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Journal of Clinical Microbiology, April 2007, p. 1325-1329, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.02437-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Hetero-Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Isolate from a Medical Center in Las Cruces, New Mexico{triangledown}

Alejandro Delgado,1 James T. Riordan,1,2 Reena Lamichhane-Khadka,1 David C. Winnett,1 Jennifer Jimenez,3 Kim Robinson,4 Frances G. O'Brien,5 Stephanie A. Cantore,1 and John E. Gustafson1,2*

Department of Biology,1 Molecular Biology Program, New Mexico State University, Las Cruces, New Mexico 88003-8001,2 Mountain View Regional Medical Center, Las Cruces, New Mexico 88001,3 Memorial Medical Center, Las Cruces, New Mexico 88011,4 Gram-positive Bacteria Typing and Research Unit, Curtin University of Technology, and Royal Perth Hospital, Perth, Western Australia 60005

Received 4 December 2006/ Returned for modification 16 January 2007/ Accepted 22 January 2007


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ABSTRACT
 
A survey of 152 methicillin-resistant Staphylococcus aureus (MRSA) strains from medical centers in Las Cruces, NM, and El Paso, TX, revealed the presence of spa types 2 and 24 (clone USA100) and spa type 1 (clone USA300-0114). Las Cruces MRSA displayed relatively high vancomycin MICs, and one hetero-vancomycin-intermediate S. aureus strain was identified.


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TEXT
 
Staphylococcus aureus is the leading cause of nosocomial infections (2), and methicillin-resistant S. aureus (MRSA) is a common cause of community skin and soft-tissue infections (10). The USA100 MRSA clone represents up to 44% of MRSA isolates in the United States (8), and a unique community-associated clone, USA300-0114, is widely disseminated (6, 8, 10, 17). The use of vancomycin for the treatment of serious MRSA infections is threatened by the appearance of vancomycin-intermediate S. aureus (VISA) and hetero-VISA (hVISA) (1, 3, 5, 18). hVISA can present initial vancomycin MICs of ≤2 µg/ml, yet upon exposure to vancomycin, VISA subpopulations appear (MICs of ≥4 µg/ml) (15, 19). VISA and vancomycin-resistant S. aureus (MIC of ≥32 µg/ml) are often USA100 derivatives (4, 8, 15).

A survey of two medical centers in El Paso, TX, revealed that 71% of the MRSA isolates were related USA100 clones (12). Northwest from El Paso (~50 miles) lies Las Cruces, NM (population ~80,000), a popular U.S. retirement destination. In order to understand MRSA epidemiology in a Southern New Mexico border city with a large Hispanic (~52%) and growing retirement population, we have analyzed MRSA isolates collected from Las Cruces medical centers.

(This work was presented in part at poster sessions of the 105th [Atlanta, GA, 2005] and 106th [Orlando, FLA, 2006] General Meetings of the American Society for Microbiology).

Seventy-four MRSA strains from the Memorial Medical Center and 67 MRSA strains from the Mountain View Regional Medical Center (isolated 15 September 2003 to 20 August 2004; "MM" and "MV" strains, respectively), both located in Las Cruces, ~3.5 miles apart, as well as 11 El Paso MRSA isolates ("LP" strains [12]) were analyzed (Fig. 1; Table 1). Kirby-Bauer antibiograms and fusidic acid and inducible clindamycin resistance were determined by using disc diffusion (11, 12). Vancomycin MICs were determined by using AB BIODISK E-test strips (Remel, Lenexa, KS) and agar dilution (11), and vancomycin resistance population analyses were preformed as described previously (13). VISA mutants of hVISA strain MM66 were isolated on brain heart infusion agar (Becton-Dickinson and Co., Sparks, MD) containing 3 µg/ml vancomycin (Sigma Chemical Co., St Louis, MO) by inoculating these plates with 100 µl of brain heart infusion broth culture (18 h, 200 rpm, 37°C). Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA (14) was carried out by using the CHEF DR III system (Bio-Rad Laboratories, Inc., Hercules, CA), and SmaI restriction fragment length polymorphism similarities were determined (12). Chromosomal DNA for spa typing was isolated using the Qiamp DNA Mini kit (QIAGEN, Inc., Valencia, CA), and spaA polymorphic X regions were amplified (9) and sequenced. spa polymorphic X sequences were analyzed using eGenomics, and spa type numbers were assigned (7) (Table 1).


Figure 1
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  		FIG. 1. PFGE patterns of SmaI-restricted chromosomal DNA and dendrogram of percent relatedness. Bars indicate "MM" strains, and dotted lines indicate "MV" strains (if within a strain group, LP strains are included under the respective lines). "LP" strains are boxed, and MM66 is indicated with an arrow.


