耐甲氧西林金黄色葡萄球菌的脉冲场凝胶电泳分型研究

目的　探讨耐甲氧西林金黄色葡萄球菌 (MRSA)的分子流行病学特点。方法　采用脉冲场凝胶电泳 (PFGE)技术对我院在 2 0 0 1年 6月～ 2 0 0 2年 4月临床分离的 5 0株MRSA作同源性分析。结果　 5 0株MRSA的PFGE图谱分为 6个组 (A～F型 )。以A型 (2 7株 )、B型 (10株 )、C型 (10株 )流行为主。 5 0株MRSA中共有 17株和重症监护室 (ICU)相关 ,占 34%。流行菌株在各病房之间传播。结论　ICU是MRSA的高发区域和疫源地 ,神经外科、肝胆胰外科和干部病房中MRSA的分离率也较高。在同一病人不同时间不同部位所采集的菌株同源性不尽相同     

Epidemiological typing of methicillin-resistant Staphylococcus aureus outbreak isolates by pulsed-field gel electrophoresis and antibiogram

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens. In April 1997, there were five MRSA-infected patients among 16 patients in the Neonatal Intensive Care Unit (NICU), Seoul National University Hospital, which is a tertiary-care hospital with 1,500 beds. The infections had spread from twin patients with MRSA who had transferred from Hospital C. MRSA was isolated from the axilla of 15 (94%) of the 16 patients, including the two patients with obvious infections. Three (19%) of 16 doctors and nine (30%) of 30 nurses had MRSA colonization of the anterior nares. Six different PFGE patterns (A through F) were identified in the 53 isolates of MRSA tested. Twelve of 13 isolates from infected sites of five patients showed pattern F. Three MRSA strains obtained from hospital C showed closely or possibly related pattern F. MRSA of type F was isolated from three of 16 patients' axilla, and one of 3 doctors' and three of 30 nurses' nasal swabs. The antibiogram code for 12 of 13 MRSA isolates from five infected patients was 66,754. PFGE patterns of these isolates were either F, F1, F2 or Fa. Only one of three strains isolated from clinical specimens of patients in Hospital C showed the antibiogram code 66754, although they were all PFGE types F1 and Fa. In conclusion, the presumptive sources of the outbreak of MRSA infection in NICU were the twin patients transferred from hospital C. Antibiogram correlated reasonably well to the PFGE type. An effective notification system is needed when a MRSA-infected patient is transferred to another hospital to control the spread of the infection.     

Epidemiological validation of pulsed-field gel electrophoresis patterns for methicillin-resistant Staphylococcus aureus

To determine the stability of pulsed-field gel electrophoresis (PFGE) patterns of methicillin-resistant Staphylococcus aureus in the nosocomial setting, we analyzed isolates from long-term carriers (>1 month) and from patients involved in well-defined nosocomial epidemics. The number of fragment differences between the first isolate and subsequent isolates in long-term carriers showed a bimodal distribution, with one group having 0 to 6 fragment differences and the other group having 14 to 24 fragment differences. The PFGE patterns of isolates involved in epidemics also presented a similar bimodal distribution of the number of fragment differences. Typing these isolates with another molecular method (inter-IS256 PCR) showed that isolates of the first group (i.e., with 1 to 6 fragment differences) were clonally related, whereas the second group (with 14 to 24 fragment differences) could be considered genetically different. Among long-term carriers with clonally related isolates, 74 of 84 (88%) of consecutive isolates showed indistinguishable patterns, whereas 10 of 84 (12%) showed related patterns differing by one to six fragments. Moreover, the frequency of apparition of related patterns is higher when the time between the first and the subsequent isolate is longer. During seven nosocomial epidemics lasting from 1 to 15 months, only 2 of 120 isolates (1.7%) showed a pattern which was different, although related, from the predominant one involved in each of these outbreaks.     

Comparison of pulsed-field gel electrophoresis and coagulase gene restriction profile analysis techniques in the molecular typing of Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) and coagulase gene restriction profile (CRP) analysis techniques were used to analyze 71 Staphylococcus aureus isolates recovered from nine food-borne disease outbreaks. Twenty-two PFGE profiles and 11 CRPs were identified, with discrimination indices of 0.86 and 0.72, respectively. In addition, the variable regions of the coagulase genes of 39 isolates were sequenced and showed extensive identity, indicating that this is not an efficient alternative for the molecular typing of S. aureus.     

