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.

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrugresistant 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 SNPbased 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. In the last 2 decades, Enterococcus faecium has emerged as an important multidrug-resistant nosocomial pathogen causing an increasing number of bloodstream infections, mainly in debilitated patients (1-3). Currently, Ͼ90% of the E. faecium strains causing infections have acquired ampicillin resistance, and an increasing number of health care-associated infections and outbreaks are caused by E. faecium strains that are resistant to both ampicillin and vancomycin (4). The global emergence of ampicillin-and vancomycin-resistant E. faecium (VRE) as nosocomial pathogens started in the United States in the late 1980s/early 1990s and occurred in other parts in the world 1 or 2 decades later. In Europe, more particularly in France, Germany, Denmark, and the Netherlands, VRE first spread among livestock due to the use of the vancomycin analog avoparcin as a growth promoter, and it only relatively recently became an important nosocomial pathogen (5, 6). Different molecular typing methods have been used to study the epidemiology of E. faecium, ranging from fingerprint-based methods, like pulsed-field gel electrophoresis (PFGE) (7), ribotyping (8), and amplified fragment length polymorphism (9), to PCR-based methods, like multilocus variable-number tandemrepeat analysis (10) and the sequenced-bas...

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Enterococci currently account for approximately 10% of all bacteraemias, reflecting remarkable changes in their epidemiology. However, population-based data of enterococcal bacteraemia are scarce. A population-based cohort study comprised all patients with a first episode of Enterococcus faecalis or Enterococcus faecium bacteraemia in two Danish regions during 2006-2009. We used data collected prospectively during clinical microbiological counselling and hospital registry data. We determined the incidence of mono- and polymicrobial bacteraemia and assessed clinical and microbiological characteristics as predictors of 30-day mortality in monomicrobial bacteraemia by logistic regression analysis. We identified 1145 bacteraemic patients, 700 (61%) of whom had monomicrobial bacteraemia. The incidence was 19.6/100 000 person-years (13.0/100 000 person-years for E. faecalis and 6.6/100 000 person-years for E. faecium). The majority of bacteraemias were hospital-acquired (E. faecalis, 45.7%; E. faecium, 85.2%). Urinary tract and intra-abdominal infections were the predominant foci for the two species, respectively. Infective endocarditis (IE) accounted for 25% of patients with community-acquired E. faecalis bacteraemia. Thirty-day mortality was 21.4% in patients with E. faecalis and 34.6% in patients with E. faecium. Predictors of 30-day mortality included age, co-morbidity and hospital-acquired bacteraemia. In addition, intra-abdominal infection, unknown focus and high-level gentamicin resistance were predictors of mortality in E. faecalis patients. E. faecium was associated with increased risk of mortality compared with E. faecalis. The study emphasizes the importance of enterococci both in terms of incidence and prognosis. The frequency of IE in patients with E. faecalis bacteraemia emphasizes the importance of echocardiography, especially in community-acquired cases.

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.

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.

Invasive Listeria monocytogenes infections carry a high mortality despite antibiotic treatment. The rareness of the infection makes it difficult to improve antibiotic treatment through randomized clinical trials. This observational study investigated clinical features and outcome of invasive L. monocytogenes infections including the efficacy of empiric and definitive antibiotic therapies. Demographic, clinical and biochemical findings, antibiotic treatment and 30-day mortality for all episodes of L. monocytogenes bacteraemia and/or meningitis were collected by retrospective medical record review in the North Denmark Region and the Capital Region of Denmark (17 hospitals) from 1997 to 2012. Risk factors for 30-day all-cause mortality were assessed by logistic regression. The study comprised 229 patients (median age: 71 years), 172 patients had bacteraemia, 24 patients had meningitis and 33 patients had both. Significant risk factors for 30-day mortality were septic shock (OR 3.0, 95% CI 1.4-6.4), altered mental state (OR 3.6, 95% CI 1.7-7.6) and inadequate empiric antibiotic therapy (OR 3.8, 95% CI 1.8-8.1). Cephalosporins accounted for 90% of inadequately treated cases. Adequate definitive antibiotic treatment was administered to 195 patients who survived the early period (benzylpenicillin 72, aminopenicillin 84, meropenem 28, sulfamethoxazole/trimethoprim 6, and piperacillin/tazobactam 5). Definitive antibiotic treatment with benzylpenicillin or aminopenicillin resulted in a lower 30-day mortality in an adjusted analysis compared with meropenem (OR 0.3; 95% CI 0.1-0.8). In conclusion, inadequate empiric antibiotic therapy and definitive therapy with meropenem were both associated with significantly higher 30-day mortality.

Objectives: From 2012-2015, a sudden huge increase of 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 SNPanalysis 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 approximately 29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5,905 genes. Conclusions:Most likely, VREfm emerged due to import of a successful VREfm clone which has 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 adaption to new environmental challenges.