International Clones of Methicillin-Resistant Staphylococcus aureus in Two Hospitals in Miami, Florida

A total of 202 methicillin-resistant Staphylococcus aureus (MRSA)single-patient isolates recovered between January and June 1998in two hospitals in Miami, Florida, were characterized by acombination of several molecular typing techniques: multilocussequence typing, spaA typing, pulsed-field gel electrophoresis,and determination of the structure of the SCCmec element. Theoverwhelming majority of the isolates—187of 202, or 93%—belongedto one of three internationally spread epidemic clones whichwere identified on the basis of their multilocus sequence type(ST) as E-MRSA-16 (ST36), the New York clone V (ST8), and theNew York/Japan clone (ST5; SCCmec II) and its single- and double-locusvariants. The rest of the isolates (15 of 202, or 7%) were moregenetically diverse and were each recovered from a few patientsonly. Of the 23 MRSA strains isolated from confirmed human immunodeficiencyvirus-positive patients, as many as 17 (or 70%) belonged toa single ST8 clone carrying SCCmec type IV. The data providefurther evidence for the conclusion of earlier studies thatmost MRSA disease in hospitals is caused by relatively few pandemicclones.

INTRODUCTION

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Introduction

The introduction of molecular typing techniques into epidemiologicalinvestigations has provided powerful new tools for trackingthe origin and routes of dissemination of methicillin-resistantStaphylococcus aureus (MRSA) strains not only among patientsin a hospital but also among hospitals in different countriesor even across different continents. One of the interestingconclusions that have emerged from large international surveillancestudies using such molecular typing techniques is that a relativelyfew MRSA clones have been responsible for a disproportionatelylarge fraction of all MRSA disease worldwide (8, 16). In additionthese studies have also indicated a considerable degree of geographicspecificity in the spread of various MRSA lineages. For instance,the Iberian clone of MRSA was found to be the dominant clonein hospitals in Southern Europe (16), while the clone referredto as E-MRSA-16 was most frequently recovered in hospitals inthe United Kingdom (11), Canada (24), and Mexico (2). In surveillanceof MRSA disease in 12 New York City hospitals in 1996 (18),as many as 42% of all MRSA isolates belonged to the New York/Japanclone. In a follow-up study conducted at 29 hospitals and/orhealth centers in the tristate area of New Jersey, Pennsylvania,and Connecticut in 1998 (19), the same clonal type was representedin 92% of the MRSA isolates from the participating centers inPennsylvania, 65% of MRSA isolates from New Jersey, and 39%of MRSA isolates from Connecticut. The purpose of the studydescribed here was to sample hospitals in the United Statesat a geographic site distant from the Northeast. Two hospitals,both located in Miami, Florida, were selected for such a study,and the basic observations are described in this communication.

MATERIALS AND METHODS

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Materials and Methods

The first of the two sites selected for the study was The JacksonMemorial Hospital (hospital C), a 1,550-bed hospital that servesthe population of Miami-Dade County. Services include generalacute care, a trauma center, and multiorgan transplantationprograms. The second site was the Miami Veterans Affairs MedicalCenter (hospital D), a 220-bed acute care facility serving theveterans of southeastern Florida. Both hospitals are closelyaffiliated with the University of Miami School of Medicine.During the calendar year of 1998, the frequency of methicillinresistance among S. aureus isolates was 38% in hospital C andabout 50% in hospital D.

Of the MRSA isolates recovered in these two hospitals between1 June and 23 December 1998, a total of 202 isolates were availablefor molecular typing: 113 from hospital C and 89 from hospitalD. Information was obtained on the source of each isolates (blood,the respiratory tract, wounds, urine, or other [including thenose, stool, and skin]), whether the isolate was collected withinthe first 72 h of hospitalization (community acquired) or after72 h of hospitalization (nosocomial), and whether the isolatewas from a patient with human immunodeficiency virus infection(if that information was available). The major clinical sourcesof the MRSA isolates were blood (n = 32), the respiratory tract(n = 53), wounds (n = 25), urine (n = 35), and other sources(n = 57). The great majority of isolates were of nosocomialorigin. Species identification and antibiograms were performedat the clinical microbiology laboratories of the hospitals.Cultures were frozen in tryptic soy broth containing 10% glycerol(Difco, Detroit, Mich.) and were stored at -70°C until transferto the Laboratory of Microbiology at The Rockefeller University,New York, N.Y., for molecular typing.

