Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis

Claudia Oancea, Ingo Klare, Wolfgang Witte and Guido Werner*

Robert Koch Institute, Wernigerode Branch, Burgstr. 37, 38855 Wernigerode, Germany

Received 20 November 2003; returned 5 January 2004; revised 5 February 2004; accepted 25 March 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis.

Materials and methods: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR.

Results: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis.

Conclusion: The esp gene is transferable by conjugation among enterococcal isolates.

Keywords: enterococci , pathogenicity island , mating


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Enterococci are facultative pathogens. Isolates from infections are enriched in (putative) virulence factors, such as cytolysin, aggregation substance, specific surface proteins, and hyaluronidase,1,2 some of which are encoded by genes within a recently identified pathogenicity island (PAI) in Enterococcus faecalis.3 A similar genetic arrangement seems probable in Enterococcus faecium.4 An enterococcal surface protein gene, esp, also part of this PAI was found to be enriched among human E. faecalis and clinical E. faecium strains from outbreaks, suggesting esp is a marker for epidemic strains at least among the latter species.5 Acquired antibiotic resistance genes spread horizontally among enterococci mainly by conjugative transfer.6 The questions remain, if, how, and to what extent chromosomally encoded virulence factors, such as esp, are transferred between enterococcal strains. Attempts to date to show a transfer of esp or the entire PAI have failed.3 We describe here transfer of esp triggered by a conjugative event and co-selected by transfer of antibiotic resistance determinants.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains

All esp-positive donor strains were from clinical samples (infection and colonization) and isolated between October 1998 and August 2001. Antibiotic susceptibilities were determined by micro-broth dilution according to Werner et al.7 All E. faecium were vancomycin-resistant, whereas all E. faecalis were vancomycin-susceptible. All strains were resistant to antibiotics of at least two independent classes, positive for esp by PCR and clonally diverse (Table 1).


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Table 1. Strains used, mating parameters and features of resulting transconjugants

 
Mating experiments

Mating experiments, plasmid preparations and PFGE were carried out as described elsewhere.7 E. faecium 64/3 and E. faecalis JH2-2, both high-level rifampicin- and fusidic acid-resistant (MICs > 256 mg/L) were chosen as recipients for matings. Selection was done on agar plates supplemented with antibiotics according to the resistance pattern of the donors (erythromycin, tetracycline or vancomycin) and the recipient (rifampicin or fusidic acid). Mating rates were calculated as transconjugants per recipient cell. Concentrations of antibiotics in the agar plates were: erythromycin 20 mg/L, tetracycline 15 mg/L, vancomycin 5 mg/L, rifampicin 30 mg/L and fusidic acid 20 mg/L.

Molecular studies

PCR for esp was carried out using 100 pM of primer 1 (5'-ACGTGGATGTAGAGTTTGC) and primer 2 (5'-GAATATGTCACTACAACCGTAC), 10 ng DNA, and 100 µM dNTPs. Parameters were as follows: initial step of 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min; final step of 72°C for 4 min. RAPD typing PCR was carried out with primer RAPD 10 (5'-TGCTCTGCCC) and 50 ng DNA.7

Labelling was done by incorporating digoxigenin-labelled dUTP (Roche Biochemicals, Mannheim, Germany) directly during PCR in a ratio dTTP/DIG dUTP = 4:1. Hybridization was carried out using buffers, kits and conditions as recommended by the manufacturer (DIG Easy Hyb Granules, DIG buffer system, Attophos, Roche Biochemicals). Real-time PCR was carried out according to the manufacturer’s recommendations (Applied Biosystems, Darmstadt, Germany). Two primers binding outside the integration site of the PAI in the reference isolate MMH594 were chosen to amplify the intact integration site in PAI(–) isolates (5'-CCTCTTGTATAAATAATAGCGGAGTGCTA, 5'-GCA TAGGGATTCGAACCCTAGA). A cytolysin operon is located on the PAI in MMH594 including the cylM gene which was used as an internal marker gene for PAI(+) isolates (primers 5'-GATGCGTATTACTGTTGTTAGAATGAGAT and 5'-GAGTCTCCCTGTGATTCTGATATAGAGTT). Specific Taqman-MGB probes were chosen (FAM-labelled 5'-CATCTGACTCTTAATCAGAG-NFQ-MGB probe for the integration site product, VIC-labelled 5'-AACATACAATCCTCAGAGCT-NFQ-MGB probe for cylM). All primers were identified using the Primer Express software (Applied Biosystems). Both products were amplified in a single approach in an allelic discrimination assay using standard conditions. Positive control strains for PAI(+) (MMH594) and PAI(–) (OG1X) were included, test strain was UW3114.3

