Plant and Microbial Science Department, University of Canterbury, Christchurch, New Zealand
Received 15 January 2002; returned 7 May 2002; revised 18 June 2002; accepted 8 July 2002
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Abstract |
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Introduction |
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The prevalence of either mechanism tends to vary with geographical location. In the USA, the M phenotype is more prevalent,2 while in Europe the MLSB phenotype dominates.3 Previous studies have determined the prevalence of pneumococcal resistance to erythromycin to be 11% in New Zealand,4 but studies on the mechanisms of resistance have not yet been performed. The current study describes the prevalence of two macrolide resistance determinants [erm(A) and mef(B)] in New Zealand, as assessed by PCR analysis. Furthermore, DNA macrorestriction profiling was performed on selected isolates to determine whether clonal expansion was responsible for increased resistance.
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Materials and methods |
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erm(B) and mef(A) genes were amplified by PCR using primers and conditions described previously.6,7 PFGE was performed essentially as described elsewhere,8 and interpreted in accordance with published criteria.9 The 330 bp PCR products of each of the erm(B) and mef(A) genes were sequenced to confirm their identities, and subsequently used as probes for Southern hybridization analysis. Southern hybridizations were performed using the DIG Easyhyb kit (Roche) in accordance with the manufacturers instructions.
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Results and discussion |
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PCR detection of the macrolide resistance determinants (Table 1) detected mef(A) in 83 (66.9%) isolates and erm(B) in 118 (95.2%) isolates. Both mef(A) and erm(B) were detected in 77 (62.1%) isolates. All 77 isolates containing both genes were multi-resistant; the most frequently associated combination of resistances was penicillin, erythromycin, co-trimoxazole and tetracycline, which was noted in 74 (96%) of these isolates. In all but one instance, when both genes were detected in the same isolate, the erythromycin MIC was 128 mg/L. This high-level resistance was presumably imparted by a functional erm(B) gene. The single isolate with low-level erythromycin resistance in which both resistance genes were detected (isolate 413) was also susceptible to clindamycin. This isolate may contain a deleted or otherwise defective erm(B) gene, and the low level of resistance resulted from the product of a functional mef(A) gene, imparting the M phenotype.
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Although an uncommon genotype, a recent report from South Africa found 36 of 118 (30.5%) erythromycin-resistant isolates tested contained both erm(B) and mef(A) genes, using PCR.10 DNA fingerprinting of these 36 serotype 19F isolates revealed that 30 isolates belonged to a single clone. To determine whether the Christchurch isolates were clonal, selected isolates were examined by macrorestriction analysis of chromosomal DNA. Of the 77 isolates from which both the erm(B) and mef(A) genes had been amplified, the first 40 consecutive isolates were analysed by PFGE; 38 belonged to a clonal group (Figure 1), and two (isolates 221 and 4) were unique. Of the 38 isolates assigned to a clonal group, 37 profiles were identical, whereas one was a subtle variant of the major clone (isolate 28). Serotype data were available for only 16 of the 40 isolates; 15 isolates belonging to the major clonal group were found to be serogroup 19 (four typed as 19F), and one isolate not belonging to the clonal group (isolate 4) was found to be 23F.
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The results from this study show that the most predominant erythromycin resistance genotype in Christchurch is erm(B) mef(A). This combination of genes imparts a phenotype essentially identical to that imparted by erm(B) alone. The majority of the erm(B) mef(A) isolates belonged to a multi-resistant serotype 19F clone, which has much in common with a multi-resistant 19F clone recently described in South Africa.10 This report suggests the possible global spread of this clone.
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Acknowledgements |
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Footnotes |
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Present address. School of Molecular Biosciences, Washington State University, Pullman, WA, USA.
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References |
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