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.

Comparison of morbidity and mortality after bloodstream infection with vancomycin-resistant versus -susceptible Enterococcus faecium: a nationwide cohort study in Denmark, 2010-2019.

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.

Risk factors for methicillin-resistant Staphylococcus aureus colonization in a level-IV neonatal intensive care unit: a retrospective study.

Objective: To identify risk factors associated with methicillin-resistant Staphylococcus aureus (MRSA) colonization in neonatal patients during an MRSA outbreak to minimize future outbreaks. Design: Retrospective case-control study. Setting: Level-IV neonatal intensive care unit (NICU) at Copenhagen University Hospital, Rigshospitalet, Denmark. Patients: Neonates with either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA). Methods: Methicillin-resistant Staphylococcus aureus-positive neonates were matched with those colonized or infected with MSSA in a 1:1 ratio. The control group was selected from clinical samples, whereas MRSA-positive neonates were identified from clinical samples or from screening. A total of 140 characteristics were investigated to identify risk factors associated with MRSA acquisition. The characteristics were categorized into three categories: patient, unit, and microbiological characteristics. Results: Out of 1,102 neonates screened for MRSA, between December 2019 and January 2022, 33 were MRSA positive. They were all colonized with an MRSA outbreak clone (spa type t127) and were included in this study. Four patients (12%) had severe infection. Admission due to respiratory diseases, need for intubation, need for peripheral venous catheters, admission to shared rooms with shared toilets and bath facilities in the aisles, and need for readmission were all correlated with later MRSA colonization (P < 0.05). Conclusion: We identified clinically relevant diseases, procedures, and facilities that predispose patients to potentially life-threatening MRSA infections. A specific MRSA reservoir remains unidentified; however, these findings have contributed to crucial changes in our NICU to reduce the number of MRSA infections and future outbreaks.

Emergence of linezolid-resistant Enterococcus faecium in a tertiary hospital in Copenhagen.

Linezolid is used as first-line treatment of infections caused by vancomycin-resistant Enterococcus faecium. However, resistance to linezolid is increasingly detected. The aim of the present study was to elucidate the causes and mechanisms for the increase in linezolid-resistant E. faecium at Copenhagen University Hospital - Rigshospitalet. We therefore combined patient information on linezolid treatment with whole-genome sequencing data for vancomycin- or linezolid-resistant E. faecium isolates that had been systematically collected since 2014 (n=458). Whole-genome sequencing was performed for multilocus sequence typing (MLST), identification of linezolid resistance-conferring genes/mutations and determination of phylogenetically closely related strains. The collection of E. faecium isolates belonged to prevalent vancomycin-resistant MLST types. Among these, we identified clusters of closely related linezolid-resistant strains compatible with nosocomial transmission. We also identified linezolid-resistant enterococcus isolates not genetically closely related to other isolates compatible with de novo generation of linezolid resistance. Patients with the latter isolates were significantly more frequently exposed to linezolid treatment than patients with related linezolid-resistant enterococcus isolates. We also identified six patients who initially carried a vancomycin-resistant, linezolid-sensitive enterococcus, but from whom vancomycin-resistant, linezolid-resistant enterococci (LVRE) closely related to their initial isolate were recovered after linezolid treatment. Our data illustrate that linezolid resistance may develop in the individual patient subsequent to linezolid exposure and can be transmitted between patients in a hospital setting.

Within-patient horizontal transfer of pOXA-48 from a hypervirulent Klebsiella pneumoniae SL218 to Serratia marcescens following spread of the K. pneumoniae isolate among hospitalised patients, Denmark, 2021.

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing bla NDM-1 and a plasmid containing bla OXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.

Vancomycin-resistant Enterococcus faecium: should we screen on admission?

