JAC Advance Access published online on February 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl530
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Transfer of plasmid and chromosomal glycopeptide resistance determinants occurs more readily in the digestive tract of mice than in vitro and exconjugants can persist stably in vivo in the absence of glycopeptide selection
1 Department of Microbiology and Virology, University of Tromsø, Tromsø, Norway 2 Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway 3 Unite d'Ecologie et de Physiologie du Systeme Digestif, INRA, Jouy en Josas, France 4 Laboratory of Medical Microbial Ecology, Karolinska Institutet, Stockholm, Sweden
* Corresponding author. Tel: +47-77-64-63-51; Fax: +47-77-64-53-50; E-mail: kristind{at}fagmed.uit.no
Received 29 September 2006; returned 18 October 2006; revised 4 December 2006; accepted 6 December 2006
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OBJECTIVES AND METHODS: The transferability of vanA and vanB glycopeptide resistance determinants with a defined plasmid (n = 9) or chromosomal (n = 4) location between Enterococcus faecium strains of human and animal origins was compared using filter mating (in vitro) and germ-free mice (in vivo) as experimental models. Moreover, the stability of exconjugants in vivo in the absence of antibiotic selection was examined.
RESULTS: Higher transfer rates were observed in vivo for four of six vanA and five of six vanB donor strains. For plasmid-encoded resistance, several log higher transfer frequencies were observed in vivo for some strains. Moreover, the in vivo model supported transfer of plasmid-encoded vanB (1 x 10–7 exconjugants/donor) when repeated in vitro experiments were negative (estimated < 1 x 10–9 exconjugants/donor). Readily detectable transfer of plasmid-located vanA and vanB as well as large chromosomal (>200 kb) vanB elements was observed after 24 h. The number of plasmid-mediated vanA exconjugants generally decreased markedly after 3 days. However, exconjugants containing a plasmid harbouring the vanA transposon Tn1546 linked to the post-segregational killing system 
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persisted stably in vivo in the absence of glycopeptides for more than 20 days.
CONCLUSIONS: The overall results support the notion that the in vitro model underestimates the transfer potential. Rapid transfer of vanA plasmids from poultry- and pig-derived strains to human faecal E. faecium shows that even transiently colonizing strains may provide a significant reservoir for transfer of resistance genes to the permanent commensal flora. Newly acquired resistance genes may be stabilized and persist in new populations in the absence of antibiotic selection.
Key Words: vanA , vanB , vancomycin , Enterococcus
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Introduction |
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The mammalian gastrointestinal tract is an important environment for inter- and intra-species gene flux.1,2 This microenvironment is a meeting place for various bacterial species, including the normal residents, transient colonizers as well as other prokaryotic guests passing through.3 Our current understanding of the extent of prokaryotic gene flux between and within these reservoirs and of barriers for their transfer and expression is limited. Increased knowledge of the in vivo transmissibility, stability of antimicrobial resistance markers, and a better understanding of the transfer mechanisms involved will help to predict the outcome of different strategies for controlling the spread of antimicrobial resistance.
