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Gram-Positive Typing and Research Unit, Curtin University of Technology, and Royal Perth Hospital, Perth, Western Australia 6000, Australia
Microbiology Group, Department of Biology, New Mexico State University, Las Cruces, New Mexico 88003-8001
Las Palmas Medical Center, El Paso, Texas 77902
ABSTRACT
Seventy-one percent of 76 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from two medical centers in El Paso, Texas, represent three similar pulsed-field gel electrophoresis types. Overall, six pulsed-field types were identified represented by multilocus sequence/staphylococcal chromosomal cassette DNA mec (SCCmec) types: ST5-MRSA-II; ST36-MRSA-II; ST8 (untypeable SCCmec); and a newly described clonal cluster 8 strain, ST507-MRSA-IV. This study demonstrates the presence of multiple-antibiotic-resistant epidemic MRSA clones in El Paso.
TEXT
Staphylococcus aureus is the leading cause of nosocomial infections (5). Today, methicillin-resistant Staphylococcus aureus (MRSA) resistant to multiple antibiotics is commonly isolated, and vancomycin-intermediate (9) and vancomycin-resistant (4) strains have been described. Only a few epidemic MRSA clones are responsible for a large percentage of MRSA disease worldwide (8, 21). Methicillin resistance in staphylococci is mediated by mecA (1), which is carried on a larger chromosomal element referred to as the staphylococcal chromosomal cassette DNA mec (SCCmec) (13). SCCmec consists of the mec gene complex, containing mecA and the mecA regulatory genes, and the ccr complex, which encodes a recombinase(s) responsible for SCCmec movement (12). To date, five SCCmec allotypes have been described on the basis of the mec gene complex, the ccr gene complex, and other genetic elements (10, 11, 15).
El Paso, Texas, is the largest United States-Mexican border city (population of 565,000) and 77% of the population is of Hispanic or Latino origin. Immediately south of El Paso lies the Mexican city of Ciudad Juarez (population of 1,200,000), where antibiotics are readily purchased over the counter (3). In order to understand MRSA epidemiology in a United States-Mexican border city with a large Hispanic or Latino population, we have performed a survey of MRSA strains collected in two medical centers in El Paso.
Typing of MRSA isolates. Seventy-six clinical MRSA isolates (Table 1) were collected from the Las Palmas (27 isolates) and Del Sol (49 isolates) medical centers in El Paso, Texas, from June to August 2002. Antibiograms were performed using the Kirby-Bauer method according to the CLSI (formerly NCCLS) (17) using antibiotic disks obtained from Oxoid (Basingstoke, England) or Remel (Lenexa, KS). Resistance to fusidic acid was determined using a 10-μg disk (Oxoid). Inducible clindamycin resistance was determined by a disk diffusion assay by the method of McDougal et al. (16). Strains resistant to three or more classes of antibiotics were considered multiply antibiotic resistant (16). Vancomycin MICs were determined with AB BIODISK Etest strips (Remel, Lenexa, KS) according to the manufacturer's instructions. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA was performed as previously described (18) using the CHEF DR III system (Bio-Rad Laboratories, Inc., Hercules, Calif.). Chromosomal patterns were analyzed by scanning with a Fluor-S MultiImager and analyzing them with Multi-Analyst/PC (Bio-Rad). PFGE SmaI restriction fragment length polymorphism patterns were grouped according to the criteria of Tenover et al. (23), and strains with 80% or greater pattern similarity were considered clonal. Multilocus sequence typing (MLST) was performed on a LI-COR Long Reader 4200 sequencer (LI-COR, Inc., Lincoln, NE) with primers and PCR protocols previously established (7). LI-COR sequences were collected with Base Imager software (LI-COR) and manipulated with AssemblyLIGN (Accelrys, Cambridge, England). Sequences were then submitted to http://www.mlst.net, and a sequence type (ST) was assigned. SCCmec allotyping (14) and phage typing (2) using the basic international, Australian MRSA, and international MRSA phage sets were determined according to published procedures.
