Vancomycin-resistant enterococci in humans and imported chickens in Japan.
ABSTRACT: The phenotypes and genotypes of 22 VanA-type vancomycin-resistant enterococci that had been isolated in Japan were examined. The VanA resistance determinant was plasmid mediated in each of the 22 strains. Of the 22 strains, 8 were isolated from different patients and 11 and 3 were obtained from different samples of chickens imported from Thailand and France, respectively. Three of the strains that were isolated from patients and the 11 strains isolated from the Thai chickens showed high-level vancomycin resistance (MICs, 512 to 1,024 micro g/ml) and low-level teicoplanin resistance (MICs, 0.5 to 4 micro g/ml). Each of these strains had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. L50 was converted to V, E54 was converted to Q, and Q69 was converted to H compared to the vanS gene sequence of Tn1546.
Project description:Acquired VanA- and VanB-type glycopeptide resistance in enterococci is due to synthesis of modified peptidoglycan precursors terminating in D-lactate. As opposed to VanA-type strains which are resistant to both vancomycin and teicoplanin, VanB-type strains remain teicoplanin susceptible. We have determined the sequence of a 7,160-bp DNA fragment associated with VanB-type resistance in Enterococcus faecalis V583 that contains seven open reading frames. The distal part encoded the VanH (B), VanB, and VanX (B) proteins that are highly similar to the putative VanH, VanA, and VanX proteins responsible for VanA-type resistance. Upstream from the structural genes for these proteins were the vanY(B) gene encoding a D,D-carboxypeptidase and an open reading frame vanW with an unknown function. The proximal part of the gene cluster coded for the apparent VanS(B)-VanR (B) two-component regulatory system. VanR (B) was related to response regulators of the OmpR subclass, and VanS (B) was related to membrane-associated histidine protein kinases. Analysis of transcriptional fusions with a reporter gene and promoter mapping indicated that the VanR B-VanS B two-component regulatory system activates a promoter located immediately downstream from the vanS B gene. Vancomycin, but not teicoplanin, was an inducer, which explains teicoplanin susceptibility of VanB-type enterococci.
Project description:We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.
Project description:Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.
Project description:OBJECTIVES:Staphylococcus aureus is a common cause of human infection, and emergence of vancomycin-resistance S. aureus is a great concern for treatment of methicillin-resistant S. aureus,(MRSA) in recent years (MRSA). Here, we report the isolation of high-level VRSA. MATERIALS AND METHODS:S. aureus was isolated from foot ulcer of a diabetic woman in Tehran, Iran. Antibiotic susceptibility was determined according to CLSI guidelines. VanA gene cluster PCR was carried out and PCR amplicon of vanA was sequenced. RESULTS:S. aureus had high-level vancomycin-resistant (MIC 512 ? µg/ml). Patient's history revealed that VRSA isolate was acquired through community transmission. Only vanA, vanR and vanS genes were amplified in our isolate. Sequencing revealed that the vanA sequence had high similarity to the vanA sequence of Tn1546. CONCLUSION:Although VRSA infection continues to be rare, isolation of community-acquired VRSA is a significant issue and it needs the efforts of public health authorities.
Project description:Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR two-component regulatory system encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS two-component regulatory system activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.
Project description:The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.
Project description:Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.
Project description:VanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system in Streptomyces coelicolor as a model, we have undertaken a series of in vivo studies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with the d-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essential d-Ala-d-Ala ligase activity by constitutive expression of vanA encoding a bifunctional d-Ala-d-Ala and d-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containing d-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance of d-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating in d-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask the d-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting with d-Ala-d-Ala residues, failed to induce van gene expression. Activation of resistance by a vancomycin-d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating in d-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.
Project description:Although enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant Enterococcus faecium (VRE) isolates in the nephrology department at Bicêtre Hospital (K.-Bicêtre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 microg/ml, whereas teicoplanin MICs ranged from 4 to 48 microg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a vanA gene. Plasmid extraction and mating-out experiments indicated that the vanA gene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The vanA gene was part of a vanA-type operon for expression of resistance located on a Tn1546-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS16 in the vanY gene that encodes a d,d-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any.
Project description:Due to Australia's management of antimicrobial use in poultry, particularly the discontinued use of avoparcin for nearly 20 years, it is hypothesized that vancomycin-resistant enterococci associated with human disease are not derived from poultry isolates. This study evaluated antimicrobial resistance (AMR) of five enterococcal species isolated from Australian meat chickens, genomic features of Enterococcus faecium and Enterococcus faecalis, and the phylogenetic relationship of the poultry-derived E. faecium with isolates from human sepsis cases. All enterococcal isolates from chicken ceca were subjected to antimicrobial susceptibility testing. E. faecium and E. faecalis underwent whole-genome sequencing. E. faecium was compared at the core genome level to a collection of human isolates (n = 677) obtained from cases of sepsis over a 2-year period spanning 2015 to 2016. Overall, 205 enterococci were isolated consisting of five different species. E. faecium was the most frequently isolated species (37.6%), followed by E. durans (29.7%), E. faecalis (20%), E. hirae (12.2%), and E. gallinarum (0.5%). All isolates were susceptible to vancomycin and gentamicin, while one isolate was linezolid resistant (MIC 16 mg/liter). Core genome analysis of the E. faecium demonstrated two clades consisting predominantly of human or chicken isolates in each clade, with minimal overlap. Principal component analysis for total gene content revealed three clusters comprised of vanA-positive, vanB-positive, and both vanA- and vanB-negative E. faecium populations. The results of this study provide strong evidence that Australian chicken E. faecium isolates are unlikely to be precursor strains to the currently circulating vancomycin-resistant strains being isolated in Australian hospitals.