Comparison of pulsed-field gel electrophoresis and PCR analysis of polymorphisms on the mec hypervariable region for typing methicillin-resistant Staphylococcus aureus

Two hundred fifty-four methicillin-resistant Staphylococcus aureus (MRSA) strains typed by pulsed-field gel electrophoresis (PFGE) were tested by PCR for the mec-associated hypervariable region (HVR-PCR) to determine their number of direct repeat units (DRUs). Eight different groups of repeats were found among the MRSA strains and compared to 28 pulsotypes classified by PFGE. Some MRSA strains belonging to the same pulsotype showed different numbers of DRUs. HVR-PCR was rapid, easy to perform, and reproducible and has the ability to obtain an unambiguous positive result for each isolate analyzed. However, this technique shows a discriminatory power inferior to that of PFGE. We conclude that PFGE is a more reliable method of typing MRSA than HVR-PCR.     

Comparison of the DiversiLab repetitive element PCR system with spa typing and pulsed-field gel electrophoresis for clonal characterization of methicillin-resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become an increasing problem worldwide in recent decades. Molecular typing methods have been developed to identify clonality of strains and monitor spread of MRSA. We compared a new commercially available DiversiLab (DL) repetitive element PCR system with spa typing, spa clonal cluster analysis, and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power and concordance. A collection of 106 well-defined MRSA strains from our hospital was analyzed, isolated between 1994 and 2006. In addition, we analyzed 6 USA300 strains collected in our institution. DL typing separated the 106 MRSA isolates in 10 distinct clusters and 8 singleton patterns. Clustering analysis into spa clonal complexes resulted in 3 clusters: spa-CC 067/548, spa-CC 008, and spa-CC 012. The discriminatory powers (Simpson's index of diversity) were 0.982, 0.950, 0.846, and 0.757 for PFGE, spa typing, DL typing, and spa clonal clustering, respectively. DL typing and spa clonal clustering showed the highest concordance, calculated by adjusted Rand's coefficients. The 6 USA300 isolates grouped homogeneously into distinct PFGE and DL clusters, and all belonged to spa type t008 and spa-CC 008. Among the three methods, DL proved to be rapid and easy to perform. DL typing qualifies for initial screening during outbreak investigation. However, compared to PFGE and spa typing, DL typing has limited discriminatory power and therefore should be complemented by more discriminative methods in isolates that share identical DL patterns.     

Comparison of DNA sequencing of the protein A gene polymorphic region with other molecular typing techniques for typing two epidemiologically diverse collections of methicillin-resistant Staphylococcus aureus

The aim of this study was to compare the recently developed typing approach for methicillin-resistant Staphylococcus aureus (MRSA) based on the DNA sequencing of the protein A gene polymorphic region (spaA typing) with a combination of three well-established molecular typing techniques: ClaI-mecA vicinity polymorphisms, ClaI-Tn554 insertion patterns, and SmaI pulsed-field gel electrophoresis (PFGE) profiles. In order to evaluate the applicability of this typing technique in different types of studies, two groups of MRSA clinical isolates were analyzed: a collection of 185 MRSA isolates circulating in Hungary recovered from 17 hospitals in seven cities during a 3-year period (1994 through 1996), and a selection of 53 MRSA strains isolated in a single hospital in Hungary between 1997 and 1998. The 238 MRSA clinical strains from Hungary were first classified in clonal types (defined as ClaI-mecA::ClaI-Tn554::SmaI-PFGE patterns), and 65 of the 238 strains, representing major MRSA clones and some sporadic clones, were further analyzed by spaA typing. Our results showed that the lineages most recently introduced in the hospital setting showed little variability in spaA types, whereas the MRSA clones circulating for a longer period of time and spread among several hospitals showed a higher degree of variability. The implementation of the spaA typing method was straightforward, and the results obtained were reproducible, unambiguous, and easily interpreted. This method seems to be adequate for outbreak investigations but should be complemented with other techniques in long-term surveillance or in studies comparing distant clonal lineages.     

