BACKGROUND:ESBL-producing Enterobacteriaceae (ESBL-E) are observed in many reservoirs. Pets might play an important role in the dissemination of ESBL-E to humans since they live closely together. OBJECTIVES:To identify prevalence, risk factors, molecular characteristics, persistence and acquisition of ESBL-E in dogs and cats, and co-carriage in human-pet pairs belonging to the same household. METHODS:In a nationwide study, one person per household was randomly invited to complete a questionnaire and to submit a faecal sample. Dog and cat owners were invited to also submit a faecal sample from their pet. Repeated sampling after 1 and 6 months was performed in a subset. ESBL-E were obtained through selective culture and characterized by WGS. Logistic regression analyses and random forest models were performed to identify risk factors. RESULTS:The prevalence of ESBL-E carriage in these cohorts was 3.8% (95% CI: 2.7%-5.4%) for human participants (n=550), 10.7% (95% CI: 8.3%-13.7%) for dogs (n=555) and 1.4% (95% CI: 0.5%-3.8%) for cats (n=285). Among animals, blaCTX-M-1 was most abundant, followed by blaCTX-M-15. In dogs, persistence of carriage was 57.1% at 1 month and 42.9% at 6 months. Eating raw meat [OR: 8.8, 95% CI: 4.7-16.4; population attributable risk (PAR): 46.5%, 95% CI: 41.3%-49.3%] and dry food (OR: 0.2, 95% CI: 0.1-0.5; PAR: 56.5%, 95% CI: 33.2%-66.6%) were predictors for ESBL-E carriage in dogs. Human-dog co-carriage was demonstrated in five households. Human-cat co-carriage was not observed. CONCLUSIONS:ESBL-E prevalence was higher in dogs than in humans and lowest in cats. The main risk factor for ESBL-E carriage was eating raw meat. Co-carriage in dogs and household members was uncommon.

Abstract 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.

Background A high prevalence of colistin resistance among E. cloacae isolates in two intensive care units (ICU) (of 16 and 6 beds) using selective digestive decontamination (SDD) since 1990 instigated a retrospective and prospective investigation to quantify the role of clonal transmission. SDD is topical application of colistin and tobramycin and systemic use of cefotaxime during the first days of ICU-admission. Methods Multi-resistant E. cloacae (MREb) was defined as ESBL production and/or tobramycin non-susceptibility and/or colistin non-susceptibility. Incidence of acquisition and prevalence of carriage with MREb was determined from microbiological culture results. Results Colistin-resistant E. cloacae was first detected in November 2009 and carriage was demonstrated in 141 patients until October 2014. Mean incidence of MREb acquisition was 4.61 and 1.86 per 1000 days at risk in ICUs 1 and 2, respectively, and the mean monthly prevalence of MREb in both ICUs was 7.0 and 3.1%, respectively, without a discernible trend in time. Conversion rates from carriage of colistin-susceptible to resistant E. cloacae were 0.20 and 0.13 per 1000 patient days, respectively. Whole genome sequencing of 149 isolates revealed eight clusters, with the number of SNPs of the largest two clusters ranging between 0 and 116 for cluster 1 ( n = 49 isolates), and 0 and 27 for cluster 2 ( n = 36 isolates), among isolates derived between 2009 and 2014. Conclusions This study demonstrates a stable low-level endemicity of MREb in two Dutch ICUs with prolonged use of SDD, which was characterized by the persistent presence of two clusters, suggesting incidental clonal transmission.

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.

Objectives ESBL/AmpC-producing Enterobacteriaceae are an emerging public health concern. As households with preschool children may substantially contribute to the community burden of antimicrobial resistance, we determined the prevalence, risk factors and co-carriage of ESBL/AmpC-producing bacteria in preschool children and their parents. Methods From April 2013 to January 2015, each month 2000 preschool children were randomly selected from Dutch population registries. The parents were invited to complete an epidemiological questionnaire and to obtain and send a faecal sample from the selected child and from one parent. Samples were tested for ESBL/AmpC-producing bacteria. Logistic regression was used to identify risk factors for ESBL/AmpC carriage in children and parents, and findings were internally validated by bootstrapping. Results In total, 1016 families were included and ESBL/AmpC prevalence was 4.0% (95% CI 3.2%–5.0%); 3.5% (95% CI 2.5%–4.8%) in children and 4.5% (95% CI 3.4%–6.0%) in parents. Attending a daycare centre (DCC) was the only significant risk factor for children (OR 2.1, 95% CI 1.0–4.3). For parents, the only significant risk factor was having one or more children attending DCCs (OR 2.2, 95% CI 1.2–4.8). For parents of ESBL/AmpC-positive children the OR for ESBL/AmpC carriage was 19.7 (95% CI 9.2–42.4). Co-carriage of specific ESBL/AmpC genotypes in child and parent occurred more often than expected by chance (14.6% versus 1.1%, P < 0.001). Conclusions In this study, intestinal carriage with ESBL/AmpCs was detected in ∼4% of households with preschool children. DCC attendance was a risk factor in both children and parents and co-carriage of specific genotypes frequently occurred in child–parent pairs. These findings suggest household transmission or/and family-specific exposure to common sources of ESBL/AmpC-producing bacteria.