PFGE of SmaI-digested chromosomal DNA revealed the presence of five pulsed-field type (PFT) groups and one unique strain (PFT-F) (Fig. 1). PFT-A represents the largest PFT group that was further subdivided into three subgroups, while PFT-B was the second largest group. MLST revealed that strains LP60 (PFT-A1), LP35 (PFT-E), and LP57 (PFT-F) were all ST5 (USA 100 or New York/Japan) clones, strain LP8 (PFT-B) had an ST36 (USA 200 or EMRSA-16) signature, LP13 (PFT-C) was of ST8 (USA 500 or New York V) ancestry, and LP3 (PFT-D) represents a new ST signature ST507 (3,3,74,58,4,4,3), in clonal complex 8 (CC8) (8). SCCmec typing revealed that strains LP60, LP8, LP35, and LP57 harbored SCCmec allotype II, while LP3 possessed a type IV SCCmec and LP13 possessed a class A mec complex, but the ccr complex could not be typed with the primer sets utilized. Phage typing was not useful in determining isolate relatedness, and many strains proved untypeable by this method (Table 1).
Therefore, 71% of the MRSA strains collected from two medical centers in El Paso, Texas, are represented by related PFTs, PFT-A, -E, and -F, which were resistant to ciprofloxacin and erythromycin and constitutively or inducibly resistant to clindamycin. Various strains in these PFT groups were also singly or multiply resistant to gentamicin, mupirocin, rifampin, tetracycline, and/or trimethoprim. Representative strains of these PFTs were all ST5-MRSA-II, and the presence of three diverged PFTs among these clones may indicate that these clones have been introduced on multiple occasions or are evolving in the medical centers investigated. ST5-MRSA-II clones represent 44% of all MRSA strains isolated in the United States (16), and in 1 year following introduction, an ST5-MRSA-II clone completely replaced an existing endemic clone in a Mexican hospital (24). Numerous vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are derived from ST5-MRSA-II clones (8, 16, 17). Since this clone is endemic in El Paso, the stage is set for the selection of VISA or VRSA.
The strains in the second largest PFT group, PFT-B, were all resistant to ciprofloxacin, clindamycin, and erythromycin and represent 17% of the isolates investigated. Representative PFT-B strain LP8 is an ST36-MRSA-II clone, which is the second most commonly isolated health care-associated MRSA in the United States (16).
The PFT-C strains were resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin, and mupirocin. Representative PFT-C strain LP13 is an ST8 clone with an untypeable SCCmec, suggesting the presence of a unique SCCmec. ST8 clones demonstrate variation in their SCCmec (6, 8), and some MRSA contain ccr gene complexes that do not amplify with the primers used for SCCmec allotyping (14, 19). ST8 clones are the second most common clone isolated in two hospitals in Miami, Florida, and 70% of MRSA strains isolated from confirmed human immunodeficiency virus-positive patients are ST8-MRSA-IV (6).
The PFT-D strains were resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem, tetracycline, and trimethoprim. PFT-D representative strain LP3 is a new ST type, ST507-MRSA-IV located in CC8, which encompasses ST8 strains as well (8). It is possible that LP3 has evolved from an ST8 strain (LP13) endemic in our area.
It is of interest to note that the PFT-B strains demonstrated the lowest and most variable (0.38 to 1.5 mg/liter) vancomycin MICs (Table 1), while all other strains demonstrated vancomycin MICs of 1.5 to 2 mg/liter.
In conclusion, we have demonstrated the presence of multiple-antibiotic-resistant epidemic MRSA clones in two medical centers in El Paso, Texas, located at a unique ethnic crossroads. All MRSA investigated were susceptible to synercid, linezolid, and fusidic acid, and no VISA or VRSA was detected.
ACKNOWLEDGMENTS
We acknowledge NIH grant S06 GM08136-29 (J.E.G.), NIH grant R15 AI054382-01 (J.E.G.), MBRS-NIH-RISE GM612222 (NMSU), and NIH-MARC GMO-7666726 (NMSU).
We acknowledge Denise Lionbarger for preliminary experiments and early administration of this project.
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