Comparative genomics of ESBL-producing Escherichia coli (ESBL-Ec) reveals a similar distribution of the 10 most prevalent ESBL-Ec clones and ESBL genes among human community faecal and extra-intestinal infection isolates in the Netherlands (2014-17).

INTRODUCTION: The human gut microbiota is an important reservoir of ESBL-producing Escherichia coli (ESBL-Ec). Community surveillance studies of ESBL-Ec to monitor circulating clones and ESBL genes are logistically challenging and costly. OBJECTIVES: To evaluate if isolates obtained in routine clinical practice can be used as an alternative to monitor the distribution of clones and ESBL genes circulating in the community. METHODS: WGS was performed on 451 Dutch ESBL-Ec isolates (2014-17), including 162 community faeces and 289 urine and blood isolates. We compared proportions of 10 most frequently identified STs, PopPUNK-based sequence clusters (SCs) and ESBL gene subtypes and the degree of similarity using Czekanowski's proportional similarity index (PSI). RESULTS: Nine out of 10 most prevalent STs and SCs and 8/10 most prevalent ESBL genes in clinical ESBL-Ec were also the most common types in community faeces. The proportions of ST131 (39% versus 23%) and SC131 (40% versus 25%) were higher in clinical isolates than in community faeces (P < 0.01). Within ST131, H30Rx (C2) subclade was more prevalent among clinical isolates (55% versus 26%, P < 0.01). The proportion of ESBL gene blaCTX-M-1 was lower in clinical isolates (5% versus 18%, P < 0.01). Czekanowski's PSI confirmed that the differences in ESBL-Ec from community faeces and clinical isolates were limited. CONCLUSIONS: Distributions of the 10 most prevalent clones and ESBL genes from ESBL-Ec community gut colonization and extra-intestinal infection overlapped in majority, indicating that isolates from routine clinical practice could be used to monitor ESBL-Ec clones and ESBL genes in the community.

Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches.

BACKGROUND: Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. AIMS: This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. SOURCES: Personal collection of relevant publications. CONTENT: When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. IMPLICATIONS: As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation.

Intestinal carriage of ampicillin- and vancomycin-resistant Enterococcus faecium in humans, dogs and cats in the Netherlands.

Background: The prevalence of ampicillin- and/or vancomycin-resistant Enterococcus faecium (AREf and VREf) has increased in hospitalized patients in the Netherlands. Objectives: To quantify the prevalence, risk factors and co-carriage of AREf and VREf in humans, cats and dogs in the Dutch population. Methods: From 2014 to 2015, ∼2000 inhabitants of the Netherlands each month were randomly invited to complete a questionnaire and provide a faecal sample. Subjects owning pets were also asked to submit one dog or cat sample. Faecal samples were screened for AREf and VREf. The genetic relatedness of isolates was determined using core genome MLST. Logistic regression analysis was used to determine risk factors. Results: Of 25 365 subjects, 4721 (18.6%) completed the questionnaire and 1992 (42.2%) human, 277 dog and 118 cat samples were submitted. AREf was detected in 29 human (1.5%), 71 dog (25.6%) and 6 cat (5.1%) samples. VREf (vanA) was detected in one human and one dog. AREf/VREf co-carriage was not detected in 388 paired samples. The use of antibiotics (OR 4.2, 95% CI 1.7-11.2) and proton pump inhibitors (OR 2.7, 95% CI 1.1-6.3) were risk factors for AREf carriage in humans. In dogs, these were the use of antibiotics (OR 2.3, 95% CI 1.1-4.6) and eating raw meat (OR 3.2, 95% CI 1.4-6.6). Core genome MLST-based phylogenetic linkage indicated clonal relatedness for a minority of human (16.7%) and pet AREf isolates (23.8%) in three clusters. Conclusions: Intestinal carriage with AREf or VREf is rare in the Dutch general population. Although AREf carriage is high in dogs, phylogenetic linkage between human and pet AREf isolates was limited.

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.