Enterococci are major residents of the human and animal bowel flora.4 They possess a broad spectrum of mobile genetic elements, including conjugative plasmids and conjugative transposons. Conjugative transposons have been shown to mobilize co-resident plasmids, excise and mobilize unlinked integrated elements as well as transfer themselves,5,6 illustrating their promiscuous gene transfer potential. Enterococci have a striking ability to acquire new antimicrobial resistance traits of which glycopeptide-resistant enterococci (GRE) have received most concern.7 VanA and VanB phenotypes are the most common forms of acquired glycopeptide resistance in enterococci. The vanA gene cluster, encoding high-level resistance to both vancomycin and teicoplanin, is carried by the Tn3-related transposon Tn1546 (or related elements), and is most often located on conjugative or mobilizable plasmids.8,9 The vanB operon can be divided into three subtypes, vanB1–B3, based on sequence differences.10 The vanB2 operon encoding resistance to vancomycin and susceptibility to teicoplanin seems to be universally linked to the putative conjugative transposon Tn5382-like and is the most widespread vanB subtype.11–16 The intra- and inter-species transfer of the vanB operon by the movement of large (90–250 kb) chromosomal elements or conjugative plasmids has been described.12,13,17–21
Various model systems have been used for qualitative and quantitative examinations of horizontal gene transfer; in vitro filter mating,22,23 broth matings24 as well as in vivo models using gnotobiotic animals25,26 and heteroxenic mice (germ-free mice containing human microbiota).27 To our knowledge, no extensive comparison has been performed to correlate the transfer results of plasmid- and chromosomally-located resistance determinants between in vitro and in vivo models. The aims of this study were to relate the transferability of well-characterized, clinically relevant glycopeptide determinants with a defined genetic support (plasmid or chromosomal location) between various Enterococcus faecium strains in vitro and in vivo and to examine the persistence of exconjugants in vivo in the absence of antibiotic selection.
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Materials and methods |
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Bacterial strains
Bacterial strains and their characteristics are given in Table 1. Briefly, a total of 12 glycopeptide-resistant E. faecium strains, 6 vanB and 6 vanA strains, were examined for intra-species transfer of their glycopeptide resistance determinants. The collection included human clinical and faecal strains, but also animal faecal strains from avoparcin-associated reservoirs of VanA-type GRE.9,10,17,28–30 The plasmid-free human faecal E. faecium 64/331 and E. faecium BM4105-RF32 were used as recipient strains. The vanA strains contained an intact Tn1546.29
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DNA isolation
Total DNA was isolated using the GenomicPrepTM Cells and Tissue DNA Isolation Kit (Amersham Biosciences, Piscataway, NJ, USA) with modification of the lysis step as described previously.11 Plasmid DNA was isolated as described by Werner et al.30
DNA transfer and hybridization
Southern transfer of DNA was performed as previously described.11 Probes were labelled using the PCR DIG probe synthesis kit and detection performed using the DIG Luminescent Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). Total DNAs from the following bacteria were used as templates for probe synthesis: vanA probe from E. faecalis CDC A256, vanB probe from E. faecalis V583, 23S rDNA probe from E. faecium ATCC19434, and 16S rDNA probe from E. faecalis DS16C2.
Determination of chromosomal or plasmid location of van genes in donor strains
Hybridization of the vanA or vanB probe to plasmid DNA indicated a plasmid localization of the van operon, whereas co-hybridization of the vanA or vanB and either 16S rDNA or 23S rDNA probes to I-CeuI PFGE fragments was interpreted as a chromosomal location of the van cluster. The intron-encoded endonuclease I-CeuI recognizes a 23 bp sequence specific for 23S rRNA genes ensuring that all chromosomal fragments contain either 16S or 23S rRNA genes, or both, depending on the relative orientation of adjacent 16S-23S rDNA copies. I-CeuI digestion and PFGE of genomic DNA was performed as described previously.12
Filter matings (three to four parallels) were done as described by Dahl et al.11 with a donor/recipient ratio of 19:1 and/or 1:1. A pilot study using 19:1, 1:1 and 1:19 donor/recipient ratios using TUH4-62 as donor and BM4105-RF as recipient showed a similar transfer frequency (1–7 x 10–6) for all three ratios. However, we chose to use the 19:1 ratio in most experiments to make sure recipient cells were in contact with an excess of donor cells in this static environment. Various dilutions of resuspended bacteria were spread on brain heart infusion (BHI) agar containing either 8 mg/L vancomycin (to enumerate donors) or 20 mg/L rifampicin and 10 mg/L fusidic acid and 8 mg/L vancomycin (exconjugants). Transfer frequencies were expressed as number of exconjugants per donor cell.
