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Objective: To investigate the association between apolipoprotein E (Apo E) genotype in multiple sclerosis (MS) and acute monosymptomatic optic neuritis (ON) in a genetically homogeneous population with a high frequency of the Apo ℇ4 allele.Background: The association between heterozygosity of Apo ℇ4 and the development of MS is thoroughly investigated, while the association between homozygosity of Apo ℇ4 and the development of MS is insufficiently studied. The association between Apo E genotype and disease progression remains controversial.Methods: 475 patients were included, 385 with MS and 90 with ON, consecutively seen in the MS clinic in the County of Copenhagen. Clinical data were obtained from medical records and degree of disability was determined prospectively using the Kurtzke expanded disability status scale (EDSS). Blood samples were used for Apo E genotyping. Disease progression was evaluated by the progression index (PI–EDSS/disease duration). Apo E genotype distribution was compared with 361 healthy controls.Results: The Apo ℇ genotype distribution in the MS and ON groups was similar to the controls. The rate of disease progression in the group of MS patients with a disease duration of 10 years or less was significantly faster in the Apo ℇ4 positive group (heterozygosity and homozygosity for Apo ℇ4) (PI=1.41) compared to the Apo ℇ4 negative group (PI=0.92) (P=0.009). Observing the MS subgroups, we found that the group of patients with RRMS had a faster rate of disease progression in the Apo ℇ4 positive group (PI=1.12) compared to the Apo ℇ4 negative group (PI=0.77) (P=0.024).Conclusions: Apo E genotypes do not influence the development of MS and ON. The Apo ℇ4 allele seems to predispose carriers with MS to a faster progression of disease. [ABSTRACT FROM AUTHOR]

Objectives: To develop and validate a real-time PCR assay detecting the sequence bridging Tn 1549 and the Enterococcus faecium chromosome in the emerging vanB vancomycin-resistant E. faecium (VREfm) clone (ST80/CT2406)., Methods: The Tn 1549 insertion site was determined on routinely sequenced VREfm isolates. The outer boundaries of Tn 1549 and adjoining host bacterial sequences were determined using a BLAST search in the silent information regulator gene sir2 . Next, the primers and probe were developed, targeting the sequence bridging Tn 1549 and the E. faecium chromosome. Finally, the PCR assay was validated on well-characterized strains and prospectively performed on rectal screening samples submitted to our laboratory., Results and Conclusions: The PCR assay proved to be accurate and provide rapid diagnosis of the emerging vanB VREfm in rectal screening samples., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The continuous emergence of SARS-CoV-2 variants of concern has rendered many therapeutic monoclonal antibodies (mAbs) ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the recently circulating Omicron sub-lineages BA.2.86 and JN.1. Here, we report the isolation of broad and potent neutralizing human mAbs (HuMabs) from a healthcare worker infected with SARS-CoV-2 early in the pandemic. These include a genetically unique HuMab, named K501SP6, which can neutralize different Omicron sub-lineages, including BQ.1, XBB.1, BA.2.86 and JN.1, by targeting a highly conserved epitope on the N terminal domain, as well as an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants that was escaped by the more recent Omicron sub-lineages through spike F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies., (© 2024. The Author(s).)

Coinfections with human pegivirus 1 (HPgV-1) are common in chronic hepatitis C virus (HCV) patients. However, little is known about whether HPgV-1 is affected by direct-acting antivirals during HCV treatment. Metagenomic analysis and reverse transcriptase-quantitative PCR (RT-qPCR) were performed on RNA from the plasma of 88 selected chronic HCV patients undergoing medical treatment. Twenty (23%) of these HCV patients had HPgV-1 coinfections and were followed by RT-qPCR during treatment and follow-up to investigate HPgV-1 RNA titers. Recovered sequences could be assembled to complete HPgV-1 genomes, and most formed a genotype 2 subclade. All HPgV-1 viral genomic regions were under negative purifying selection. Glecaprevir/pibrentasvir treatment in five patients did not consistently lower the genome titers of HPgV-1. In contrast, a one log 10 drop of HPgV-1 titers at week 2 was observed in 10 patients during treatment with sofosbuvir-containing regimens, sustained to the end of treatment (EOT) and in two cases decreasing to below the detection limit of the assay. For the five patients treated with ledipasvir/sofosbuvir with the inclusion of pegylated interferon, titers decreased to below the detection limit at week 2 and remained undetectable to EOT. Subsequently, the HPgV-1 titer rebounded to pretreatment levels for all patients. In conclusion, we found that HCV treatment regimens that included the polymerase inhibitor sofosbuvir resulted in decreases in HPgV-1 titers, and the addition of pegylated interferon increased the effect on patients with coinfections. This points to the high specificity of protease and NS5A inhibitors toward HCV and the more broad-spectrum activity of sofosbuvir and especially pegylated interferon., Importance: Human pegivirus 1 coinfections are common in hepatitis C virus (HCV) patients, persisting for years. However, little is known about how pegivirus coinfections are affected by treatment with pangenotypic direct-acting antivirals (DAAs) against HCV. We identified human pegivirus by metagenomic analysis of chronic HCV patients undergoing protease, NS5A, and polymerase inhibitor treatment, in some patients with the addition of pegylated interferon, and followed viral kinetics of both viruses to investigate treatment effects. Only during HCV DAA treatment regimens that included the more broad-spectrum drug sofosbuvir could we detect a consistent decline in pegivirus titers that, however, rebounded to pretreatment levels after treatment cessation. The addition of pegylated interferon gave the highest effect with pegivirus titers decreasing to below the assay detection limit, but without clearance. These results reveal the limited effect of frontline HCV drugs on the closest related human virus, but sofosbuvir appeared to have the potential to be repurposed for other viral diseases.