High-density fecal Enterococcus faecium colonization in hospitalized patients is associated with the presence of the polyclonal subcluster CC17.

Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successful spread of these pathogens. We aimed to quantify the enterococcal intestinal colonization densities in stool samples from AREfm-colonized and non-colonized patients using fluorescent in situ hybridization (FISH). Stool samples were collected from AREfm-colonized (n = 8) and non-colonized (n = 8) patients. The relative number of Enterococcus faecalis and E. faecium was determined by FISH using specific 16S rRNA probes, while the total amount of bacterial cells was counted by staining the sample with 4',6-diamidino-2-phenylindole (DAPI). The median bacterial cell numbers in fecal samples, counted by DAPI staining, were 7.7 × 10(9) and 4.8 × 10(9) cells/g for AREfm-colonized and non-colonized patients, respectively (p = 0.34). The E. faecium densities in AREfm-colonized patients, accounting for 0.5-7% of all fecal bacterial cells, exceeded E. faecalis levels by over ten-fold. E. faecium was not detected in non-colonized patients. This study demonstrated high E. faecium cell densities in stool samples from patients colonized with AREfm. Increased cell densities may contribute to host-to-host transmission and environmental contamination, facilitating the spread of AREfm in the hospital setting.

Comparison of the identification of coagulase-negative staphylococci by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tuf sequencing.

The increasing incidence of coagulase-negative staphylococci (CoNS) in hospital-acquired infections underlines the need for an accurate and simple identification of Staphylococcus isolates at the species level. Sequencing of the tuf gene has been shown to be the most accurate for the species identification of CoNS. We determined the species of 62 consecutive clinical and 31 reference CoNS isolates by tuf gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Species assignment by MALDI-TOF-MS and tuf sequencing was congruent in all cases. We conclude that MALDI-TOF-MS is accurate for identifying CoNS in routine clinical practice. The study also identified an unexpectedly high number of cases of Staphylococcus capitis infections among 62 consecutive CoNS isolates in 2009 at the University Medical Center Utrecht, the Netherlands.

Clinical impact of a highly prevalent Pseudomonas aeruginosa clone in Dutch cystic fibrosis patients.

Studies suggest that infection with highly prevalent Pseudomonas aeruginosa clones in cystic fibrosis (CF) is associated with an unfavourable clinical outcome. We studied the clinical characteristics of patients infected with a recently described, highly prevalent P. aeruginosa clone (ST406) in two CF centres in The Netherlands. Multilocus sequence typing data were available for 219 patients, of whom 40 (18.3%) were infected with ST406 and 179 with other sequence types. ST406 infection was independently associated with age, having a sibling with ST406 infection and use of inhaled antibiotics, but not with unfavourable clinical outcome, suggesting that high transmissibility is not necessarily associated with high virulence.

Aspergillus fumigatus colonization in cystic fibrosis: implications for lung function?

Aspergillus fumigatus is commonly found in the respiratory secretions of patients with cystic fibrosis (CF). Although allergic bronchopulmonary aspergillosis (ABPA) is associated with deterioration of lung function, the effects of A. fumigatus colonization on lung function in the absence of ABPA are not clear. This study was performed in 259 adults and children with CF, without ABPA. A. fumigatus colonization was defined as positivity of >50% of respiratory cultures in a given year. A cross-sectional analysis was performed to study clinical characteristics associated with A. fumigatus colonization. A retrospective cohort analysis was performed to study the effect of A. fumigatus colonization on lung function observed between 2002 and 2007. Longitudinal data were analysed with a linear mixed model. Sixty-one of 259 patients were at least intermittently colonized with A. fumigatus. An association was found between A. fumigatus colonization and increased age and use of inhaled antibiotics. In the longitudinal analysis, 163 patients were grouped according to duration of colonization. After adjustment for confounders, there was no significant difference in lung function between patients colonized for 0 or 1 year and patients with 2-3 or more than 3 years of colonization (p 0.40 and p 0.64) throughout the study. There was no significant difference in lung function decline between groups. Although colonization with A. fumigatus is more commonly found in patients with more severe lung disease and increased treatment burden, it is not independently associated with lower lung function or more severe lung function decline over a 5-year period.

