Dataset Information

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

ABSTRACT: The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.

SUBMITTER: Lim SK

PROVIDER: S-EPMC1610277 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

Lim Suk-Kyung SK Tanimoto Koichi K Tomita Haruyoshi H Ike Yasuyoshi Y

Applied and environmental microbiology 20061001 10

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to van ...[more]

PMID: 17021204

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Project description:A total of seventy VanA-type vancomycin-resistant enterococci (VRE) isolates obtained in Taiwan in the early 2000s were retrospectively characterized. Forty isolates were obtained from human patients and thirty from livestock. Of these VRE isolates, twenty-three (57.5%) of the human VRE and thirty (100%) of the livestock VRE were Enterococcus faecalis, and the remaining seventeen (42.5%) of the human VRE were E. faecium. Of the 53 E. faecalis isolates, twenty-two (96%) of the human VRE and thirty (100%) of the livestock VRE exhibited a high level of resistance to vancomycin and sensitivity to teicoplanin. They also had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. The vancomycin resistance of all of the 22 human isolates, and 20 of the 30 livestock isolates, transferred to E. faecalis FA2-2 at a frequency of 10-5 to 10-3 per donor cell in broth. Each of the transconjugants responded to E. faecalis pheromone (i.e., E. faecalis FA2-2 culture filtrate), indicating that the conjugative plasmids were pheromone-responsive plasmids. Three of the conjugative plasmids originated from human isolates, and five plasmids from livestock isolates were corresponded and classified as type A plasmid. Two plasmids originated from human isolates and six plasmids from livestock isolates were corresponded and classified as type B plasmid. E. faecalis FA2-2 containing either the type A or type B plasmid responded to the synthetic pheromone cAD1. The type A and type B plasmids transferred between E. faecalis FA2-2 and JH2SS at a frequency of about 10-2 per donor cell and conferred vancomycin, bacitracin, and erythromycin resistances. The complete DNA sequence of the representative type A plasmid pTW9 (85,068 bp) showed that the plasmid carried a Tn1546-like element encoding vanA-type resistance, erythromycin resistance (ermB), and bacitracin resistance (bcrABDR). The plasmid contained the regulatory region found in the pheromone-responsive plasmid and encoded the genes traA, traD and iad1, which are the key negative regulatory elements, and traE1, a key positive regulator of plasmid pAD1, indicating that plasmid pTW9 was pAD1-type pheromone-responsive plasmid. PFGE analysis of SmaI-digested chromosomal DNAs showed that several E. faecalis strains harboring an identical type A pheromone-responsive plasmid were indistinguishable, and that these were identified both in human and livestock isolates, indicating the transmissions of the VRE strains between livestock and humans. These data showed that the multiple-drug-resistant pheromone-responsive conjugative plasmids have been widely spread in both human and livestock VRE, and there was high potential for transfers of VRE from food animals to humans in Taiwan in the early 2000s.

Project description:The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

Publications

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

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