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Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation. Vancomycin-resistant Enterococcus faecium is an important healthcare-associated pathogen and genomic analyses could inform targeted interventions. Here, the authors optimise an analysis pipeline for identification of putative transmission events using core genome multilocus sequence type clustering and split kmer analysis.

We investigated genomic evolution of vancomycin-resistant Enterococcus faecium (VREF) during an outbreak in Shenzhen, China. Whole-genome sequencing revealed 2 sequence type 80 VREF subpopulations diverging through insertion sequence-mediated recombination. One subpopulation acquired more antimicrobial resistance and carbohydrate metabolism genes. Persistent VREF transmission underscores the need for genomic surveillance to curb spread.

Background and objectives Vancomycin-resistant Enterococcus faecium (VRE) infections have increased in the last years. Hospital outbreaks have been described with a challenging microbiological diagnosis and control of the transmission. Methods This is a prospective study of a nosocomial outbreak of VRE in a conventional hospitalization ward. Three clinical samples of VRE genotype vanB (vanB VRE) were detected in two surgical wards. Epidemiological control measures were implemented, including contact isolation, patients from clean surgery ward transferred to other wards, staff training, weekly screening with rectal swab and environmental study. Results In a 3-month follow-up period, rectal screening was performed on a total of 314 patients, being positive for vanB VRE 51 patients (16.2%). A study of the surface of the common areas with exclusive use by healthcare personnel detected vanB VRE in 28% of the samples. All the strains of VRE analyzed by MLST were ST117, which belongs to clonal complex 17. Hand hygiene observations show proper adherence in 56% of the events monitored. Notwithstanding the large number of colonized patients, just one patient had a relevant infection requiring treatment, with good evolution. Conclusions The survival of VRE on surfaces and the poor adherence to hand hygiene might have contributed to repetitively infect surfaces, perpetuating the outbreak. After 10 months without positive clinical samples, it was decided to suspend the screening, even though there were still screening positive results. Despite its spread, the clinical impact was low, possibly because the outbreak took place in a ward without severe immunosuppressed patients. The frequent isolation of VRE on surfaces which were exclusive from healthcare personnel areas, indicates how important is the disinfection of these areas.

The spread of vancomycin-resistant enterococci (VRE) is a major threat in nosocomial settings. A large-scale multiclonal VRE outbreak has rarely been reported in Japan due to low VRE prevalence. We evaluated the transmission of vancomycin resistance in a multiclonal VRE outbreak, conducted biological and genomic analyses of VRE isolates, and assessed the implemented infection control measures. In total, 149 patients harboring VanA-type VRE were identified from April 2017 to October 2019, with 153 vancomycin-resistant Enterococcus faecium isolated being grouped into 31 pulsotypes using pulsed-field gel electrophoresis, wherein six sequence types belonged to clonal complex 17. Epidemic clones varied throughout the outbreak; however, they all carried vanA -plasmids (pIHVA). pIHVA is a linear plasmid, carrying a unique structural Tn 1546 containing vanA ; it moves between different Enterococcus spp. by genetic rearrangements. VRE infection incidence among patients in the “hot spot” ward correlated with the local VRE colonization prevalence. Local prevalence also correlated with vancomycin usage in the ward. Transmission of a novel transferrable vanA -plasmid among Enterococcus spp. resulted in genomic diversity in VRE in a non-endemic setting. The prevalence of VRE colonization and vancomycin usage at the ward level may serve as VRE cross-transmission indicators in non-intensive care units for outbreak control.

