JAC Advance Access originally published online on March 9, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):1005-1009; doi:10.1093/jac/dkm057
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Co-transfer of vanA and aggregation substance genes from Enterococcus faecalis isolates in intra- and interspecies matings
1 Institute of Microbiology and Biomedical Sciences, Polytechnic University of Marche, 60100 Ancona, Italy 2 Department of Technologies and Health, Istituto Superiore di Sanità, 00161 Rome, Italy 3 Department of Biology, University of Genova, 16132 Genova, Italy
* Corresponding author. Tel: +39-071-2206-296; Fax: +39-071-2206-293; E-mail: b.facinelli{at}univpm.it
Received 17 October 2006; returned 21 November 2006; revised 23 January 2007; accepted 31 January 2007
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Abstract |
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Objectives: The study was undertaken to investigate vancomycin-resistant (vanA) Enterococcus faecalis isolates carrying aggregation substance (AS) gene(s) for their ability to co-transfer vanA and AS genes.
Methods: Six vanA clumping-positive E. faecalis isolates (five human and one food sample) carrying one or more AS genes (prgB, asa1, asa373) were analysed for co-transfer of vanA and AS genes to E. faecalis JH2-2 and Enterococcus faecium 64/3.
Results: E. faecalis isolates harboured one or multiple plasmids carrying vanA, one or more AS gene(s) or both. vanA was transferred to JH2-2 (frequencies of 10–3–10–6) from all donors and to 64/3 (10– 6–10– 8) only from donors from humans. AS genes were detected in 51/60 (85%) of JH2-2 and in 20/50 (40%) of 64/3 vanA transconjugants (prgB, asa1, asa373 or prgB asa373), of which a total of 53.6% were clumping-positive. The plasmid content of JH2-2 transconjugants from the same donor was either identical to that of the donor or it was completely different, suggesting different mechanisms for co-transfer (location on the same pheromone plasmid, mobilization of vanA plasmids by the pheromone-inducible conjugation system, rearrangement of plasmid content during matings). After induction with pheromones, a marked increase in adhesion to Caco-2 cells was observed in four isolates and in some JH2-2 transconjugants, all clumping-positive.
Conclusions: Findings suggest that co-transfer of vanA and AS genes may be a common feature of E. faecalis isolates. Since AS is recognized as a virulence factor, this feature might contribute to the emergence of strains with enhanced ability to cause infection and disease in humans.
Keywords: E. faecalis , prgB , asa1 , asa373
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Introduction |
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Over the last two decades, vancomycin-resistant enterococci (VRE) have emerged as major causes of nosocomial infections in clinical settings.1 Vancomycin resistance is most commonly found in Enterococcus faecium, but is increasingly being seen in Enterococcus faecalis, the predominant enterococcal species in humans.1 Clonal expansion has contributed significantly to the increasing incidence of VRE, e.g. the E. faecium clonal complex-17 (CC17) and the E. faecalis CC2 and CC9, which are associated with hospital outbreaks worldwide.2 VanA (vanA) expresses inducible, high-level vancomycin resistance and cross-resistance to teicoplanin, and is prevalently detected in E. faecium.1 VanA-type resistance in enterococcal clinical isolates is mediated by genetic elements, identical or closely related to Tn1546, that are generally carried by self-transferable plasmids.3,4 Conjugal transfer of vanA plasmids appears to be responsible for the spread of vanA resistance in enterococci.3,4
Aggregation substance (AS) of E. faecalis is a pheromone-inducible surface protein encoded by pheromone plasmids that facilitates conjugative exchange, by mediating strong binding between cells and formation of cell aggregates (clumps).1 AS also contributes to pathogenicity by enhancing cell adhesion and internalization as well as favouring intracellular survival within macrophages.1 Although pheromone plasmids are frequently found in E. faecalis, the presence of AS genes in vancomycin-resistant isolates of this species has rarely been reported.4–7
The present study was undertaken to investigate recently characterized vanA E. faecalis isolates carrying one or more AS genes (F. Biavasco, G. Foglia, C. Paoletti, G. Zandri, G. Magi, E. Guaglianone, A. Sundsfjord, C. Pruzzo, G. Donelli and B. Facinelli, unpublished data) for their ability to co-transfer vanA and AS genes in intra- and interspecies matings.
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Materials and methods |
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Strains
Six vanA E. faecalis isolates (five human and one food sample) carrying one or more AS genes (prgB, asa1, asa373), all clumping-positive, were studied (Table 1). These strains have recently been detected among vanA enterococci collected in Italy and investigated for vanA location, Tn1546 type, and presence of virulence determinants (F. Biavasco, G. Foglia, C. Paoletti, G. Zandri, G. Magi, E. Guaglianone, A. Sundsfjord, C. Pruzzo, G. Donelli and B. Facinelli, unpublished results); prgB with an additional EcoRI site (prgB*) was detected in two isolates. Growth in the presence of pheromones gave rise to a strong clumping reaction rated as +++ (two strains) or ++ (four strains). Plasmid analysis and hybridization experiments demonstrated that all harboured plasmid DNA strongly hybridizing to a probe specific for the vanA resistance determinant; SmaI restriction analysis of total DNA resulted in different PFGE profiles.
