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Journal of Antimicrobial Chemotherapy (2004) 53, 814-817
© 2004 The British Society for Antimicrobial Chemotherapy

erm(C) is the predominant genetic determinant for the expression of resistance to macrolides among methicillin-resistant Staphylococcus aureus clinical isolates in Greece

I. Spiliopoulou1,*, E. Petinaki2, P. Papandreou1 and G. Dimitracopoulos1

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


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Macrolide, lincosamide and streptogramin type B (MLSB) resistance was determined in Staphylococcus aureus clinical isolates from two University Hospitals.

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


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Infections caused by multi-resistant Staphylococcus aureus strains still remain a problem in many health care institutions.1 Methicillin-resistant S. aureus (MRSA) represents a major cause of nosocomial infections, and the emergence of strains with intermediate susceptibility and resistance to glycopeptides reinforces the potential for spread of resistance determinants among bacteria and emphasizes the need for alternative therapeutic options.2 Macrolide, lincosamide and streptogramin B (MLSB) antibiotics are chemically distinct, but have similar effects on bacterial protein synthesis.3,4 Treatment of staphylococcal infections using MLSB antibiotics is commonplace but it is often accompanied by increased numbers of resistant strains.3,4

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.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Bacterial isolates

The study included 1477 S. aureus clinical isolates collected during a 3 year period (1999–2001) 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 Mueller–Hinton 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 macrolides–lincosamides 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


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Of the 1477 S. aureus isolates, 173 (11.7%) expressed resistance to erythromycin (Table 1). The constitutive phenotype was detected in 106 isolates (61.3%), the inducible phenotype in 53 (30.6%) and the MS phenotype in 13 isolates (7.5%). Sixty isolates were MSSA (14 from Patras and 46 from Thessalia) and 113 MRSA (73 from Patras and 40 from Thessalia) (Table 1). No isolate expressed resistance to quinupristin/dalfopristin (MICs <= 1 mg/L).3


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  		Table 1.. The distribution of methicillin- and erythromycin-resistant S. aureus isolates during the study period
 
MSSA isolates expressed the inducible or the MS phenotypes (Table 2). Isolates bearing the inducible MLSB phenotype possessed the erm(A) gene (58.3%) or the erm(C) gene (20%). The 13 isolates carrying the msr(A) gene expressed the MS phenotype (Table 2). It is worth mentioning that we identified six MSSA with erythromycin MICs of 1–3 mg/L, expressing inducible resistance and harbouring the erm(A) or erm(C) genes (Table 2). Martineau et al.8 have also reported the identification of four S. aureus isolates susceptible by disc diffusion that carried the erm(C) gene conferring resistance to macrolides.


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  		Table 2.. Resistance phenotypes and genotypes of erythromycin-resistant methicillin-susceptible S. aureus isolates
 
Among the MRSA, only six expressed inducible MLSB resistance, whereas the rest expressed the constitutive MLSB phenotype.3,4,8 Most of the MRSA isolates carried the erm(C) gene (109 isolates, see Table 3), followed by erm(A) (three isolates), whereas in other studies, even though it was predominant in the total S. aureus population, erm(C) was common among MSSA as well as coagulase-negative staphylococci.9,10 One isolate carried both erm(A) and erm(C) genes, a genotype that has been infrequently reported.9 No msr(A)-positive MRSA was identified.


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  		Table 3.. Resistance phenotypes and genotypes of erythromycin- and methicillin-resistant S. aureus isolates
 
Most of the S. aureus isolates in our study carried the erm(C) gene (70%) followed by the erm(A) gene (22%) and the msr(A) gene (7.5%), and one isolate carried both the erm(A) and erm(C) genes (0.6%) (Tables 2 and 3). This finding is in agreement with the results from 15 German University Hospitals which identified predominance of the erm(C) gene among S. aureus,10 whereas most studies report erm(A) as the most frequent genetic determinant.3,8,9 In this collection, no isolate was found to be positive for the erm(B) and msr(B) genes.3,9

