1 Department of Microbiology, School of Medicine, University of Patras, Rion 26500, Patras; 2 Department of Microbiology, School of Medicine, University of Thessalia, Larissa, Greece
Received 17 September 2003; returned 8 December 2003; revised 14 January 2004; accepted 24 February 2004
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Abstract |
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Methods: Antibiotic resistance was investigated by double disc diffusion and MIC determination. Resistance determinants were detected by PCR and DNA hybridization, while clonal types were identified by pulsed-field gel electrophoresis (PFGE) analysis of SmaI DNA fragments.
Results: Among methicillin-susceptible S. aureus (MSSA) isolates, inducible and MS phenotypes were detected, with the predominance of the erm(A) gene, followed by the msr(A) and erm(C) genes. The majority of methicillin-resistant S. aureus (MRSA) isolates expressed the constitutive phenotype and carried the erm(C) gene. PFGE revealed the dissemination of two major clones among the MRSA in both hospitals.
Conclusions: erm(C) is the predominant genetic determinant for the expression of MLSB resistance among S. aureus isolates, especially MRSA, in Greece. This is due to the spread of two major clones in the country.
Keywords: erythromycin, clindamycin, staphylococci, mechanisms, typing
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Introduction |
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The mechanisms responsible for resistance to erythromycin in staphylococci are target site modification and active drug efflux.3,4 Target site modification is mediated by the presence of erm genes [erm(A), erm(B) and erm(C)] conferring resistance to MLSB antibiotics.3,4 Phenotypic expression of MLSB resistance can be inducible or constitutive.3,4 On the other hand, macrolide efflux is effected by membrane proteins encoded by the msr(A)/msr(B) genes and is specific for the 14- and 15-membered macrolides and streptogramin B (MS phenotype); lincosamide and streptogramin A antibiotics remain unaffected.3,4
The purpose of this study was to investigate the prevalence of erythromycin resistance in S. aureus clinical isolates in Greece, to examine the genetic mechanisms of resistance and to analyse clonality by molecular methods.
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Materials and methods |
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The study included 1477 S. aureus clinical isolates collected during a 3 year period (19992001) in the University Hospitals of Patras and Thessalia in the southwestern and central parts of Greece. Duplicate isolates from the same patients, even if the site of infection was different, were excluded. Isolates were characterized to the species level by Gram stain, growth on mannitol salt agar (BBL, Becton Dickinson, MD, USA), catalase and coagulase production (Staphyloslide latex test; BBL, Becton Dickinson) and by biochemical tests (API Staph; bioMérieux, SA Lyon, France).
Susceptibility testing
Disc diffusion tests and the differentiation between MS and MLSB phenotypes were carried out for all isolates previously identified as S. aureus by a modified double disc diffusion test.5 Discs (BBL, Becton Dickinson) containing oxacillin (1 µg), erythromycin (15 µg), clindamycin (2 µg) and lincomycin (2 µg) were applied to inoculated MuellerHinton agar plates (Becton Dickinson) and incubated for 24 h at 35°C. S. aureus ATCC 25923 was used as a negative control.
Minimal inhibitory concentrations (MICs) of oxacillin, erythromycin, clindamycin and quinupristin/dalfopristin were determined by Etest (Biodisk, Solna, Sweden) and the results were interpreted according to NCCLS recommendations.6
PCR
Primers were chosen for the antibiotic resistance genes mecA (sense: 5'-GGTCCCATTAACTCTGAAG-3' and anti-sense: 5'-AGTTCTGCAGTACCGGATTTGC-3'),7 erm(A) (sense: 5'-GTTCAAGAACAATCAATACAGAG-3' and anti-sense: 5'-GGATCAGGAAAAGGACAT TTTAC-3'), erm(B) (sense: 5'-CCGTTTACGAAATTGGAACAGGT AAAGGGC-3' and anti-sense: 5'-GAATCGAGACTTGAGTGTGC-3'), erm(C) (sense: 5'-GCTAATATTGTTTAAATCGTCAATTCC-3' and anti-sense: 5'-GGATCAGGAAAAGGACATTTTAC-3'), msr(A) (sense: 5'-GGCACAATAAGAGTGTTTAAAGG-3' and anti-sense: 5'-AAGTTATATCATGAATAGATTGTCCTGTT-3') and msr(B) (sense: 5'-TATGATATCCATAATAATTATCCAATC-3' and anti-sense: 5'-AAGTTATATCATGAATAGATTGTCCTGTT-3').3 Total DNA was extracted from all isolates expressing resistance to macrolideslincosamides and PCR was carried out.3 The presence of mecA, erm(A), erm(B), erm(C) and msr(A)/msr(B) genes was identified by agarose gel electrophoresis of PCR products. Three erythromycin-susceptible S. aureus isolates were included as negative controls in all experiments. Streptococcus pyogenes O2C 1064 and S. pyogenes O2C 1061 were used as positive controls for the detection of erm(A) and erm(B) genes, respectively.
