Methicillin-Resistant Staphylococcus aureus Sequence Type 239-III, Ohio, USA, 2007–2009

Identification of virulent strains emphasizes the need for molecular surveillance.


As part of a study supported by the Prevention Epicenter
Program of the Centers for Disease Control and Prevention (Atlanta, GA, USA), which evaluated transmission of MRSA among hospitals in Ohio, molecular typing identifi ed 78 (6%) of 1,286 patients with MRSA ST239-III infections. Ninety-fi ve percent (74/78) of these infections were health care associated, and 65% (51/78) of patients had histories of invasive device use. The crude case-fatality rate was 22% (17/78). Identifi cation of these strains, which belong to a virulent clonal group, emphasizes the need for molecular surveillance.
S taphylococcus aureus is a major human pathogen that possesses multiple toxins and virulence mechanisms (1). Antimicrobial drug resistance in S. aureus has added to the complexity of treating serious infections caused by this bacteria, and methicillin-resistant S. aureus (MRSA) appears to have greater virulence than methicillin-susceptible strains (2,3). Most MRSA strains in the United States are pulsed-fi eld gel electrophoresis (PFGE) types USA100 and USA300, corresponding to multilocus sequence typing (MLST) ST5 and ST8, respectively (4). MRSA belonging to MLST ST239 and harboring staphylococcal cassette chromosome mec (SCCmec) type III (MRSA ST239-III) are associated with infections in health care settings, outbreaks, increased resistance to antimicrobial drugs, and capacity for invasive disease (5)(6)(7).
MRSA ST239-III has a history of successful dissemination in many regions, leading to a diverse array of regionally prevalent clones. These clones include the Brazilian; British Epidemic 1, 4, 7, 9, and 11; Canadian Epidemic 3/Punjab; Czech; Eastern Australian 2 and 3; Georgian; Hungarian; Lublin; Nanjing/Taipei (ST241); Portuguese; and Vienna clones (8,9). Although it is common worldwide, MRSA ST239-III has not played any predominant role in the United States; infections with MRSA ST239-III have been rarely reported in the United States since the 1990s (9)(10)(11)(12)(13). Recently, only 2 reports of this strain in the United States involving sporadic nasal colonization and bloodstream infections have been published (13,14).
In this study, we describe clinical epidemiologic characteristics and molecular analysis of clinical infections with MRSA ST239-III in the midwestern United States. Identifi cation of a strain from such a virulent clonal group in the United States with wide dissemination in other parts of the world represents a potential public health concern.

Sampling Method
As part of Centers for Disease Control and Prevention (CDC) (Atlanta, GA, USA)-sponsored Prevention Epicenter Program study evaluating the transmission of MRSA between hospitals in Ohio, USA, molecular analysis was performed on a group of clinical MRSA isolates collected from The Ohio State Health Network (OSHN). The OSHN consists of The Ohio State University (OSU) Wexner Medical Center (WMC), which is a tertiary care medical center, and 7 smaller community hospitals located 30-120 miles from OSU. All MRSA specimens from community hospitals and selected OSU MRSA blood isolates and isolates from patients residing in the catchment areas of the outreach hospitals were prospectively collected during March 2009-February 2010 for genotyping by using a repetitive element PCR (rep-PCR). Among archived MRSA isolates from OSUWMC from January 2007 through February 2009, only a selection of isolates was chosen for genotyping (not a randomized sampling). The total number of isolates and the total number of ST239 from each time period was collected.

Data Collection
We performed medical record reviews for 1,286 patients. Patient demographic characteristics (presence of health care-related risk factors during the preceding 12 months, presence of an invasive device during the previous 7 days, and concurrent conditions) were collected. Patientlevel data, including addresses for geocoding, were entered into a secure database within the OSUWMC Information Warehouse.