All procedures were carried out in accordance with the European guidelines for the care and use of laboratory animals. For in vivo conjugation, germ-free male NMRI mice (average age 3 months) kept as previously described33 were used. The mice were fed an autoclaved standard pellet diet for rodents (Lactamin R36, Ewos AB, Södertälje, Sweden) and given free amounts of autoclaved germ-free water; they were colonized intragastrically with 106 cfu of vancomycin-susceptible E. faecium BM4105-RF or E. faecium 64/3 1–3 days prior to the addition of 108 cfu of a donor strain. Each mating pair combination was inoculated in two animals. Animals colonized with different bacteria were kept in separate isolators. Preliminary studies had shown a similar frequency of transfer in three different segments (ileum, caecum and colon) of the mice intestine (data not shown). We thus examined colon material from mice sacrificed 1 day or 3 days after colonization with the donor strain except for mating using donors TUH44-29 and TUH44-39, where faecal material was examined. Dilutions of homogenized colon material were spread on selective BHI agar and incubated at 37°C for 48 h. Samples from a non-colonized germ-free animal and animals colonized with the recipient or each of the donors were used as controls. Recipients were enumerated on plates containing 20 mg/L rifampicin and 10 mg/L fusidic acid. Donors and exconjugants were enumerated as for the in vitro experiments. Transfer frequencies were expressed as number of exconjugants per donor or recipient cell.
Verification of exconjugants by PCR amplification and PFGE
Five vancomycin-resistant exconjugants from each experiment were analysed for the presence of vanA34 or vanB10 by PCRs and by PFGE of SmaI-digested total DNA11 followed by hybridization with a vanA or vanB probe.
The exconjugants obtained in vivo for couple TUH44-39 x 64/3 and exconjugants obtained in the in vivo persistence study described below were enumerated on plates containing 10 mg/L vancomycin and 25 mg/L fusidic acid. The numbers of exconjugants, donor and recipient obtained in vivo for couple TUH44-29 x 64/3 are calculated from Figure 3 and correspond to sampling time day 1 of the persistence study. Six randomly picked clones per mouse at each sampling time were confirmed to be exconjugants by the following methods: (i) bacterial colonies were streaked on BHI agar plates containing 25 mg/L rifampicin, allowing secondary discrimination between the recipient/exconjugant strain and the donor strain; (ii) DNA purified from the bacterial colonies using the GeneReleaser® kit (BioVentures Inc., Murfreesboro, TN, USA) was used for PCR amplification of a vanA gene fragment. Oligonucleotides primers vanA-1 (5'-TCTGCAATAGAGATAG CCGC-3') and vanA-2 (5'-GGAGTAGCTATCCCAGCATT-3') were used for PCR amplification of a 377 bp fragment of the vanA gene.35
In vivo persistence of the VanA phenotype
All procedures were carried out in accordance with the European guidelines for the care and use of laboratory animals. A long-term in vivo conjugation experiment was performed to study the course of colonization and persistence of donor, recipients and exconjugants. Five germ-free C3H/He mice were first orogastrically inoculated with 250 µL of a 10-fold diluted fresh overnight culture of the donor strain TUH44-29 (approximately 2 x 107 cfu/animal). Mice were then kept for at least 1 week for colonization and stabilization of the bacterial population before inoculation of the recipient strain 64/3 (approximately 2 x 109 cfu/animal). Faeces from at least three animals were separately collected at days 0.25, 1, 2, 3, 8, 13 and 21, and were treated as described above. The stability of a newly acquired glycopeptide resistance plasmid (pVEF1) was also investigated after inoculation of one exconjugant isolated from mating between strains TUH44-29 and 64/3. Faeces from 10 mice drinking either water (n = 5) or water containing 100 mg/L vancomycin (n = 5) were collected over a period of 30 days. Appropriate dilutions of resuspended bacteria were spread on non-selective BHI plates or on selective plates supplemented with 10 mg/L vancomycin. After 48 h of incubation at 37°C, 100 to 450 clones grown on BHI plates were subsequently replicated with a toothpick on non-selective and selective plates. In vivo persistence of the VanA phenotype was evaluated by determination of the ratio between clones grown on selective plates and clones grown on non-selective plates.