The emergence of bloodstream infections (BSI) caused by vancomycin-resistant Enterococci (VRE) has caused concern. Nonetheless, it remains unclear whether these types are associated with an excess risk of severe outcomes when compared with infections caused by vancomycin-susceptible Enterococci (VSE). This cohort study included hospitalized patients in Denmark with Enterococcus faecium -positive blood cultures collected between 2010 and 2019 identified in the Danish Microbiology Database. We estimated 30-day hazard ratio (HR) of death or discharge among VRE compared to VSE patients adjusted for age, sex, and comorbidity. The cohort included 6071 patients with E. faecium BSI (335 VRE, 5736 VSE) among whom VRE increased (2010-13, 2.6%; 2014-16, 6.3%; 2017-19; 9.4%). Mortality (HR 1.08, 95%CI 0.90-1.29; 126 VRE, 37.6%; 2223 VSE, 37.0%) or discharge (HR 0.89, 95%CI 0.75-1.06; 126 VRE, 37.6%; 2386 VSE, 41.6%) was not different between VRE and VSE except in 2014 (HR 1.87, 95% CI 1.18-2.96). There was no interaction between time from admission to BSI (1-2, 3-14, and >14 days) and HR of death ( P  = 0.14) or discharge ( P  = 0.45) after VRE compared to VSE, despite longer time for VRE patients (17 vs. 10 days for VSE, P  < 0.0001). In conclusion, VRE BSI was not associated with excess morbidity and mortality. The excess mortality in 2014 only may be attributed to improved diagnostic- and patient-management practices after 2014, reducing time to appropriate antibiotic therapy. The high level of mortality after E. faecium BSI warrants further study.

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.

Introduction: During the 2022 outbreak of mpox (previously called monkeypox), which primarily affected Gay, Bisexual, and other Men who have Sex with Men (GBMSM), testing was mainly limited to individuals with symptoms of infection. Although sporadic cases of mpox continue to be diagnosed in Denmark, the feasibility of screening asymptomatic high-risk populations, such as those using HIV pre-exposure prophylaxis (PrEP), is still unknown., Methods: During the autumn of 2022, a rectal swab test for mpox PCR was included in the routine sexually transmitted infections (STI) screening for PrEP users., Results: The screening included 224 asymptomatic men with a median age of 36.5 years. One patient (0.4%) tested positive for mpox. Ten (4.5%) and nine (4.0%) had chlamydia and gonorrhea, respectively., Discussion: Our study demonstrates that screening for mpox is feasible in two Danish PrEP clinics., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To investigate the role of E. coli virulence-associated genes (VAGs) in predicting urinary tract infection (UTI) as the source of bacteremia in two distinct hospital populations, one with a large general catchment area and one dominated by referrals., Methods: E. coli bacteremias identified at Department of Clinical Microbiology (DCM), Hvidovre Hospital and DCM, Rigshospitalet in the Capital Region of Denmark from October to December 2018. Using whole genome sequencing (WGS), we identified 358 VAGs from 224 E. coli bacteremia. For predictive analysis, VAGs were paired with clinical source of UTI from local bacteremia databases., Results: VAGs strongly predicting of UTI as primary infection source of bacteremia were primarily found within the pap gene family. papX (PPV 96%, sensitivity 54%) and papGII (PPV 93%, sensitivity 56%) were found highly predictive, but showed low sensitivities. The strength of VAG predictions of UTI as source varied significantly between the two hospital populations. VAGs had weaker predictions in the tertiary referral center (Rigshospitalet), a disparity likely stemming from differences in patient population and department specialization., Conclusion: WGS data was used to predict the primary source of E. coli bacteremia and is an attempt on a new and different type of infection source identification. Genomic data showed potential to be utilized to predict the primary source of infection; however, discrepancy between the best performing profile of VAGs between acute care hospitals and tertiary hospitals makes it difficult to implement in clinical practice., (© 2024. The Author(s).)

As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8 + T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty ® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8 + T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8 + T cells were the only responses detected after vaccination and non-spike-specific CD8 + T cells were only detected after infection. Both spike-specific and non-spike-specific CD8 + T cells were found at much lower frequencies than CD8 + T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty ® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8 + T cell responses, which are maintained longitudinally., Competing Interests: Authors KJ, DI, and LB are employed by the company Immudex ApS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Underwood, Sølund, Jacobsen, Binderup, Fernandez-Antunez, Mikkelsen, Inekci, Villadsen, Castruita, Pinholt, Fahnøe, Ramirez, Brix, Weis and Bukh.)

