JAC Advance Access originally published online on March 24, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):832-839; doi:10.1093/jac/dkl069
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Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry
1 Danish Institute for Food and Veterinary Research, 1790 Copenhagen V, Denmark; 2 Center for Biological Sequence Analysis, The Technical University of Denmark, 2800 Lyngby, Denmark
* Corresponding author. Tel: +45-72-34-60-00; Fax: +45-72-34-60-01; E-mail: ya{at}dfvf.dk
Received 7 October 2005; returned 16 November 2005; revised 10 February 2006; accepted 14 February 2006
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Abstract |
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Objectives: To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark.
Methods: A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates.
Results: In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements.
Conclusions: This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.
Keywords: Enterococcus , tetA(M) diversity , Tn5397
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Enterococci isolated from animals, humans and other sources are often resistant to tetracyclines, and several classes of tetracycline resistance genes have been identified in enterococci. In a previous study, the tet(M) gene was found in 95% of Enterococcus faecium and Enterococcus faecalis from humans, pigs and broilers.1 This gene is widely distributed and has to date been found in 42 genera of Gram-positive and Gram-negative species.2 This is probably due to the association of the tet(M) gene with conjugative elements.2,3 In enterococci and other species, tet(M) has been found to be associated with conjugative transposons related to the Tn916/Tn1545 family.3 Other conjugative transposons such as Tn5397 from Clostridium difficile have been found to harbour tet(M) as well.4 Tn5397 is a 21 kb tetracycline resistance-encoding conjugative transposon found originally in C. difficile.4,5 Tn5397 was shown to be transferred by a conjugation-like process from C. difficile to Bacillus subtilis and back to C. difficile and between C. difficile strains.5 Tn5397 is related to Tn916; the central regions that are involved in conjugation of these two elements are very similar.4,5 However, Tn5397 can be distinguished from Tn916 by at least two important characteristics: first, Tn5397 contains a group II intron inserted into a gene almost identical to orf14 from Tn916, and, second, the DNA sequences at the ends of Tn5397 are completely different from those of Tn916.4 Instead of possessing the int and xis genes that have been shown to be required for integration and excision of Tn916, Tn5397 contains the gene tndX, which encodes a putative protein not related to Int or Xis but belonging to the large resolvase subgroup of site-specific recombinases.4,6
Although, tet(M) is widely distributed among bacteria in different environments, only a few studies concerning the diversity and phylogenetic relationship of the genes have been performed. In one analysis, based on high-resolution restriction analysis of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae, six allele types were identified, and these were all in isolates containing the int-Tn gene of the Tn916/Tn1545 conjugative transposons.7
In another study, restriction enzyme analysis and partial sequencing of the tet(M) gene in Lactobacillus isolated from different types of fermented dry sausage revealed two different allele types.8 The tet(M) genes were mainly located on plasmids.8 Recently, Huys et al.9 characterized enterococci isolated from food. All isolates that contained tet(M) had the xis-Tn gene from Tn916/Tn1545 family transposons. The tet(M) genes were classified into four distinct groups.9
The present study was conducted to characterize and determine the occurrence of mobile elements associated with tet(M) in E. faecium and E. faecalis isolated from humans, pigs and broilers in Denmark, and to see whether there is a correlation between the presence of certain mobile elements and the diversity of tet(M) genes at the nucleotide level by comparison with tet(M) sequences submitted to the GenBank database.
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Bacterial strains
A total of 76 bacterial isolates from broilers, pigs and humans were obtained from the continuous surveillance programme for antimicrobial resistance in Denmark (DANMAP), as described previously.10 Of the 76 isolates, 40 were E. faecalis isolates (13 from humans, 15 from pigs and 12 from broilers) and 36 were E. faecium isolates (10 from humans, 13 from pigs and 13 from broilers). The 17 enterococcal isolates (Table 1) included in the phylogenetic analysis had very distinct PFGE patterns, indicating the absence of any clonal relationship between these strains.