Wastewater-based surveillance (WBS) could complement clinical data and be used as an early warning tool for population-level monitoring of priority pathogens such as vancomycin-resistant Enterococcus faecium (VREfm). In this pilot study, we isolated VREfm using CHROMAgar VRE from 77 composite wastewater influent samples from ten wastewater treatment plants (WWTPs), assessed their phenotypic antibiotic susceptibility profiles using the broth microdilution assay, and used core genome MLST (cgMLST) to examine the genetic relatedness of human and wastewater isolates in Finland. VREfm was isolated from 17 samples (22%), with no significant difference in the isolation rate across the ten WWTPs ( p  = 0.407). The phenotypic antimicrobial susceptibility testing (AST) revealed that all isolates were resistant to ampicillin, ciprofloxacin, erythromycin, teicoplanin, and vancomycin and were susceptible to chloramphenicol, daptomycin, and linezolid. All 17 VREfm isolates belonged to CC17, and 12 of them were sequence type ST80. Altogether, 76% ( n  = 13) of the wastewater isolates harboured the van A gene, 18% ( n  = 3) had the van B gene, and 6% ( n  = 1) had both van A and van B genes. cgMLST revealed that most VREfm strains from wastewater clustered closely (< 10 allelic differences) with those from human surveillance isolates across the country. The ST80 isolates belonged to the clusters identified during 2022–2023 as part of national VRE surveillance of screening samples. Therefore, WBS closely aligns with what is observed through human surveillance. We believe that this pilot supports the utility of WBS as a sensitive tool for AMR surveillance. This study also contributes to the One Health approach, needed to fully understand the transmission dynamics of pathogenic bacteria and effectively manage the antimicrobial resistance (AMR) challenge.

Gastrointestinal colonization by the nosocomial pathogen vancomycin-resistant Enterococcus faecium (VREfm) can lead to bloodstream infections with high mortality rates. Shifts in VREfm lineages found within healthcare settings occur, but reasons underlying these changes are not understood. Here we sequenced 710 VREfm clinical isolates collected between 2017 and 2022 from a large tertiary care centre. Genomic analyses revealed a polyclonal VREfm population, although 46% of isolates formed genetically related clusters, suggesting a high transmission rate. Comparing these data to a global collection of 15,631 publicly available VREfm genomes collected between 2002 and 2022 identified replacement of the sequence type (ST) 17 VREfm lineage by emergent ST80 and ST117 lineages at the local and global level. Comparative genomic and functional analyses revealed that emergent lineages encoded bacteriocin T8, which conferred a competitive advantage over bacteriocin T8-negative strains in vitro and upon colonization of the mouse gut. Bacteriocin T8 carriage was also strongly associated with strain emergence in the global genome collection. These data suggest that bacteriocin T8-mediated competition may have contributed to VREfm lineage replacement. Genomic and functional analyses of healthcare-associated vancomycin-resistant Enterococcus faecium reveal that bacteriocin T8 is enriched in emergent lineages and provides a competitive advantage in vitro and in vivo.

Introduction: Carbapenem-resistant Klebsiella pneumoniae (CRKP) and vancomycin-resistant Enterococcus (VRE) pose significant threats in neonatal intensive care units (NICUs) due to their high transmission potential and limited treatment options. Identifying the source of colonization and detection of predominant antibiotic-resistant genes are crucial for effective infection control measures. Methodology: In this study, we analyzed 20 carbapenem-susceptible Klebsiella pneumoniae (CSKP) and 20 Vancomycin-susceptible Enterococcus (VSE) of meconium versus 20 CRKP and 20 VRE of rectal swabs isolates, respectively, from 20 preterm infants hospitalized in the NICU. Pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) were used for molecular epidemiological analysis. The presence of carbapenemase (blaOXA-48, blaNDM, blaKPC) and Vancomycin-resistance (vanA, vanB, vanC) genes was investigated by multiplex PCR. Results: No predominant outbreak strain was detected, and isolates exhibited high genetic diversity, indicating an exogenous source of colonization of both CRKP and VRE. PFGE analysis revealed 24 distinct genotypes among CRKP and 25 among VRE isolates, with a clustering rate of 57.5%. The most commonly detected resistance gene in CRKP isolates was blaOXA-48 (50%), followed by blaNDM (35%) and blaKPC (10%). Among VRE isolates, only the vanA gene was present (85%). Conclusions: The absence of a clonal outbreak and the detection of resistance genes primarily on plasmids indicate healthcare-associated transmission rather than endogenous selection. This highlights the critical role of hand hygiene and strict infection control measures in preventing multidrug-resistant pathogen colonization in vulnerable preterm infants.