A putative arrangement of esp in structures similar to the PAI described in MMH594 was sought in the E. faecalis donors. Two primers binding outside the PAI integration site in MMH594 (translation direction inside the PAI; EF1: 5'-CCATGTTCAGCGAAGTTGCC, ER2: 5'-GCTGATTTATTATGGTTCTC)3 and two primers binding inside both ends of PAI (translation direction outside the PAI; ER1 5'-ATTCAAGAATGGCTGGGAC, EF2 5'-CCAAAAAGCAA- CTTTCAACC) were used in various combinations. A PCR amplified product for the primer combinations EF1/ER1 and EF2/ER2 (positive control MMH594) and no product for EF1/EF2 demonstrate an arrangement similar to MMH594. In addition, classical PCR for cylM (representing the cytolysin operon) was carried out amplifying another part of the PAI.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Mating experiments

Fourteen esp-positive donors (nine E. faecium, five E. faecalis) were tested for a conjugative transfer of esp in filter-matings (Table 1). Five of nine E. faecium and two of five E. faecalis pairs yielded antibiotic-resistant transconjugants. Transconjugants were subsequently tested for growth on agar plates with the donor-selective antibiotic, the second selective marker of the recipient and were checked by RAPD PCR for clonal relatedness with the recipient. Transconjugants from the mating with strains UW3183 and UW2840 revealed a pattern identical to the corresponding donor strains and were assessed as mating-negative (Table 1). Transconjugants from three mating pairs (one E. faecium, two E. faecalis) were positive for esp in PCR (Table 1). In each case, only a subpopulation of transconjugants acquired the esp gene indicating that the transferred antibiotic resistance determinants were not directly linked with the esp gene. This is not surprising since antibiotic resistance determinants have not been identified in the PAI structures reported to date in enterococci.3,4,9 The three donors (E. faecalis 3410 and 3114, E. faecium 3308) generating esp-positive transconjugants were used for mating experiments with recipients of the other species. Only matings with the E. faecalis donors yielded transconjugants (E. faecalis 3114 x E. faecium 64/3—mating rate 5.81 x 10–8; E. faecalis 3410 x E. faecium 64/3—mating rate 3.87 x 10–9). The identification and antibiotic susceptibility profiles of transconjugants were checked (not shown in detail). PCR for esp with transconjugant DNA of E. faecalis 3410 transconjugants and E. faecalis 3114 transconjugants revealed positive signals in 0/1 (only one transconjugant available) and 1/14 cases, suggesting that only a single E. faecium transconjugant acquired the esp determinant from E. faecalis 3114.

Each single pair of E. faecium and E. faecalis (UW3308, UW3114) and their corresponding esp-positive and negative transconjugants derived from matings between donors and recipients of the same species were chosen for a further detailed molecular analysis.