Objectives: To determine if vancomycin-resistant Enterococcus faecium (VREfm) carriers carry the same VREfm clone after a minimum follow-up of 365 days. For those carrying the same clone, we investigated the genomic evolution per year per genome., Methods: We used WGS results to assign VREfm clones to each isolate and determine clone shifts. Finally, we calculated distance in core-genome MLST alleles, and the number of SNPs between consecutive VREfm isolates from patients carrying the same VREfm clone., Results: In total, 44.2% of patients carried the same VREfm clone, and the genomic evolution was 1.8 alleles and 2.6 SNPs per genome per year., Conclusions: In our population of long-term carriers, we calculated a molecular clock of 2.6 SNPs., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The aim of this review was to assess the efficacy and safety of Lacticaseibacillus rhamnosus GG (LGG) (previously known as Lactobacillus rhamnosus GG) for the eradication of vancomycin-resistant Enterococcus faecium (VREfm) in colonized carriers. We searched Cochrane Central, EMBASE, and the PubMed Library from inception to 21 August 2023, for randomized controlled trials (RCTs) investigating the effectiveness of LGG for the eradication of gastrointestinal carriage of VREfm. An initial screening was performed followed by a full-text evaluation of the papers. Out of 4076 articles in the original screening, six RCTs (167 participants) were included in the review. All were placebo-controlled RCTs. The meta-analysis was inconclusive with regard to the effect of LGG for clearing VREfm colonization. The overall quality of the evidence was low due to inconsistency and the small number of patients in the trials. We found insufficient evidence to support the use of LGG for the eradication of VREfm in colonized carriers. There is a need for larger RCTs with a standardized formulation and dosage of LGG in future trials.

Objective: The purpose of this systematic review and meta-analysis was to synthesize the literature on eating disorders and eating disorder symptomatology among transgender individuals and to summarize the existing literature on gender-affirming treatment and the prevalence of eating disorder symptomatology., Method: The literature search for this systematic review and meta-analysis was performed in PubMed, Embase.com, and Ovid APA PsycInfo. We searched for "eating disorders" and "transgender" using both controlled vocabularies and natural language terms for their synonyms. The PRISMA statement guidelines were followed. Quantitative data from studies on transgender individuals and eating disorders assessed with relevant assessment tools was included., Results: Twenty-four studies were included for the qualitative synthesis, and 14 studies were included in the meta-analysis. The results revealed higher levels of eating disorder symptomatology among transgender individuals compared with cisgender individuals, especially cisgender men. Transgender men tend to display higher levels of eating disorder symptomatology than transgender women; however, transgender women seem to have higher levels of eating disorder symptomatology than cisgender men and, interestingly, this study also noted a trend toward transgender men having higher levels of eating disorders than cisgender women. Gender-affirming treatment seems to alleviate the presence of eating disorder symptomatology in transgender individuals., Discussion: The body of research on this subject is extremely limited, and transgender individuals are underrepresented in the eating disorder literature. More research investigating eating disorders and eating disorder symptomatology in transgender individuals and the relationship between gender-affirming treatment and eating disorder symptomatology is needed., (© 2023. The Author(s).)

Synbiotics combine probiotics and prebiotics and are being investigated for potential health benefits. In this single-group-design trial, we analyzed changes in the gut microbiome, stool quality, and gastrointestinal well-being in 15 healthy volunteers after a synbiotic intervention comprising Lacticaseibacillus rhamnosus (LGG), Lactobacillus acidophilus (LA-5), Lacticaseibacillus paracasei subsp. paracasei (L. CASEI 431), and Bifidobacterium animalis subsp. lactis BB-12 and 20 g of chicory-derived inulin powder consumed daily for 4 weeks. Fecal samples were collected at baseline and at completion of the intervention, and all participants completed a fecal diary based on the Bristol Stool Scale and recorded their gastrointestinal well-being. No adverse effects were observed after consumption of the synbiotic product, and stool consistency and frequency remained almost unchanged during the trial. Microbiome analysis of the fecal samples was achieved using shotgun sequencing followed by taxonomic profiling. No changes in alpha and beta diversity were seen after the intervention. Greater relative abundances of Bifidobacteriaceae were observed in 12 subjects, with indigenous bifidobacteria species constituting the main increase. All four probiotic organisms increased in abundance, and L. rhamnosus, B. animalis, and L. acidophilus were differentially abundant, compared to baseline. Comparison of the fecal strains to the B. animalis subsp. lactis BB-12 reference genome and the sequenced symbiotic product revealed only a few single-nucleotide polymorphisms differentiating the probiotic B. animalis subsp. lactis BB-12 from the fecal strains identified, indicating that this probiotic strain was detectable after the intervention. IMPORTANCE The effects of probiotics/synbiotics are seldom investigated in healthy volunteers; therefore, this study is important, especially considering the safety aspects of multiple probiotics together with prebiotic fiber in consumption by humans. The study explores at the potential of a synbiotic intervention with lactobacilli, bifidobacteria, and inulin in healthy volunteers and tracks the ingested probiotic strain B. animalis subsp. lactis .