Genome-based insights into the evolution of enterococci.

It is now 15 years since the first genome of a free-living organism was sequenced. Subsequent to this milestone, a veritable avalanche of genome sequence data has revolutionized many aspects of microbiology. In this review, we discuss recent progress on the genomics of Enterococcus faecalis and Enterococcus faecium, which are the two enterococcal species that cause the large majority of enterococcal infections. We focus on the genome-based analysis of enterococcal diversity and phylogeny. Studies based on comparative genome hybridization have shown that both species exhibit considerable inter-strain genomic diversity, which is mainly linked to the variable presence of phages, plasmids, pathogenicity islands and conjugative elements. We also discuss how the advent of next-generation sequencing technologies allows for a comprehensive characterization of the gene repertoire of multiple isolates, which can be used for extremely robust analyses of diversity and population structure.

Molecular characterisation of outbreak-related strains of vancomycin-resistant Enterococcus faecium from an intensive care unit in Beijing, China.

We investigated an outbreak of vancomycin-resistant Enterococcus faecium affecting 14 patients in a 20-bed intensive care unit (ICU) between September 2006 and August 2007 (incidence: 3.56 cases per 1000 ICU patient days). Eighteen isolates of vanA type E. faecium were analysed by pulsed field gel electrophoresis, which showed 14 types overall. Multilocus sequence typing (MLST) identified eight different sequence types (STs) (ST78, ST117, ST203, ST316, ST362, ST363, ST364 and ST365), including four new types (ST362, ST363, ST364 and ST365) and 17 strains belonged to clonal complexes CC17. Sixteen of these carried the esp gene. Eighteen Tn1546-like elements encoding vanA-type VRE were classified into three types (types I to III) and all of them contained both IS1216V and IS1542 insertions. Vancomycin resistance of 14 vanA type E. faecium isolates was transferred at a frequency of 1.3 x 10(-6) to 6.4 x 10(-5) between E. faecium strains during filter mating. Our findings indicate that conjugative dissemination of Tn1546-like elements among CC17 E. faecium occurred during the outbreak in this ICU.

Genotyping and preemptive isolation to control an outbreak of vancomycin-resistant Enterococcus faecium.

BACKGROUND: Control of vancomycin-resistant Enterococcus faecium (VRE) in European hospitals is hampered because of widespread asymptomatic carriage of VRE by healthy Europeans. In 2000, our hospital (The University Medical Center Utrecht, Utrecht, The Netherlands) was confronted with a large outbreak of VRE. INTERVENTION: On the basis of genotyping (by pulsed-field gel electrophoresis), epidemic and nonepidemic VRE strains were distinguished, and infection-control measures were exclusively targeted toward epidemic VRE. The outbreak was retrospectively divided into 3 periods of different infection-control measures. Compliance with use of alcohol-based hand rubs was enforced during all periods. Period I involved active surveillance, isolation of carriers, and cohorting (duration, 4 months); preemptive isolation of high-risk patients for VRE colonization was added in period II (7 months); and cohorting and preemptive isolation were abandoned in period III (18 months). METHODS: When the outbreak was identified, 27 patients in 6 wards were colonized; 93% were colonized with an epidemic VRE strain. Detection rates of nonepidemic VRE were 3.5%, 3.0%, and 2.9% among 683, 810, and 977 screened patients in periods I, II, and III, respectively, comparable to a prevalence of 2% (95% confidence interval [CI], 1%-3.5%) among 600 nonhospitalized persons. The relative risks of detecting epidemic VRE in periods II and III, compared with period I, were 0.67 (95% CI, 0.41-1.10) for period II and 0.02 (95% CI, 0.002-0.6) for period III. Infection-control measures were withheld for patients colonized with nonepidemic VRE (76 [54%] of 140 patients with a test result positive for VRE). Use of alcohol-based hand rubs increased by 31%-275% in outbreak wards. CONCLUSION: Genotyping-targeted infection control, isolation of VRE carriers, enhancement of hand-hygiene compliance, and preemptive isolation successfully controlled nosocomial spread of epidemic VRE infection.