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All isolates were tested for susceptibility to bacitracin, chloramphenicol, erythromycin, gentamicin, kanamycin, penicillin, streptomycin, quinupristin/dalfopristin, tetracycline and vancomycin and for the presence of selected resistance genes, including tet(M), in a previous study.1 The isolates from pigs and broilers were collected during the first 9 months of 1998. Only one isolate per flock or herd was included in the collection.1 Bacterial isolates from humans were obtained from stool samples received at Statens Serum Institut, Denmark, for diagnostic purposes, as described previously.1
Detection of Tn916-like and Tn5397-like conjugative transposons
PCR was used to demonstrate the presence of the Tn916-like or Tn5397-like transposons in the 76 isolates containing tet(M). For the detection of Tn916-like transposons, the xis-Tn gene of Tn916 was amplified using the primers Tn916-1 and Tn916-2 (PCR conditions: 3 min hot start at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 45°C, 1 min at 72°C and a final extension for 10 min at 72°C). Eleven of the xis-Tn gene positive isolates were checked by linking the essential xis-Tn gene of Tn916 to the tet(M) gene using primers Tn916-2 and ReversTetM-2, as described previously (PCR conditions: 3 min hot start at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 45°C, 3 min at 72°C and a final extension for 10 min at 72°C).12
For the detection of Tn5397-like transposons, the tndX gene of Tn5397 was amplified using PCR with primers Tn5397-tndx-1 and Tn5397-tndx-2 (PCR conditions: 3 min hot start at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 53°C, 3 min at 72°C and a final extension for 10 min at 72°C). DNA sequencing was used to verify the identity of the gene products in two randomly selected isolates (9830414-1 and 9830359-1).13 In all tndX-positive isolates tndX was linked to the tet(M) gene using four PCR sets with the following primers: (i) ReversTetM-2 + Tn5397; (ii) Tn5397-6 + Tn5397-7; (iii) Tn5397-4 + Tn5397-5; and (iv) Tn5397-tndX-1 + Tn5397-tndX-2. The PCR products of 9830414-1 were sequenced, assembled and submitted to GenBank (accession no. DQ206711 [GenBank] ). The ability of Tn5397 and Tn916 to excise from the genome to form a circular form was demonstrated using the following primer sets: REO + LEO and 2tn5397 + 3tn5397, amplifying the circular form of Tn916-like and Tn5397-like transposons, respectively.
A reference strain containing the respective transposon was included as a positive control for each PCR assay (Table 1). For negative controls both samples containing the PCR mixture with no DNA and DNA from the reference strain containing another conjugative transposon were used. The primers used are listed in Table 2.
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Filter-mating experiments
Filter-mating experiments were performed as described previously using four donors (E. faecalis 9839133-1 and E. faecium 9830470-4, 9830409-1 and 9830414-1) to the recipients E. faecium BM4105 and E. faecalis JH2-2.14 Transconjugants were selected on brain heart infusion agar supplemented with tetracycline 8 mg/L, rifampicin 25 mg/L (Sigma-Aldrich, Brøndby, Denmark) and fusidic acid 25 mg/L (Sigma-Aldrich). tet(M), Tn5397 and Tn916 were verified in the transconjugants as described above and the PFGE patterns of selected transconjugants were compared with the PFGE patterns of the respective recipient and donor.
PCR amplification of full-length tet(M)
The upstream part of the gene was amplified using PCR in one fragment (primers: TetM-upstream and TetM-up, PCR product 1, Table 2), and in isolates where tet(M) was located on Tn916-like transposons overlapping with the upstream PCR fragment, the downstream part of the gene was amplified also in one fragment (primers: ReversTetM-2; Tn916-2, PCR product 2a, Table 2) (Figure 1).12
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In one isolate where the downstream part of tet(M) could not be determined, the DNA was digested using restriction enzyme HindIII and ligated. The circularized fragment was used as the template for PCR with primers pointing out of the gene (primers: ReversTetM-1; ReversTetM-2, Table 2) in order to determine the sequence downstream of tet(M). The DNA sequence downstream of tet(M) revealed using this method showed 99.6% homology to the corresponding sequence of transposon Tn5397 from C. difficile, position 16 571–17 045 (GenBank accession no. AF333235 [GenBank] ). The sequence was used to design a new primer (Tn5397) and the downstream fragment PCR 2b was amplified from isolates with this sequence (Figure 1).
Sequencing of the tet(M) gene
DNA sequencing of PCR products was preformed on an ABI 377A automatic sequencer using the PRISM BigDye terminator kit (Applied Biosystems, Foster City, CA, USA) with the primers listed in Table 2, as described previously.1,13 The tet(M) gene in isolates containing Tn1545/Tn916-like or Tn5397-like elements was sequenced as illustrated in Figure 1 using the primers listed in Table 2. All sequences submitted to GenBank were sequenced twice.