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Results and discussion |
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Localization of the van-containing elements
All isolates were genomically diverse10 (this study) except TUH2-18 and TUH2-19, which are closely related as demonstrated by PFGE.17 The strains were characterized with regard to plasmid or chromosomal localization of the van gene cluster by Southern hybridization of plasmid DNA and I-CeuI PFGE fragments. In TUH44-29 and TUH44-39 the plasmid localization has been confirmed by plasmid DNA sequencing.36 The results are summarized in Table 1. All vanA strains and two vanB strains had a plasmid-located van operon (data not shown). A chromosomal location of the vanB operon was found in three strains, while one strain (TUH4-64) showed hybridization both to a 23S rRNA-containing I-CeuI PFGE fragment and to plasmid DNA (data not shown) indicating that this strain has both a plasmid and a chromosomal copy of the vanB operon.
The vanA exconjugants were confirmed by vanA PCR and PFGE similarity to BM4105-RF or 64/3 (data not shown), confirming horizontal transfer of DNA segments containing Tn1546. All exconjugants after mating using vanB donors harboured vanB and had PFGE patterns related to that of the recipient BM4105-RF, as illustrated for representative strains in Figure 1. The exconjugants from mating using the donor TUH7-15 containing a chromosomal vanB copy showed several patterns similar to the recipient strain (Figure 1a, lanes 2–6). The exconjugants seem to have lost a 290 kb fragment present in the recipient strain and gained one or more fragments of different sizes. The exconjugant pattern in lane 5 immediately seems very similar to the recipient pattern. However, a closer comparison with the recipient pattern shows that this exconjugant has gained a double band at approximately 80–85 kb and the 290 kb fragment is replaced by a slightly smaller fragment hybridizing to vanB. The various PFGE patterns of different exconjugants may result either from insertion of fragments of different sizes or from rearrangements after insertion into the recipient strain. Investigations not presented here suggest that the various patterns among exconjugants are due to rearrangements by homologous recombinations after insertion of large fragments into the genome.37 All exconjugants from mating using TUH7-55 containing a plasmid copy of vanB as donor showed the same PFGE patterns as the recipient BM4105-RF (Figure 1a, lanes 6 and 8–13) and vanB hybridized to the well of both donor and exconjugants (Figure 1b, lanes 7–13) as expected for plasmids containing no SmaI sites. Circular forms of plasmids typically reside in the well of the pulsed-field gels.
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Transfer of van-containing elements in vitro
Mating between the van donors and the plasmid-free recipients E. faecium BM4105-RF and/or E. faecium 64/3 were performed. Major differences in transfer frequencies from the different vanA (<3 x 10–10 to 5 x 10–5 exconjugants/donor cell) and vanB (7 x 10–9 to 2 x 10–5 exconjugants/donor cell) donors to BM4105-RF (Table 2) and from the different vanA donors to 64/3 (1 x 10–9 to 3 x 10–6 exconjugants/donor cell) (Table 3) were observed. Transfer frequencies in vitro were similar when mating the same donors with the different recipient strains (Tables 2 and 3). Thus, the donor potential seems to determine transfer frequencies in vitro in this study. Transfer of chromosomal vanB elements was obtained for three E. faecium donors (vanB2 TUH2-18 and TUH7-15; vanB1 TUH4-65) into E. faecium BM4105-RF at a frequency varying between 7 x 10–9 and 2 x 10–7 exconjugants/donor cell (Table 2). The poultry vanA donors in general showed lower frequencies of transfer (
4 x 10–9 exconjugants/donor cell) than the human- or pig-related donors (
7 x 10–7 exconjugants/donor cell) independently of which recipient was used (Tables 2 and 3).