Background The prevention of vancomycin-resistant Enterococcus faecium (VREfm) infections and transmissions poses a major challenge to hospitals. Vancomycin resistance can be plasmid encoded; however, as the analysis of plasmids is challenging, so far only a few reports have provided a detailed characterization of plasmids in nosocomial VREfm transmission. Here we describe a nosocomial VREfm outbreak caused by a vanA positive ST80 isolate. vanA plasmid sequence data was used to distinguish outbreak-associated isolates from sporadic VREfm cases and to investigate the spread of this plasmid within the local VREfm population. Methods 446 VREfm isolates were collected from routine surveillance between 01/2022 and 02/2024 and analyzed using long-read whole genome sequencing (lrWGS). Genetic relatedness of isolates was evaluated based on core genome multilocus sequence typing (cgMLST). Genetically similar vanA plasmids were identified using a Mash based approach. Results 30 genetically similar VREfm isolates were identified in patients’ screening and environmental samples. Infection control evaluation confirmed transmission through shared hospital rooms. All outbreak-related VREfm isolates, including environmental samples, carried a highly similar vanA plasmid (Mash distance of < 0.001) with an identical replicon type. After enhanced infection control measures were established, no new transmissions were detected. Comparison with additional VREfm isolates from the respective department showed no evidence for further plasmid transmission. Conclusions Our study illustrates how vanA plasmid analysis can support the evaluation of VREfm transmission in hospitals. The outbreak-associated vanA plasmids were genetically highly similar, but could be clearly distinguished from other vanA plasmids in the local hospital population. Taken together, detailed analysis of hospital-associated vanA plasmids can improve our understanding of VREfm transmission and epidemiology.

Vancomycin-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. Experimental 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. 8 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. GeneXpert vanA/vanB can diagnose VRE with high-accuracy and shows greater accuracy in diagnosing vanA.

Vancomycin-resistant and (VRE) are increasingly recognized as major opportunistic pathogens in immunocompromised patients, where they may cause bloodstream infections (BSIs). The present study aimed to characterize a cohort of immunocompromised patients colonized or infected with VRE, performing genomic analysis of these isolates. Additionally, we investigated the impact of bacterial culture supernatants on Caco-2 epithelial cells, focusing on adhesion and cytotoxicity to elucidate mechanisms related to epithelial dysfunction and bacterial translocation. We conducted a retrospective study including 46 VRE from two Italian hospitals. Clinical and epidemiological data were collected, and isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing. Four representative isolates ( ST80, ST117, ST28, and ST179) and two reference strains (ATCC 29212™ and ATCC 51299™) were selected for analyses. Adhesion to Caco-2 monolayers was quantified, while cytotoxicity was assessed using MTT assays with bacterial cell-free supernatants (CS). Hydrogen peroxide (H O ) production was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. The majority of isolates were (78.3%), predominantly ST80 and ST117, possessed multiple resistance determinants. isolates displayed greater sequence type diversity with a ST28 predominance, carrying virulence genes as , , and . , bloodstream-derived isolates ( 51, 52) and reference strain ATCC 29212™ adhered more strongly to Caco-2 cells than other isolates. CS from invasive isolates and ATCC 51299™ significantly reduced epithelial cell viability at 24 h (p < 0.01). In these isolates, H O higher quantification was documented in a cellular model. Our findings highlighted the convergence of antimicrobial resistance and virulence traits in VRE, alongside functional evidence of strain-dependent adhesion and secretion of cytotoxic metabolites. Elevated H O production provides a possible path between enterococcal secretomes and epithelial injury, suggesting oxidative stress as a contributor to epithelial dysfunction and potential translocation. These insights expand current understanding of VRE pathogenesis and point to novel therapeutic approaches aimed at preserving epithelial integrity and mitigating oxidative damage in high-risk patients.