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E. faecalis OG1RF strains containing different pheromone plasmids (pCF10, prgB; pAD1, asa1; pAM373, asa373) were used to generate specific probes; fusidic acid- and rifampicin-resistant E. faecalis JH2-2, a pheromone-producer, and E. faecium 64/3 were used as recipients. Test strains also included four transconjugants obtained in another study using E. faecalis HC-VI4 as donor (G. Foglia, M. J. Flores, D. Mater, C. Paoletti, C. Vignaroli, E. Guaglianone, B. Facinelli, G. Cortier and F. Biavasco, unpublished results). Brain–heart infusion (BHI) broth and agar and blood agar base (BAB; Oxoid, Basingstoke, UK) supplemented with 5% defribinated sheep blood were used for routine growth. Isolates were maintained in glycerol at –70°C and subcultured twice in BHI agar before testing.
Conjugation experiments and clumping assay
Conjugal transfer and clumping assays were performed as described previously.8
Plasmid analysis and detection of vanA and AS genes
Plasmid analysis and PCR detection of vanA were done as described previously.8 AS genes were detected by using primers internal to highly conserved regions in asa1 and prgB (5'-AAGAAAAAGAAGTAGACCAAC; 3'-AAACGGCAAGACAAGTAAATA), and to asa373 (5'-GGACGCACGTACACAAAGCTAC; 3'-CTGGGTGTGATTCCGCTGTTA).9,10 EcoRI digestion of amplicons was performed according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim, Germany). Hybridization experiments were performed using biotin-labelled prgB, asa1 and asa373 probes and the BrightStar BioDetect kit (Ambion, Huntingdon, UK).
Adhesion was studied using Caco-2 cells, a human enterocyte-like cell line, as previously described.8 Bacterial adhesion efficiency was quantified by determining the ratio of cell-associated cfu to the total cfu of the initial inoculum and was defined as high (>10%), intermediate (1–10%) or low (<1%). Each strain was tested in at least three separate assays on different days, each assay representing the average of triplicate wells.
Fisher's exact test was applied with the Fisher test procedure of the S-Plus statistical package (S-PLUS 6.1 for Windows, Professional Edition, Release 1).
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Results |
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Plasmid location of vanA and AS genes in E. faecalis isolates
Plasmid analysis and hybridization experiments performed on the donors demonstrated that they harboured 1–3 plasmids (27–62 kb) carrying vanA, AS gene(s) or both (Table 1). vanA and AS genes (prgB, prgB*) were located on the same plasmid in HI-AN23, HC-AN21 and F-PM25, and on different plasmids in the remaining strains. In HC-VI4, prgB and asa1 were found on one plasmid, which was different from both the vanA plasmid and the asa373 plasmid. A vanA plasmid and two different prgB plasmids were detected in HC-AN22.
Co-transfer of vanA and AS genes in intra- and interspecies matings
vanA was successfully transferred from all E. faecalis isolates to E. faecalis JH2-2 (transfer frequencies of 10–3–10–6), whereas it was transferred to E. faecium 64/3 only when isolates from humans were used as donors (transfer frequencies of 10–6–10–8) (Table 1). To test co-transfer of vanA and AS genes, 10 vanA transconjugants from each mating experiment—for a total of 110 vanA transconjugants (60 from intra- and 50 from interspecific matings)—were randomly chosen and tested for the presence of AS genes by PCR and EcoRI restriction analysis of PCR products and for clumping activity (Table 1).
AS genes were detected in 71/110 (64.5%) vanA transconjugants [in 51/60 (85%) and in 20/50 (40%) of E. faecalis JH2-2 and E. faecium 64/3, respectively]. A weak (+) clumping reaction was detected in 59/110 (53.6%) vanA transconjugants [in 47/60 (78.3%) and in 12/50 (24%) of E. faecalis JH2-2 and E. faecium 64/3, respectively]. EcoRI restriction analysis made it possible to recognize different AS genes: prgB was the one most frequently detected in transconjugants, either alone or with asa1 or asa373. prgB was detected in 40/51 E. faecalis JH2-2 and in 19/20 E. faecium 64/3 transconjugants, which were either clumping-positive (36/40 JH2-2 transconjugants and 11/19 E. faecium 64/3 transconjugants) or clumping-negative (4/40 E. faecalis JH2-2 transconjugants and 8/19 E. faecium 64/3 transconjugants). When using vanA donors carrying more than one AS gene (HC-VI4 and HC-AN21), transconjugants carrying prgB or asa1 or asa373 or prgB asa373 were detected, all prgB transconjugants being clumping-negative. Moreover, transfer of prgB* (bearing an additional EcoRI site) from HC-AN21 to E. faecium 64/3 resulted in transconjugants carrying prgB (Table 1).