Table 1 lists the MRSA isolates according to their moleculartypes and antibiotypes. Multilocus sequence typing (MLST) (6,7), determination of the spaA type (15, 22), and pulsed-fieldgel electrophoresis (PFGE) of SmaI-digested chromosomal DNA(5) were performed by published methods. A multiplex PCR techniquewas used to identify the type of the mec element (SCCmec type)carried by the bacterium (17).

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TABLE 1. Molecular types and antibiotypes of MRSA isolates recovered at two Miami hospitals

RESULTS AND DISCUSSION

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Results and Discussion

We chose a combination of several molecular typing techniquesfor characterization of the MRSA isolates recovered in the twoMiami hospitals, since these methods can complement one anotherboth in terms of expedience and in terms of the type of informationthey provide. While superior in resolving power, PFGE may blurevolutionary relationships among strains which may be revealedby MLST, particularly when the structure of the SCCmec typeis also determined. Experience with the relatively simple sequence-basedspaA typing technique, recently introduced, indicates that thismethod can frequently predict the MLST type (14, 16). Whileshort-term epidemiological investigations need high-resolutiontracking techniques such as PFGE, additional characterizationof strains by MLST and other sequence-based techniques allowsone to recognize issues that transcend the geographic boundariesof a hospital and permits insights into the global epidemiologyand population biology of S. aureus as well. From this perspective,the molecular typing data generated in the two Miami hospitalsallow one to draw several conclusions.

The most frequent MRSA clone in hospital C and the second mostfrequent clone in hospital D was E-MRSA-16 (ST36) (Fig. 1).All MRSA isolates belonging to this sequence type in both hospitalswere homogeneous: they had identical MLSTs, carried SCCmec typeII, and showed a uniform PFGE pattern as well as an invariantspaA type (Fig. 2). E-MRSA-16 is widespread in the United Kingdom(11) and has also been detected in Greece (3), Scandinaviancountries (20, 21), Switzerland and Belgium (12), Mexico (2),and Canada (24). Our identification of this clone in the twoMiami hospitals represents the first detection of E-MRSA-16in the United States. The uniformity of MRSA isolates belongingto this clone may be interpreted as an indication that E-MRSA-16has arrived relatively recently at the Miami hospitals.

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FIG. 1. Molecular types of MRSA isolates recovered in two Miami hospitals. MRSA isolates recovered in hospitals C and D were characterized by MLST, and the frequencies of the various sequence types (ST) are plotted as percentages of the total number of MRSA isolates. The number of strains with the indicated genotype at the indicated hospital is given above each bar.

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FIG. 2. (Top) PFGE patterns of the various sequence types of MRSA isolates recovered in the two Miami hospitals. Chromosomal DNAs were isolated, restricted by SmaI, and separated by PFGE. (Bottom) The various strains used as sources of the chromosomal DNA are identified by their MLST, sequence type (ST), and spaA type. The hospital source of each isolate is indicated by the letter C or D in the strain designation; strains NTCT8325 and BK2464 are included for comparison.

The second most frequent MRSA clone was the New York clone V(14), which accounted for 47% of all MRSA isolates in hospitalD and was the second most frequent clone (36%) in hospital C(Fig. 1). This clone was first identified as an epidemic MRSAclone in a surveillance study in New York, N.Y., where it wasassociated with SCCmec IV and was assigned the multilocus sequencetype ST8 (14). However, the ST8 genetic background was proposedto represent the predicted ancestor of the very first EuropeanMRSA strain (8). Furthermore, a single-locus variant of ST8,MRSA clonal type ST247, also known as the Iberian clone (6),is one of the most widely spread MRSA clones in Southern andWestern Europe (16). In the two Miami hospitals, most of thestrains belonging to ST8 (68 of 73) carried SCCmec type IV but5 strains of this sequence type were associated with SCCmectype I, which was shown to be present in the first MRSA isolatesfrom the United Kingdom and Denmark (6, 8). Variation in theSCCmec types associated with this genetic background has beendemonstrated previously (8).

MRSA strains belonging to the New York clone V and carryingSCCmec type IV were recovered with relatively high frequency(70%) from human immunodeficiency virus-positive patients inthe two Miami hospitals, and a similar association was notedin a surveillance study in New York (18, 19). The significanceof these observations is not clear at present. Unlike that ofSCCmec type I, the structure of SCCmec type IV lacks the plsgene, which may be involved in colonization by S. aureus (16).

In contrast to the MRSA isolates belonging to the E-MRSA-16clone, MRSA isolates belonging to the New York clone V showedvariations in spaA type (Fig. 2).