Classifi cation of MRSA Infections
All MRSA cases were classifi ed into 3 categories on the basis of accepted epidemiologic defi nitions (11). The fi rst category was health care-associated, defi ned as a culture obtained >48 hours after admission. The second category was health care-associated community onset, defi ned as a culture obtained <48 hours after admission with identifi ed health care-associated risk factors. The third category was community-associated, defi ned as a culture obtained <48 hours after admission without health care-associated risk factors. Health care-associated risk factors comprised presence of an invasive device, history of MRSA infection or colonization, surgery, hospitalization, dialysis, or residence in a long-term care facility in the 12 months preceding the culture.
Outcomes for MRSA infection were categorized as cure (complete resolution after antimicrobial drug treatment); failure (persistence of infection and change in antimicrobial drug regimen); relapse (resolution of infection after complete treatment with subsequent development of new symptoms); recurrent (redevelopment of MRSA at same or other site >2 weeks after completion of treatment for initial MRSA infection); indeterminate (unknown outcome); and death (death <30 days after diagnosis of MRSA infection because of any cause or during the same hospitalization). Destination after hospital discharge, such as home or skilled nursing facility, was also noted.

Drug Susceptibility Testing
The respective OSHN Clinical Microbiology Laboratories initially identifi ed all MRSA isolates by using standard microbiological methods. Antimicrobial drug susceptibility testing was performed at each institution, and results were interpreted according to Clinical and Laboratory Standards Institute break point guidelines (15). At OSUWMC, antimicrobial drug susceptibility testing was performed by using the automated Micro-Scan method (Siemens Diagnostics, Sacramento, CA, USA), and only constitutive clindamycin testing was performed. Linezolid MIC >4 mg/L were confi rmed by using the Etest method (bioMérieux, Marcy l'Etoile, France).

Genotyping
The MRSA ST239 III isolates were genotyped initially by using rep-PCR, followed by PFGE, staphylococcal protein A sequencing (spa typing), SCCmec typing, and mec-associated direct repeat unit (dru) typing. Selected isolates were also characterized by MLST and singlenucleotide polymorphism (SNP) typing. Detection of genes encoding Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin (TSST), arginine catabolic mobile element (ACME), and high-level mupirocin resistance (mupA) was also performed. Brief descriptions of each testing method are outlined below.

rep-PCR
The DiversiLab System (bioMérieux, Durham, NC, USA) was used for rep-PCR analysis according to described methods (16). Isolates belonging to designated rep-PCR clusters shared >95% similarity. In addition, comparison of matching patterns in the DiversiLab System library was initially used to infer the PFGE and SCCmec types, which were later validated by using appropriate testing methods. The numeric classifi cation system used for rep-PCR analysis is unique to OSUWMC.

PFGE
The PulseNet protocol for molecular subtyping of S. aureus was followed. Salmonella enterica serotype Braenderup DNA was digested with XbaI (Roche, Indianapolis, IN, USA) and used as the normalization standard for gel analysis. S. aureus chromosomal DNA was digested with SmaI (Roche). Fragments were separated in a clamped homogenous electric fi eld mapper unit (Bio-Rad Laboratories, Hercules, CA, USA). Fingerprint images were analyzed by using Bionumerics software version 4.61 (Applied Maths NV, Sint-Martens-Latem, Belgium). The traditional classifi cation of PFGE subtypes was not used because we were not analyzing for an outbreak (17). Thus, interpretations of possibly or probably related PFGE subtypes between strains obtained by PFGE band patterns were not made. Each PFGE band difference was classifi ed as a unique PFGE pattern.

SCCmec Typing
SCCmec typing was performed on all MRSA isolates by using a described multiplex real-time PCR (20). This PCR is specifi c for 2 essential gene complexes (ccr and mec) found in all SCCmec elements.
dru Typing MRSA isolates were also characterized by sequencing the hypervariable dru repeat region within the SCCmec element (21) and using DruID software (9). New dru types were submitted to www.dru-typing.org.

SNP Typing
A panel of 43 SNPs for describing the global population structure of MRSA ST239-III (9) was used to identify the haplotypes of 22 isolates. These SNPs were typed by using Golden-Gate Genotyping Assay (Illumina, San Diego, CA, USA) and conventional Sanger sequencing.

Statistical Analysis
All patient demographic, clinical, and molecular typing data were aggregated in tabular format, and descriptive statistics were generated by using SAS version 9.2 (SAS Institute Inc., Cary NC, USA), for demographic and risk factor history. A signifi cant difference between ST239 and other strains (US300, US100, and all other strains) was examined by using χ 2 tests for categorical variables and t-tests for continuous variables. An α level of 0.5 was used.

