Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

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.

Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023.

BackgroundThe war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients.AimTo investigate the genomic epidemiology of New Delhi metallo-ß-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries.MethodsWhole-genome sequencing of 66 isolates sampled in 2022-2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire.ResultswgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam-avibactam. Patients with data on age (n = 41) were 19-74 years old; of 49 with information on sex, 38 were male.ConclusionXDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

Contaminated dicloxacillin capsules as the source of an NDM-5/OXA-48-producing Enterobacter hormaechei ST79 outbreak, Denmark and Iceland, 2022 and 2023.

From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Investigation of the introduction and dissemination of vanB Enterococcus faecium in the Capital Region of Denmark and development of a rapid and accurate clone-specific vanB E. faecium PCR.

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.

Taxonomic reassessment of the genus Pseudocitrobacter using whole genome sequencing: Pseudocitrobacter anthropi is a later heterotypic synonym of Pseudocitrobacter faecalis and description of Pseudocitrobacter vendiensis sp. nov.

The taxonomic status of all Pseudocitrobacter species was re-evaluated by comparative genomics based on whole genome sequencing. As a result, it is obvious that Pseudocitrobacter anthropi is a later heterotypic synonym of Pseudocitrobacter faecalis. In addition, genome-based analysis of strain CPO20170097T, isolated from a patient in northern Denmark was allocated to the genus Pseudocitrobacter. This strain showed significant genotypic and phenotypic differences from P. faecalis and it is proposed that this strain represents a novel species of the genus, for which the name Pseudocitrobacter vendiensis sp. nov. is proposed with the type strain CPO20170097T (=CCUG 73096T=LMG 31042T).

Surveillance of OXA-244-producing Escherichia coli and epidemiologic investigation of cases, Denmark, January 2016 to August 2019.

BackgroundCarbapenemase-producing Escherichia coli are increasing worldwide. In recent years, an increase in OXA-244-producing E. coli isolates has been seen in the national surveillance of carbapenemase-producing organisms in Denmark.AimMolecular characterisation and epidemiological investigation of OXA-244-producing E. coli isolates from January 2016 to August 2019.MethodsFor the epidemiological investigation, data from the Danish National Patient Registry and the Danish register of civil registration were used together with data from phone interviews with patients. Isolates were characterised by analysing whole genome sequences for resistance genes, MLST and core genome MLST (cgMLST).ResultsIn total, 24 OXA-244-producing E. coli isolates were obtained from 23 patients. Among the 23 patients, 13 reported travelling before detection of the E. coli isolates, with seven having visited countries in Northern Africa. Fifteen isolates also carried an extended-spectrum beta-lactamase gene and one had a plasmid-encoded AmpC gene. The most common detected sequence type (ST) was ST38, followed by ST69, ST167, ST10, ST361 and ST3268. Three clonal clusters were detected by cgMLST, but none of these clusters seemed to reflect nosocomial transmission in Denmark.ConclusionImport of OXA-244 E. coli isolates from travelling abroad seems likely for the majority of cases. Community sources were also possible, as many of the patients had no history of hospitalisation and many of the E. coli isolates belonged to STs that are present in the community. It was not possible to point at a single country or a community source as risk factor for acquiring OXA-244-producing E. coli.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin.

OBJECTIVES: Fully sequenced IncI1 plasmids obtained from CTX-M-1-producing Escherichia coli of human and animal origin were compared. METHODS: Twelve E. coli isolates sharing identical ESBL genes and plasmid multilocus STs sequenced on Illumina and MinION platforms were obtained from the Danish antimicrobial resistance surveillance programme, DANMAP. After de novo assembly, the sequences of plasmids harbouring blaCTX-M-1 were manually curated and ORFs annotated. Within-group comparisons were performed separately for the IncI1 ST3 plasmid type and the IncI1 ST7 plasmid type. The IncI1 ST3 plasmid group was obtained from 10 E. coli isolates (2 from patients with bloodstream infections, 6 from food and 2 from animals). The IncI1 ST7 plasmids originated from E. coli isolates obtained from a patient with bloodstream infection and from a pig. Sequences of IncI1 ST3 and IncI1 ST7 plasmids harbouring blaCTX-M-1 with determined origin were retrieved from GenBank and used for comparison within the respective group. RESULTS: The 10 IncI1 ST3 blaCTX-M-1 plasmids were highly similar in structure and organization with only minor plasmid rearrangements and differences in the variable region. The IncI1 ST7 blaCTX-M-1 plasmids also showed high similarity in structure and organization. The high level of similarity was also observed when including plasmids from E. coli of animal origin from Australia, Switzerland, the Netherlands and France. CONCLUSIONS: This study shows broad spread of a very successful CTX-M-1-producing IncI1 type plasmid among E. coli of both human and animal origin.