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In vivo colonization
In the gnotobiotic mice model system a stable level of approximately 1 x 109 cfu/g colon sample was observed after 1 day when the strains were given separately to control animals (data not shown). When given together, both donor and recipient strains colonized at a level of approximately 1 x 109 cfu/g colon sample, except for the pig strain UW4, which colonized at a 2-log-fold lower level (Table 2). Non-specific host defences, competitive effects of the first colonizers, and the ability to utilize nutrition from the mucus layer may limit colonization by exogenous bacteria.3 On the other hand, it has been shown that the in vivo persistence level of introduced populations can be higher under conventional conditions compared with gnotobiotic conditions as species diversity may create new niches promoting persistence.38 UW4 colonized at a lower level only when given together with the recipient strain (data not shown). Thus, this strain seems to have a competitive disadvantage with respect to the recipient. If we consider these experiments to mimic natural settings, the newly incoming bacteria may experience a washout situation compared with the recipient, which represents the established flora. The exconjugant to donor ratio will be comparably higher for this strain than the other ratios. Slight competitive effects are also observed for the recipient 64/3 in co-habitation with some donors (Table 3). This is not the first time this has been observed. Inoculation of E. faecium 64/3 cells to germ-free mice resulted in rapid colonization of the digestive tract and stabilization of the bacterial population in less than 48 h at 
1010 cfu/g faeces. After inoculation of an E. faecium donor strain of clinical origin, recipient 64/3 strain showed an ecological disadvantage (it was recovered between 7 x 107 and 8 x 108 cfu/g faeces) (M. J. Flores, D. D. G. Mater and G. Corthier, unpublished results).
In vivo versus in vitro transfer frequency
Strains supporting transfer in vitro and strains with non-transferable plasmid- and chromosomal-borne van were selected for in vivo transfer studies. We observed increased in vivo transfer frequencies for four of six vanA strains and five of six vanB strains including plasmid transfer from TUH4-64, which did not support in vitro transfer of vanB in repetitive experiments (Table 2).
The poultry vanA donor TUH4-34 and the human vanA donor TUH4-62 showed very high in vivo frequencies of transfer (1 x 10–1 exconjugants/donor cell) to 64/3 after 1 day. After 3 days, the transfer frequencies decreased markedly but in vivo transfer frequencies after 3 days were still 50–3000-fold higher than the in vitro frequencies measured after 18 h of mating (Figure 2 and Tables 2 and 3). No qualitative differences were shown between in vitro and in vivo exconjugants by Southern hybridization of PFGE fragments (data not shown). These observations indicate that the conditions in the mammalian intestinal tract favour horizontal gene transfer and that failure to demonstrate transfer of a particular marker under laboratory conditions does not mean that the marker is non-transmissible but may be due to lack of favourable environmental factors. The in vivo model supported transfer of chromosomal vanB elements as shown in vitro (Table 2). However, plasmid transfer was 50–1 x 108-fold higher in vivo when measured as cfu/g colon material (Figure 2 and Tables 2 and 3), indicating an underestimation of gene transfer rates for some strains by in vitro methods. The higher rates of plasmid transfer in vivo could be due to constant mixing of bacteria by the peristaltic movements in the gastrointestinal tract ensuring that donor bacteria will have more accessible recipients than during filter mating where the bacterial position is more fixed. This would in particular be an advantage for transfer of plasmids, which is considered more rapid than transfer of large chromosomal elements. A prolonged generation time in vivo versus in vitro,38 may favour the latter. In addition, horizontal gene transfer primarily occurs in niches characterized by high densities such as biofilms where the likelihood of cell-to-cell contact is high. Thus, the greater population size and relative densities within the intestine compared with the in vitro situation may have influenced either the transfer rate or the clonal expansion of exconjugants.39 We further acknowledge the possibility that continuous re-transfer of van determinants during the 3 day transfer studies could influence the outcome of the comparison of in vitro and in vivo results. However, the results after 3 days were consistently lower than on day 1 (Figure 2 and Tables 2 and 3).