Construction of phylogenetic trees
The 15 sequences obtained in this study were compared with full-length and partial tet(M) DNA sequences retrieved from GenBank using the Blast program 2.0. All partial sequences covering positions 286 to 1543 from the translation start site were included. DNA sequences from uncultured samples or sequences with no description of the organism or origin of the DNA were not included.
Phylogenetic trees were reconstructed using Bayesian techniques as implemented in the program MrBayes, version 3.0B4.15 The program MrModeltest was used to find the most appropriate model for the process.16 In both the reconstructed trees [full-length and partial tet(M) sequences, respectively] the best model was found to be HKY + I + G, the so-called HKY model, with a proportion of invariable sites and gamma-distributed rate variation across sites.17 MCMC sampling was performed for 5 000 000 generations with four chains.
Convergence was confirmed by comparing the results of two independent runs. The program Tracer was used to determine burn-in and for further confirmation of proper mixing and adequate run-length.18
Detection of signs for recombination within the tet(M) group
To search for signs of recombination we used several independent tools. The maxchi2 program implements a modification of the maximum chi-square method.19 When assessing whether sequence mosaics were more pronounced than expected for random reasons, 1000 alignment permutations were used. We also used the program TOPALi, which implements a number of different methods.20 Specifically, we used the probabilistic divergence measure method of Husmeier and Wright21 and the Hidden Markov model method of Husmeier and McGuire.22 In addition to these specialized methods, we also searched for local sequence similarities using the BLAST local alignment and database search program.23
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Detection of Tn916-like and Tn5397-like conjugative transposons
A total of 39 tet(M)-positive E. faecium and 37 E. faecalis isolated from humans, pigs and broilers were screened for the presence of the xis-Tn gene from Tn916-like transposons and the resolvase gene tndX from the transposon Tn5397 (Table 3). One of the two conjugative transposons could be detected in 62 (82%) of the isolates. The xis-Tn gene from Tn916-like transposons was present in both E. faecium and E. faecalis from all three sources (humans, pigs and broilers). Of the isolates from humans, 21 (91%) contained the xis-Tn gene from Tn916-like transposons. E. faecium from broilers contained the lowest fraction of the xis-Tn gene from Tn916-like transposons, present in four isolates (31%), and the resolvase gene tndX from the transposon Tn5397 was present in eight (62%) of the isolates. The PCR products obtained from two isolates (9830414-1 and 9830359-1) were sequenced and revealed 100% identity to the corresponding sequence in C. difficile (GenBank accession no. AF333235 [GenBank] , position 19115–19704). One of the E. faecium isolates (9830133-1) from broilers contained both the xis-Tn gene from Tn916-like transposons and tndX from transposon Tn5397. Both genes could be linked to tet(M). The highest fraction of isolates [seven isolates (47%)] with no detectable mobile elements was found among E. faecalis from pigs.
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Characterization of Tn5397-like transposons
The eight isolates containing tndX were further characterized. tet(M) could be linked to tndX and gave the expected size of product for all PCR set-ups except in two isolates (9830133-1 and 9830359-1) where the PCR product (tn5397-6; tn5397-7) gave a band 
1500 bp larger than expected. In seven isolates the circular form of Tn5397 was detected. No PCR product of the circular form of Tn5397 was detected in the isolate with both transposons (9830133-1), but the circular form of Tn916 was detected. Isolate 9830133-1 was used as a donor in filter-mating experiments, and horizontal gene transfer of tet(M) was demonstrated to both recipients (E. faecium BM4105 2.1 x 10–8 transconjugant/donor and E. faecalis 10–8 transconjugant/donor), but all transconjugants contained only Tn916.
The E. faecium isolate (9830414-1) from broilers with a Tn5397-like element was partially sequenced. The sequence including tet(M) and tndX genes (5070 bp) had 97% identity to the corresponding sequence of C. difficile (GenBank accession no. AF333235 [GenBank] , position 14631–19700). Isolate 9830414-1 was used as a donor in filter-mating experiments and Tn5397 was transferred to E. faecium BM4105 (7 x 10–8 transconjugant/donor) but not to E. faecalis JH2-2 (detection limit >1.1 x 10–8 transconjugant/donor).