Evaluation of temocillin for phenotypic carbapenemase screening of Escherichia coli and Salmonella enterica isolates in relation to the presence of genes encoding ESBLs and carbapenemase production.

BACKGROUND: The expression of enzymes of the OXA-48 carbapenemase group is difficult to detect by phenotypic methods owing to frequent low levels of carbapenem resistance and negative results with some screening methods. Temocillin has been shown to be a good option for phenotypic screening as it is hydrolysed by the OXA-48-group enzymes, whereas ESBLs, AmpC and some other carbapenemases have a lower hydrolytic effect on this antimicrobial. However, no epidemiological cut-off for temocillin is available. OBJECTIVES: To evaluate temocillin MICs in relation to the presence or absence of genes encoding ESBLs and carbapenemases in Escherichia coli and Salmonella enterica. METHODS: In this study, 111 E. coli and 102 S. enterica isolates, including WT and well-characterized ESBL-, AmpC- or carbapenemase-producing isolates, were tested by three independent laboratories. MICs were determined according to the CLSI guidelines by agar dilution with the test range from 0.5 to 512 mg/L temocillin and WGS was performed and analysed with ResFinder. RESULTS: Some overlap was detected between temocillin MICs for WT and ESBL- or AmpC-producing isolates. However, isolates carrying genes encoding carbapenemases showed a broader range of MICs for both E. coli and S. enterica. Higher MICs were observed for the OXA-48 group, VIM and some NDM-producing isolates, whereas isolates harbouring KPC enzymes showed low MICs. CONCLUSIONS: The results indicate that temocillin MICs enable phenotypic distinction between strains producing OXA-48-group enzymes and both WT susceptible and ESBL/AmpC-carrying isolates, whereas the distinction from other carbapenemases likely requires genotypic testing.

LRE-Finder, a Web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.

OBJECTIVES: In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. METHODS: LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. RESULTS: LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. CONCLUSIONS: A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online.

ST131 fimH22 Escherichia coli isolate with a blaCMY-2/IncI1/ST12 plasmid obtained from a patient with bloodstream infection: highly similar to E. coli isolates of broiler origin.

OBJECTIVES: This study compares the genome of an ST131 CMY-2-producing Escherichia coli isolate from a Danish patient with other ST131 CMY-2-producing E. coli isolates of both human and animal origin. METHODS: In 2016, an ST131 CMY-2-producing E. coli isolate (ESBL20160056) was obtained from a patient with a bloodstream infection. The genome of the ESBL20160056 isolate was compared with genomes from six ST131 CMY-2-producing E. coli isolates obtained from broiler meat imported to Denmark, 15 ST131 CMY-2-producing E. coli isolates obtained from Enterobase (http://enterobase.warwick.ac.uk) and two ST131 CMY-2-producing E. coli from European collaborators. The plasmid from ESBL20160056 was sequenced using a MinION Mk1B (Oxford Nanopore Technologies). RESULTS: The E. coli isolate from the Danish patient clustered together with 13 other fimH22 ST131 CMY-2-producing E. coli isolates in a distinct clade. The clade consisted of genomes from six E. coli isolates from humans collected in Denmark, Spain, Cambodia and the USA, six E. coli isolates obtained from broiler meat samples imported to Denmark from France, the Netherlands and Germany, and two E. coli isolates obtained from broilers in Belgium and Luxembourg. The 101.5 kb plasmid with blaCMY-2 from ESBL20160056 had an IncI1 replicon and belonged to ST12 using the plasmid MLST scheme. In total, 10 of the 14 ST131 E. coli isolates belonging to the fimH22 clade carried an IncI1 ST12 plasmid with blaCMY-2. CONCLUSIONS: From our data, it seems plausible that the ST131 fimH22 CMY-2-producing E. coli isolate obtained from the Danish patient could have a zoonotic broiler origin.