Denmark has experienced an increase in the proportion of invasive vancomycin-resistant Enterococcus faecium (VRE) since 2002 (e.g. <4% in 2015, 7.1% in 2017 and 12% in 2018). At Rigshospitalet, we employ active screening at departments with high prevalence or in case of outbreaks. This includes the collection of rectal swabs specifically for VRE screening. Our purpose was to describe the carrier prevalence of vancomycin-resistant enterococci among acute patients admitted to the Neurointensive Care Unit, Department of Neuroanaesthesiology, Rigshospitalet, Copenhagen, Denmark (NICU). Between April 2018 and January 2019, we investigated 99 consecutive rectal swabs from patients admitted to NICU. The primary outcome was prevalence of VRE carriage. The median age was 64 years (range 23-87) and gender was equally distributed (Female = 47, Male = 46). 26 (28%) had previously been admitted within 179 days and 67 patients (72%) had no hospital admissions within 180 days prior to the admission to NICU. Of the 93 rectal swabs, 2 (2%, 95% CI 0.26-7.55%) were positive for vanA and none were positive for vanB. Routine screening of all patients at admission may be effective in hospital settings with high VRE prevalence, whereas the benefit of screening for VRE in hospitals with a low prevalence may be restricted to specific patient populations.

Efficacy of piperacillin-tazobactam and cefotaxime against Escherichia coli hyperproducing TEM-1 in a mouse peritonitis infection model.

OBJECTIVES: Piperacillin-tazobactam (TZP) is a frequently prescribed antibiotic in hospital settings. Reports suggest in vivo efficacy of TZP, despite in vitro resistance of isolates susceptible to cephalosporins. Escherichia coli (E. coli) isolates hyperproducing TEM-1 ß-lactamase possess this phenotype. This study investigated the influence of tazobactam (TAZ) concentration on piperacillin (PIP) inhibition of such isolates and compared the in vivo efficacy of TZP with cefotaxime (CTX) in an infection model. METHODS: The PIP MICs for E. coli isolates, either hyperproducing TEM-1 because of promoter substitutions (n = 4) or because of gene amplification (n = 2) or producing an inhibitor-resistant TEM-35 (IRT) (n = 1), were determined using increasing concentrations of TAZ in a checkerboard setup. Furthermore, the efficacy of TZP and CTX against the isolates was investigated in a mouse peritonitis model using antibiotic exposures mimicking human conditions. Isolates producing either OXA-48 or CTX-M-15 ß-lactamases were included as controls. RESULTS: Using TAZ concentrations ≤ 64 mg/L, one isolate hyperproducing TEM-1 had a PIP MIC of 8 at TAZ 16 mg/L and two additional isolates at TAZ 64 mg/L. In the mouse peritonitis infection model, reduction of bacterial load in the peritoneum was larger for TZP than CTX only for the CTX-M-15-producing isolate. Larger reductions in bacterial load were observed after CTX treatment than TZP treatment for seven of the eight remaining test isolates. CONCLUSIONS: Piperacillin-tazobactam treatment of E. coli isolates hyperproducing TEM-1 was less effective than CTX treatment and may, for some isolates, be comparable with TZP treatment of isolates producing established resistance markers as IRT or OXA-48.

Microbiome Compositions and Resistome Levels after Antibiotic Treatment of Critically Ill Patients: An Observational Cohort Study.

Hospitalization and treatment with antibiotics increase the risk of acquiring multidrug-resistant bacteria due to antibiotic-mediated changes in patient microbiota. This study aimed to investigate how broad- and narrow-spectrum antibiotics affect the gut microbiome and the resistome in antibiotic naïve patients during neurointensive care. Patients admitted to the neurointensive care unit were treated with broad-spectrum (meropenem or piperacillin/tazobactam) or narrow-spectrum antibiotic treatment (including ciprofloxacin, cefuroxime, vancomycin and dicloxacillin) according to clinical indications. A rectal swab was collected from each patient before and after 5-7 days of antibiotic therapy (N = 34), respectively. Shotgun metagenomic sequencing was performed and the composition of metagenomic species (MGS) was determined. The resistome was characterized with CARD RGI software and the CARD database. As a measure for selection pressure in the patient, we used the sum of the number of days with each antibiotic (antibiotic days). We observed a significant increase in richness and a tendency for an increase in the Shannon index after narrow-spectrum treatment. For broad-spectrum treatment the effect was more diverse, with some patients increasing and some decreasing in richness and Shannon index. This was studied further by comparison of patients who had gained or lost >10 MGS, respectively. Selection pressure was significantly higher in patients with decreased richness and a decreased Shannon index who received the broad treatment. A decrease in MGS richness was significantly correlated to the number of drugs administered and the selection pressure in the patient. Bray-Curtis dissimilarities were significant between the pre- and post-treatment of samples in the narrow group, indicating that the longer the narrow-spectrum treatment, the higher the differences between the pre- and the post-treatment microbial composition. We did not find significant differences between pre- and post-treatment for both antibiotic spectrum treatments; however, we observed that most of the antibiotic class resistance genes were higher in abundance in post-treatment after broad-spectrum treatment.