Plasmid extraction was carried out in all erm(C)-positive isolates (Qiagen midi plasmid purification kit; Qiagen, Valencia, CA, USA) and the presence of the gene was investigated by PCR.
Hybridization
Total DNA was extracted from the erythromycin-resistant S. aureus isolates into agarose discs as described previously.1 The presence of the resistance genes was verified by hybridization of ClaI DNA digests with the specific mecA and erm(A) probes and the Tn554 transposon labelled by the chemiluminescence ECL kit (Amersham, Pharmacia Biotech, Buckinghamshire, UK). The mecA and erm(A)-specific DNA probes were prepared after amplification of chromosomal DNA of S. aureus BB270 (mecA-positive) and plasmid DNA from E. coli RN7951 carrying Tn554, using the aforementioned primers. PCR products were purified by the Wizard DNA purification system (Promega, Madison, WI, USA) and used as DNA probes.
Clonal types
Pulsed-field gel electrophoresis (PFGE) of SmaI DNA digests was carried out as described previously.1 One to six band differences defined a PFGE subtype and seven or more band differences defined a distinct PFGE type. Clones of MRSA were defined by the combination of ClaI-mecA types ClaI-Tn554 polymorphisms and PFGE patterns,1 after comparison with previously identified MRSA clones in Patras University Hospital and reference strains.1
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Results and discussion |
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PFGE analysis classified the MSSA into eight types (Figure 1). Seventeen of the 60 MSSA, including five isolates with erythromycin MICs of 13 mg/L, belonged to PFGE type A, a pulsotype already identified among MRSA strains of Patras University Hospital from 1993.1 The remaining clonal types of MSSA were not related to previously identified clones (Table 2). MRSA isolates expressing the inducible MLSB phenotype belonged to PFGE types A and C endemic in the same hospital (Figure 1).1 Ninety-nine of 104 MRSA with the constitutive MLSB phenotype carrying the erm(C) gene belonged to two multi-resistant clonesIII'::KK::B and X'::KK::B (Table 3).1 These clones were distributed among strains of both University Hospitals. MRSA isolates carrying the erm(A) gene belonged to different clones (one strain each).
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To our knowledge, this is the first surveillance study in Greece for erythromycin resistance in S. aureus. The overall erythromycin resistance rate was lower compared with those in other countries,9,10 possibly due to the low use of MLS antibiotics in the hospitals. Erythromycin resistance was higher among MRSA (42% with MIC 4 mg/L) compared with MSSA (5%), with moderate variation during the 3 year period. Multicentre studies have already shown that resistance to macrolides is higher among MRSA, reaching 82%.9,10
Even though the predominance of erm(C) as the genetic determinant for the expression of resistance to MLS antibiotics among the total S. aureus population has been previously published, this is the first report of erm(C) predominance among MRSA, most likely due to the selection and dissemination of two major clones in the country.
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Acknowledgements |
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Footnotes |
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References |
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2
.
Weigel, L. M., Clewell, D. B., Gill, S. R. et al. (2003). Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302, 156971.
3
.
Lina, G., Quaglia, A., Reverdy, M. E. et al. (1999). Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrobial Agents and Chemotherapy 43, 10626.
4
.
Roberts, M. C., Sutcliffe, J., Courvalin, P. et al. (1999). Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants. Antimicrobial Agents and Chemotherapy 43, 282330.
5 . Stirnimann, G., Droz, S., Matter, L. et al. (1997). Phenotypes of resistance to macrolide and lincosamide antibiotics in Staphylococcus aureus. Clinical Microbiology and Infection 3, 7025.[Medline]
6 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Dilution Antimicrobial Susceptibility TestsFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
7
.
Petinaki, E., Arvaniti, A., Dimitracopoulos, G. et al. (2001). Detection of mecA, mecR1 and mecI genes among clinical isolates of methicillin-resistant staphylococci by combined polymerase chain reactions. Journal of Antimicrobial Chemotherapy 47, 297304.
8
.
Martineau, F., Picard, F. J., Lansac, N. et al. (2000). Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 44, 2318.
9
.
Schmitz, F. J., Sadurski, R., Kray, A. et al. (2000). Prevalence of macrolide-resistance genes in Staphylococcus aureus and Enterococcus faecium isolates from 24 European university hospitals. Journal of Antimicrobial Chemotherapy 45, 8914.
10 . Schmitz, F.-J., Petridou, J., Fluit, A. C. et al. (2000). Distribution of macrolide-resistance genes in Staphylococcus aureus blood-culture isolates from fifteen German University Hospitals. European Journal of Clinical Microbiology and Infectious Diseases 19, 3857.[CrossRef][ISI][Medline]