Emergence of virulent methicillin-resistant Staphylococcus aureus strains carrying Panton-Valentine leucocidin genes in The Netherlands.

Methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the Panton-Valentine leucocidin (PVL) genes have been reported worldwide and are a serious threat to public health. The PVL genes encode a highly potent toxin which is involved in severe skin infections and necrotizing pneumonia, even in previously healthy individuals. We assessed the prevalence of PVL-positive MRSA in The Netherlands for two periods of time: (i) 1987 through 1995 and (ii) 2000 and 2002, and determined their characteristics by using multilocus sequence typing and staphylococcal chromosome cassette (SCCmec) typing. It was found that up to 15% of all MRSA isolates detected in The Netherlands harbored the PVL genes. Most PVL-positive MRSA isolates were obtained from severe soft tissue infections in relatively young individuals. The first PVL-positive MRSA described in The Netherlands, isolated in 1988, was a single-locus variant of the "Berlin" epidemic MRSA clone. The 20 PVL-positive MRSA isolates studied in 2000 and 2002 consisted of five different sequence types (STs) that belonged to four clonal complexes. One of the STs, ST80, is considered to be a widespread European clone and was the most predominant ST (60%) in this study, while ST37 had never been found to be associated with PVL-positive MRSA. Most isolates harbored SCCmec type IV, a supposed marker for community-acquired MRSA. The number and type of virulence-associated genes varied among the different STs.

Widespread dissemination in The Netherlands of the epidemic berlin methicillin-resistant Staphylococcus aureus clone with low-level resistance to oxacillin.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and represents a growing public health burden due to the emergence and spread of epidemic strains, particularly within the hospital environment. An epidemic MRSA clone, with characteristic low-level resistance to oxacillin, emerged in the year 2000 and became endemic in the Netherlands. Multilocus sequence typing characterized the strain as sequence type 45, which was previously designated the Berlin epidemic MRSA clone. In 2 years, this strain has become the predominant MRSA clone in the Netherlands.

Detection of enterococcal surface protein gene (esp) and amplified fragment length polymorphism typing of glycopeptide-resistant Enterococcus faecium during its emergence in a Greek intensive care unit.

The emergence of glycopeptide-resistant Enterococcus faecium (GREF) in a Greek intensive care unit was studied by amplified fragment length polymorphism analysis and esp gene detection. Three GREF clones harboring the esp gene were recovered from 17 out of 21 patients, indicating the dissemination of genetically homogenous and virulent strains of GREF.

Molecular epidemiology of Enterococcus faecalis in liver transplant patients at University Hospital Groningen.

We report the molecular epidemiology of Enterococcus faecalis in liver transplant patients transplanted at the University Hospital Groningen (The Netherlands) as determined by amplified fragment length polymorphism (AFLP) typing. A total of 133 E. faecalis isolates were cultured from the faeces and throat (95 isolates) or clinical sites (35 isolates) of 43 liver transplant patients. Among these 133 isolates, 15 different AFLP types could be identified with 90% AFLP similarity. Of these 15 groups, nine contained isolates from more than one patient, which may indicate transmission of E. faecalis isolates between patients. In five of these groups transmission could be explained by the fact that patients carrying identical strains were staying in the same ward at the same time. One of these epidemic isolates (AFLP type K) distinguished itself by colonizing 23 liver transplant patients during 15 months. Antimicrobial susceptibility testing did not reveal any multi-resistant isolates. This study showed that transmission of susceptible E. faecalis isolates occurs frequently on the liver transplant wards. Detection of this transmission and understanding of the mechanism is important, as it might also be an indicator of possible transmission of enterococci resistant to antibiotics.