Molecular characterization

Genomic and plasmid DNA were isolated from the donors. Non-digested plasmid patterns and HindIII-digested genomic DNA were resolved in a single agarose gel, blotted onto a nylon membrane and hybridized with a labelled esp probe. None of the plasmids showed a signal, whereas in all the genomic preparations definite bands could be visualized (not shown). This was as expected, as all the esp clusters identified so far in both enterococcal species have been exclusively chromosomally located.3,4,9 Association of esp in structures similar to the PAI in E. faecalis MMH594 was identified for all five E. faecalis donors using primers amplifying cylM and both ends of the PAI. All but one possessed identically sized PCR products when compared with MMH594 (not shown). Isolate 3410 showed no product for cylM and the left end structure of the PAI, suggesting deletion of fragment(s) upstream of the esp gene of the putative PAI in this strain. When HindIII-digested plasmids from both donor/transconjugant (esp+) pairs were compared, several band differences were identified (Figure 1a). Whereas in the E. faecium mating pair, the transconjugant showed more fragments than the donor, in E. faecalis, the picture was reversed. After Southern hybridization, a single band was labelled corresponding to a novel DNA fragment in the esp-positive E. faecium transconjugant. The most likely explanation is that the esp gene was mobilized from the donor chromosome, integrated into a conjugative plasmid which was then transferred into recipient 64/3, giving an esp-positive transconjugant (Figure 1b). When plasmids in the E. faecalis pair were digested, none of the fragments showed a signal (Figure 1b). Since esp was not on a plasmid either in the E. faecalis donor or in the esp-positive transconjugant, we postulated that chromosome-to-chromosome transmission had occurred. To test this hypothesis, we carried out SmaI macrorestriction analysis (PFGE) with DNA from the donor, the recipient and both an esp-positive and an esp-negative transconjugant (Figure 1c). The patterns of the recipient and the esp-negative transconjugant were identical, while there was a single band loss in the pattern of the esp-positive transconjugant compared to the former two strains and a completely different donor pattern. The picture became clearer after Southern hybridization with a labelled esp probe. The largest band in the donor and the esp-positive transconjugant’s pattern showed a signal (Figure 1d), but no signal appeared in the other two lanes. The largest band giving a signal with the esp probe in the esp-positive transconjugant’s pattern also appeared in the patterns of the esp-negative transconjugant and the recipient. This indicated that, in the former case, the band comprised two fragments of similar size, which were not resolved in PFGE, with one of them possessing esp. The size difference between the lost and the new fragment is about 150–180 kb, which is approximately the size of the entire PAI in E. faecalis.1 However, we were unable to investigate the possibility that the entire PAI had been transferred for a number of reasons. When we carried out PCR screening for acquisition of additional markers of this PAI (cytolysin operon genes, PCR for the integration site of the PAI using primers PAI164/167 as suggested by Shankar et al.3), we have shown that parts of the PAI were already present in E. faecalis JH2-2 used as the recipient. JH2-2 is widely used as a recipient in mating experiments, mainly with pheromone-related plasmid transfer models. The finding that this strain already possesses parts of the PAI (although not esp) was not previously known. PCR with primers binding outside and inside the PAI at both ends of the PAI in JH2-2 (primers EF1/ER1 and EF2/ER2) generated a PCR product in both cases similar to MMH594 (not shown).



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Figure 1. Molecular analyses of esp gene transfer. (a) HindIII-digested plasmid patterns from (1) E. faecium donor UW3308 and (2) the corresponding esp-positive transconjugant, (3) E. faecalis donor UW3114 and (4) the corresponding esp-positive transconjugant. M1 and M2 indicate different digoxigenin-labelled size markers. (b) Corresponding Southern blot hybridized with a labelled esp gene probe. (c) SmaI macrorestriction patterns of (1) E. faecalis donor UW3114, (2) a corresponding esp-positive transconjugant, (3) a corresponding esp-negative transconjugant and (4) the recipient JH2-2. M3 indicates SmaI-digested genomic DNA of Staphylococcus aureus NCTC 8325 serving as a size marker. (d) Corresponding Southern blot hybridized with a labelled esp gene probe.

