection systems,RPN3040 (Amersham International, Little Chalfont, U.K.), fol-lowing the manufacturer's recommendation.

RESULTS AND DISCUSSION

Experimental evidence summarized in Fig. 1 and Table 1 in-dicates that the multidrug-resistant Iberian clone of MRSA hasspread to several of the hospitals and ambulatory units analyzedin Rome and also to hospitals that provided MRSA samplesfrom the western part of Scotland. The clonal fingerprints ofthese strains are shown in Fig. 1 and Table 2.

Out of the total of 53 MRSA strains analyzed from the Ital-ian sources, 22 isolates showed simple subtype variants of thePFGE pattern A characteristic of the Iberian MRSA. A subsetof this group of 22 strains—12 out of the 22—carried not onlythe PFGE type A but the mecA polymorph I and Tn554 type Ealso, i.e., these 12 strains showed the typical clonal profile ofthe Iberian MRSA.

From the other 10 out of the 22 MRSA isolates that sharedthe common PFGE type A, four isolates carried the mecA poly-morph I in combination with either the Tn554 type J (corre-sponding to two copies of Tn554; in three of the isolates) or

the Tn554 type £ (a new Tn554 type) present in one isolate.The remaining six isolates in this subset had the followingclonal assignments (mecA::Tn554::PFGE) II::NH::A (four iso-lates), II::E::A (one isolate), and II::-n:A (one isolate, a new

Tn554 type).The 12 representatives of the Iberian clone (clonal type

I::E::A) were identified in the following hospitals and ambula-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18

FIG. 1. Pulsed-field gel electrophoresis patterns of methi-cillin-resistant Staphylococcus aureus Iberian clone. Represen-tative strains from different countries. Lane 1 : high molecularweight \ ladder; lanes 2 and 10: NCTC8325 (control strain);lane 3: HPV107, pattern A2 (from Portugal); lane 4: HDG89,pattern A3 (from Portugal); lane 5: PER34, pattern Al (fromSpain); lane 6: ITA7, pattern A4 (from Italy); lane 7: ITA8,pattern A5 (from Italy); lane 8: ITA32, pattern A6 (from Italy);lane 9: ITA43, pattern A8 (from Italy); lane 11-Scl, pattern A9(from Scotland); lane 12: Sc4, pattern A12 (from Scotland);lane 13: Sc5, pattern A13 (from Scotland); lane 14; Scl2, pat-tern A16 (from Scotland); lane 15: 82, pattern A17 (from Eu-ropean Study Group); lane 16: 83, pattern A18 (from EuropeanStudy Group); lane 17: 238/93, pattern A19 (from EuropeanStudy Group); lane 18: BK1953, pattern A20 (from New York).

tory services providing MRSA isolates for the study: 4 out ofthe 8 hospitals (Ospedale San Giovanni, Ospedale Santo Eu-genio, Policlinico Umberto I, and Istituto Dermopatico del-l'lmmacolata) and three out of the four ambulatory services(Ospedale Santo Spirito, Ospedale San Giovanni, Lab BIOS).

The number of MRSA samples examined from the Italianhospitals was too small to allow definitive epidemiological con-

clusions. Nevertheless, the MRSA isolates were picked at ran-

dom (40 MRSA recovered in eight hospitals and 13 MRSA re-

covered in four ambulatory units) and the fact that as many as

four of the hospitals and three of the ambulatory units harbouredrepresentatives of the Iberian clone suggests that this multire-sistant MRSA is not only widely spread, but is probably alsopresent at high frequency in each of the hospitals and ambula-tory sources.

Out of the small sample of 13 MRSA isolates recovered inten hospitals located in western Scotland, eight shared variantsof PFGE type A, and seven of these eight isolates also carriedmecA polymorph I in combination with a variety of Tn554 types(one isolate each with Tn554 types J, £, v, tr, and three isolateswith Tn554 type E). This clonal analysis showed that threeout of the 13 MRSA from Scotland showed the clonal profileof I::E::A, i.e., the typical clonal assignment of the IberianMRSA.

110

Table 2. Clonal Characterization of MRSA Italian andScotland Isolates and Iberian Clone Representative Strains

MATO ET AL.

Country IsolatesCla/-mecA

typeCla/-7n554

typePFGEPattern

No.isolates

Clonal Type(mecA: :Tn55A::PFGE)

Italy (Rome) 7,36,48,49,528,9,10,11,12,453238,39,5133301534,43,4421

Scotland 1311245612

Iberian Clone Representative StrainsSpain PER34Portugal HPV 107

HDG89

VI

EEEJ

ENHNH

TEEEJ7<i>v

TT

EEE

A4A5A6A5A5A5A7A8A8A9AllA15A10A12A13A14A16

AlA2A3

I::E::A

I::J::AI::6::AII::E::AII::NH::A

II::t::AI::E::A

I::J::AVI::y::AIII

1II

:<p: : A:v::A:it : : A

12

n = 3n = 1n = 1n = A

n = 1n = 3

n = 1n = 1n = 1n = 1n = 1

Tn554 insertion patterns not described before: £, r, y, <p, v, it;n = number of isolates.

NH—no homology with the Tn554 transposon.

Portugal(10)

FIG. 2. Geographic spread of methicillin-resistant Staphylococcus aureus Iberian clone. (The numbers in parenthesis indicatethe number of hospitals where the Iberian clone was identified.)

MRSA IBERIAN CLONE SPREAD 111

Figure 1 shows the PFGE profiles of the Italian and ScottishMRSA together with eight control strains: two from Portugal(strains HPV10716 and HDG8915), one from Spain (strainPER34),5 three from European countries (European StudyGroup coordinated by Dr. Marc Struelens),17 and one from NewYork,13 each representing variants of the Iberian MRSA.

Table 1 summarizes the relevant properties of the Italian andScottish isolates including their antibiotic resistance profile,clonal assignments, as well as the molecular sizes of the DNAfragments reacting with the mecA and Tn554 DNA probes. Thesmall zone size around the 1 mg methicillin disc shown in Table1 indicates that in all except two of the strains (ITA21 andITA48) the strains had a highly and homogeneously resistantmethicillin phenotype. All strains were resistant to ciproflox-acin, gentamicin, oxacillin, penicillin, and tobramycin. Thegreat majority of the strains was also resistant to erythromycin,rifampin, pefloxacin, and cephalosporins, while most or all ofthe strains remained susceptible to sulfamethoxazole/trimetho-prim, fosfomycin, teicoplanin, mupirocin, and vancomycin.This antibiotype is also typical of most representatives of theIberian clone examined so far in Portuguese and Spanish hos-pitals.

These observations further document the capacity of the Iber-ian MRSA for extensive geographic spread. Figure 2 shows a

map illustrating the geographic spread of the Iberian MRSAclone in Europe and more recently to New York.13

ACKNOWLEDGMENTS

This work was partial supported by the CEM/NET initiative-CEM/NET Project 31 from IBET, PRAXIS XXI 2/2.1/SAU/1295/95 and PECS/C/SAU/145/95 from Junta Nacional de In-vestigaçâo Científica (Portugal) awarded to H. de Lencastre. R.Mato was supported by a grant (PRAXIS XXI/BPD/6077/95)from Junta Nacional de Investigaçâo Científica (Portugal). Theauthors express their gratitude to Dr. Alexander Tomasz forstimulating discussions and critical reading of the manuscript,to Drs. Marc Struelens and W. Witte for the gift of the strainsof Belgium and Germany, and to Drs. Richard Roberts andBarry Kreiswirth for the strain from New York.

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New York NY 10021