Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested.This research was funded by the European Union Horizon 2020 Research and Innovation Program under grant agreement No. 773830: One Health European Joint Program, JRP13-AMRSH5-WORLDCOM project.info:eu-repo/semantics/publishedVersio

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.Peer reviewe

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Mapping and Analysing Potential Sources and Transmission Routes of Antimicrobial Resistant Organisms in the Environment using Geographic Information Systems—An Exploratory Study

Antimicrobial resistance (AMR) is one of the leading threats to human health worldwide. The identification of potential sources of antimicrobial resistant organisms (AROs) and their transmission routes in the environment is important for improving our understanding of AMR and to inform and improve policy and monitoring systems, as well as the identification of suitable sampling locations and potential intervention points. This exploratory study uses geographic information systems (GIS) to analyse the spatial distribution of likely ARO sources and transmission routes in four local authority areas (LAAs) in Ireland. A review of relevant spatial data in each LAA, grouped into themes, and categorised into sources and transmission routes, was undertaken. A range of GIS techniques was used to extract, organise, and collate the spatial data into final products in the form of thematic maps for visual and spatial analysis. The results highlight the location of ‘clusters’ at increased risk of harbouring AMR in each LAA. They also demonstrate the relevance of aquatic transmission routes for ARO mobility and risk of human exposure. The integration of a GIS approach with expert knowledge of AMR is shown to be a useful tool to gain insights into the spatial dimension of AMR and to guide sampling campaigns and intervention points

Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs

Increasing prevalence of esbl production among irish clinical enterobacteriaceae from 2004 to 2008: an observational study

Background: Extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae infections are associated with delayed initiation of appropriate treatment, poor outcomes and increased hospital stay and expense. Although initially associated with healthcare settings, more recent international reports have shown increasing isolation of ESBLs in the community. Both hospital and community ESBL epidemiology in Ireland are poorly defined. Methods: This report describes clinical and laboratory data from three hospitals over 4.5 years. All significant isolates of Enterobacteriaceae were subjected to standardized antimicrobial susceptibility testing and screening for ESBL production. Available patient data from hospital databases were reviewed. Results: The database included 974 ESBL producing organisms from 464 patients. Urine and blood isolates represented 84% and 3% of isolates respectively. E. coli predominated (90.9%) followed by K. pneumoniae (5.6%). The majority of patients (n = 246, 53.0%) had been admitted to at least one of the study hospitals in the year prior to first isolation of ESBL. The overall 30-day all-cause mortality from the date of culture positivity was 9.7% and the 1 year mortality was 61.4%. A Cox regression analysis showed age over 60, male gender and previous hospital admissions were significant risk factors for death within 30 days of ESBL isolation. Numbers of ESBL-producing E. coli isolated from urine and blood cultures increased during the study. Urine isolates were more susceptible than blood isolates. Co-resistance to other classes of antimicrobial agents was more common in ESBL producers from residents of long stay facilities (LSF) compared with hospital inpatients who lived at home. Conclusions: This work demonstrates a progressively increasing prevalence of ESBL Enterobacteriaceae in hospital, LSF and community specimens in a defined catchment area over a long time period. These results will improve clinician awareness of this problem and guide the development of empiric antimicrobial regimens for community acquired bloodstream and urinary tract infections