Human Subjects Protection
We obtained approval for this study from the OSU Offi ce of Responsible Research Practices' Biomedical Institutional Review Board. The OSU Information Warehouse has established honest broker status with the OSU Institutional Review Board, enabling storage of fully identifi able data and presentation of patient data to investigators in a coded format that maintains patient confi dentiality.
These strains were fi rst recognized by rep-PCR as possible SCCmecA type III isolates from the DiversiLab System library with imputed Brazilian PFGE types. Additional molecular typing identifi ed MRSA ST239-III in a clade different from that containing the Brazilian strains.
Comparison of clinical characteristics of MRSA ST239-III with those of PFGE types USA100 and USA300 and other non-ST239 infections are shown in the Table. Most ST239 isolates were health care-associated MRSA and had characteristics and concurrent conditions similar to those associated with USA100. MRSA ST239-III did not have virulent determinants often associated with health care-associated or community-associated MRSA strains, such as PVL, TSST, ACME, or mupA.
A total of 33 unique genotypic combinations from 78 patients infected with MRSA ST239-III were represented among isolates with complete SCCmec, rep-PCR, PFGE, spa, and dru data. This information and year of isolation are shown in Figure 2. The genotype cluster sizes ranged from 2 to 18 isolates; 23 of the isolates were unique. In contrast, SNP typing of 22/78 isolates with a panel of 43 SNPs showed them to be indistinguishable from each other. All isolates tested belonged to haplotype 9 (H9) within MRSA ST239-III clade II (Figure 3). This clade was composed of isolates from many continents, including many from sources in Asia (9).