In March 2022, we observed samples with a negative fluorescent signal (60.5%, n = 43) for the influenza A matrix gene and a stronger positive signal for subtype A(H3N2). Forty-three samples were positive in InfA (H3N2) (mean Cq 30.9, range 23.9-35.1), and 26 of the 43 samples were negative in InfA matrix (mean Cq 28.0, range 23.2-30.6). Our multiplex test is a laboratory-developed four-target, four-color influenza A reverse-transcription PCR assay targeting the matrix gene, subtypes A(H3N2) and A(H1N1)pdm09. Several samples were negative when retested on commercial influenza Point-of-Care assays. As the matrix gene is a stand-alone target in most commercial diagnostic assays, we caution against false-negative subtype A test results., (© 2022 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology.)

Daptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here, we analyzed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 50 days of daptomycin and linezolid combination therapy. A total of seven isogenic VREfm isolates from the same patient (daptomycin-susceptible and daptomycin-resistant) were analyzed using Illumina whole genome sequencing, and two isolates were further characterized with Nanopore sequencing. One nonsynonymous SNP in the rpoC gene previously shown to harbor mutations in daptomycin-resistant VREfm was identified in the daptomycin-resistant isolates. Whole genome comparative analysis identified the loss of a 46.5 kb fragment, duplication of a 29.7 kb fragment, and integration of two plasmids upon acquisition of daptomycin resistance. Transmission electron microscopy showed similar alterations in cell morphology and cell wall structure as have previously been described in daptomycin-resistant E. faecalis., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Syndromic testing for lower respiratory tract infections with BioFire® FilmArray® Pneumonia Panel Plus (BF) detects 27 pathogens with a turn-around-time of one hour. We compared the performance of BF with culture. Samples from 298 hospitalized patients with suspected pneumonia routinely sent for culture were also analyzed using BF. Retrospectively, patients were clinically categorized as having "pneumonia" or "no pneumonia." BF and culture were compared by analytical performance, which was evaluated by pathogen concordance, and by clinical performance by comparing pathogen detections in patients with and without pneumonia. The BF results for viruses and atypical bacteria were not included in the performance analysis. In 298 patient samples, BF and culture detected 285 and 142 potential pathogens, respectively. Positive percent agreement (PPA) was 88% (125/142). In patients with community-acquired pneumonia (CAP), clinical sensitivity was 70% and 51%, and specificity was 43% and 71% for BF and culture, respectively. In patients with hospital-acquired pneumonia, the corresponding numbers were 55% and 23%, and 47% and 68%. There was no significant improvement of performance, when only high-quality sputum samples were considered. Efficacy of both BF and culture was low. Both tests are best used in CAP patients for whom the diagnosis has already been clinically established. Indiscriminate use may be clinically misleading and a cause of improper use of antibiotics., (© 2022 Scandinavian Societies for Medical Microbiology and Pathology.)

The purpose of this trial was to evaluate the efficacy of a 4-week supplementation of Lactobacillus rhamnosus GG (LGG) in eliminating the gastrointestinal carrier state of vancomycin-resistant Enterococcus faecium (VREfm) in hospitalized adults. The primary outcome of the study was the number of patients with cleared VREfm colonization after the 4-week intervention. Secondary outcomes were clearance of VREfm colonization at weeks 8, 16, and 24, number of VREfm infections (isolated from nonintestinal foci), and changes in fecal microbiome diversity after the intervention. The trial was a multicenter, randomized, double-blind, placebo-controlled trial in hospitalized adult VREfm carriers. Patients were enrolled and randomized to receive 60 billion CFU of LGG daily or placebo for 4 weeks. For a subgroup of patients, rectal swabs for VREfm were collected also at 8, 16, and 24 weeks and analyzed using shotgun metagenomics. Patients ingesting a minimum of 50% of the probiotic during the 4-week intervention were included in subsequent outcome analyses (48 of 81 patients). Twelve of 21 patients in the LGG group (57%) compared to 15 of 27 patients in the placebo group (56%) cleared their VREfm carriage. Eighteen patients completed the entire 24-week intervention with the same minimum compliancy. Of these, almost 90% in both groups cleared their VREfm carriage. We found a statistically significant difference between VREfm clearers and nonclearers regarding metronidazole and vancomycin usage as well as length of hospitalization after inclusion. The microbiome analyses revealed no significant difference in alpha diversity between the LGG and the placebo group. Beta diversity differed between the groups and the different time points. This study did not show an effect of LGG in eradication of VREfm after a 4-week intervention. IMPORTANCE Whereas other studies exploring the effect of L. rhamnosus in clearing VREfm from the intestine included children and adults, with a wider age range, our study consisted of a geriatric patient cohort. The natural clearance of VREfm in this study was almost 60% after 4 weeks, thus much higher than described previously. Also, this study characterizes the microbiome of VREfm patients in detail. This article showed no effect of the probiotic L. rhamnosus in clearing VREfm from the intestine of patients.