The third most frequent MRSA clone in both Miami hospitals wasthe New York/Japan clone (multilocus sequence type ST5), togetherwith its single-locus variants ST105 and ST83 and its double-locusvariant ST231 (Fig. 1). All these strains carried SCCmec typeII. MRSA isolates belonging to this clone are most frequentin the northeastern United States (7, 18, 19), Canada (24),and Japan (1). All MRSA isolates identified so far in the UnitedStates and Japan with reduced susceptibility to vancomycin (so-calledVISA strains) belong to this clone (8, 23). Variations bothin spaA type and in PFGE pattern were detected among representativesof this clone (Fig. 2).

The capacity of each of these three major MRSA clonal typesto cause staphylococcal disease is documented by their frequentrecovery from all major clinical sites (Fig. 3).

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FIG. 3. Recovery of the three major MRSA clonal types and sporadic MRSA sequence types (STs) from various clinical sites of disease in the two Miami hospitals. The frequencies of association of the MRSA clonal types ST36 (light shading), ST8 (solid), and ST5 (105, 231, 83) (dark shading) and of sporadic STs (open) with the different clinical sites of disease are shown. Resp, respiratory tract.

In contrast to the majority of MRSA isolates represented bythe three dominant clones, the rest of the isolates (sporadicMRSA isolates; n =15) were more genetically diverse: they included9 different sequence types in association with several SCCmectypes and 12 distinct PFGE patterns. Three of these geneticallydiverse isolates were representatives of the Brazilian MRSAclone (ST239 and ST241)—a major internationally spreadMRSA lineage (4, 16). An additional group of five MRSA isolatesbelonged to ST45—a clone widely spread both as methicillin-susceptibleS. aureus (MSSA) and as MRSA in Western Europe, Scandinavia,Canada, and the northeastern United States (8; www.mlst.net).Two MRSA isolates, with ST107 (SCCmec type IV) and ST106 (SCCmectype unknown), represent novel genetic backgrounds not describedpreviously. One isolate, with ST72 (SCCmec type IV), has previouslybeen described in Spain (17) and Cuba (www.mlst.net). Two isolatesbelonging to ST9 (SCCmec type unknown) have been reported inthe United Kingdom but only as MSSA (www.mlst.net). A singleMRSA isolate of ST15 (SCCmec type unknown) and another MRSAisolate belonging to ST97 (SCCmec type IV) have been reportedpreviously from several countries, but only as MSSA strains(www.mlst.net). The SCCmec types that gave no PCR products,namely, those identified in ST106, ST9, and ST15 MRSA strains,are likely to represent new structural types or structural rearrangementsof the mec element (10). It is also interesting that three ofthe genetic backgrounds identified in MRSA isolates from theMiami hospitals, namely, ST9, ST15, and ST97, were reportedonly as backgrounds of MSSA strains until now. The fact thatthese genetic backgrounds were identified in MRSA strains inthe Miami hospitals is consistent with the notion of the relativelyfrequent acquisition of the SCCmec element by S. aureus (9,16). It is noteworthy that most of these 15 genetically morediverse isolates were associated with hospital C, the patientpopulation of which—in contrast to that of hospital D—ismore likely to represent a geographically diverse group (frominternational patient referrals, immigrants, and tourists visitingthe Miami area) that may have been the source of these diverseMRSA isolates. Thus, at least some of the sporadic MRSA isolatesidentified in hospital C may not have a strictly nosocomialorigin. It is interesting in this respect that a few of thesporadic isolates belonging to ST45 had the genetic backgroundalso seen in some so-called community-acquired MRSA isolates(13).

With the continued and increasing mobility of human populationsthrough international travel, it is expected that the geographicboundaries of dominance among MRSA clones will become blurredin time, and the appearance of the genetically diverse sporadicisolates in hospital C may represent this trend. Why such alarge proportion of MRSA disease is caused by so few clonaltypes of bacteria still remains a puzzle, but one componentof the extensive geographic spread of these few pandemic MRSAclones may be related to a combination of particular genetictraits in the genetic background of the bacteria (16).

ACKNOWLEDGMENTS

Partial support for these investigations came from a grant fromthe U.S. Public Health Service (1 RO1 AI45738) and from thePalm Beach Institute of Infectious Diseases (to Istvan Krisko).

We thank Carmen Bermudez for assistance with this project.

FOOTNOTES

* Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688. E-mail: tomasz{at}mail.rockefeller.edu.

REFERENCES

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