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The relative low colonization number of the UW4 donor strain (1 x 107 cfu/g colon sample; Table 2) did not influence the transfer rate markedly, suggesting that transient colonizing bacteria may transfer resistance plasmids readily in the gastrointestinal tract. The high numbers of bacteria and the rich nutritional resources of most commensal niches make the gastrointestinal tract an ideal setting for gene exchange. Thus, bacteria entering the gastrointestinal tract may transfer resistance genes to the commensal microbiota even without colonizing. This was recently confirmed in a study reporting in vivo transfer of vanA among E. faecium in the intestines of human volunteers.40
Exconjugants ability to establish in the digestive tract
Initially, we investigated the number of exconjugants 1 and 3 days after donor inoculation using TUH4-62 and TUH4-34 as vanA plasmid donors and E. faecium 64/3 as recipient strain (Table 3). The high transfer frequency (1 x 10–1 exconjugants per donor; Table 3) observed for both strains 1 day after inoculation support the rapid transfer of vanA and erm(B) determinants reported in gnotobiotic mice41 and of vanA in heteroxenic mice containing human microbiota27 (data not shown) to the same recipient E. faecium 64/3. The authors observed exconjugants 5 or 6 h after inoculation of donor or recipient, respectively. Plasmid transfer kinetics within the intestine are in general similar to transfer obtained in biofilm with an initial brief period of rapid transfer followed by no further increase in number of exconjugants, probably due to inefficient mixing within the biofilm and thus fewer cell collisions necessary to trigger mating pair formation. It is believed that transfer within the intestine mainly occurs within the bacterial fraction constituting a biofilm in the mucus layer due to a more stable cell–cell contact and more favourable growth conditions than within the gut content.42,43 Indeed material scraped out of the colon in general showed higher transfer frequencies (Table 2 and matings using donors TUH4-62 and TUH4-34 in Table 3) compared with samples of faecal material (matings using donors TUH44-29 and TUH44-39 in Table 3), indicating that colon scrapings may give a better estimate of transfer frequency as this material may contain more biofilm-associated cells than faecal material.
Moubareck et al.41 reported an impressively high level of exconjugants/g faeces (1 x 107) using the UW7 donor strain throughout the observation period of 20 days, even without antibiotic selection. Long-term persistence of antibiotic-resistant enterococcal exconjugants without selection has also been observed in gnotobiotic rats or mice using satA44 or vanA,45 respectively, as resistance markers. These observations clearly contrast with the marked decrease in number of exconjugants in our study on the third day after donor inoculation, even though the recipient and donor numbers stayed at similar high levels (Table 3).
The difference in the persistence of exconjugants between these studies may reflect a biological cost associated with the newly acquired van plasmids leading to a competitive disadvantage (i.e. reduced fitness) relative to the recipient and donors. It is also possible that the plasmids were highly unstable in the absence of glycopeptide selective pressure. We thus performed a long term in vivo conjugation experiment using the strain TUH44-29 (isolated from a Norwegian poultry farmer) with a fully sequenced vanA-containing plasmid (pVEF1) as a donor strain, and 64/3 as a recipient (Figure 3). The pVEF1 plasmid contains an intact Tn1546 linked to a putative post-segregational killing system recently hypothesized to play an important role in GRE persistence in antibiotic-free environments.9,46 We observed a stable level of exconjugants (1 x 104 cfu/g faeces) from day 1 until day 21 without antibiotic selection and with stable donor and recipient levels. Interestingly, the relative ratios between exconjugants and plasmid-free recipients appeared to be rather stable suggesting a very low, if any, overall biological cost associated with the newly acquired pVEF1. Enne et al.47 recently demonstrated that a newly acquired Escherichia coli antibiotic resistance plasmid reduced fitness both in vivo and in vitro, and after 20 days in pigs, the relative ratios between exconjugants and recipients were low suggesting a reduced biological cost associated with plasmid carriage.
