a Department of Microbiology, Hippokration General Hospital, Thessaloniki; b Department of Medical Microbiology, University of Thessalia, Larissa; c Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece
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Abstract |
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Introduction |
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Materials and methods |
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PCR was performed on MRSA isolates in order to amplify a 449 bp product from within the mecA gene.7 PCR was also performed on the multi-susceptible MRSA isolates, for amplification of genes ermA (139 bp product), aadD (165 bp product) and aacAaphD (220 bp product) using published primers.8,9
The erythromycin-susceptible isolates were typed by pulsed-field gel electrophoresis (PFGE) after genomic DNA macrorestriction. Chromosomal DNA was prepared and digested with SmaI nuclease as described previously.5 Digested chromosomal DNA was separated with a CHEF DRIII apparatus (Bio-Rad, Birmingham, UK) for 20 h with ramp pulse times ranging from 5 to 34.9 s. Banding patterns of the strains were compared visually, on the basis of up to three band differences for related isolates.
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Results |
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A 449 bp product of the mecA gene was detected in all 76 isolates. PCR failed to amplify any product for genes ermA and aadD in all erythromycin-susceptible MRSA isolates, while a 220 bp product for gene aacAaphD was amplified in the 47 aminoglycoside-resistant isolates. The latter gene was not detected in any of the 29 aminoglycoside-susceptible isolates (Figure 1). The MRSA isolates belonged to the same major PFGE type, which included two subtypes (Ia and Ib) differing from each other by two bands (Figure 2
). The isolates within each subtype were indistinguishable. Subtype Ia corresponded to the predominant aminoglycoside-resistant strain, while subtype Ib included the aminoglycoside-susceptible isolates. The PFGE pattern of subtype Ia included a band of approximately 340 kb, while with the pattern of subtype Ib this band was substituted by a smaller band of approximately 295 kb.
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Discussion |
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The isolates that exhibited susceptibility to all non-ß- lactam antibiotics differed from the aminoglycosideresistant isolates by two bands, and belonged to the same clone. Multi-susceptible MRSA isolates have spread in our hospital during 1999.5 However, aminoglycoside-resistant multi-susceptible MRSA isolates were scarcely detected previously but gradually became predominant, and currently represent more than 60% of the multi-susceptible isolates. This observation could suggest that the aminoglycoside-resistant strain possibly resulted from the susceptible strain after a genetic event, obtaining an element that contained the gene aacAaphD. This shift is possibly caused by the substitution of a smaller by a larger PFGE band. It should be noted that strains with a PFGE pattern identical to that of the subtype Ib have been identified previously in another hospital, suggesting the circulation of this strain in our region.5 In contrast, strains with the antibiotic resistance phenotype of subtype Ia have not been identified in other northern Greek hospitals (J. Douboyas, personal communication). Thus, it could be postulated that the aminoglycoside-susceptible strain might have been the ancestor of the aminoglycoside-resistant strain and the genetic event possibly occurred intra-hospitally. The gradual predominance of isolates belonging to subtype Ia might have been facilitated by the high consumption of aminoglycoside antibiotics in our setting. It is also of interest that the PFGE patterns of Greek strains seem to differ substantially from those of other multi-susceptible MRSA clones,3,4 although these strains were not run under the same conditions.
The clone of the present report, predominated in a hospital with a high prevalence of MRSA, representing 69.1% of the total MRSA. The remaining MRSA isolates exhibited various multi-resistant phenotypes. It seems that in our hospital, the susceptible clone has replaced the classical multi-resistant MRSA clones, as the overall frequency of MRSA among S. aureus has not changed considerably during the last decade (data not shown). Changes in use of antibiotics cannot in themselves account for the emergence of this clone and the contribution of factors influencing the colonization or resistance of this particular strain to environmental stress cannot be excluded.
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Acknowledgments |
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Notes |
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References |
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Received 9 February 2001; returned 10 April 2001; revised 4 May 2001; accepted 10 May 2001