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Antimicrobial Agents and Chemotherapy, September 2006, p. 3207-3211, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.00059-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

High-Level Mupirocin Resistance within Methicillin-Resistant Staphylococcus aureus Pandemic Lineages

Eduardo Pérez-Roth,¹ Celeste López-Aguilar,¹ Julia Alcoba-Florez,² and Sebastián Méndez-Álvarez^1,3,4*

Laboratorio de Biología Molecular, Unidad de Investigación,¹ Unidad de Microbiología, Hospital Universitario Nuestra Señora de Candelaria,² Departamento de Biología Celular y Microbiología, Universidad de La Laguna, Santa Cruz de Tenerife,³ Investigador Asociado, Centro de Investigaciones Biológicas del Consejo Superior de Investigaciones Científicas, Madrid, Spain⁴

Received 13 January 2006/ Returned for modification 24 March 2006/ Accepted 2 July 2006

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The methicillin-resistant Staphylococcus aureus (MRSA) population in the Hospital Universitario Nuestra Señora de Candelaria over a 5-year period (1998 to 2002) was marked by shifts in the circulation of pandemic clones. Here, we investigated the emergence of high-level mupirocin resistance (Hi-Mup^r). In addition to clonal spread, transfer of ileS2-carrying plasmids played a significant role in the dissemination of Hi-Mup^r among pandemic MRSA lineages.

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Most hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) isolates are members of five MRSA pandemic lineages or clonal complexes (CCs), namely, CC5, CC8, CC22, CC30, and CC45 (6, 14). Mupirocin constitutes the cornerstone of avoidance of MRSA carriage and ulterior infection, but resistance has emerged, and its spreading is worrisome, with transferable high-level mupirocin resistance (Hi-Mup^r) being of clinical significance (3, 4, 5, 11, 23, 25). Hi-Mup^r is associated with an additional isoleucyl-tRNA synthetase that is encoded by the ileS2 gene (8, 10). The ileS2 gene was commonly reported on plasmids that differed in size, restriction patterns, and ability to be transferred in conjugation experiments (12, 13, 16, 21, 24, 26).

We reported that 95.5% of the 375 MRSA isolates obtained from patients at Hospital Universitario Nuestra Señora de Candelaria (located in Tenerife, Canary Islands, Spain) between 1998 and 2002 belonged to six clones fitting in pandemic lineages (i.e., CC5, CC8, CC22, and CC30) (20). In the present work, we investigated Hi-Mup^r in such a staphylococcal population.

(Parts of this work were presented during the 1st International Workshop for Origin and Evolution of Bacterial Pathogens, Baeza, Spain, October 2004, and during the XX Congreso Nacional de Microbiología, Cáceres, Spain, September 2005.)

Isolates were screened for mupirocin resistance by the diskdiffusion and Etest methods (7, 15). The ileS2 gene was detected by a multiplex PCR (17, 18). Plasmid DNA was extracted with the QIAprep spin plasmid kit (QIAGEN, Hilden, Germany) with the addition of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) and digested with EcoRI and HindIII. Curing of plasmids and conjugation experiments were performed as previously described (24). The primers used are listed in Table 1.

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TABLE 1. Amplification and sequencing primers used in this study

The ileS2 locus was associated with conjugative plasmids in each Hi-Mup^r isolate. Hi-Mup^r was manifested by 48 MRSA isolates (12.8%) (Table 2) and increased significantly from 0% in 1998 to 15.6% in 2002 (P < 0.001). Each Hi-Mup^r isolate belonged to one of the six major MRSA clones and amplified the ileS2 gene fragment. Non-Hi-Mup^r isolates did not show the ileS2 amplicon. Pulsed-field gel electrophoresis (PFGE) subtypes shown by Hi-Mup^r isolates, excepting PFGE subtype B4, also included mupirocin-susceptible isolates, suggesting intrahospital acquisition of Hi-Mup^r by circulating clones.

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TABLE 2. Phenotypic and genotypic characteristics of high-level mupirocin-resistant MRSA isolates

HindIII restriction endonuclease analysis of plasmid DNA (REAP) revealed 16 patterns (i.e., I to XVI) (Table 2), each hybridizing with the ileS2-specific probe. Thus, the ileS2 gene was harbored by plasmids; this result was consistent with data from previous studies (12, 21). The different sizes of ileS2-hybridizing plasmid fragments permitted five polymorphs of the ileS2 locus (i.e., A to E) to be distinguished. Polymorphs A, B, D, and E were characterized by a single HindIII-hybridizing fragment, while polymorph C was defined by two hybridizing fragments (Tables 2 and 3).

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TABLE 3. Structural group, ileS2 locus polymorph, and clonal complex distribution of Hi-Mupr plasmid types

One Hi-Mup^r isolate of each restriction endonuclease analysis pattern (n = 16) was randomly selected for conjugation assays. The Hi-Mup^r was transferred from each isolate (Table 2). Overall, nine different ileS2-carrying plasmids (i.e., pMUP1 to pMUP9) were identified in transconjugants (Fig. 1). Susceptibility tests and curing experiments support that idea that these plasmids mediate Hi-Mup^r. Plasmids differed from each other by at least one HindIII restriction fragment, but some bands in common permitted four structural groups (i.e., S1 to S4) to be distinguished (Table 3). EcoRI digestions corroborated these results (data not shown). Plasmids included in each group displayed the same ileS2 locus polymorph (Fig. 1A1). The exception was group S2, given that plasmids pMUP3 and pMUP7 showed different polymorphs due to an additional ileS2-hybridizing fragment in pMUP7, which appeared as a double-intensity ca. 4.7-kb HindIII band, suggesting the existence of two copies of the ileS2 gene in pMUP7 (Fig. 1A, lane 7). Plasmids belonging to distinct groups differed from each other by at least five bands and showed different polymorphs.