 
There is considerable evidence that PAIs in bacteria are exchanged.10 However, the mechanisms by which these structures are transferred are not understood.10 To investigate putative excision of the PAI in E. faecalis from the chromosome as a triggering factor for a transfer, we carried out real-time PCR. Non-replicative transfer of the PAI would result in the generation of an intact integration site after excision.3 The relation between PAI-positive and PAI-negative isolates within a population of isogenic donor strains can be quantified by a real-time approach amplifying a fragment which is specific for an intact integration site in relation to a fragment specific for PAI. As a positive control marker for the existence of the PAI, a fragment of the cylM gene was chosen. We tested 5 ng DNA per run (identical to 1.44 x 106 cells; http://molbiol.ru/eng/scripts/01_07.html). No amplification for an intact integration site was identified within 40 cycles, neither in the control strain MMH594 nor in the test strain UW3114. The differences between the delta Ct values for cylM (delta Ct(MMH594) = 21.02; delta Ct(UW3114) = 21.44) and PAI integration site (delta Ct > 40) were >18.98 and >18.56, suggesting a putative excision of the PAI in less than one among 4.89 x 105 and 3.7 x 105 cells, respectively (data not shown). We confirmed with our in vitro approach the results from Shankar et al. for MMH594 using classical quantitative PCR and who did not identify a deletion of the PAI in one among 105 cells.3 UW3114 showed the same results in Taqman PCR as MMH594, a strain not able to transfer esp.3 Therefore, we suggest that excision of the PAI is not a triggering factor for esp transfer.

In conclusion, this is, to our knowledge, the first experimental proof that chromosomally encoded virulence traits, such as esp, can be exchanged between enterococcal strains. Transfer of esp was triggered by selecting for conjugative transfer of antibiotic resistance determinants. Mechanisms by which esp was transferred varied between the species, involving integration into a conjugative plasmid in E. faecium and by a chromosome-to-chromosome transmission in E. faecalis. Further experiments are needed to clarify the detailed molecular mechanism(s) by which esp and other chromosomal markers are exchanged between enterococci.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Excellent technical assistance by B. Hildebrandt is kindly acknowledged.


    Footnotes
 
* Corresponding author. Tel/Fax: +49-3943-679-207; E-mail: wernerg{at}rki.de


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Hancook, L. E. & Gilmore, M. S. (2000). Pathogenicity in enterococci. In Gram-positive Pathogens (Fischetti, V. A., Novick, R. P., Ferretti, J. J. et al., Eds), pp. 251–8. ASM Press, Washington, DC, USA.

2 . Klare, I., Werner, G. & Witte, W. (2001). Enterococci—Habitats, infections, virulence factors, resistance to antibiotics, transfer of resistance determinants. In Emerging Bacterial Pathogens (Muehldorfer, I. & Schaefer, K. P., Eds), Contributions to Microbiology, Vol. 8, pp. 108–22. Karger, Basle, Switzerland.

3 . Shankar, N., Baghdayan, A. S. & Gilmore, M. S. (2002). Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis. Nature 417, 746–50.[CrossRef][ISI][Medline]

4 . Leavis, H., Top, J., Shankar, N. et al. (2003). A novel putative pathogenicity island linked to esp virulence gene of Enterococcus faecium and associated with epidemicity. Journal of Bacteriology 186, 672–82.[CrossRef][ISI]

5 . Willems, R. J. L., Homan, W., Top, J. et al. (2001). Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals. Lancet 357, 853–5.[CrossRef][ISI][Medline]

6 . Tenover, F. C. (2001). Development and spread of bacterial resistance to antimicrobial agents: An overview. Clinical Infectious Diseases 33, Suppl. 3, S108–15.[CrossRef][ISI][Medline]

7 . Werner, G., Willems, R. J. L., Hildebrandt, B. et al. (2003). Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium. Journal of Clinical Microbiology 41, 1499–506.[Abstract/Free Full Text]

8 . Klare, I., Konstabel, C., Badstuebner, D. et al. (2003). Occurrence and spread of antibiotic resistance in Enterococcus faecium. International Journal of Food Microbiology 88, 269–90.[CrossRef][ISI][Medline]

9 . Eaton, T. J. & Gasson, M. J. (2002). A variant enterococcal surface protein Espfm in Enterococcus faecium: distribution among food, commensal, medical, and environmental isolates. FEMS Microbiology Letters 216, 269–75.[CrossRef][ISI][Medline]

10 . Kaper, J. B. & Hacker, J. (1999). Pathogenicity Islands and Other Mobile Virulence Elements, 1st edn. ASM Press, Washington, DC, USA.