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We also evaluated in vivo persistence of the VanA phenotype in the absence of selection pressure for one exconjugant isolated from a mating between strains TUH44-29 and 64/3. Thirty days after inoculation of germ-free mice, more than 99.3% of clones recovered from faeces were vancomycin resistant (data not shown), indicating a very high stability of the VanA phenotype. Thus, the observed stability of pVEF1-containing exconjugants in vivo underlines the recent notion that plasmid addiction systems might support the persistence of GRE in non-selective environments.9
Taken together, the evidence presented above suggests that different plasmids have different fates in a novel host. Whereas newly acquired glycopeptide resistance determinants from TUH4-62 and TUH4-34 were unable to persist at their initial high level in glycopeptide-free environments, possibly due to a competitive disadvantage, the TUH44-29 x 64/3 exconjugant was able to persist and demonstrated a remarkable stability. TUH4-62 exconjugants seem to persist better in E. faecium BM4105-RF45 (Table 2) than in the E. faecium 64/3 background (Table 3) suggesting a more favourable association between the vanA determinant and recipient BM4105-RF. However, TUH4-34 exconjugants show a similar persistence in both backgrounds after 3 days (Tables 2 and 3). It is clear from these observations that potential transfer and persistence of a given plasmid in glycopeptide-free environments is dependent on both plasmid and recipient factors.
Transfer between E. faecium of animal and human origins
Transfer of the vanA operon from a strain with porcine origin to the human faecal E. faecium 64/3 in vivo has been reported.41 In vivo transfer in the intestine of human volunteers of vanA from a poultry-originating strain into the human faecal E. faecium BM4105-RF has also been reported.40 In this study we confirmed transfer of vanA-containing plasmids from poultry- and porcine-originating strains to human faecal recipients in vivo. The early transfer of vanA plasmids from poultry- and pig-derived strains to human faecal E. faecium, indeed suggests that even transiently colonizing strains of animal origin would allow enough time for glycopeptide transfer to the faecal flora. Moreover, the observed transfer in the gastrointestinal tract 1 day after inoculation of donor includes vanB-containing chromosomal elements (Table 2) as well as vanA-carrying plasmids (Table 3). Hence, 1 day of passage was sufficient time for readily detectable transfer of both plasmid and chromosomally encoded resistance elements, even though the size of the transferable chromosomal vanB elements can be extensive (200 kb, data not shown).
Our comparison of in vitro versus in vivo transfer of plasmid- and chromosomal-located van resistance determinants in E. faecium confirms that in vitro transfer is reproducible in vivo. Moreover, in vitro transfer might in fact underestimate the potential for gene transfer as shown by a significantly higher transfer rate in vivo, most notably for plasmid-located markers, but also for chromosomal determinants. For the strain with both plasmid- and chromosomally-located vanB, detectable transfer was only observed in vivo. Detection of transfer in vivo but not in vitro, and reproducible differences observed between in vivo and in vitro settings, demonstrates that quantitative comparison of in vitro and in vivo transfer seems to be relevant.
The rapid occurrence and high number of exconjugants without antibiotic selection indicate that even transient colonizers may provide a significant donor potential for transfer of resistance genes to the permanent commensal flora. Transfer of plasmid-mediated Tn1546 from animal strains to human-derived recipients underlines the potential transfer of resistance genes from animals to humans through the food chain. Finally, newly acquired resistance genes may be stabilized and persist in new populations even in the absence of antibiotic selection.
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None to declare.
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Footnotes |
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Present address: Department of Pharmacology, University of Tromsø, Tromsø, Norway 
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Acknowledgements |
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We thank S. Harthug and A. Digranes, M. A. Pfaller and S. A. Marshall, C. Poyart, and W. Witte for providing strains. We also thank Bjørg C. Haldorsen, Eirik W. Lundblad, Anna-Karin Persson, Torunn Pedersen and Ewa Österlund for excellent technical assistance. The work was in part supported by the European Commission, contract QLK2-CT-2002-00843 "ARTRADI", Northern Norway Regional Health Authority Medical Research Programme and the Norwegian Research Council, project no. 165997/V40.
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References |
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