Escherichia coli Causing Recurrent Urinary Tract Infections: Comparison to Non-Recurrent Isolates and Genomic Adaptation in Recurrent Infections.

Recurrent urinary tract infection (rUTI) remains a major problem for many women and therefore the pursuit for genomic and phenotypic traits which could define rUTI has been ongoing. The present study applied a genomic approach to investigate recurrent urinary tract infections by comparative analyses of recurrent and non-recurrent Escherichia coli isolates from general practice. From whole-genome sequencing data, phylogenetic clustering and genomic traits were studied on a collection of isolates which caused recurrent infection compared to non-recurrent isolates. In addition, genomic variation between the 1st and following infection was studied on a subset of the isolates. Evidence of limited adaptation between the recurrent infections based on single nucleotide polymorphism analyses with a range of 0-13 non-synonymous single nucleotide polymorphisms (SNPs) between the paired isolates. This included an overrepresentation of SNPs in metabolism genes. We identified several genes which were more common in rUTI isolates, including nine fimbrial genes, however, not significantly after false-discovery rate. Finally, the results show that recurrent isolates of the present dataset are not distinctive by variation in the core genome, and thus, did not cluster distinct from non-rUTI isolates in a SNP phylogeny.

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.

Catalase Protects Biofilm of Staphylococcus aureus against Daptomycin Activity.

Daptomycin is recommended for the treatment of Staphylococcus aureus infections due to its bactericidal activity. However, its mechanism of action is poorly understood. The involvement of reactive oxygen species (ROS) in the bactericidal activity of daptomycin has been proved against planktonic S. aureus, but not against the biofilm of S. aureus. Therefore, we evaluated if ROS contributes to the effect of daptomycin against biofilm of S. aureus. Biofilms of wild type, catalase deficient and daptomycin-resistant S. aureus strains were grown in microtiter-plates. After three days, the biofilms were exposed to daptomycin with or without thiourea in the presence of a ROS indicator. After overnight incubation, the amount of ROS and the percentage of surviving bacteria were determined. The bacterial survival was higher and the amount of ROS was lower in the wild type than in the catalase deficient biofilm, demonstrating a protective effect of catalase against daptomycin. The induction of cytotoxic ROS formation by daptomycin was verified by the addition of thiourea, which reduced the amount of ROS and protected the wild type biofilm against high concentrations of daptomycin. Accordingly, only the highest concentration of daptomycin reduced the bacterial survival and increased the ROS formation in the resistant biofilm. In conclusion, daptomycin induced the production of cytotoxic levels of endogenous ROS in S. aureus biofilm and the presence of catalase protected the biofilm against the lethality of the induced ROS.

Asymptomatic Bacteriuria (ABU) in Elderly: Prevalence, Virulence, Phylogeny, Antibiotic Resistance and Complement C3 in Urine.