2CS-CHXT Operon Signature of Chlorhexidine Tolerance among Enterococcus faecium Isolates.

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years that has recently acquired higher relevance in nosocomial infection control worldwide. Despite this, CHX tolerance among Enterococcus faecium bacteria, representing one of the leading agents causing nosocomial infections, has been poorly understood. This study provides new phenotypic and molecular data for better identification of CHX-tolerant E. faecium subpopulations in community and clinical contexts. The chlorhexidine MIC (MICCHX) distribution of 106 E. faecium isolates suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of the 2CS-CHXT operon, identified here. It encodes glucose and amino acid-polyamine-organocation family transporters, besides the DNA-binding response regulator ChtR, with a P102H mutation previously described only in CHX-tolerant clade A1 E. faecium, and the ChtS sensor. 2CS-CHXT seems to be associated with three regulons modulating diverse bacterial biological functions. Combined data from normal MIC distribution and 2CS-CHXT operon characterization support a tentative epidemiological cutoff (ECOFF) of 8 mg/liter to CHX, which is useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds calls for the prudent use of CHX in multiple contexts.IMPORTANCE Chlorhexidine is one of the substances included in the World Health Organization's list of essential medicines, which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic in health care (skin, hands, mouthwashes, eye drops) since the 1950s, its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here, we provide a comprehensive study on chlorhexidine tolerance among strains of Enterococcus faecium, one of the leading nosocomial agents worldwide, and identify a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Our data allowed for the proposal of a tentative epidemiological cutoff of 8 mg/liter, which is useful to detect tolerant E. faecium populations in surveillance studies in community and clinical contexts. The prediction of 2CS-CHXT regulons will also facilitate the design of future experimental studies to better uncover chlorhexidine tolerance among E. faecium bacteria.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clones and national spread of a vancomycin-variable vanA Enterococcus faecium ST1421-CT1134 clone, Denmark, 2015 to March 2019.

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.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D ß-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D ß-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Development of a Web Tool for Escherichia coli Subtyping Based on fimH Alleles.

The aim of this study was to construct a valid publicly available method for in silico fimH subtyping of Escherichia coli particularly suitable for differentiation of fine-resolution subgroups within clonal groups defined by standard multilocus sequence typing (MLST). FimTyper was constructed as a FASTA database containing all currently known fimH alleles. The software source code is publicly available at https://bitbucket.org/genomicepidemiology/fimtyper, the database is freely available at https://bitbucket.org/genomicepidemiology/fimtyper_db, and a service implementing the software is available at https://cge.cbs.dtu.dk/services/FimTyper FimTyper was validated on three data sets: one containing Sanger sequences of fimH alleles of 42 E. coli isolates generated prior to the current study (data set 1), one containing whole-genome sequence (WGS) data of 243 third-generation-cephalosporin-resistant E. coli isolates (data set 2), and one containing a randomly chosen subset of 40 E. coli isolates from data set 2 that were subjected to conventional fimH subtyping (data set 3). The combination of the three data sets enabled an evaluation and comparison of FimTyper on both Sanger sequences and WGS data. FimTyper correctly predicted all 42 fimH subtypes from the Sanger sequences from data set 1 and successfully analyzed all 243 draft genomes from data set 2. FimTyper subtyping of the Sanger sequences and WGS data from data set 3 were in complete agreement. Additionally, fimH subtyping was evaluated on a phylogenetic network of 122 sequence type 131 (ST131) E. coli isolates. There was perfect concordance between the typology and fimH-based subclones within ST131, with accurate identification of the pandemic multidrug-resistant clonal subgroup ST131-H30. FimTyper provides a standardized tool, as a rapid alternative to conventional fimH subtyping, highly suitable for surveillance and outbreak detection.

WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

Objectives: To evaluate a genome-based surveillance of all Danish third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec ) from bloodstream infections between 2014 and 2015, focusing on horizontally transferable resistance mechanisms. Methods: A collection of 552 3GC-R Ec isolates were whole-genome sequenced and characterized by using the batch uploader from the Center for Genomic Epidemiology (CGE) and automatically analysed using the CGE tools according to resistance profile, MLST, serotype and fimH subtype. Additionally, the phylogenetic relationship of the isolates was analysed by SNP analysis. Results: The majority of the 552 isolates were ESBL producers (89%), with bla CTX-M-15 being the most prevalent (50%) gene, followed by bla CTX-M-14 (14%), bla CTX-M-27 (11%) and bla CTX-M-101 (5%). ST131 was detected in 50% of the E. coli isolates, with the remaining isolates belonging to 73 other STs, including globally disseminated STs (e.g. ST10, ST38, ST58, ST69 and ST410). Five of the bloodstream isolates were carbapenemase producers, carrying bla OXA-181 (3) and bla OXA-48 (2). Phylogenetic analysis revealed 15 possible national outbreaks during the 2 year period, one caused by a novel ST131/ bla CTX-M-101 clone, here observed for the first time in Denmark. Additionally, the analysis revealed three individual cases with possible persistence of closely related clones collected more than 13 months apart. Conclusions: Continuous WGS-based national surveillance of 3GC-R Ec , in combination with more detailed epidemiological information, can improve the ability to follow the population dynamics of 3GC-R Ec , thus allowing for the detection of potential outbreaks and the effects of changing treatment regimens in the future.

Emergence of vanA Enterococcus faecium in Denmark, 2005-15.

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.

Prevalence of antimicrobial resistant Escherichia coli from patients with suspected urinary tract infection in primary care, Denmark.

BACKGROUND: Escherichia coli is the most common pathogen causing Urinary Tract Infections (UTI). Data from the current National Surveillance program in Denmark (DANMAP) may not accurately represent the prevalence of resistant E. coli in primary care, because only urine samples from complicated cases may be forwarded to the microbiological departments at hospitals for diagnostic examination. The aim of this study was to assess the prevalence of resistant E. coli to the most commonly used antimicrobial agents in primary care in a consecutive sample of patients from general practice. METHODS: Observational study carried out from December 2014 to December 2015. Thirty-nine general practices from The Capital Region of Denmark included adult patients with urinary tract symptoms and suspected UTI. All urine samples were sent to the central laboratory Statens Serum Institut (SSI). Significant bacteriuria was interpreted according to the European Urinalysis Standards. Susceptibility testing was performed and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. RESULTS: From the 39 general practices 505 patients were recruited. Completed data were obtained from 485 (96%) patients. According to the European Urinalysis Standards, 261 (54%) patients had positive bacteriuria. The most common uropathogen in patients with uncomplicated (uUTI) and complicated (cUTI) urinary tract infection was E. coli 105 (69%) and 76 (70%), respectively. Eighty-two (45%) of 181 E. coli isolates were resistant to at least one of the tested antibiotics and 50 out of 82 isolates were resistant to two or more antimicrobial agents. The highest resistance-rate was found against ampicillin 34% (95% CI 24;42) in uUTI and 36% (24;46) in cUTI. There were no differences in the distribution of resistance between uncomplicated and complicated cases. The prevalence of resistance was similar to the one reported in DANMAP 2014. CONCLUSION: In E. coli from uUTI there is high resistance rates to antimicrobial agents commonly used in primary care. There was no difference in the distribution of resistant E. coli in suspected uUTI vs cUTI. In Denmark, data from the National Surveillance program DANMAP can guide the decision for choice of antibiotic in patients with suspected UTI seeking care in primary care. TRIAL REGISTRATION: ClinicalTrials.gov NCT02249273 .