Plasmid analysis performed on 31 vanA and AS gene-carrying JH2-2 transconjugants derived from four donors showed that the plasmid content of transconjugants, derived from the same donor and carrying the same AS gene, was either identical to that of the donor or it was completely different (Figure 1).
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Induction with pheromones and adhesion to Caco-2 cells of E. faecalis isolates and transconjugants carrying vanA and AS genes
E. faecalis isolates and nine vanA transconjugants (six JH2-2 and three 64/3 carrying asa1, prgB and/or asa373) were tested for their ability to adhere to Caco-2 cells (Table 1).
In the absence of pheromones, adhesion efficiency was high in HI-AN23, low in HC-AN22 and HC-UD6, and intermediate in the remaining isolates, in the recipients and in the transconjugants (the only exception being the JH2-2 transconjugant, derived from HI-AN23, where adhesion efficiency was as high as in the donor).
After induction with JH2-2 pheromones, a significant association between the presence of AS genes and enhanced cell adhesion was noted (P < 0.01). Adhesion efficiency was increased in four (66.7%) isolates (HI-AN23, HC-VI4, HC-UD6 and F-PM25) and in three (50%) JH2-2 prgB or asa1 transconjugants (from HI-AN23, HC-AN21 and F-PM25); these proportions are not significantly different (P = 0.98). The increase was 2.2- to 6.0-fold in isolates and 1.7- to 6.6-fold in transconjugants Adhesion efficiency remained unaffected in all E. faecium 64/3 (the recipient and its transconjugants).
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Discussion |
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Co-transfer of pheromone response and antibiotic resistance has been reported in enterococci.7,8,11,12 In E. faecalis, co-transfer of AS and VanA resistance has been described recently,7 whereas in E. faecium it was demonstrated in 1996.8,11 In the present study, six vanA E. faecalis isolates from human and food samples harbouring one or more AS genes were investigated for their ability to co-transfer vanA and AS genes to E. faecalis and E. faecium recipients. All isolates, independently of their source, were capable of co-transferring vanA and AS genes, suggesting that the ability to co-transfer these genes may be a common feature of E. faecalis. Since AS promotes colonic mucosal invasion by E. faecalis, this feature might contribute to the emergence of strains with enhanced ability to cause infection and disease in humans.
vanA was successfully transferred from all donors to E. faecalis JH2-2, albeit with different transfer frequencies, while it was transferred to E. faecium 64/3 only when isolates from humans were used as donors, and then at lower frequencies. About two-thirds of vanA transconjugants acquired one or two AS genes, co-transfer being more frequent in intra- than interspecies matings; when present, clumping was less evident in transconjugants. prgB was co-transferred more frequently in intraspecific than in interspecific matings and, when transferred, it was not always expressed, asa1 was transferred alone in both intra- and interspecies matings and was always expressed, whereas asa373 was co-transferred either alone or with prgB only in intraspecies matings. When using donor HC-AN21 carrying prgB*, a reversion to prgB was detected in transconjugants, suggesting that the donor could have multiple prgB plasmids with polymorphic sequences.
After induction with pheromones, a marked increase in adhesion to Caco-2 cells was observed in intestinal, blood, bile and food vanA isolates and in half of E. faecalis transconjugants, all clumping-positive. Adhesion was not increased in E. faecium transconjugants, irrespective of clumping ability. These results demonstrated a significant association between the presence of AS genes and enhanced cell adhesion in E. faecalis; no significant differences were noted between donors and transconjugants.
Overall, our results demonstrated that co-transfer of vanA and AS genes is a fairly complicated process. In some E. faecalis isolates, vanA and AS genes were located on the same pheromone plasmid, whereas in others they were detected on different plasmids; moreover, two different AS genes could be located on the same plasmid, or the same AS gene could be found on two different plasmids. In addition, transconjugants derived from the same donor and carrying identical AS genes displayed different profiles that were also different from that of the donor. Data suggest that vanA and AS genes were co-transferred via different mechanisms: (i) location on the same pheromone plasmid; (ii) mobilization of vanA plasmids and (iii) rearrangement of the plasmid content during matings. In this regard, mobilization of plasmids by the pheromone-inducible conjugation system has recently been reported,13 and there is strong evidence for the importance of co-integrate formation between multiple plasmids in the transfer of vancomycin resistance.1
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None to declare.
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Acknowledgements |
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E. faecium 64/3 was kindly provided by A. Sundsfjord (University of Trømso, Norway). This work has been performed with financial support from the Commission of the European Union, specific RTD programme ‘Quality of Life and Management of Living Resources’, contract no. QLK2-CT 2002-00843, ARTRADI. It does not necessarily reflect the views of the Commission and in no way anticipates its future policy in this area.
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References |
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