SummaryBackground Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it diffi cult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the fi rst structured survey on the occurrence of carbapenemaseproducing Klebsiella pneumoniae and Escherichia coli in European hospitals.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.

Antimicrobial resistance is a major public health concern. Carbapenemase-producing Enterobacterales (CPE) represent a significant health threat as some strains are resistant to almost all available antibiotics. The aim of this research was to examine hospital effluent and municipal wastewater in an urban area in Ireland for CPE. Samples of hospital effluent ( n = 5), municipal wastewater before (n = 5) and after ( n = 4) the hospital effluent stream joined the municipal wastewater stream were collected over a nine-week period (May–June 2017). All samples were examined for CPE by direct plating onto Brilliance CRE agar. Isolates were selected for susceptibility testing to 15 antimicrobial agents in accordance with EUCAST criteria. Where relevant, isolates were tested for carbapenemase-encoding genes by real-time PCR. CPE were detected in five samples of hospital effluent, one sample of pre-hospital wastewater and three samples of post-hospital wastewater. Our findings suggest hospital effluent is a major contributor to CPE in municipal wastewater. Monitoring of hospital effluent for CPE could have important applications in detection and risk management of unrecognised dissemination of CPE in both the healthcare setting and the environment.

In this study, New Delhi metallo-beta-lactamase (NDM)-producing Enterobacteriaceae were identified in Irish recreational waters and sewage. Indistinguishable NDM-producing Escherichia coli by pulsed-field gel electrophoresis were isolated from sewage, a fresh water stream and a human source. NDM-producing Klebsiella pneumoniae isolated from sewage and seawater in the same area were closely related to each other and to a human isolate. This raises concerns regarding the potential for sewage discharges to contribute to the spread of carbapenemase-producing Enterobacteriaceae.We report the finding of New Delhi metallo-beta-lactamase (NDM)-producing Enterobacteriaceae in fresh water and seawater samples collected at two beaches located near an untreated human sewage ocean discharge. Isolates of NDM-producing Escherichia coli derived from the sewage collection system, the sewage storage tank and the outflow were 100% identical by pulsed-field gel electrophoresis (PFGE) to those derived from a fresh water stream on one of the beaches, and to a clinical isolate. Recreational water and sewage sample sitesIn 2016, we identified a beach (Beach A) in Ireland, used for bathing and recreation, which is crossed by two fresh water streams (Stream A and Stream B), flowing from the surrounding countryside. These streams were examined for the presence of carbapenemaseproducing Enterobacteriaceae (CPE). The detection of NDM-producing E. coli in these waters prompted subsequent additional sampling of the streams. As untreated human sewage was being discharged into the sea in the vicinity of the beach, and the fresh water streams can become immersed in seawater at high tide, sewage was evaluated as a potential source. Sewage samples included samples from the collection system, the storage tank and the outflow. Sampling was performed in the period May to September, 2016. The sewage system is not linked to any hospital or long-term care facility that we are aware of. Further sampling of the fresh water streams and sewage sites was carried out in January 2017. In addition to this, seawater from Beach A and from a second beach (Beach B), ca 950 m in a direct line from Beach A were examined. Figure 1 shows a schematic diagram of the sampling points and their location relative to each other. Processing of samplesWe applied a previously described method (CapE), to examine large volumes of water (30L) from both the fresh water streams and the seawater, for the presence of CPE [1]. Following filtration and overnight enrichment, the samples were sub-cultured onto Brilliance CRE agar (Oxoid). Sewage samples were examined by direct plating onto Brilliance CRE agar. Following purification, presumptive isolates were identified to species level by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and antimicrobial susceptibility testing was performed and interpreted in accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria [2]. Carbapenemase-encoding genes were detected ...

Background Escherichia coli (E. coli) comprise part of the normal vaginal microflora. Transfer from mother to neonate can occur during delivery resulting, sometimes, in neonatal bacterial disease. Here, we aim to report the first outbreak of CTX-M ESBL-producing E. coli with evidence of mother-to-neonate transmission in an Irish neonatal intensive care unit (NICU) followed by patient-to-patient transmission.MethodsInvestigation including molecular typing was conducted. Infection was defined by clinical and laboratory criteria and requirement for antimicrobial therapy with or without positive blood cultures. Colonisation was determined by isolation without relevant symptoms or indicators of infection.ResultsIndex case was an 8-day-old baby born at 34 weeks gestation who developed ESBL-producing E. coli infections at multiple body sites. Screening confirmed their mother as colonised with ESBL-producing E. coli. Five other neonates, in the NICU simultaneously with the index case, also tested positive. Of these, four were colonised while one neonate developed sepsis, requiring antimicrobial therapy. The second infected neonate’s mother was also colonised by ESBL-producing E. coli. Isolates from all eight positive patients (6 neonates, 2 mothers) were compared using pulsed-field gel electrophoresis (PFGE). Two distinct ESBL-producing strains were implicated, with evidence of transmission between mothers and neonates for both strains. All isolates were confirmed as CTX-M ESBL-producers. There were no deaths associated with the outbreak.ConclusionsResources were directed towards control interventions focused on hand hygiene and antimicrobial stewardship, which ultimately proved successful. Since this incident, all neonates admitted to the NICU have been screened for ESBL-producers and expectant mothers are screened at their first antenatal appointment. To date, there have been no further outbreaks.