Pheromone responses and high-level aminoglycoside resistance of conjugative plasmids of Enterococcus faecalis from Greece

S. Pournarasa, A. Tsakrisb,*, M.-F. I. Palepouc, A. Papab, J. Douboyasa, A. Antoniadisb and N. Woodfordc

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fifteen of 22 clinical isolates of Enterococcus faecalis with high-level aminoglycoside resistance isolated in Greece, which were tested for mating ability, co-transferred pheromone response genes together with aminoglycoside resistance determinants to a sensitive recipient strain. Nine of them belonged to the same pulsotype, while the remaining six isolates were genetically unrelated. The prgB gene, which encodes aggregation substance, was detected in all the clinical isolates and transconjugants by both PCR and DNA hybridization but prgA, which encodes the surface exclusion protein, was only detected in two isolates, whereas it is present in most pheromone response plasmids from other sources.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococci have become a significant cause of hospital-acquired infections. High-level resistance to aminoglycosides (HLAR), specifically gentamicin and streptomycin, results in failure of the bactericidal synergic action normally seen when these drugs are combined with cell wall-active agents. HLAR and resistance to various other antimicrobial classes may be transferred horizontally among enterococci, and conjugation has been considered one of the major mechanisms responsible.1,2 In enterococcal strains, transfer of resistance to susceptible recipient strains is highly efficient in the presence of the sex pheromone system.1,3 In the generally accepted model of this system,1 chromosomally encoded oligopeptides (pheromones) are secreted into the surrounding medium by recipient strains lacking a corresponding pheromone-responsive plasmid. Potential ‘donor’ strains respond by producing an adhesin called aggregation substance, which is encoded by a pheromone-response gene, prgB. The prgB gene is carried by pheromone-responsive plasmids, and is conserved among plasmids responding to distinct pheromones.1 A second conserved pheromone-response gene, prgA, encodes a surface exclusion protein, responsible for the prevention of plasmid transfer between aggregated donor cells.1

Clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin and/or streptomycin are commonly recovered in Greece. During 1993–94, the prevalence of high-level gentamicin resistance (HLGR) was 20.6%, whereas 2 years later (1996–97) 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 138 HLAR E. faecalis isolates were obtained consecutively from separate patients hospitalized in AHEPA University Hospital, Thessaloniki, Greece, during 1995–97. Of the 138 isolates, eight exhibited HLGR only (MIC > 500 mg/L), 39 high-level streptomycin resistance only (MIC > 1000 mg/L) and 91 resistance to both aminoglycosides. The isolates were identified to the species level with standard biochemical tests. The specimens were obtained from patients hospitalized in various wards of the hospital, and included wound exudates, bronchial aspirates, urine and blood. MICs of gentamicin, streptomycin, chloramphenicol, ciprofloxacin, erythromycin, penicillin and trimethoprim were determined by an agar dilution method.5 Twenty-two of the 138 clinical isolates were selected randomly for mating experiments. These isolates were used as donors in filter mating experiments3 with E. faecalis strain JH2-2 (fusidic acid and rifampicin resistant), a plasmid-free reference strain that secretes multiple pheromones as the recipient. Transconjugant colonies were selected on brain–heart infusion agar plates containing rifampicin (100 µg/mL), fusidic acid (25 µg/mL) together with either gentamicin (500 µg/mL) or streptomycin (1000 µg/mL).

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ten of the 22 E. faecalis isolates transferred high-level resistance to both gentamicin and streptomycin to E. faecalis JH2-2, and five isolates transferred high-level streptomycin resistance. Conjugation frequencies varied between 5.2 x 10–3 and 9.7 x 10–8 transconjugants per donor (TableGo). In all but two isolates, resistance to other unrelated antibiotics was linked to HLAR. In two cases, the ability to cause ß-haemolysis of horse blood was also transferred with antibiotic resistance traits. The bifunctional aminoglycoside resistance gene, aac(6')-aph(2''), was amplified from all donors and transconjugants that exhibited HLGR. E. faecalis isolates with transferable aminoglycoside resistance were divided into seven distinct PFGE types (I–VII, TableGo) on the basis of more than three band differences. One major type included nine isolates; six of these were indistinguishable (type Ia) and three differed from them by two or three bands (Ib and Ic). The remaining six isolates were unrelated (II–VII).


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Table. Characteristics of pheromone-responsive plasmids encoding high-level aminoglycoside resistance
 
All the donor and transconjugant strains exhibited a clumping response upon exposure to a cell-free solution of pheromones, comprising an E. faecalis JH2-2 culture filtrate, thus indicating that the isolates contained pheromone-responsive plasmids. In support of this conclusion, a product of c. 420 bp was amplified by PCR with prgB- specific primers from all donors and transconjugants. However, only two donors (strains 7 and 10, TableGo), and the transconjugants derived from them, yielded the expected c. 700 bp product when tested with prgA-specific primers. All the other strains gave no products with these primers.

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 (TableGo). Resistance to streptomycin or to streptomycin plus chloramphenicol was transferred from two donors at a relatively low frequency (9.7 x 10–8 and 8.1 x 10–7, 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.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The pheromone system plays an important role in the horizontal spread of genes between strains of E. faecalis, including those genes encoding antibiotic resistance and virulence traits.1 This is possibly the explanation for the widespread dissemination of the bifunctional enzyme gene amongst enterococcal isolates from different continents.7 In the present study, identical and clonally related E. faecalis isolates were revealed by PFGE, indicating an intra-hospital transmission of such strains. Unrelated strains carrying HLAR determinants were also observed. Furthermore, both genetically related and distinct strains were positive in clumping experiments and contained pheromone-responsive conjugative determinants, suggesting that the pheromone system possibly contributed to the high prevalence of aminoglycoside resistance in our setting. Frequency of HLAR transfer was relatively low in some enterococcal isolates and a similar observation has also been reported previously.2

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.


    Notes
 
* Corresponding author. Tel: +30-31-999-091; Fax: +30-31-999-149; E-mail: atsakris{at}med.auth.gr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Wirth, R. (1994). The sex pheromone system of Enterococcus faecalis. More than just a plasmid-collection mechanism? European Journal of Biochemistry 222, 235–46.[Abstract]

2 . 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, 2889–92.[Abstract/Free Full Text]

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, 177–83.[ISI][Medline]

4 . Tsakris, A., Pournaras, S. & Douboyas, J. (1997). Changes in antimicrobial resistance of enterococci isolated in Greece. Journal of Antimicrobial Chemotherapy 40, 735–7.[Free Full Text]

5 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth 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 Microbiology—Principles and Applications (Persing, D. H., Smith, T. F., Tenover, F. C. & White, T. J., Eds), pp. 547–52. 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, 681–4.[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, 777–83.[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, 2059–63.[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, 5574–81.[Abstract]

Received 3 April 2000; returned 17 June 2000; revised 14 July 2000; accepted 19 August 2000





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