Background: The incidence of asymptomatic bacteriuria (ABU) increases with age and is most common for persons 80 years of age and above and in elderly living in nursing homes. The distinction between ABU and urinary tract infection (UTI) is often difficult, especially in individuals, who are unable to communicate their symptoms, and there is a lack of objective methods to distinguish between the two entities. This can lead to overuse of antibiotics, which results in the selection and dissemination of antibiotic resistant isolates. Materials and methods: From voided midstream urine samples of 211 participants ≥60 years old from nursing homes, an activity center and a general practitioners clinic, we collected 19 ABU, 16 UTI and 22 control urine samples and compared them with respect to levels of complement component C3 in urine as determined by an ELISA assay relative to creatinine levels in the same urine samples, as measured by a creatinine assay. Further, we studied all Escherichia coli isolates for selected virulence genes by multiplex PCR, and by whole-genome sequencing (WGS) for genotypes and phylogenetic clustering. Antibiotic susceptibility was determined by microtiter broth dilution. Results: We identified a prevalence of ABU of 18.9% in nursing home residents, whereas ABU was only found in 4% of elderly living in the community (p < 0.001). E. coli from ABU patients were significantly more antibiotic resistant than E. coli from UTIs (p = 0.01). Prevalence of classical virulence genes, detected by multiplex PCR, was similar in E. coli isolates from ABU and UTI patients. Whole-genome sequencing of the E. coli isolates showed no specific clustering of ABU isolates compared to UTI isolates. Three isolates from three different individuals from one of the nursing homes showed signs of transmission. We demonstrated a significantly increased level of C3/creatinine ratio in ABU and UTI samples compared to healthy controls; however, there was no significant difference between the ABU and UTI group with respect to C3 level, or virulence factor genes. Conclusion: ABU was significantly more prevalent in the elderly residing in nursing homes than in the elderly living at home. Antibiotic resistance was more prevalent in E. coli from nursing homes than in UTI isolates, but there was no difference in prevalence of virulence associated genes between the two groups and no phylogenetic clustering, as determined by WGS relative to the two types of E. coli bacteriuria. The similar complement C3 response in ABU and UTI patients may indicate that ABU should be reconsidered as an infection albeit without symptoms.

In vitro Relative Fitness, in vivo Intestinal Colonization and Genomic Differences of Escherichia coli of ST131 Carrying bla CTX-M-15.

INTRODUCTION: Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli have become prevalent worldwide, with E. coli of sequence type 131 (ST131) as the dominant genotype. E. coli ST131 predominantly exhibits the serotype O25, is associated with the ESBL CTX-M-15 and belongs to a well-defined subclade within the FimH30-R clade, FimH30-Rx/C2. Multidrug resistance may have fitness costs for the bacteria. The aim of the current study was to investigate the fitness burden compared to a susceptible ST131 isolate without resistance genes in vitro and in vivo and describe genetic differences between fit and less fit isolates. MATERIALS AND METHODS: From a collection of clinical ESBL and non-ESBL E. coli isolates from urinary tract infection, we selected 16 bla CTX-M-15-positive isolates of ST131. The in vitro fitness was examined, and relative bacterial fitness (fit t ) was determined by direct competition with a fully susceptible ST131 isolate and illustrated in percent, with <100% resulting in a lower fitness, compared to the susceptible reference isolate. The isolates were subjected to whole-genome sequencing and analyzed for resistance markers, plasmids, phage content, and serotype. In vivo competition was tested in a mouse colonization model. RESULTS: The majority (12 out of 16) of the CTX-M-15-producing isolates had a slightly lower relative fitness compared to the susceptible ST131 isolate (mean, 97.6%; range, 82.6-108%) in vitro. Three isolates had a better fitness than the susceptible ST131 isolate, and one isolate had an identical fitness to the susceptible ST131 isolate. The in vitro fitness showed no correlation to the number of plasmids, number of phages, number of resistances, or genome size. For the in vivo competition assays, all three ESBL-producing isolates showed better colonization of the ESBL-resistant ST131 isolates compared to the susceptible ST131 isolate. CONCLUSION: This study shows that ESBL-producing ST131/H30-Rx are not necessarily burdened by multidrug resistance, however, have a better in vitro fitness than the susceptible isolate. These data contribute to the understanding of the success of ST131/H30-Rx, although they do not indicate ways to overcome this highly fit, virulent, and antimicrobial-resistant clone.

A snapshot of diversity: Intraclonal variation of Escherichia coli clones as commensals and pathogens.

Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyses of single nucleotide polymorphisms (SNPs) and gene-content variation in ten isolates belonging to the same clone and isolated from rectal swabs or urine samples. We identified only one clone in eight of the nine urines sampled (89 %). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0-2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2-15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider.

