a Institute for Medical Microbiology and Virology, Heinrich-Heine Universität Düsseldorf, Universitätsstraße 1, Geb. 22.21, 40225 Düsseldorf, Germany; b Eijkman-Winkler Institute for Clinical Microbiology, University Medical Center Utrecht, The Netherlands
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Abstract |
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Introduction |
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In an attempt to update our knowledge of the status of MLS resistance in Europe, we recently investigated the prevalence of resistance to macrolides, clindamycin and quinupristin/dalfopristin.5 The present investigation was undertaken to study the prevalence of the macrolide resistance genes, ermA, ermB, ermC, msrA/msrB, ereA and ereB, using the polymerase chain reaction (PCR), in the first 851 unrelated clinical isolates of erythromycin-resistant S. aureus and the first 75 unrelated clinical isolates of erythromycin-resistant Enterococcus faecium. These isolates were sent from 24 different European university hospitals as part of the SENTRY Antimicrobial Surveillance Programme.
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Material and methods |
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The study included clinical isolates collected since the initiation of the European SENTRY programme in April 1997 through to December 1998. The protocol for this study has been described previously.5
PCR for the detection of macrolide resistance genes
Every isolate of S. aureus and E. faecium demonstrating resistance to erythromycin was screened for the presence of macrolide resistance genes. In total, 851 erythromycin-resistant S. aureus (358 methicillin-susceptible S. aureus (MSSA) and 493 methicillin-resistant S. aureus (MRSA)) and 75 erythromycin-resistant E. faecium isolates were analysed. In addition, 50 random isolates that were fully susceptible to macrolides (40 S. aureus and 10 E. faecium) were screened for the presence of macrolide resistance genes as described below.
Oligonucleotide primers for ermA, ermB, ermC, msrA/ msrB, ereA and ereB for use in the PCRs were selected from the DNA sequences published by Sutcliffe et al.3 The specificity of each set of primers was tested using DNA extracts of reference strains containing ermA, ermB, ermC, msrA/msrB, ereA and ereB (ermA: S. aureus RN 1389; ermB: Streptococcus pyogenes AC1/pAC1; ermC: S. aureus RN4220/pE194 and msrA: S. aureus RN4220/pAT10) all kindly supplied by Joyce Sutcliffe (Central Research Division, Pfizer, Groton, CT, USA). In addition, strains containing ereA (Escherichia coli/pIP1100) and ereB (E. coli/pAT72) kindly supplied by Patrice Courvalin (Institute Pasteur, Paris, France) were used. A random sample of PCR products with each set of primers was sequenced. Primers specific for conserved regions of the 16S rRNA gene were used as additional internal controls.6
Genomic DNA was isolated and two multiplex PCRs (primer set for ermA, ermB and ermC, together with msrA/msrB, as well as a primer set for ereA and ereB in a second separate PCR) were performed as described by Sutcliffe et al.3 The expected PCR products for ermA, ermB and ermC were between 639 and 645 bp. Therefore, after confirmation of the presence of an erm gene, single PCRs were performed in order to verify the class of the erm gene, either ermA, ermB or ermC.
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Results and discussion |
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This present survey constitutes the largest collection of clinical isolates of S. aureus and E. faecium studied for the prevalence of macrolide resistance genes. The multiplex PCRs described by Sutcliffe et al.3 for research purposes can also be routinely applied to survey macrolide resistance mechanisms present in large collections of clinical isolates. Using their method we failed to detect non-specific PCR products. Furthermore, macrolide resistance genes were not detected in any of the 50 erythromycin-sensitive S. aureus and E. faecium isolates tested. As shown in Table I, the most prevalent resistance gene in S. aureus was ermA (571/851; 67%), followed by ermC (192/851; 23%) and msrA/msrB (48/851; 6%). Less common were ermB and ereB, each occurring in 0.6% of the erythromycin-resistant S. aureus isolates tested. The ereA gene was not detected in any of the isolates.
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The ereB gene, coding for a macrolide-inactivating enzyme, was only found in MRSA isolates expressing the constitutive MLSB phenotype (5/458; 1%) (Table I). Neither ereA nor ereB in combination with other macrolide resistance determinants was found. We are not aware of any other surveillance study describing the prevalence of ereA and ereB in erythromycin-resistant S. aureus isolates.
Macrolide resistance by efflux due to the msrA/msrB gene was only found in MSSA isolates (14/358; 13%). This is in contrast to the results of Lina et al.,7 who detected the msrA/msrB gene in both MSSA and MRSA isolates. Moreover, they found the gene in only 2.1% of the 144 S. aureus strains tested. In line with their observations, however, we found no combination of msrA/msrB with other macrolide resistance determinants. To date, three S. aureus isolates have been found to harbour esterase activity-hydrolysing macrolides and a macrolide efflux system.4
The ermB gene was the most prevalent resistance determinant found in erythromycin-resistant E. faecium isolates, followed by ermA (93% versus 4%). The combination of ermA and ermB was detected in two of 75 isolates (3%). Jensen and colleagues recently analysed 113 erythromycin-resistant enterococcal isolates of human and animal origin and found the ermB gene to be present in 88%.11
The frequency of isolates that displayed erythromycin resistance in the absence of one of the six resistance genes tested for ranged between 4.5% and 8.6% in the five groups of isolates analysed. This implies that other mechanisms contribute to macrolide resistance in S. aureus and E. faecium.
In summary, resistance to erythromycin in S. aureus isolates from French hospitals was due mainly to the presence of ermA and ermC genes. The ermA gene was more common in MRSA isolates, mainly in strains with a constitutive MLSB expression, than in MSSA isolates, whereas ermC was more common in MSSA isolates, mainly in strains with inducible expression. Only a few strains had the ereB or ermB gene, while macrolide resistance by efflux due to the msrA gene was more common, but only detectable in MSSA. In contrast to S. aureus, erythromycin resistance in E. faecium was almost exclusively due to the presence of the ermB gene.
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Acknowledgments |
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Notes |
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References |
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2 . Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S. & Wootton, J. C. (1990). Inducible erythromycin-resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Molecular Microbiology 4, 120714.[ISI][Medline]
3 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 25626.[Abstract]
4 . Wondrack, L., Massa, M., Yang, B. V. & Sutcliffe, J. (1996). Clinical strain of Staphylococcus aureus inactivates and causes efflux of macrolides. Antimicrobial Agents and Chemotherapy 40, 9928.[Abstract]
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10 . Eady, E. A., Ross, J. I., Tipper, J. L., Walters, C. E., Cove J. H. & Noble, W. C. (1993). Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pump in epidemiologically distinct groups of staphylococci. Antimicrobial Agents and Chemotherapy 31, 21117.
11 . Jensen, L. B., Frimodt-Moller, N. & Aarestrup, F. M. (1999). Presence of erm gene classes in gram-positive bacteria of animal and human origin in Denmark. FEMS Microbiology Letters 170, 1518.[ISI][Medline]
Received 13 October 1999; returned 2 December 1999; revised 4 January 2000; accepted 24 January 2000