Dataset Information

VanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174.

ABSTRACT: Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl-pentapeptide[D-Ser] for cell wall assembly and prevent synthesis of peptidoglycan precursors ending in D-Ala. The vanC cluster of Enterococcus gallinarum BM4174 consists of five genes: vanC-1, vanXY(C), vanT, vanR(C), and vanS(C). Three genes are sufficient for resistance: vanC-1 encodes a ligase that synthesizes the dipeptide D-Ala-D-Ser for addition to UDP-MurNAc-tripeptide, vanXY(C) encodes a D,D-dipeptidase-carboxypeptidase that hydrolyzes D-Ala-D-Ala and removes D-Ala from UDP-MurNAc-pentapeptide[D-Ala], and vanT encodes a membrane-bound serine racemase that provides D-Ser for the synthetic pathway. The three genes are clustered: the start codons of vanXY(C) and vanT overlap the termination codons of vanC-1 and vanXY(C), respectively. Two genes which encode proteins with homology to the VanS-VanR two-component regulatory system were present downstream from the resistance genes. The predicted amino acid sequence of VanR(C) exhibited 50% identity to VanR and 33% identity to VanR(B). VanS(C) had 40% identity to VanS over a region of 308 amino acids and 24% identity to VanS(B) over a region of 285 amino acids. All residues with important functions in response regulators and histidine kinases were conserved in VanR(C) and VanS(C), respectively. Induction experiments based on the determination of D,D-carboxypeptidase activity in cytoplasmic extracts confirmed that the genes were expressed constitutively. Using a promoter-probing vector, regions upstream from the resistance and regulatory genes were identified that have promoter activity.

SUBMITTER: Arias CA

PROVIDER: S-EPMC89929 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174.

Arias C A CA Courvalin P P Reynolds P E PE

Antimicrobial agents and chemotherapy 20000601 6

Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl-pentapeptide[D-Ser] for cell wall assembly and prevent synthesis of peptidoglycan precursors ending in D-Ala. The vanC cluster of Enterococcus gallinarum BM4174 consists of five genes: vanC-1, vanXY(C), vanT, vanR(C), and vanS(C). Three genes are sufficient for resistance: vanC-1 encodes a ligase that synthesizes the dipeptide D-Ala-D-Ser for addition to UDP-MurNAc-tripeptide, vanXY(C) encodes a D,D-dipeptidase-carboxypep ...[more]

PMID: 10817725

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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.

Project description:Vancomycin-resistant Enterococcus (VRE) is a leading cause of hospital-acquired infections and continues to spread despite widespread implementation of pathogen-targeted control guidelines. Commensal gut microbiota provide colonization resistance to VRE, but the role of gut microbiota in VRE acquisition in at-risk patients is unknown. To address this gap in our understanding, we performed a case-control study of gut microbiota in hospitalized patients who did (cases) and did not (controls) acquire VRE. We matched case subjects to control subjects by known risk factors and "time at risk," defined as the time elapsed between admission until positive VRE screen. We characterized gut bacterial communities using 16S rRNA gene amplicon sequencing of rectal swab specimens. We analyzed 236 samples from 59 matched case-control pairs. At baseline, case and control subjects did not differ in gut microbiota when measured by community diversity (P = 0.33) or composition (P = 0.30). After hospitalization, gut communities of cases and controls differed only in the abundance of the Enterococcus-containing operational taxonomic unit (OTU), with the gut microbiota of case subjects having more of this OTU than time-matched control subjects (P = 0.01). Otherwise, case and control communities after the time at risk did not differ in diversity (P = 0.33) or community structure (P = 0.12). Among patients who became VRE colonized, those having the Blautia-containing OTU on admission had lower Enterococcus relative abundance once colonized (P = 0.004). Our results demonstrate that the 16S profile of the gut microbiome does not predict VRE acquisition in hospitalized patients, likely due to rapid and profound microbiota change. The gut microbiome does not predict VRE acquisition, but it may be associated with Enterococcus expansion, suggesting that these should be considered two distinct processes.IMPORTANCE The Centers for Disease Control and Prevention estimates that VRE causes an estimated 54,000 infections and 539 million dollars in attributable health care costs annually. Despite improvements in hand washing, environmental cleaning, and antibiotic use, VRE is still prevalent in many hospitals. There is a pressing need to better understand the processes by which patients acquire VRE. Multiple lines of evidence suggest that intestinal microbiota may help some patients resist VRE acquisition. In this large case-control study, we compared the 16S profile of intestinal microbiota on admission in patients that did and did not subsequently acquire VRE. The 16S profile did not predict subsequent VRE acquisition, in part due to rapid and dramatic change in the gut microbiome following hospitalization. However, Blautia spp. present on admission predicted decreased Enterococcus abundance after VRE acquisition, and Lactobacillus spp. present on admission predicted Enterococcus dominance after VRE acquisition. Thus, VRE acquisition and domination may be distinct processes.

Dataset Information

VanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174.

Publications

vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174.

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