The role of the aquatic environment in the spread of antimicrobial resistance of public health significance

Abstract

Antimicrobial resistance (AMR) is one of the greatest threats to human health of the 21st Century, with certain antimicrobial resistant organisms classified as being a greater threat to public health than others. At the top of that list are carbapenemase-producing Enterobacterales (CPE) and extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). It is well recognised that a ‘One Health’ approach is necessary when addressing the growing problem of AMR, as the health of humans, animals and the environment are interconnected. However, the environment remains the most under investigated of the three, and the role it may play in the emergence, persistence and dissemination of AMR is largely unknown. The aim of this thesis was therefore to add to current knowledge in this area, by assessing recreational waters and sewage for the presence of CPE and ESBL-PE and comparing the genomes of the isolates obtained to those originating from humans. Two sites in the West of Ireland were examined in this study. Sampling points at the first site consisted of two beaches (seawater samples), a river and two streams (fresh water samples), a sewage collection system and a sewage outflow where sewage is released untreated into the ocean (at a point between the two beaches). Sampling points at the second site consisted of a beach (seawater samples) and a nearby secondary wastewater treatment plant (untreated influent and treated effluent sewage samples). Following collection, samples were examined for the presence of CPE and ESBL-PE. Preliminary characterisation was subsequently performed on the isolates obtained, which consisted of antimicrobial susceptibility testing and real-time PCR. The genomes of a subset of isolates were sequenced using an Illumina HiSeq platform and the genomes compared to collections of clinical CPE and ESBL-PE. The same two strains of NDM-19-producing Enterobacterales (Escherichia coli ST167 and Klebsiella pneumoniae ST11) were detected repeatedly in recreational water samples taken at the first site. These findings represented the first identification of CPE in seawater in Europe. An untreated sewage discharge was identified as the probable source of CPE at this site. Findings indicated that three clinical NDM-19-producing Enterobacterales harboured blaNDM-19 on a similar IncX3 plasmid, with two of those isolates (one E. coli and one K. pneumoniae) found to be closely related to the two environmental strains of NDM-19-producing Enterobacterales. ESBL-producing E. coli (ESBL-PEc) was commonly identified among seawater, fresh water and sewage samples at this site (in 31/53 of samples (58.5%)). Comparison of these genomes to a collection of clinical ESBL-PEc identified 12 of the same AMR-associated genes and five plasmid replicon types among ESBL-PEc originating from all four sample types (seawater, fresh water, sewage and clinical samples), with blaCTX-M-15, found to be the most common variant of blaCTX-M detected. ST131 was the most commonly identified sequence type among all sample types. OXA-48-like-producing E. coli ST131 and OXA-48-like-producing K. pneumoniae ST101 were detected in seawater samples collected at the second site. These isolates harboured blaOXA-48 on similar mobile genetic elements to those identified in a clinical collection (pOXA-48 fragment in E. coli and IncL(pOXA-48) plasmid in K. pneumoniae). The source of the CPE at this site was not determined. In conclusion, multiple similar strains of CPE and ESBL-PEc and/or their mobile genetic elements were identified among isolates originating from humans and the aquatic environment. The findings of this thesis highlight the need for the environment’s role in the emergence, persistence and dissemination of antimicrobial resistant organisms to be further investigated.