BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon.
METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments.
RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020).
CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

BACKGROUND: Vancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the acquisition of vancomycin-resistant S. aureus strains by the integration of an enterococcal plasmid containing the vanA operon into the S. aureus chromosome via homologous recombination involving a specific integration site called locus L2.
METHODS: To characterise all mechanisms of acquisition of vanA, this study analysed the 15 706 S. aureus genomes to look for vanA and described its genetic environment.
RESULTS: A complete vanA operon was found in 25 S. aureus strains isolated from 12 patients, including nine co-isolated with vancomycin-resistant Enterococcus strains. VanA was found within transposon Tn1546-like elements on 17 plasmids and eight chromosomes. VanA might be acquired through conjugation of enterococcal and staphylococcal plasmids, transposition of Tn1546 carrying vanA and plasmid integration into the chromosome. Further, L2 was detected in 2087 genomes (13.3%) of S. aureus strains across different continents. Six potential chromosomal hotspots for integration of the entire vanA-containing enterococcal plasmid were identified by homologous recombination via L2.
CONCLUSIONS: These findings suggest that the recently described scenario in a New York patient could be reproduced anywhere. Surveillance of this possibility is mandatory, especially in patients with vancomycin-resistant Enterococcus infection or colonisation.

OBJECTIVE: We analysed 4 y of laboratory data to characterise the species and determine the antimicrobial susceptibility profiles of enterococci as human pathogens in Fiji. The study also investigated the molecular epidemiology amongst the subset of vancomycin-resistant enterococci (VRE).
METHODS: This retrospective study reviewed bacteriological data from Colonial War Memorial Hospital (CWMH) and other healthcare facilities in the Central and Eastern divisions of Fiji. Phenotypic, antimicrobial susceptibility and vanA and vanB PCR testing were performed using locally approved protocols. The first clinical isolates per patient with antimicrobial susceptibility testing results in a single year were included in the analysis. Data was analysed using WHONET software and Microsoft Excel.
RESULTS: A total of 1817 enterococcal isolates were reported, 1415 from CWMH and 402 from other healthcare facilities. The majority of isolates, 75% (n = 1362) were reported as undifferentiated Enterococcus spp., 17.8% (n = 324) were specifically identified as Enterococcus faecalis and 6.7% (n = 122) as E. faecium. Overall, 10% of the enterococci isolates were from blood cultures. Among isolates from CWMH, <15% of E. faecium were susceptible to ampicillin, and 17.2% were vancomycin resistant. Overall, 874 enterococcal isolates (including the undifferentiated species) were tested against vancomycin, of which 4.8% (n = 42) were resistance. All of the VRE isolates tested (n = 15) expressed vanA genes.
CONCLUSIONS: This study demonstrates the clinical importance of VRE, particularly van A E. faecium in the national referral hospital in Fiji. Enhanced phenotypic and molecular surveillance data are needed to better understand enterococci epidemiology and help guide specific infection prevention and control measures and antibiotic prescribing guidelines.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole-genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control.

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Vancomycin-resistant Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.

We aimed to evaluate whether the FilmArray blood culture identification (BCID) panel holds the ability to detect vanM-type vancomycin-resistant enterococci (VRE) clinical isolates effectively.
Twenty VRE clinical strains, including 10 vanA-type VRE and 10 vanM-type VRE, were collected from patients in five tertiary hospitals, Shanghai, China. By conventional PCR and sequencing, the strains were identified and van genotypes were confirmed. All VRE strains were investigated using the FilmArray BCID panel. All results, including enterococcus assay, vanA/B assay, DNA melting curves and melting temperature (Tm), were recorded. We also compared these results with those obtained via the conventional PCR and sequencing.
According to the instructions of the FilmArray BCID panel, the Enterococcus assay is used to identify species and vanA/B assay is used to detect van genes. In all vanA-type VRE, the Enterococcus assay and vanA/B assay were positive. The results correctly showed that the tested strains were VRE. However, in 10 vanM-type VRE, the Enterococcus assay was positive and vanA/B assay were negative. The results mistakenly showed that the tested strains were vancomycin-sensitive enterococci (VSE). In the vanA/B assay, the melting curves of vanM-type VRE were similar to that of vanA-type VRE, but the Tm values were lower. The Tm values were then compared against the expected Tm range for the vanA/B assay. The Tm values of vanM-type VRE fall outside the assay-specific Tm range, resulting in negative reports. Thus, by adjusting the expected Tm range for the Enterococcus assay, the FilmArray BCID panel holds the ability to detect vanM-type VRE.
The vanM-type VRE isolates can be effectively detected by optimizing the expected Tm range for the vanA/B assay.