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FIG. 1. Analysis of ileS2-carrying plasmid types by restriction endonuclease analysis and Southern hybridization. (A) HindIII restriction patterns of plasmid types extracted from S. aureus transconjugants of Hi-Mupr MRSA isolates. , ileS2-hybridizing fragments. (A1) Southern hybridization with the ileS2-containing DNA as a probe, showing the different ileS2 polymorphs (A to E). (A2) Southern hybridization using the traK DNA fragment as probe. Lanes 1 through 9, plasmid types pMUP1 to pMUP9. , lambda phage DNA digested with HindIII and digoxigenin labeled as a molecular size standard. Numbers on the right correspond to the molecular sizes (in base pairs) of DNA HindIII restriction fragments.

ileS2-carrying plasmids are related to the pSK41 family of staphylococcal multiresistance plasmids. It has been suggested that mupirocin resistance plasmids belong to the pSK41 family of staphylococcal conjugative plasmids (12) (the pSK41 sequence was obtained from GenBank, accession no. NC_005024). The putative relationship of pSK41 to ileS2-carrying plasmids was examined at the traK gene level. The traK gene of pSK41 is involved in the tra gene system for conjugation (2, 9). The traK gene was detected in each ileS2-carrying plasmid (Fig. 1A2). The nucleotide sequence of the open reading frame (1,641 bp) in the ca. 1.8-kb traK PCR products from plasmids included in groups S1 and S2 was identical to the pSK41 traK gene sequence. Group S3 plasmids showed 41 nucleotide differences, plus the insertion of 6 nucleotides, compared to the pSK41 traK sequence (>97% identity). The three plasmid types included in group S4 showed one nucleotide difference (>99% identity). These results indicated that the traK gene of pSK41 was conserved in ileS2-carrying plasmids. The plasmids included in groups S1, S2, and S4 had a single 4.7-kb traK-hybridizing fragment. The plasmids in group S3 presented a ca. 9-kb hybridization band. Additionally, the traD-traE region was detected in each Hi-Mup^r isolate and transconjugants. Our results are consistent with the concept of ileS2-carrying plasmids being members of the pSK41 family of conjugative plasmids.

Distribution of Hi-Mup^r plasmids among pandemic lineages (CCs). Some of the ileS2-carrying plasmids were detected in the background of different MRSA clones belonging to different CCs (Tables 2 and 3), indicating plasmid transfer. Furthermore, we found plasmids of the four structural groups in the collection of Hi-Mup^r ST36-II epidemiologically related isolates. The emergence of Hi-Mup^r in ST36-II is of health concern because it is the predominant clone, completely replacing the Iberian clone (ST247-IA) (19, 20, 22). Unlike ST36-II isolates, Hi-Mup^r isolates belonging to clones ST125-IVA and ST22-IV shared a single, unique ileS2-carrying plasmid, which indicates clonal dissemination in the spread of Hi-Mup^r.

Conclusions. All major MRSA pandemic clones already circulating in the hospital acquired ileS2-carrying plasmids, resulting in a multiclonal population structure. In addition to clonal spread and plasmid transfer, the finding of the ileS2 gene in plasmids differing in size and restriction patterns show that gene transposition constitutes an important mode of dissemination of Hi-Mup^r. Intervention strategies in the current scenario should identify dissemination of MRSA clones and/or ileS2-carrying mobile elements.

Nucleotide sequence accession numbers. The nucleotide sequences of the entire traK homolog genes from mupirocin resistance plasmids pMUP1, pMUP2, pMUP3, pMUP4, pMUP5, pMUP6, pMUP7, pMUP8, and pMUP9 identified in this study have been deposited in GenBank under accession numbers DQ232628, DQ232629, DQ232630, DQ232631, DQ232632, DQ232633, DQ232634, DQ232635, and DQ232636, respectively.

 
We are grateful to Mark Enright and Manuel Espinosa for critical reading of the manuscript, Warren B. Grubb for providing the S. aureus WBG541 strain, and Ninive Batista and Francisco Javier González-Paredes for their technical assistance in susceptibility tests and traK gene sequencing, respectively. We thank SmithKline Beecham Pharmaceuticals (United Kingdom) for the gift of mupirocin powder.

This study was supported by grants 2001/020 from the Consejería de Educación, Cultura y Deportes, FUNCIS 2002/038 and 2005/50 from the Canary Islands Autonomous Government, 2001/3150 from the Fondo de Investigación Sanitaria, and BIO2002/00953 from the Ministerio de Ciencia y Tecnología, Government of Spain, to S.M.-A. E.P.-R. was supported by a grant from the Consejería de Educación, Cultura y Deportes, and C.L.-A. was supported by a grant from FUNCIS, Canary Islands Autonomous Government. S.M.-A. was partially supported by Fondo de Investigación Sanitaria contract 99/3060.

 
* Corresponding author. Mailing address: Unidad de Investigación, Hospital Ntra. Sra. de Candelaria, Ctra. del Rosario s/n, 38010 Santa Cruz de Tenerife, Spain. Phone: 34-922-600080. Fax: 34-922-600562. E-mail: smenalv{at}gobiernodecanarias.org.

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Antimicrobial Agents and Chemotherapy, September 2006, p. 3207-3211, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.00059-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pérez-Roth, E. Articles by Méndez-Álvarez, S. Search for Related Content PubMed PubMed Citation Articles by Pérez-Roth, E. Articles by Méndez-Álvarez, S.