Comparison of an automated repetitive sequence-based PCR microbial typing system to pulsed-field gel electrophoresis for analysis of outbreaks of methicillin-resistant Staphylococcus aureus

Rapid and sensitive methods for accurate strain delineation are essential for monitoring and preventing transmission of methicillin-resistant Staphylococcus aureus (MRSA). Pulsed-field gel electrophoresis (PFGE) has been the standard technique for strain typing most bacterial species including MRSA. The goal of this study was to compare the performance of the DiversiLab microbial typing system (Bacterial BarCodes, Inc., Houston, TX) (rep-PCR) to that of PFGE for typing MRSA isolates from five well-defined outbreaks. The DiversiLab rep-PCR assay is a rapid, semiautomated method based on PCR amplification of specific regions between noncoding repetitive sequences in the bacterial genome. rep-PCR was performed according to the manufacturer's recommendations, and the results were analyzed and dendrograms were generated using the DiversiLab analysis software (version 2.1.66 a). PFGE was performed and interpreted according to published procedures. rep-PCR results using similarity indices (SI) of 80%, 85%, and 90% were compared to PFGE analysis. In addition, intra- and interrun reproducibility was determined for rep-PCR. Overall, correct assignment to outbreak versus nonoutbreak clusters occurred for 91 of 109 isolates (85% agreement) when using a SI of 85%. For each specific outbreak, concordance between rep-PCR and PFGE ranged from 73% to 100%. There were 18 discrepant results (17%). Fourteen isolates were unique by PFGE, but they were placed in clusters by rep-PCR; the other 4 were placed in clusters different from those assigned by PFGE. Intra- and interrun reproducibility was excellent. Times to results were 12 to 24 h for rep-PCR compared to 2 to 4 days for PFGE. Rapid, standardized results and excellent reproducibility make rep-PCR a valuable tool for use in MRSA investigations. However, since rep-PCR was less discriminatory than PFGE, we recommend that it be used to screen isolates, followed by testing isolates which share the same rep-PCR pattern with a more sensitive method, such as PFGE or multilocus sequence typing.     

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis as tools for typing Staphylococcus aureus isolates in a microepidemiological setting

Multilocus sequence typing (MLST) of Staphylococcus aureus is well suited to the study of global or long-term epidemiology, but its role in local epidemiology has not been defined. The present study has compared MLST with pulsed-field gel electrophoresis (PFGE) by using S. aureus isolates associated with carriage and disease in a busy regional renal unit. One hundred forty-four patients were prospectively recruited, of whom 103 were receiving hemodialysis and 41 were on continuous ambulatory peritoneal dialysis. Three nasal swab specimens were obtained 1 month apart on entering the study. A nasal swab was positive for S. aureus on at least one occasion in 50 patients (35%). Typing of the 104 carriage isolates demonstrated 21 PFGE types and 21 sequence types (STs). Thirty-one carriers had two or more positive nasal swabs; of these, the isolates in all swabs from a given carrier had identical PFGE types for 29 carriers; the isolates in all of the same 29 swabs had identical STs. The carriage strain in two patients changed both PFGE type and STs during the period of swabbing. Eight patients (6%) had an episode of S. aureus bacteremia during the 12-month study period, and two of these were nasal carriers. One of these invasive isolates had the same PFGE type and ST as the carriage isolate. There were no differences between Simpson's index of diversity for PFGE and Simpson's index of diversity for MLST for both invasive and carriage isolates, suggesting that the two methods have very similar discriminatory abilities. We conclude that PFGE and MLST performed equally in this study.     

Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus.

Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best method of typing methicillin-resistant S. aureus strains.     

Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus using pulsed-field gel electrophoresis

A panel of 24 methicillin-resistant Staphylococcus aureus strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.     

Epidemiologic genotyping of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis at a university hospital and comparison with antibiotyping and protein A and coagulase gene polymorphisms

A total of 124 methicillin-resistant Staphylococcus aureus (MRSA) isolates were ascertained at the University Hospital of the Canary Islands between January 1997 and April 2000. Genotyping included pulsed-field gel electrophoresis (PFGE) (SmaI digestion) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for the coagulase (coa) and protein A (spa) genes. Antibiotic resistance was the main phenotypic marker correlated with genotyping results. Three main PFGE types were detected: A (with 12 subtypes), B (with 2 subtypes), and C. PFGE type A1 was the most commonly found (61% of isolates) and the one responsible for all the epidemic outbreaks. Other genetics markers used (coa and spa RFLPs) were significantly correlated with the PFGE types detected (P < 0.001). These PCR-RFLP assays were useful as molecular markers for a quick, preliminary study of MRSA outbreaks.     

Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus.     

Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates.

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.     

Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains.

Analysis of sequences in the fragments of the 16S-23S rRNA intergenic spacer region by the ribosome spacer PCR (RS-PCR) can differentiate strains of methicillin-resistant Staphylococcus aureus (MRSA). We compared this technique with pulsed-field gel electrophoresis (PFGE) for typing MRSA strains and its application during an investigation of an outbreak. A total of 180 isolates of MRSA collected from various hospital laboratories within the United Kingdom and elsewhere were typed by PFGE and RS-PCR. PFGE identified 17 different types among the 180 strains examined, and RS-PCR generated 13 different types. PFGE could detect minor genetic variations among the isolates and could identify the variants which were not discriminated by RS-PCR. Four unique strain types detected by PFGE were not detected by RS-PCR. When applied to typing the outbreak-related strains from the vascular surgery unit at the General Infirmary at Leeds, the results of RS-PCR were identical to those of PFGE. Our results have shown that RS-PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as PFGE for typing MRSA isolates and should be useful in the local investigation of MRSA outbreaks.     

Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates

Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staphylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the results produced by MLVA and the three other typing methods. As in our earlier study, MLVA turned out to have discriminatory power similar to that of PFGE. Comparison of data obtained by the two approaches allowed us to propose a 70% or ca. 80% cutoff value of the similarity between two MLVA patterns, depending on a cutoff level applied to interpret the PFGE results, 75% or ca. 90%, respectively. The cutoff values corresponded to the difference of up to six or four bands, respectively, among maximum 14 bands in total produced by two isolates in the analysis. The MLVA clusters matched well those obtained by PFGE, and they were also consistent in general with clusters generated by spa typing and MLST, these latter methods characterized lower resolution. Our results suggest that MLVA may be reliable in shorter-term S. aureus epidemiological studies, including analyses of outbreaks and hospital-to-hospital strain transmission events. Well-known advantages of typing methods based on PCR (low cost, short time, and easiness of performance) make MLVA a method that may be useful in a variety of laboratories, including those performing routine microbiological analyses within medical centers.     

Epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis.

To evaluate the usefulness of phenotypic and genotypic analyses for the epidemiologic typing of methicillin-resistant Staphylococcus aureus (MRSA), we characterized 64 epidemic MRSA isolates and 10 sporadic methicillin-susceptible S. aureus isolates from a university hospital and 18 MRSA isolates from hospitals in different geographical areas. Chromosomal DNA macrorestriction analysis with SstII was resolved by pulsed-field gel electrophoresis and compared with antibiotype analysis, phage type analysis, and standard genomic DNA restriction analysis with BglII. Indices of the discriminatory ability of these methods were 0.982, 0.959, 0.947, and 0.959, respectively. Macrorestriction patterns of 94% of MRSA isolates from patients, personnel, and the environment associated with a nosocomial outbreak were closely related (similarity coefficient, 85 to 100%). In contrast, methicillin-susceptible S. aureus isolates showed a marked diversity of macrorestriction patterns (median similarity, 41%). MRSA isolates from other geographical areas showed diverse macrorestriction patterns, with the exception of four isolates displaying identical or closely related patterns; these isolates were associated with concurrent outbreaks in four other Belgian hospitals. A concordance of genomic DNA macrorestriction typing with phenotypic methods was observed for 60 to 65% of MRSA isolates, and a concordance with standard DNA restriction analysis was found for 79 to 98% of these isolates. In conclusion, genomic DNA macrorestriction analysis was a useful complement to phenotypic methods for delineating epidemic isolates of MRSA, for identifying their nosocomial reservoirs, and for tracing their intra- and interhospital spread. The genetic relatedness of MRSA isolates, as estimated by this technique, appeared to correlate with their space-time clustering.     

Multilocus sequence typing compared to pulsed-field gel electrophoresis for molecular typing of Pseudomonas aeruginosa

For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa.     

Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional "violations" of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.     

Characterization of isolates of methicillin-resistant Staphylococcus aureus from Hong Kong by phage typing, pulsed-field gel electrophoresis, and fluorescent amplified-fragment length polymorphism analysis

The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major "clones" at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.