Nucleotide sequence of IS1678, an insertion sequence in the vanA cluster of enterococci.
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ABSTRACT:
SUBMITTER: Jung WK
PROVIDER: S-EPMC1068654 | biostudies-literature | 2005 Apr
REPOSITORIES: biostudies-literature
Publications
Nucleotide sequence of IS1678, an insertion sequence in the vanA cluster of enterococci.
Jung Woo Kyung WK Hong Soon Keun SK Koo Hye Cheong HC Kwon Nam Hoon NH Park Yong Ho YH
Antimicrobial agents and chemotherapy 20050401 4
Similar Datasets
Project description:We report an outbreak of vancomycin-variable vanA(+) enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA(+) Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.
| S-EPMC4914660 | biostudies-literature
Project description:The molecular characterization of five clinical isolates of vanA-containing vancomycin-resistant enterococci with altered resistance to glycopeptides was examined. One strain represented an IS1216V insertion accompanied by partial deletion of the reading frame of vanX following a transposition event. The other four strains represented IS1216V within the vanX-vanY intergenic region associated with deletion of vanY or vanZ.
| S-EPMC375328 | biostudies-other
Project description:In several strains of Enterococcus faecium isolated in Europe, the cluster of genes encoding high-level resistance to vancomycin (VanA phenotype) resides on a 10.85-kb transposon, Tn1546, or closely related elements. To determine whether Tn1546 was conserved in recent enterococcal isolates from the northeastern United States, seven strains were compared by restriction mapping and DNA hybridization with probes from within the van cluster. Two of the seven strains contained intact Tn1546-like sequences; however, in five of the strains, the organization of the van cluster differed from that of Tn1546. Three of the five strains with variations harbored a novel DNA segment within the van gene cluster. This 1,496-bp segment was similar to IS1165 of Leuconostoc mesenteroides and IS1181 of Staphylococcus aureus and was flanked by 24- and 23-bp imperfect inverted repeats and 8-bp direct repeats. On the basis of these findings, we propose that this element comprises a novel insertion-like sequence, IS1251. Multiple copies of IS1251 were also present at other sites in both resistant and susceptible clinical isolates. Our findings suggest that the van cluster in recent isolates from the northeastern United States differs from that present in the early European VanA phenotype strains.
| S-EPMC162544 | biostudies-other
Project description:Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistantE. faecium(VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra- and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated ≥10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed thatvanAupregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. ThevanHpromoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. UsingvanHreporter experiments withBacillus subtilisand deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion ofvanRdid not result in increased chlorhexidine susceptibility, demonstrating thatvanHAXinduction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.
| S-EPMC4808233 | biostudies-literature
Project description:Twenty seven isolates of vancomycin resistant Enterococci based on the disk diffusion and E- test have been screened; being found eight (0.3%) clinical isolates of vanA & vanB through Taq Man Real Time PCR assay. This study shows the presence of both vanA & vanB genotypes in vanA phenotypes clinical isolates in the three hospitals in Iran.
| S-EPMC4512060 | biostudies-literature
Project description:Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid.IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.
| S-EPMC7142295 | biostudies-literature
Project description:PurposeVancomycin-resistant enterococci infection is a worrying worldwide clinical problem. To evaluate the accuracy of GeneXpert vanA/vanB in the diagnosis of VRE, we conducted a systematic review in the study.MethodsExperimental data were extracted from publications until May 03 2021 related to the diagnostic accuracy of GeneXpert vanA/vanB for VRE in PubMed, Embase, Web of Science and the Cochrane Library. The accuracy of GeneXpert vanA/vanB for VRE was evaluated using summary receiver to operate characteristic curve, pooled sensitivity, pooled specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio.Results8 publications were divided into 3 groups according to two golden standard references, vanA and vanB group, vanA group, vanB group, including 6 researches, 5 researches and 5 researches, respectively. The pooled sensitivity and specificity of group vanA and vanB were 0.96 (95% CI, 0.93-0.98) and 0.90 (95% CI, 0.88-0.91) respectively. The DOR was 440.77 (95% CI, 37.92-5123.55). The pooled sensitivity and specificity of group vanA were 0.86 (95% CI, 0.81-0.90) and 0.99 (95% CI, 0.99-0.99) respectively, and those of group vanB were 0.85 (95% CI, 0.63-0.97) and 0.82 (95% CI, 0.80-0.83) respectively.ConclusionGeneXpert vanA/vanB can diagnose VRE with high-accuracy and shows greater accuracy in diagnosing vanA.
| S-EPMC8575182 | biostudies-literature
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.
| S-EPMC229654 | biostudies-other
Project description:AimThe aim of this study was to investigate the VRE frequency and the rate of each gene in isolated enterococci from patients with intestinal infection in the central region of Iran.BackgroundEnterococci infections are a public health growing concern due to the glycopeptide antibiotics resistance especially vancomycin. Genes, vanA, B, and H contribute to the influence of vancomycin-resistant enterococci (VRE).Patients and methodsThis study was conducted from January to July 2014 in Shahrood university hospital. Enterococci isolation and its antibacterial susceptibility were performed by culturing in Aesculin Azide agar and Kirby-Bauer method, respectively. Vancomycin-resistant genes were screened through conventional PCR, and subsequently sequenced.ResultsAmong 265 specimens, 100 isolates revealed enterococci, in which E. faecalis (91%) and E. faecium (9%). The isolated enterococci were resistant to vancomycin (6%) and chloramphenicol (21%), whereas their large proportions (94% to 100%) were multi-drug resistant. All VRE isolates belonged to E. faecalis, conversely, the E. faecium were susceptible to the same antibiotic. Both vanA and vanH genes were identified in all VRE isolates, although, no vanB gene was indicated. Homology analysis of sequenced amplicons verified the full length compatibility to the worldwide reported genes.ConclusionThe present study revealed VR E.faecalis in gastroenteritis patients and resistance factor for vanA and vanH genes are coordinated. Since enterococci isolates were all multidrug resistance, increase in VR E.faecalis vanA / vanH in this area could be expected.
| S-EPMC5118856 | biostudies-literature
Project description:d-alanine-d-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-A resolution. This structure shows that the active site has unexpected interactions and is distinct from previous models for d-alanyl-d-lactate ligase mechanistic studies. It appears that the preference of the enzyme for lactate as a ligand over d-alanine could be mediated by electrostatic effects and/or a hydrogen-bonding network, which principally involve His-244. The structure of d-alanyl-d-lactate ligase provides a revised interpretation of the molecular events that lead to vancomycin resistance.
| S-EPMC16797 | biostudies-literature