DNA-based techniques are frequently used to confirm the relatedness of putative outbreak isolates. These techniques often lack the discriminatory power when analyzing closely related microbes such as E. coli. Here the value of Raman spectroscopy as a typing tool for E. coli in a clinical setting was retrospectively evaluated.

OBJECTIVES:Patients can acquire extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households. METHODS:Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model. RESULTS:In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa. CONCLUSION:There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community.

The prevalence of patients colonized with extended-spectrum beta-lactamase (ESBL)-producing bacteria increases, especially in long-term-care facilities (LTCFs). Identification of ESBL carriers at hospital admission is relevant for infection control measures and antibiotic therapy for nosocomial infections. We aimed to develop a prediction rule for ESBL carriage at hospital admission for patients admitted from home and LTCFs, and to quantify incidences of nosocomial infections caused by ESBL-producing bacteria. The ESBL-carrier status was determined of patients admitted from LTCFs and from home settings in four hospitals in the Netherlands using perianal swabs obtained within 48 hours of admission. Risk factors for ESBL carriage were assessed. Infections caused by ESBL-producing bacteria were identified retrospectively. Among 1351 patients, 111 (8.2%) were ESBL carriers at admission: 50/579 (8.6%) admitted from LTCFs and 61/772 (7.9%) from home settings (p 0.63). Previous ESBL carriage and previous hospital admission were risk factors for ESBL carriage in multivariable analysis. The area under the curve of the receiver operating characteristic curve of the model was 0.64 (95% CI 0.58-0.71). Presence of ≥1 risk factor (n = 803; 59%) had sensitivity of 72%. Incidences of nosocomial infections caused by ESBL-producing bacteria were 45.5/10,000 and 2.1/10,000 admission days for ESBL carriers and non-carriers, respectively (p <0.05). In conclusion, prevalence of ESBL carriage at hospital admission was 8.2%, and was comparable among patients admitted from LTCF and home. A clinically useful prediction rule for ESBL carriage at admission could not be developed. The absolute incidence of nosocomial infections by ESBL-producing bacteria was low, but higher among patients carrying ESBL-producing bacteria at the time of hospital admission.

On 31 May 2011, after notification of Klebsiella pneumoniae (KP)(OXA-48;CTX-M-15) in two patients, nosocomial transmission was suspected in a Dutch hospital. Hospital-wide infection control measures and an outbreak investigation were initiated. A total of 72,147 patients were categorised into groups based on risk of OXA-48 colonisation or infection, and 7,527 were screened for Enterobacteriaceae(OXA-48) by polymerase chain reaction (PCR). Stored KP isolates (n=408) were retrospectively tested for OXA-48 and CTX-M-1 group extended-spectrum beta-lactamases (ESBL). 285 KP isolates from retrospective and prospective patient screening were genotyped by amplified fragment length polymorphism (AFLP). 41 isolates harbouring different Enterobacteriaceae species were analysed by plasmid multilocus sequence typing (pMLST). No nosocomial transmission of Enterobacteriaceae(OXA-48) was detected after 18 July 2011. Enterobacteriaceae(OXA-48) were found in 118 patients (KP (n=99), Escherichia coli (n=56), ≥1 Enterobacteriaceae(OXA-48) species (n=52)), of whom 21 had clinical infections. 39/41 (95%) of OXA-48 containing plasmids were identical in pMLST. Minimum inhibitory concentrations (MICs) of KP(OXA-48) and E. coli(OXA-48) for imipenem and meropenem ranged from ≤1 to ≥16 mg/L, and 153/157 (97%) had MIC >0.25 mg/L for ertapenem. AFLP identified a cluster of 203 genetically linked isolates (62 KP(OXA-48;CTX-M15); 107 KP(CTX-M-15); 34 KP(OXA-48)). The ‘oldest’ KP(CTX-M-15) and KP(OXA-48) clonal types originated from February 2009 and September 2010, respectively. The last presumed outbreak-related KP(OXA-48) was detected in April 2012. Uncontrolled transmission of KP(CTX-M-15) evolved into a nosocomial outbreak of KP(OXA-48;CTX-M15) with large phenotypical heterogeneity. Although the outbreak was successfully controlled, the contribution of individual containment measures and of the hospital relocating into a new building just before outbreak notification was impossible to quantify.

The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

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.

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases.

Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1), bla(CTX-M-2), bla(SHV-2), bla(SHV-12), bla(TEM-20), bla(TEM-52)): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105).

Staphylococcus aureus is the major cause of surgical site infections, and meticillin-resistant S. aureus (MRSA) is increasingly accounting for infections worldwide. Preventing surgical site infections by screening and decolonising positive patients reduces the number of infections, but does not completely eradicate the risk. A balance between prevention, costs and the chance of mupirocin-resistant S. aureus needs to be evaluated and decolonisation strategies optimised. It is essential to know the site of S. aureus during colonisation. In this study, for the first time the exact location of S. aureus in the human nose was determined using a histological approach. We showed the presence of S. aureus in the cornified layer of squamous epithelium, associated keratin and mucous debris and within hair follicles in the vestibulum nasi. The presence of S. aureus in hair follicles suggests that this could be the niche from which relapses occur after decolonisation. Decolonisation strategies might have to be reconsidered.

Objectives: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M.Methods: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n=106 ESBL positive).Results: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates.Conclusions: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

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