PFGE analysis classified the MSSA into eight types (Figure 1). Seventeen of the 60 MSSA, including five isolates with erythromycin MICs of 1–3 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 clones—III'::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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  		Figure 1. PFGE of SmaI macrorestriction fragments of erythromycin-resistant S. aureus isolates. Lanes 1 and 29 molecular size standards (lambda oligomers); numbers on the right show molecular sizes in kilobases; letters below indicate PFGE types. Lanes 2 and 28, NCTC 8325 S. aureus; lane 3, MRSA representative of type A;1 lane 4, MRSA from Thessalia, clonal type VII'::NH::A, MLSB inducible, erm(C)-positive; lanes 5 and 6, MSSA from Patras, type A, MLSB inducible, erm(A)- and erm(C)-positive, respectively; lane 7, MRSA representative of type B;1 lane 8, MRSA from Thessalia, clonal type X'::KK::B, MLSB constitutive, erm(C)-positive; lane 9, MRSA from Patras, clonal type X'::KK::B, MLSB constitutive, erm(C)-positive; lane 10, MRSA from Patras, clonal type XII::B::B, MLSB constitutive, erm(A)- and erm(C)-positive; lane 11, MRSA representative of type C;1 lane 12, MRSA from Thessalia, clonal type I::KK::C, MLSB inducible, erm(A)-positive; lane 13, MRSA from Patras, clonal type II::NH::C, MLSB constitutive, erm(C)-positive; lane 14, MRSA from Patras, clonal type II::NH::C, MLSB inducible, erm(C)-positive; lane 15, MSSA from Patras, type F, MLSB inducible, erm(C)-positive; lanes 16–18, MSSA from Patras, type G, MLSB inducible, erm(A)-positive; lane 19, MSSA from Patras, type H, MLSB inducible, erm(A)-positive; lane 20, MSSA from Patras, type H, MLSB inducible, erm(C)-positive; lane 21, MSSA from Thessalia, type H, MS phenotype, msr(A)-positive; lane 22, MSSA from Patras, type I, MLSB inducible, erm(A)-positive; lanes 23 and 24, MSSA from Thessalia, type K, MLSB inducible, erm(A)-positive; lane 25, MSSA from Thessalia, type K, MLSB inducible, erm(C)-positive; lane 26, MSSA from Thessalia, type L, MLSB inducible, erm(A)-positive; lane 27, MSSA from Patras, type M, MS phenotype, msr(A)-positive.

 
The transfer of erm(A), which is a part of Tn554, was shown to occur via transposition and one should expect the dissemination of a limited number of clones among erm(A)-positive isolates.6,10 In our collection, it seems that erm(A)-positive S. aureus strains are distinct, since they belong to different clones. erm(C) is located in small plasmids that cannot be transferred by conjugation or mobilization by larger conjugative plasmids.8,10 Our results showed that the majority of erm(C)-positive MRSA isolates belonged to two clones even though they were isolated in two distinct hospitals. The prevalence of erm(C)-positive isolates is most likely due to the selection and dissemination of these two multi-resistant MRSA clones in Greece. The ClaI-Tn554 type of these clones (named KK) represents strains with one hybridization band with the transposon.1 This fact can be explained by the loss of a large part of a previously integrated whole Tn554, carrying erm(A), and the acquisition of erm(C) by these strains, as a result of antibiotic selective pressure.

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.


   Acknowledgements
 
We thank Professor H. de Lencastre for providing the reference strain RN7951 and Professor B. Berger-Bächi for providing the MRSA strain BB270. Reference strains for ClaI-Tn554 types belonged to the collection of the Molecular Genetics Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB/UNL), Oeiras, Portugal. We also thank C. Bartzavali for technical assistance.


   Footnotes
 
* Corresponding author. Tel: +30-2610-993978; Fax: +30-2610-994922; E-mail: spiliopl{at}med.upatras.gr Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Aires de Sousa, M., Bartzavali, C., Spiliopoulou, I. et al. (2003). Two international methicillin-resistant Staphylococcus aureus clones endemic in a University Hospital in Patras, Greece. Journal of Clinical Microbiology 41, 2027–32.[Abstract/Free Full Text]

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, 1569–71.[Abstract/Free Full Text]

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, 1062–6.[Abstract/Free Full Text]

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, 2823–30.[Free Full Text]

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, 702–5.[Medline]

6 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Dilution Antimicrobial Susceptibility Tests—Fifth 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, 297–304.[Abstract/Free Full Text]

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, 231–8.[Abstract/Free Full Text]

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, 891–4.[Abstract/Free Full Text]

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, 385–7.[CrossRef][Web of Science][Medline]


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