Objective: To study whether replacement of nosocomial ampicillin-resistant Enterococcus faecium (ARE) clones by vancomycin-resistant E. faecium (VRE), belonging to the same genetic lineages, increases mortality in patients with E. faecium bacteremia, and to evaluate whether any such increase is mediated by a delay in appropriate antibiotic therapy., Design: Retrospective, matched-cohort study., Setting: The study included 20 Dutch and Danish hospitals from 2009 to 2014., Patients: Within the study period, 63 patients with VRE bacteremia (36 Dutch and 27 Danish) were identified and subsequently matched to 234 patients with ARE bacteremia (130 Dutch and 104 Danish) for hospital, ward, length of hospital stay prior to bacteremia, and age. For all patients, 30-day mortality after bacteremia onset was assessed., Methods: The risk ratio (RR) reflecting the impact of vancomycin resistance on 30-day mortality was estimated using Cox regression with further analytic control for confounding factors., Results: The 30-day mortality rates were 27% and 38% for ARE in the Netherlands and Denmark, respectively, and the 30-day mortality rates were 33% and 48% for VRE in these respective countries. The adjusted RR for 30-day mortality for VRE was 1.54 (95% confidence interval, 1.06-2.25). Although appropriate antibiotic therapy was initiated later for VRE than for ARE bacteremia, further analysis did not reveal mediation of the increased mortality risk., Conclusions: Compared to ARE bacteremia, VRE bacteremia was associated with higher 30-day mortality. One explanation for this association would be increased virulence of VRE, although both phenotypes belong to the same well-characterized core genomic lineage. Alternatively, it may be the result of unmeasured confounding.

Objectives: Hospital-associated infections with vancomycin-resistant Enterococcus faecium (VREfm) have increased dramatically in Denmark. A cornerstone in infection control is effective cleaning and disinfection. This study investigated the survival and resuscitation/growth of clinical isolates of E. faecium exposed to the chlorine-releasing disinfectant, sodium dichloroisocyanurate plus detergent (NaDCC Plus)., Methods: To assess biocide efficacy, we modified a method developed to characterise the dose-time-response of bacteria to antibiotics. E. faecium isolates (n = 59) were screened in 96-well plates containing 50-1400 ppm free available chlorine. Bacteria were exposed for 10 min, after which the biocide was inactivated with a neutralizer. Cells were collected by centrifugation, new broth added, and after 20-22 h, viability was recorded as growth/no growth. For a subset of strains the impact of shorter biocide exposure times were examined, as was the influence of longer incubation times., Results: E. faecium survived exposure to relatively high concentrations of NaDCC Plus, average 415 ppm of free available chlorine (SD ± 78 ppm), compared to recommended in-use concentration (1000 ppm). "Outbreak" clones did not prove more tolerant to NaDCC Plus compared to other VREfm clones, hospital-associated vancomycin-susceptible E. faecium (VSEfm) or community-associated VSEfm. Shorter exposure time and extended incubation time in broth both significantly increased the concentration needed to eradicate E. faecium, with some isolates surviving higher concentrations than the recommended in-use concentration., Conclusion: Our results indicate that if an exposure time of 10 min is not achieved, the efficacy of the disinfectant will not be sufficient., Competing Interests: Declaration of Competing Interest The authors independently of the funding body have carried out all aspects of this study and thus declare no conflicts of interest and no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The prevalence of vancomycin-resistant Enterococcus faecium has increased rapidly, and in Denmark, we are facing an endemic outbreak situation in hospitals. The aim of this study was to use whole-genome sequencing (WGS) and core genome multilocus sequencing typing (cgMLST) to determine the duration of VREfm outbreaks and thereby evaluate the effect of our infection control strategies. We included all VREfm isolates from six hospitals in the Capital Region of Denmark that were sequenced between 2012 and 2020. Ward data were collected from our laboratory information system. A ward outbreak was defined as two patient samples from the same ward within a period of 30 days belonging to the same cgMLST cluster. cgMLST complex types were determined using Ridom SeqSphere v7.2.3, where a maximum of 20 allelic differences between isolates defines a cluster. We included 1690 patient isolates between 2012 and 2020. Our collection consisted of 45 unique clusters and 227 ward outbreaks. The median duration of outbreaks was 20 days. We reported a median outbreak duration of VREfm outbreaks based on WGS data to be 20 days, and thus concluded that our infection control precautions are adequate., (© 2022 Scandinavian Societies for Medical Microbiology and Pathology.)