Discussion
MRSA ST239-III has demonstrated epidemic potential worldwide, and identifi cation of this strain in the United States might represent a major public health concern. Although the precise time frame of emergence of this strain in Ohio is unknown, it appears to have been present before the study because it was identifi ed in archived specimens dating back to 2007. The origin of the MRSA ST239-III clonal group has been dated to the mid-20th century in 2 studies (9,27). Thus, the Ohio strain might have been introduced into the region anytime during the past 40 years. Because of incomplete sampling during the study, the transmission dynamics of Ohio MRSA ST239 III are uncertain. Whether the prevalence would have been >6% if complete sampling was conducted is unknown. Furthermore, we found no epidemiologic evidence of clustering of cases consistent with an outbreak of infection with this strain. The large number of isolates at OSUWMC merely refl ects its role as a large referral center.
Increased illness and death for pulmonary disease might be caused by an enhanced ability of MRSA ST239-III strains to produce biofi lms and adhere to airway epithelial cells, providing a biologically plausible explanation for its potential to cause pneumonia and central line-associated BSIs (28,29). The association of MRSA ST239-III isolates with pulmonary infections has also been reported in South Korea (30). Moreover, in a multicenter study involving 21 hospitals in Beijing, where MRSA ST239-III is especially prevalent, 61% of MRSA LRTI cases were attributed to MRSA ST239-III (31). The London outbreak strain of MRSA ST239-III, and most examined MRSA ST239-III strains from the People's Republic of China, belong to the same clade (clade II) as the Ohio strain of MRSA ST239-III, demonstrating the potential for this lineage to disseminate and cause disease (9,27,32). Because of incomplete collection of all MRSA strains causing LRTIs, whether MRSA ST239-III is the prevalent strain in MRSA pneumonias is unknown. A total of 17% (203/1,206) of the study isolates were pulmonary infections and 9% (19/222) of the pneumonias were caused by MRSA ST239-III.
PFGE is the standard for MRSA outbreak investigations. However, we chose a rapid PCR-based method that could serve as a potential screening surveillance tool to identify outbreaks of a particular strain. Although many institutions might be using PFGE for outbreak investigations to identify clonal clusters, methods for PFGE subtype classifi cation are often not standardized between laboratories (33,34). To maximize uniformity and enable comparisons of PFGE classifi cations between institutions, the standardized CDC protocols for performing and analyzing PFGE results were used. The initial CDC PFGE analysis classifi ed our isolates as the Brazilian clone. However, SNP typing unambiguously placed the Ohio isolates within a different clade than the Brazilian clone ( Figure 3) (9). Because PFGE typing cannot defi ne specifi c lineages for MRSA ST239-III, CDC has changed the reporting of MRSA ST239-III strains from Brazilian subtype to ST239. Laboratories will need to perform additional testing to identify specifi c clades or clonal groups. Classifi cation of the 78 MRSA ST239-III isolates by the traditional PFGE outbreak method would identify only 2 PFGE subtypes instead of the 8 observed patterns. PFGE subtype 1 would consist of isolates A-E, G, and H, and subtype 2 would consist of isolate F ( Figure  1, panel B).
Classifi cation issues also exist with respect to the DiversiLab system. Five of the 8 initial rep-PCR patterns matched isolates in the DiversiLab library, which likewise categorized them as the Brazilian clone. In contrast, 3 other rep-PCR patterns did not match any isolates in the DiversiLab library. Additional molecular tests were performed to identify these isolates as MRSA ST239-III. Because of the novel identifi cation of MRSA ST239-III strains, we performed a battery of molecular tests to better classify the strains. Although the same dru types might be found in unrelated MRSA lineages and different staphylococcal species (32), variation at the dru locus within the SCCmec III element is consistent with MRSA ST239-III phylogeny (9). Because use of this typing method is relatively new, further study of its phylogenetic utility is needed (21). The feasibility of population-based genomewide SNP datasets has been demonstrated for MRSA ST239-III by Harris et al. (27). Cluster groupings from these PFGE, rep-PCR, and dru methods were not identical, suggesting that the molecular tests are not completely interchangeable. Of the various molecular methods used to index genomic variation in MRSA, dru typing and rep-PCR appeared to be more discriminatory.
The publication of only 2 recent reports of MRSA ST239-III in the United States (13,14) might have resulted from inadequate national surveillance or low transmissibility of the strain. Institutions might not be able to perform genotyping on all their isolates because of lack of appropriate laboratory facilities or resources. An alternative method of surveillance for MRSA ST239-III might be evaluation of the drug susceptibility pattern. Most of the MRSA ST239-III strains in our study were resistant to clindamycin, tetracycline, and gentamicin. In a subset analysis, the phenotypic patterns of antimicrobial drug susceptibility and MRSA genotype prediction were determined for 798 MRSA isolates from the OSHN dataset. Ninety-four percent (63/67) of the MRSA ST239-III isolates were resistant to all 3 drugs (clindamycin, tetracycline, and gentamicin) (35). Use of phenotypic drug susceptibility patterns might alert infection control practitioners that they are dealing with a MRSA ST239-III clone. Additional molecular testing, such as spa typing in conjunction with SCCmec typing, can then be performed to confi rm the strain type.
We have genotyped and geocoded 1,286 MRSA isolates in the CDC Prevention Epicenter Study by using rep-PCR. The lack of a clear association between MRSA ST239-III molecular genotype patterns could be caused by the retrospective nature of this study, lack of complete population sampling, and the inability to gather social network history. The rep-PCR and PFGE methods might be of limited use in evaluating geographic clustering at the scale studied here unless specifi c lineages of the reference strains are included in the respective databases. Because of the clonality exhibited by MRSA ST239-III, even a relatively small panel of 43 SNPs is able to identify the same major phylogenetic lineages that are identifi ed by genome-wide SNPs. A more detailed genome-wide SNP analysis might be required to resolve geographic clustering. Additional social networking for determining spatial, temporal, and geographic relationships of patients at the medical institutions studied is under way to identify potential nosocomial interhospital and intrahospital transmission.
Recent identifi cation of a strain of MRSA ST239-III in the midwestern United States is a major public health concern. Globally, this strain has demonstrated increased virulence and widespread dissemination. Because of an inadequate national surveillance system, temporal emergence and dissemination of the strain in the United States is uncertain. The MRSA ST239-III strains in Ohio have molecular heterogeneity and geographic diversity. The SNP-based data suggest that the strains also show clonal diversity. Continued surveillance is warranted because this MRSA ST239-III strain, similar to related strains worldwide, exhibits increased antimicrobial drug resistance, capacity for causing invasive disease, potential for causing outbreaks, and resulting in illnesses and deaths. MRSA ST239-III strains might have the potential to become established locally and disseminate among health care institutions as reported in other regions. Our study underscores the value of molecular surveillance, including traditional fi ngerprinting methods and newer sequence-based typing methods, as a critical component in understanding the evolving epidemiology of MRSA. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 10, October 2012  (9). Sizes of circles indicate relative frequency of different haplotypes. Arrows indicate haplotype 5 (H5), which includes the Brazilian clone, and haplotype 9 (H9), which includes the 22 MRSA ST239-III isolates from Ohio. Relationships between haplotypes were determined by using maximum-parsimony analysis (9).