a Department of Microbiology, AHEPA University Hospital, 54 636 Thessaloniki, Greece; b Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece; c Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, London NW9 5HT, UK
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Abstract |
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Introduction |
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Clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin and/or streptomycin are commonly recovered in Greece. During 199394, the prevalence of high-level gentamicin resistance (HLGR) was 20.6%, whereas 2 years later (199697) this prevalence had more than doubled (43.7%).4 In the present study we investigated, for genetically related and distinct E. faecalis isolates, the possible contribution of the pheromone system to the conjugal transfer of aminoglycoside resistance genes and the increased prevalence of HLAR among enterococci from Greece. The presence of prgA and prgB, and phenotypic expression of the pheromone system were also determined.
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Materials and methods |
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PCR was performed on all gentamicin-resistant donors and transconjugants with primers specific for the gene encoding the bifunctional aminoglycoside modifying enzyme AAC(6')-APH(2'').6 E. faecium strain 2781,7 which expresses this enzyme, was included in the PCR assay as a positive control. All gentamicin and/or streptomycin-resistant donor strains and transconjugants were examined for their ability to aggregate when exposed to a cell-free culture filtrate of E. faecalis JH2-2. This method tests for any pheromone response, as described previously.8 The presence of prgA and prgB was investigated by PCR with published primers.3 DNA probes specific for prgA and prgB were generated by secondary PCRs to incorporate digoxigenin-11-dUTP (Roche Diagnostics Ltd, Lewes, UK) into primary amplicons. The incorporation of label was checked by comparing the electrophoretic mobilities of the primary and secondary products. The mobility of the latter was impeded by the label. Plasmid DNA was extracted from donors and transconjugants by an alkaline lysis procedure,7 separated on agarose gels and transferred to nylon membranes by capillary (Southern) blotting. Probes were hybridized to plasmid DNA under stringent conditions and the hybrids were detected colorimetrically according to the manufacturer's recommendations (Roche Diagnostics).
Clinical isolates that transferred resistance were analysed further by pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA as described previously.9 Banding patterns were compared visually and all loci were scored for the presence or absence of a band.
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Results |
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The 15 isolates that transferred HLAR contained from one to three plasmids. Transconjugants harbouring a single plasmid were derived from eight donor isolates: four donors transferred two plasmids to the recipient, and one donor transferred three plasmids (Table). Resistance to streptomycin or to streptomycin plus chloramphenicol was transferred from two donors at a relatively low frequency (9.7 x 108 and 8.1 x 107, respectively), but multiple plasmid extractions failed to detect any plasmid DNA in the transconjugants. This observation suggests possible integration of the element encoding resistance traits and a pheromone response into the recipient chromosome. A prgA-specific probe hybridized only to plasmids from the two transconjugants (derived from strains 7 and 10) that were positive for prgA by PCR. All plasmid-containing transconjugants hybridized strongly with a prgB-specific probe. The prgB probe also hybridized weakly to DNA from the chromosomal band of the two transconjugants (derived from strains 1 and 11) that were positive for pheromone response, but which did not harbour any detectable plasmids.
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Discussion |
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Enterococci possess a variety of mechanisms for transferring antibiotic resistance determinants to susceptible recipients. The pheromone-mediated conjugation systems of several plasmids have been studied, including those of pAD1, pCF10, pPD1 and pAM373.1,3,8,10 The genes encoding the surface exclusion protein (prgA) and aggregation substance (prgB) are conserved in most of the pheromone-responsive plasmids studied, although pAM373 is an exception.1 Genes involved in the regulation of the pheromone response are clustered on a 7 kb region of each plasmid.10 In the present study, eight of the 15 donors transferred a single pheromone-responsive plasmid together with high-level resistance to gentamicin and/or streptomycin, which indicates that the pheromone-responsive plasmids in our hospital frequently carried antibiotic resistance determinants. Several other pheromone-responsive plasmids that carry antibiotic resistance have been described previously.1
The hybridization of a prgB probe with chromosomal DNA of the two plasmid-free transconjugants may suggest the integration into the enterococcal chromosome of a plasmid carrying the genes that regulate the pheromone response and the antibiotic resistance determinants. These observations might indicate that E. faecalis strains in our hospital carry pheromone-responsive plasmids that harbour a gene with significant homology to prgB of other characterized plasmids, which encodes aggregation substance. Although prgA is conserved in many pheromone-responsive plasmids,1 it was not detected in the majority of isolates studied here. This suggests either the absence of a surface exclusion gene or, more likely, the presence in our strains of a surface exclusion protein encoded by a gene that has only low homology with prgA. The overall genetic similarity of the pheromone-response genes in our strains to those described previously is currently under investigation.
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Notes |
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References |
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2
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Casetta, A., Hoi, A. B., de Cespedes, G. & Horaud, D. (1998). Diversity of structures carrying the high-level gentamicin resistance gene (aac6-aph2) in Enterococcus faecalis isolated in France. Antimicrobial Agents and Chemotherapy 42, 288992.
3 . Heaton, M. P. & Handwerger, S. (1995). Conjugative mobilization of a vancomycin resistance plasmid by a putative Enterococcus faecium sex pheromone response plasmid. Microbial Drug Resistance 1, 17783.[ISI][Medline]
4
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Tsakris, A., Pournaras, S. & Douboyas, J. (1997). Changes in antimicrobial resistance of enterococci isolated in Greece. Journal of Antimicrobial Chemotherapy 40, 7357.
5 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition; Approved Standard M7-A4. NCCLS, Wayne, PA.
6 . Van de Klundert, J. A. M. & Vliegenthart, J. S. (1993). PCR detection of genes coding for aminoglycoside-modifying enzymes. In Diagnostic Molecular MicrobiologyPrinciples and Applications (Persing, D. H., Smith, T. F., Tenover, F. C. & White, T. J., Eds), pp. 54752. American Society for Microbiology, Washington, DC.
7 . Woodford, N., Morrison, D., Cookson, B. & George, R. C. (1993). Comparison of high-level gentamicin-resistant Enterococcus faecium isolates from different continents. Antimicrobial Agents and Chemotherapy 37, 6814.[Abstract]
8 . Ike, Y. & Clewell, D. B. (1984). Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen. Journal of Bacteriology 158, 77783.[ISI][Medline]
9 . Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R. & Weinstock, G. M. (1990). Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 205963.[ISI][Medline]
10 . Fujimoto, S., Tomita, H., Wakamatsu, E., Tanimoto, K. & Ike, Y. (1995). Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response. Journal of Bacteriology 177, 557481.[Abstract]
Received 3 April 2000; returned 17 June 2000; revised 14 July 2000; accepted 19 August 2000