Isolation and Characterization of a Phage to Control Vancomycin Resistant Enterococcus Faecium.
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SUBMITTER: Raza T
PROVIDER: S-EPMC7874677 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundVancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome's structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates.ResultsAnalysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity.ConclusionsFor rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period.
| S-EPMC8542323 | biostudies-literature
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.
| S-EPMC521886 | biostudies-literature
Project description:Vancomycin-resistant Enterococcus faecium carrying the vanD1 gene on plasmid pEF-D was isolated from a fecal sample of a hospitalized patient in Japan. The strain JH5687 showed moderate resistance to vancomycin (MIC, 16 μg/mL) but remained susceptible to teicoplanin (MIC, 1 μg/mL). The backbone gene organization of pEF-D was highly homologous to that of conjugative plasmid pMG1 or pHTβ. The calculated conjugation frequency of JH5687 was 10-4 to 10-5 per donor cell.
| S-EPMC9664843 | biostudies-literature
Project description:VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.
| S-EPMC149003 | biostudies-literature
Project description:Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays.
| S-EPMC5652432 | biostudies-literature
Project description:Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein, we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance, it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.
| S-EPMC5494988 | biostudies-literature
Project description:A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.
| S-EPMC1636183 | biostudies-literature
Project description:The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report, we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faeciumβ-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.
| S-EPMC9189978 | biostudies-literature
Project description:BackgroundSpread of vancomycin-resistant Enterococcus (VRE) is a global concern as a significant cause of healthcare-associated infections. A series of VRE faecium (VREf) outbreaks caused by clonal propagation due to interhospital transmission occurred in six general hospitals in Aomori prefecture, Japan.MethodsThe number of patients with VREf was obtained from thirty seven hospitals participating in the local network of Aomori prefecture. Thirteen hospitals performed active screening tests for VRE. Whole genome sequencing analysis was performed.ResultsThe total number of cases with VREf amounted to 500 in fourteen hospitals in Aomori from Jan 2018 to April 2021. It took more than three years for the frequency of detection of VRE to return to pre-outbreak levels. The duration and size of outbreaks differed between hospitals according to the countermeasures available at each hospital. Whole genome sequencing analysis indicated vanA-type VREf ST1421 for most samples from six hospitals.ConclusionsThis was the first multi-jurisdictional outbreak of VREf sequence type 1421 in Japan. In addition to strict infection control measures, continuous monitoring of VRE detection in local medical regions and smooth and immediate communication among hospitals are required to prevent VREf outbreaks.
| S-EPMC9308179 | biostudies-literature
Project description:Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bp vanB gene cluster including the vanR(B2), vanS(B2), vanY(B2), vanW(B2), vanH(B2), vanB2, and vanX(B2) genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the vanB1 gene cluster: VanR(B1), 97%; VanS(B1), 97%; VanY(B1), 96%; VanH(B1), 95%; VanB1, 96%; and VanX(B1), 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is a D-Ala-D-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published vanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 microg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.
| S-EPMC88102 | biostudies-literature