Sequencing of the tet(M) gene
In order to sequence tet(M) in isolates with Tn916-like transposons an upstream and a downstream PCR product of the gene were amplified according to the sequencing strategy illustrated in Figure 1. Eleven of the isolates could be sequenced using this strategy. In six isolates fragment 2a could not be amplified using this approach. PCR for circularized DNA from one of these isolates revealed tet(M) on a Tn5397-like element and the downstream part of the gene was sequenced. One more isolate (9830359-1) had tet(M) on a Tn5397-like element, and a downstream PCR fragment (2b, Figure 1) was amplified and sequenced from this isolate. Circularized DNA from two isolates revealed the downstream sequence of tet(M) but no transposon was detected in these isolates. Two isolates failed to be amplified and full-length tet(M) could not be sequenced. These were E. faecalis isolates from pigs.
Construction of phylogenetic trees
Full-length tet(M) sequences were obtained from 15 isolates and the sequences were aligned with 25 full-length tet(M) sequences retrieved from GenBank. This alignment was then used to construct a phylogenetic tree (Figure 2). In the resulting tree, tet(M) fell into three distinct subgroups. Subgroup I of tet(M) had a clade-credibility of 100% and contained tet(M) on Tn5397 from C. difficile (AF333235 [GenBank] ), two E. faecium isolates from broilers with Tn5397-like elements and tet(M) from E. faecium isolated from pig and broiler with tet(M) associated with 80 kb plasmids (data not shown). Subgroup II had a clade-credibility of 99% and contained 11 isolates with tet(M) on Tn916-like elements, where 8 were 100% identical to tet(M) on Tn916 from E. faecalis (GenBank accession nos. X92947 [GenBank] and M85225 [GenBank] ). These isolates were from E. faecium isolated from pigs and humans and from E. faecalis isolated from broilers and pigs. Two tet(M) genes of E. faecalis from humans were 100% identical to each other and branched separately from the other tet(M) genes associated with Tn916-like elements. The clade-credibility of subgroup III was 54%, indicating only limited support for its monophylicity. Included in the group is one tet(M) gene derived from an E. faecalis isolate of human origin (20074-s-1). This tet(M) gene is most similar to tet(M) from Tn1545 (GenBank accession no. X04388 [GenBank] ). The isolate was also resistant to erythromycin, streptomycin and kanamycin and contained the erm(B) and aphA3 genes. The resistance patterns and additional resistance genes for the isolates are listed in Table 1.
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A second phylogenetic tree was constructed from an alignment of 63 partial tet(M) sequences in GenBank and the corresponding segment of our 15 tet(M) sequences (position 286–1443 from the translation start was used). This included tet(M) of Lactobacillus from fermented sausages and enterococci primarily from cheese. The phylogenetic tree had the same structure as the full-length tree, with three major groups (data not shown). Again there was good support for the monophylicity of both group I and group II (100 and 93%, respectively), while the monophylicity of group III received only limited support (57%). The additional sequences aligned with subgroup II, except the tet(M) genes of two aquatic isolates from Vibrio sp. (GenBank accession no. AB124557 [GenBank] ) and Photobacterium damselae (GenBank accession no. AB124556 [GenBank] ), respectively, which were in subgroup III. tet(M) of two E. faecalis isolated from cheese (GenBank accession nos. AJ585083 [GenBank] and AJ585082 [GenBank] ) branched together with those of two E. faecalis isolated from humans (18854-s-1 and 20028-s-1) within subgroup II. All four isolates contained the xis-Tn gene of the Tn916/Tn1545 family.
Detection of signs for recombination within the tet(M) group
Using the maxchi2 program on the entire full-length alignment very strongly indicated that recombination was indeed present. Specifically, the null hypothesis of no recombination was clearly rejected with P < 0.001. In an attempt to pinpoint the sequences involved, we used maxchi2, methods from the TOPALi program and BLAST searches on several different subsets of the full-length alignment. These investigations confirmed that subgroup III sequences could well be the result of recombination between subgroup I and subgroup II sequences: maxchi2 and TOPALi analyses on subsets containing representatives from all three groups often gave significant results, and BLAST often found one part of a subgroup III sequence to be more similar to subgroup I sequences while the other part was more similar to sequences belonging to subgroup II. However, it should be noted that the picture was not quite this simple, as some analyses also found significant evidence of recombination not just between, but also within, subgroups I and II.
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Phylogenetic analysis divided the tet(M) genes into three major subgroups based on both partial and full-length sequences (Figure 2). Interestingly, tet(M) genes from isolates with Tn5397-like elements clustered with the Tn5397-associated tet(M) from C. difficile (AF333235 [GenBank] ) in subgroup I (Figure 2). Moreover, these tet(M) genes were clearly different from tet(M) in Tn916-like elements found in subgroup II (Figure 2).