Background: Vancomycin-resistant Enterococcus faecium (VREfm) cause a wide range of hospital infections. Ireland has had one of the highest invasive VREfm infection rates in Europe over the last decade, yet little is known about Irish VREfm., Objectives: To investigate the population structure of Irish VREfm, explore diversity by analysing the vanA transposon region and compare Irish, Danish and global isolates., Methods: E. faecium (n = 648) from five Irish hospitals were investigated, including VREfm [547 rectal screening and 53 bloodstream infection (BSI)] isolates and 48 vancomycin-susceptible (VSEfm) BSI isolates recovered between June 2017 and December 2019. WGS and core-genome MLST (cgMLST) were used to assess population structure. Genetic environments surrounding vanA were resolved by hybrid assembly of short-read (Illumina) and long-read (Oxford Nanopore Technologies) sequences., Results: All isolates belonged to hospital-adapted clade A1 and the majority (435/648) belonged to MLST ST80. The population structure was highly polyclonal; cgMLST segregated 603/648 isolates into 51 clusters containing mixtures of screening and BSI isolates, isolates from different hospitals, and VREfm and VSEfm. Isolates within clusters were closely related (mean average ≤16 allelic differences). The majority (96.5%) of VREfm harboured highly similar vanA regions located on circular or linear plasmids with multiple IS1216E insertions, variable organization of vanA operon genes and 78.6% harboured a truncated tnpA transposase. Comparison of 648 Irish isolates with 846 global E. faecium from 30 countries using cgMLST revealed little overlap., Conclusions: Irish VREfm are polyclonal, yet harbour a characteristic plasmid-located vanA region with multiple IS1216E insertions that may facilitate spread., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: The genomic relationships among Enterococcus faecium isolates are the subject of ongoing research that seeks to clarify the origins of observed lineages and the extent of horizontal gene transfer between them, and to robustly identify links between genotypes and phenotypes. E faecium is considered to form distinct groups-A and B-corresponding to isolates derived from patients who were hospitalised (A) and isolates from humans in the community (B). The additional separation of A into the so-called clades A1 and A2 remains an area of uncertainty. We aimed to investigate the relationships between A1 and non-A1 groups and explore the potential role of non-A1 isolates in shaping the population structure of hospital E faecium ., Methods: We collected short-read sequence data from invited groups that had previously published E faecium genome data. This hospital-based isolate collection could be separated into three groups (or clades, A1, A2, and B) by augmenting the study genomes with published sequences derived from human samples representing the previously defined genomic clusters. We performed phylogenetic analyses, by constructing maximum-likelihood phylogenetic trees, and identified historical recombination events. We assessed the pan-genome, did resistome analysis, and examined the genomic data to identify mobile genetic elements. Each genome underwent chromosome painting by use of ChromoPainter within FineSTRUCTURE software to assess ancestry and identify hybrid groups. We further assessed highly admixed regions to infer recombination directionality., Findings: We assembled a collection of 1095 hospital E faecium sequences from 34 countries, further augmented by 33 published sequences. 997 (88%) of 1128 genomes clustered as A1, 92 (8%) as A2, and 39 (4%) as B. We showed that A1 probably emerged as a clone from within A2 and that, because of ongoing gene flow, hospital isolates currently identified as A2 represent a genetic continuum between A1 and community E faecium . This interchange of genetic material between isolates from different groups results in the emergence of hybrid genomes between clusters. Of the 1128 genomes, 49 (4%) hybrid genomes were identified: 33 previously labelled as A2 and 16 previously labelled as A1. These interactions were fuelled by a directional pattern of recombination mediated by mobile genetic elements. By contrast, the contribution of B group genetic material to A1 was limited to a few small regions of the genome and appeared to be driven by genomic sweep events., Interpretation: A2 and B isolates coming into the hospital form an important reservoir for ongoing A1 adaptation, suggesting that effective long-term control of the effect of E faecium could benefit from strategies to reduce these genomic interactions, such as a focus on reducing the acquisition of hospital A1 strains by patients entering the hospital., Funding: Wellcome Trust., Competing Interests: We declare no competing interests., (© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

Vancomycin-resistant Enterococcus faecium (VREfm) is a globally significant nosocomial pathogen with a rapidly increasing prevalence. The objectives were to investigate VREfm outbreak duration and study the additional impact that infection control bundle strategies (ICBSs) set up to curb coronavirus disease 2019 (COVID-19) spreading had on VREfm outbreaks. Outbreak data set were collected prospectively from April 2, 2014 to August 13, 2020 at Copenhagen University Hospital Bispebjerg, Denmark. All VREfm samples had polymerase chain reaction performed for vanA / vanB genes before whole genome sequencing using the Illumina MiSeq platform. The relatedness of isolates was studied by core genome multilocus sequence typing (cgMLST) using Ridom SeqSphere. Eighty-one outbreaks had a median outbreak duration of 32.5 days (range 5-204 days) and 1,161 VREfm isolates were sequenced. The same cgMLST cluster types reappeared after outbreaks were terminated. When comparing the first 5 months of the COVID-19 pandemic with the corresponding period in 2019, we found a 10-fold decrease in VREfm outbreak patients and median outbreak duration decreased from 56 to 7 days (88%). Several COVID-19 ICBSs were implemented from March 13 through summer 2020. VREfm outbreaks lasted up to 204 days, but our findings suggest that outbreaks might last longer since the same cgMLST persisted in the same wards for years implying an endemic situation with recurrent outbreaks caused by hospital reservoirs or readmittance of unknown VREfm carriers. The sharp decline in VREfm outbreaks during the COVID-19 pandemic was most likely due to the ICBSs, resulting in a decrease in VREfm transmission.