Mutational change of CTX-M-15 to CTX-M-127 resulting in mecillinam resistant Escherichia coli during pivmecillinam treatment of a patient.

Pivmecillinam (amdinocillin pivoxil) is the recommended first-choice antibiotic used to treat urinary tract infections (UTIs) in Denmark. The frequency of mutation to mecillinam (MEC) resistance is described as high in vitro; however, treatment of UTI has a good clinical response and prevalence of mecillinam resistance in Escherichia coli remains low despite many years of use. We describe occurrence of in vivo mecillinam resistance in a clinical isolate of ESBL-producing E. coli following pivmecillinam treatment. The identified phenotypic differences in the mecillinam resistant isolate compared with the original mecillinam susceptible isolate were a full-length LPS with O-antigen (O25), mecillinam resistance and a lower MIC for ceftazidime. Regarding genotype, the resistant isolate differed with a mutation in blaCTX-M-15 to blaCTX-M-127 , loss of a part of a plasmid and a genomic island, respectively, and insertion of a transposase in wbbL, causing the rough phenotype. The observed mecillinam resistance is expected to be caused by the mutation in blaCTX-M-15 with additional contribute from the serotype shift. We continue to recommend the use of pivmecillinam as first-line treatment for UTI.

Escherichia coli belonging to ST131 rarely transfers bla ctx-m-15 to fecal Escherichia coli.

BACKGROUND: Extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) causing urinary tract infections often belong to sequence type 131 (ST131), serotype O25, carrying bla CTX-M-15. AIM: The main aim of this study was to examine the conjugational frequencies of E. coli with plasmids carrying bla CTX-M-15 to E. coli isolates from the fecal flora of healthy humans to determine whether ST131 is more likely to uptake or donate ESBL resistance compared to other E. coli clones. METHODS:  Donors and recipients were all clinical isolates and did not harbor plasmids with identical incompatibility groups (Inc-groups) based on in silico analyses of Inc-groups and restriction/modification systems (R/M-systems). The in vitro conjugation experiments were performed as filter conjugation with verification of transconjugants by random amplified polymorphic DNA (RAPD) PCR and bla CTX-M-15 PCR. RESULTS: The frequencies of conjugation with bla CTX-M-15-carrying plasmids were found to be very rare with detectable conjugation frequencies in the range of 4x10-9-7x10-7 transconjugants/recipient. Recipients of O25/ST131 type yielded significantly lower conjugation frequencies compared to recipients of other O-types (P=0.004). The applied ST131/O25 donors did not yield detectable levels of transconjugants regardless of the applied recipient. Presence of sub-MIC levels of ampicillin increased plasmid transfer frequencies x100 fold (P=0.07). CONCLUSION: The results indicate that bla CTX-M-15 is rarely transferred by conjugation to E. coli isolates of the intestinal flora, even when the gene is plasmid-borne.

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.

Selection of ESBL-Producing E. coli in a Mouse Intestinal Colonization Model.

Asymptomatic human carriage of antimicrobially drug-resistant pathogens prior to infection is increasing worldwide. Further investigation into the role of this fecal reservoir is important for combatting the increasing antimicrobial resistance problems. Additionally, the damage on the intestinal microflora due to antimicrobial treatment is still not fully understood. Animal models are powerful tools to investigate bacterial colonization subsequent to antibiotic treatment. In this chapter we present a mouse-intestinal colonization model designed to investigate how antibiotics select for an ESBL-producing E. coli isolate. The model can be used to study how antibiotics with varying effect on the intestinal flora promote the establishment of the multidrug-resistant E. coli. Colonization is successfully investigated by sampling and culturing stool during the days following administration of antibiotics. Following culturing, a precise identification of the bacterial strain found in mice feces is applied to ensure that the isolate found is in fact identical to the strain used for inoculation. For this purpose random amplified of polymorphic DNA (RAPD) PCR specifically developed for E. coli is applied. This method allows us to distinguish E. coli with more than 99.95% genome similarity using a duplex PCR method.