Two E. faecium isolates from pig and broiler, respectively (9830470-4 and 9830409-1), had tet(M) present on 80 kb plasmids (data not shown). The tet(M) genes from these isolates branched separately in subgroup I (Figure 2). We could not detect transposons in these isolates, and no horizontal transfer of tet(M) from these isolates to E. faecalis (JH2-2) or E. faecium (BM4105) was observed. One or more plasmids were detected in 8 of the 17 isolates, but since the tet(M) probe hybridized to plasmids in only 2 isolates, tet(M) was present either on the chromosome or on large plasmids (>150 kb, not extracted by this method) in the majority of isolates (data not shown).
Of the isolates with tet(M) on Tn916/Tn1545 family transposons, 11 were sequenced. Of these isolates, 10 were grouped into subgroup II and 8 were 100% identical to tet(M) from Tn916 found in E. faecalis (GenBank accession nos. M85225 [GenBank] and U09422 [GenBank] ) and were isolated from E. faecalis from pigs and broilers and from E. faecium from humans and pigs (Figure 2). Partial sequences of tet(M) from Enterococcus durans (GenBank accession no. AJ585084 [GenBank] ) and Staphylococcus aureus (GenBank accession no. AY057893 [GenBank] ) were also identical (data not shown). The 100% identical tet(M) in these isolates may indicate either recent transfer events or that no adaptation of the tet(M) has been necessary in order for the gene to be maintained in these species. Subgroup II contains several tet(M) genes associated with Tn916-like elements. For instance, a group within subgroup II includes E. faecalis and Bacillus cereus with transferable tet(M) on Tn916/Tn1545 family transposons located on the main chromosome. These bacteria were isolated from shellfish and farmland soil, respectively. However, this group also contains tet(M) associated with an element designated CW459tet(M), which contains the integrase int459 from Clostridium perfringens. Furthermore, the majority of tet(M) genes in this group are plasmid borne and often show no indication of the presence of any Tn916/Tn1545 family transposon.7 Even though the mobile elements carrying tet(M) seem very diverse in subgroup II and are from both Gram-positive and Gram-negative origin, the majority of tet(M) genes are highly related, with zero or a few base pair differences.
One E. faecalis isolate from human had highest homology to tet(M) on Tn1545 from E. faecalis (GenBank accession no. X04388 [GenBank] ) within subgroup III. This isolate had erm(B) and aphA-3 responsible for resistance to erythromycin and kanamycin, respectively, and these genes were present on Tn1545. This strain may contain tet(M) on a Tn1545-like transposon rather than on a Tn916-like transposon.
Mosaic structures resulting from recombination within the tet(M) group were observed by Oggioni et al.24 in E. faecalis, S. pneumoniae, S. aureus, Ureaplasma urealyticum and Neisseria, and they could be traced to two distinct alleles. Huang et al. found mosaic structures within tet(M) of Gardnerella with regions of homology to tet(M) gene sequences from Tn916/Tn1545 and the American type plasmid found in Neisseria gonorrhoeae.25 Recombination within the tet(M) group was also confirmed by our study. Subgroup III was peculiar not only in consisting of several tet(M) genes located on composite transposons (Tn1545, Tn2009 and Tn5251) but also in being placed more closely to the root and having relatively low clade-credibilities. These features could indicate that some or all subgroup III sequences are the result of recombination between subgroup I and/or II sequences; we therefore used a variety of different tools to investigate this hypothesis further. Significant evidence for recombination not just between but also within subgroups II and I was found. For recombination to take place between tet(M) genes on different mobile elements, both elements must be present within the same cell. We found in this study one E. faecium isolate with both Tn916/Tn1545-like and Tn5397 transposons. This is to our knowledge the first finding of tet(M) on two different mobile elements within the same isolate, and this supports the notion that recombination between tet(M) genes is possible. This finding is also interesting since studies of these two transposons within the same C. difficile cell have shown that Tn5397 can induce the loss of Tn916 when introduced into a C. difficile cell by conjugation and vice versa.6 The Tn5397 had an insert of 1500 bp downstream to the tet(M) gene, and the circular form of the transposon could not be detected and horizontal transfer of the transposon could not be demonstrated, indicating that the transposon was not functional, which could explain the presence of both transposons within the same isolate. However, this needs to be further studied.