Introduction SARS-CoV-2 outbreaks at care homes are associated with a high morbidity and mortality. We aimed to study the molecular epidemiology of a major care home outbreak in Denmark. Methods After a staff member had been tested positive on 16 November 2020, a bundle approach programme was initiated including frequent surveillance screenings of residents and staff, isolation and cohorting procedures. This approach also involved limiting the number of visitors and enhancing the use of personal protective equipment, hand hygiene, and environmental cleaning. Naso/oropharyngeal swabs were tested for SARS-CoV-2 by polymerase chain reaction. Available positive samples were sequenced and phylogenetic relationships between the outbreak and local circulating strains were reconstructed. Results In all, 50% (56/114) of residents and 26% (49/190) of staff members became infected during the 46-day outbreak period. Altogether 16% of the infected residents died within 30 days after becoming infected. A total of 44% (46/105) of the samples with SARS-CoV-2 were sequenced. and phylogenetic analysis demonstrated a dominant outbreak lineage belonging to Global Lineage B.1.1.29 containing the mutation I233V in the S gene. The outbreak lineage was detected in the community 28 days before its introduction into the care home. Conclusions Introduction of SARS-CoV-2 to care homes is associated with severe outbreaks. Initiation of a bundle approach infection control programme in addition to measures ensuring enhanced herd immunity were successful in controlling the outbreak. Genome sequencing proved to be a powerful tool to describe the relatedness of the various clones and may help focusing outbreak interventions. Funding The study was funded in part by The Poul Due Jensen Foundation and The Danish Ministry of Higher Education and Science. The authors have no conflicts of interest to report. Trial registration not relevant., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Background: During 2018-19, an increase of vanB vancomycin-resistant Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. vanA/vanB PCR performed directly on rectal swabs is accurate in detection of vanA; however, the positive predictive value for vanB-positive samples is low because of the presence of vanB in non-enterococcal gut commensals., Objectives: We investigated the epidemiology and clonal relatedness of vanB VREfm from the period 2015-19 and describe the application of a clone-specific vanB VREfm PCR assay for rapid and accurate detection of vanB VREfm in rectal screening samples., Methods: vanB VREfm were investigated using epidemiological data and WGS data. The SeqSphere+ software was used to analyse MLST and cgMLST, and de novo assemblies were annotated to determine insertion sites for the vanB transposon (Tn1549). A clone-specific vanB VREfm PCR assay was designed to detect the sequence bridging Tn1549 and the E. faecium chromosome (araA2) in the dominant cluster., Results: Two hundred and seventy-five vanB VREfm isolates were identified, of which 76% were identified in 2019. A dominant cluster (Cluster 1, n = 204, 74%), six minor clusters and 15 singletons were identified. All Cluster 1 isolates and six non-Cluster 1 isolates had Tn1549 integrated into araA2. In 2019, the PCR assay would have detected 92% of all rectal screening samples containing vanB VREfm., Conclusions: vanB VREfm increased due to the introduction and nosocomial transmission of the successful Cluster 1. The clone-specific PCR assay detected vanB VREfm outbreak isolates in rectal screening samples rapidly and accurately., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output., Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins., Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance., Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: Since 2012, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased dramatically in Copenhagen and vanA E. faecium has become endemic and polyclonal., Objectives: To examine whether a patient with a positive VRE clinical sample had the same VREfm in a preceding screening sample (within 60 days)., Methods: We performed a 30 month retrospective study. From our laboratory information system (LIS), we identified all patients with an invasive VREfm isolate and a VREfm rectal screening isolate within 60 days before infection. VREfm pairs (screening isolate and invasive isolate) were whole-genome sequenced. All isolates were analysed using SeqSphere and core-genome MLST (cgMLST) types were determined. We examined all isolates for the presence of the three most dominant vanA plasmids in the Capital Region of Denmark. Two novel vanA plasmids were closed by Nanopore/Illumina sequencing., Results: We found a total of 19 VREfm pairs. Of these, 13 patients had pairs with matching cgMLST types and vanA plasmids and a median number of 6 days from identification of carriage to clinical infection. One patient had a pair with non-matching cgMLST types but matching vanA plasmids and 24 days between identification of carriage to clinical infection. Five patients had pairs with non-matching cgMLST types and non-matching vanA plasmids and a median number of 18 days from identification of carriage to clinical infection., Conclusions: Of our 19 pairs, 13 were a match regarding cgMLST types (68%) and 1 more (5%) had matching vanA plasmids. Infection was thus preceded by colonization with the same isolates in 13 out of 19 patients. The five mismatches (26%) could be explained by the longer interval between colonization and infection., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We describe clonal shifts in vanA Enterococcus faecium isolates from clinical samples obtained from patients in Denmark from 2015 to the first quarter (Q1) of 2019. During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/ vanB genes in Q1 2019, 44% belonged to this type.