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  		TABLE 1. Characteristics of investigated strains

Five pulsed-field-type (PFT) groups containing 7 or more strains and 48 unrelated strains were identified (Fig. 1). PFT-A represented the largest PFT group (65 isolates, including 5 LP strains) and contained 13 strains of spa type 2 or 24, which diverge by only a single amino acid (B{leftrightarrow}E) (Fig. 1; Table 1). PFT-B was the second-largest group (14 isolates, including 1 LP strain) (Fig. 1; Table 1) and was represented by two spa type 2 strains. spa types 2 and 24 are also represented in PFT-E, as well as four unrelated strains (MM19, MM45, MV20, and LP20), and overall these spa types represent 74% of the strains typed (Table 1). spa types 2 and 24 are representative of the multilocus sequence/SCCmec type ST5-MRSA-II, which identifies USA100 (16). LP57 is also spa type 2 (Table 1) and was previously identified as ST5-MRSA-II (12). PFT-C (nine strains) is represented by two spa type 1 strains, and MM2 at the PFT-C boundary and MV8 below PFT-D were also spa type 1 (Fig. 1, Table). spa type 1 is a marker for the U.S. community-associated MRSA clone USA300-0114 (17).

PFT-D and -E are made up entirely of MM strains, and MV and MM strains throughout tended to group together in PFGE (Fig. 1). This demonstrates that while the major clones spreading within Las Cruces medical centers are similar, the clones within the two hospitals have evolved independently following introduction and/or the initial clone introduction was unique to each hospital.

All strains investigated were susceptible to synercid, linezolid, and fusidic acid, and no vancomycin-resistant S. aureus strains were detected. Of the 152 MRSA strains analyzed, 123 were resistant to the drugs ciprofloxacin, clindamycin, and erythromycin. Ninety percent of the Las Cruces strains demonstrated vancomycin E-test MICs of ≥2.0, and 12 exhibited a MIC of 3.0 µg/ml (Table 1). All but one of the latter group of strains (MV48) came from one medical center, and five clustered within related PFT-D and -E (Fig. 1; Table 1). The agar dilution MIC of MM66 was 4 µg/ml, and initial vancomycin resistance population analyses on all strains with E-test MICS of 3 µg/ml revealed that only MM66 produced colonies on plates containing 3 µg/ml vancomycin. hVISA strain MM66 proved 96% identical by PFT to strain MM61, yet MM61 has an additional SmaI band of 80 kb (Fig. 1) and an E-test MIC of 2.0. MM66 colonies appeared at a frequency of 1.5 x 10–6 on 3 µg/ml vancomycin, and 5 MM66 VISA colonies were picked. The agar dilution/E-test vancomycin MICs (µg/ml) for these five VISA MM66 mutants were ≥4/≥4. Colonies of hVISA MM66 did not appear above 3.0 µg/ml, while all MM66 VISA mutants produced colonies above this vancomycin concentration (4.5 to 6.0 µg/ml) (Fig. 2).


Figure 2
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  		FIG. 2. Vancomycin resistance population analysis of MM66 (parent) and MM66 VISA mutants (MM66-1 through -5).

In conclusion, these findings demonstrate that USA100 clones are spreading within medical centers in two predominantly Hispanic Southwestern cities. The predominant community-associated clone USA300-0114 was also found in Las Cruces hospitals. Furthermore, we report a preponderance of MRSA with vancomycin MICs of ≥2 µg/ml in Las Cruces medical centers and have identified the first hVISA in this ethnically unique region.


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ACKNOWLEDGMENTS
 
We are indebted for help in determining spa types and to the training of Alejandro Delgado provided by Barry N. Kreiswirth, William Eisner, and Steve Naidich (Public Health Research Institute).

We acknowledge support from the National Institutes of Health: S06 GM008136-32 (J.E.G., NMSU SCORE Program), R25 GM07667-29 (NMSU-MARC Program), S06 GM61222-05 (A.D., NMSU-MBRS-RISE Program), and P20RR16480 from the NM-INBRE Program of the National Center for Research Resources.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, New Mexico State University, MSC 3AF, P.O. Box 30001, Las Cruces, NM 88003-8001. Phone: (505) 646-5660. Fax: (505) 646-5665. E-mail: jgustafs{at}nmsu.edu Back

{triangledown} Published ahead of print on 31 January 2007. Back


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Journal of Clinical Microbiology, April 2007, p. 1325-1329, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.02437-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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