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None to declare.
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Acknowledgements |
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We would like to acknowledge Christina Aaby Svendsen, Christina Ahlblad, Anette Nielsen and Jane Larsen for excellent technical assistance. We thank Dr P. Mullany for providing us with a control strain for Tn5397 transposon and Dr A. Rambaut for kindly making the program maxchi2 available. This study was funded by a grant from the Danish Research Agency, ref. no. 274-05-0117. Part of the work was presented as a poster at the 2nd International ASM-FEMS Conference on Enterococci, 2005.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
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1. Aarestrup FM, Agersø Y, Gerner-Smidt P et al. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn Microbiol Infect Dis 2000; 37: 127–37.[CrossRef][Web of Science][Medline]
2. Roberts MC. Update on acquired tetracycline resistance genes. FEMS Microbiol Lett 2005; 245: 195–203.[CrossRef][Web of Science][Medline]
3.
Rice LB. Tn916 family conjugative transposons and dissemination of antimicrobial resistance determinants. Antimicrob Agents Chemother 1998; 42: 1871–7.
4.
Roberts AP, Johanesen PA, Lyras D et al. Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules. Microbiology 2001; 147: 1243–51.
5.
Mullany P, Wilks M, Lamb I et al. Genetic analysis of a tetracycline resistance determinant from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis. J Gen Microbiol 1990; 136: 1343–9.
6.
Wang H, Roberts AP, Lyras D et al. Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularisation is mediated by the large resolvase, TndX. J Bacteriol 2000; 182: 3775–83.
7.
Doherty N, Trzcinski K, Pickerill P et al. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob Agents Chemother 2000; 44: 2979–84.
8.
Gevers D, Danielsen M, Huys G et al. Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl Environ Microbiol 2003; 69: 1270–5.
9.
Huys G, D'Haene K, Collard JM et al. Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl Environ Microbiol 2004; 70: 1555–62.
10. Aarestrup FM, Bager F, Jensen NE et al. Surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. APMIS 1998; 106: 606–22.[Web of Science][Medline]
11. Clewell DB. Movable genetic elements and antibiotic resistance in enterococci. Eur J Clin Microbiol Infect Dis 1990; 9: 90–102.[CrossRef][Web of Science][Medline]
12. Agersø Y, Jensen LB, Givskov M et al. The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group. FEMS Microbiol Lett 2002; 214: 251–6.[Web of Science][Medline]
13. Sears LE, Moran LS, Kissinger C et al. CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase. Biotechniques 1992; 13: 626–33.[Web of Science][Medline]
14. Hammerum AM, Jensen LB, Aarestrup FM. Detection of the satA gene and transferability of virginiamycin resistance in Enterococcus faecium from food-animals. FEMS Microb Lett 1998; 168: 145–51.[CrossRef][Web of Science][Medline]
15.
Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003; 19: 1572–4.
16. Nylander JAA. MrModeltest v1.0b. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, Sweden, 2004.
17. Yang Z. Maximum likelihood estimation of phylogeny from DNA sequences when substitution rates differ over sites. Mol Biol Evol 1993; 10: 1396–401.[Abstract]
18. Rambaut A, Drummond A. Tracer. Program distributed by the authors. Oxford Evolutionary Biology Group, University of Oxford, UK, 2004.
19.
Posada D. Evaluation of methods for detecting recombination from DNA sequences: empirical data. Mol Biol Evol 2002; 19: 708–17.
20.
Milne I, Wright F, Rowe G et al. TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics 2004; 20: 1806–7.
21. Husmeier D, Wright F. Probabilistic divergence measures for detecting interspecies recombination. Bioinformatics 2001; 17: S123–31.[Abstract]
22.
Husmeier D, McGuire G. Detecting recombination in 4-taxa DNA sequence alignments with Bayesian Hidden Markov models and Markov chain Monte Carlo. Mol Biol Evol 2003; 20: 315–37.
23.
Altschul SF, Madden TL, Schaffer AA et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389–402.
24. Oggioni MR, Dowson CG, Smith JM et al. The tetracycline resistance gene tet(M) exhibits mosaic structure. Plasmid 1996; 35: 156–63.[CrossRef][Web of Science][Medline]
25.
Huang R, Gascoyne-Binzi DM, Hawkey PM et al. Molecular evolution of the tet(M) gene in Gardnerella vaginalis. J Antimicrob Chemother 1997; 40: 561–5.
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