Objectives: From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized., Methods: WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems., Results: Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in ≥99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ∼29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes., Conclusions: Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Primary screening for VRE with PCR directed against vanA allowed identification of vanA+ samples from which VRE could not be isolated when selective culture methods were used. From such a sample a vancomycin-susceptible, vanA+ Enterococcus faecium, Efm-V1511, was isolated, when vancomycin selection was not used during culture. Similar isolates with variable susceptibility to vancomycin were obtained in the following months., Objectives: To characterize Efm-V1511 and investigate the causes of variable susceptibility to vancomycin., Methods: All strains were sequenced using Illumina technology. Plasmids containing vanA were reconstructed by scaffolding to known plasmids or plasmids were sequenced using Oxford Nanopore MinION. Derived structures were verified by PCR and sequencing. Furthermore, selected vanA+ vancomycin-susceptible isolates were passaged in the presence of vancomycin and vancomycin-resistant variants obtained were sequenced., Results: Efm-V1511 belonged to ST1421 and contained a 49 696 bp plasmid pHVH-V1511 carrying a Tn1546-derived genetic element. Within this element vanX was truncated by a 252 bp 3' deletion explaining the susceptibility of Efm-V1511. Between March 2016 and April 2017, 48 isolates containing pHVH-V1511 were identified. All were ST1421. In isolates resistant to vancomycin, resistance could be attributed to changes in ddl disrupting gene function sometimes accompanied by changes in vanS, increased pHVH-V1511 copy number or the existence of an additional vanA-containing plasmid encoding a functional vanX., Conclusions: E. faecium carrying pHVH-V1511 is capable of nosocomial transmission and may develop clinical resistance to vancomycin. Strains may not be detected using standard culture methods for VRE.

Purpose: In Danish hospitals, the number of infections caused by vancomycin-resistant Enterococcus faecium (VRE faecium) has dramatically increased in recent years. Hospital disinfectants are essential in eliminating pathogenic microorganisms, and reduced susceptibility may contribute to hospital-associated infections. We have addressed whether clinical VRE faecium display decreased biocide susceptibility when compared to vancomycin-sensitive Enterococcus faecium (VSE faecium) isolates., Methodology: In total 12 VSE faecium and 37 VRE faecium isolates obtained from Danish hospitals over an extended time period were tested for susceptibility towards three commonly applied biocides, namely benzalkonium chloride, chlorhexidine and hydrogen peroxide., Results: For benzalkonium chloride, 89 % of VRE faecium strains had a minimal inhibitory concentration (MIC) of 8 mg l -1 , whereas for VSE faecium, only 25 % of the strains had an MIC of 8 mg l -1 . For chlorhexidine, the MIC of 95 % of VRE faecium strains was 4 mg l -1 or higher, while only 33 % of VSE faecium strains displayed MIC values at the same level. In contrast, both VRE and VSE faecium displayed equal susceptibility to hydrogen peroxide, but a higher minimal bactericidal concentration (MBC) was found for the former. The efflux activity was also assessed, and this was generally higher for the VRE faecium strains compared to VSE faecium., Conclusion: VRE faecium from Danish hospitals demonstrated decreased susceptibility towards benzalkonium chloride and chlorhexidine compared to VSE faecium, where the use of chlorhexidine is particularly heavy in the hospital environment. These findings suggest that biocide tolerance may characterize VRE faecium isolated in Danish hospitals.

Objectives: To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015., Methods: During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates., Results: During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was <50 isolates/year until 2012 to > 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015., Conclusions: VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals., Methods: VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid., Results: Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n = 44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon., Conclusions: This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Population-based studies conducted in single regions or countries have identified significant changes in the epidemiology of invasive group B streptococcus (GBS) infection. However, no studies have concurrently compared the epidemiology of GBS infections among multiple different regions and countries over time. The study objectives were to define the contemporary incidence and determinants of GBS bloodstream infection (BSI) and assess temporal changes in a multi-national population., Methods: Population-based surveillance for GBS BSI was conducted in nine regions in Australia, Canada, Denmark, Sweden, Finland and the UK during 2000-2010. Incidence rates were age- and gender-standardised to the EU population., Results: During 114 million patient-years of observation, 3464 cases of GBS BSI were identified for an overall annual incidence of 3.4 patients per 100,000 persons. There were marked differences in the overall (range = 1.8-4.1 per 100,000 person-year) and neonatal (range = 0.19-0.83 per 1000 live births) incidences of GBS BSI observed among the study regions. The overall incidence significantly (p = 0.05) increased. Rates of neonatal disease were stable, while the incidence in individuals older than 60 years doubled (p = 0.003). In patients with detailed data (n = 1018), the most common co-morbidity was diabetes (25%). During the study period, the proportion of cases associated with diabetes increased., Conclusions: While marked variability in the incidence of GBS BSI was observed among these regions, it was consistently found that rates increased among older adults, especially in association with diabetes. The burden of this infection may be expected to continue to increase in ageing populations worldwide.

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.

Objectives: In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm., Methods: A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE., Results: One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power., Conclusions: This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)