The natural aquatic environment is a significant contributor to the development and circulation of clinically significant antibiotic resistance genes (ARGs). The potential for the aquatic environment to act as a reservoir for ARG accumulation in areas receiving anthropogenic contamination has been thoroughly researched. However, the emergence of novel ARGs in the absence of external influences, as well as the capacity of environmental bacteria to disseminate ARGs via mobile genetic elements remain relatively unchallenged. In order to address these knowledge gaps, this scoping literature review was established focusing on the detection of two important and readily mobile ARGs, namely, extended spectrum beta-lactamase (ESBL) and carbapenemase genes. This review included 41 studies from 19 different countries. A range of different water bodies including rivers (n = 26), seawaters (n = 6) and lakes (n = 3), amongst others, were analysed in the included studies. ESBL genes were reported in 29/41 (70.7%) studies, while carbapenemase genes were reported in 13/41 (31.7%), including joint reporting in 9 studies. The occurrence of mobile genetic elements was evaluated, which included the detection of integrons (n = 22), plasmids (n = 18), insertion sequences (n = 4) and transposons (n = 3). The ability of environmental bacteria to successfully transfer resistance genes via conjugation was also examined in 11 of the included studies. The findings of this scoping review expose the presence of clinically significant ARGs in the natural aquatic environment and highlights the potential ability of environmental isolates to disseminate these genes among different bacterial species. As such, the results presented demonstrate how anthropogenic point discharges may not act as the sole contributor to the development and spread of clinically significant antibiotic resistances. A number of critical knowledge gaps in current research were also identified. Key highlights include the limited number of studies focusing on antibiotic resistance in uncontaminated aquatic environments as well as the lack of standardisation among methodologies of reviewed investigations.

Water bodies worldwide have proven to be vast reservoirs of clinically significant antibiotic resistant organisms. Contamination of waters by anthropogenic discharges is a significant contributor to the widespread dissemination of antibiotic resistance. The aim of this research was to investigate multiple different anthropogenic sources on a national scale for the role they play in the environmental propagation of antibiotic resistance. A total of 39 water and 25 sewage samples were collected across four local authority areas in the West, East and South of Ireland. In total, 211 Enterobacterales were isolated (139 water, 72 sewage) and characterised. A subset of isolates (n=60) were chosen for whole genome sequencing. Direct comparisons of the water versus sewage isolate collections revealed a higher percentage of sewage isolates displayed resistance to cefoxitin (46%) and ertapenem (32%), while a higher percentage of water isolates displayed resistance to tetracycline (55%) and ciprofloxacin (71%). Half of all isolates displayed extended spectrum beta-lactamase (ESBL) production phenotypically (n = 105/211; 50%), with blaCTX-M detected in 99/105 isolates by PCR. Carbapenemase genes were identified in 11 isolates (6 sewage, 5 water). The most common variant was blaOXA-48 (n=6), followed by blaNDM-5 (n=2) and blaKPC-2 (n=2). Whole genome sequencing analysis revealed numerous different sequence types in circulation in both waters and sewage including E. coli ST131 (n=15), ST38 (n=8), ST10 (n=4) along with Klebsiella ST405 (n=3) and ST11 (n=2). Core genome MLST (cgMLST) comparisons uncovered three highly similar Klebsiella isolates originating from hospital sewage and two nearby waters. The Klebsiella isolates from an estuary and seawater displayed 99.1% and 98.8% cgMLST identity to the hospital sewage isolate respectively. In addition, three pairs of E. coli isolates from different waters also revealed cgMLST similarities, indicating widespread dissemination and persistence of certain strains in the aquatic environment. These findings highlight the need for routine monitoring of water bodies used for recreational and drinking purposes for the presence of multi-drug resistant organisms.

The natural environment represents a complex reservoir of antibiotic-resistant bacteria as a consequence of different wastewater discharges including anthropogenic and agricultural. Therefore, the aim of this study was to examine sewage and waters across Ireland for the presence of antibiotic-resistant Enterobacterales. Samples were collected from the West, East and South of Ireland. Two periods of sampling took place between July 2019 and November 2020, during which 118 water (30 L) and 36 sewage samples (200 mL) were collected. Waters were filtered using the CapE method, followed by enrichment and culturing. Sewage samples were directly cultured on selective agars. Isolates were identified by MALDI-TOF and antibiotic susceptibility testing was performed in accordance with EUCAST criteria. Selected isolates were examined for bla_CTX-M, bla_VIM, bla_IMP, bla_OXA-48, bla_NDM, and bla_KPC by real time PCR and whole genome sequencing (n = 146). A total of 419 Enterobacterales (348 water, 71 sewage) were isolated from all samples. Hospital sewage isolates displayed the highest percentage resistance to many beta-lactam and aminoglycoside antibiotics. Extended-spectrum beta-lactamase-producers were identified in 78% of water and 50% of sewage samples. One or more carbapenemase-producing Enterobacterales were identified at 23 individual sampling sites (18 water, 5 sewage). This included the detection of bla_OXA-48 (n = 18), bla_NDM (n = 14), bla_KPC (n = 4) and bla_OXA-484 (n = 1). All NDM-producing isolates harbored the ble-MBL bleomycin resistance gene. Commonly detected sequence types included Klebsiella ST323, ST17, and ST405 as well as E. coli ST131, ST38 and ST10. Core genome MLST comparisons detected identical E. coli isolates from wastewater treatment plant (WWTP) influent and nursing home sewage, and the surrounding waters. Similarly, one Klebsiella pneumoniae isolated from WWTP influent and the surrounding estuarine water were identical. These results highlight the need for regular monitoring of the aquatic environment for the presence of antibiotic-resistant organisms to adequately inform public health policies.

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.

Antimicrobial resistance (AMR) is a global health concern related to antimicrobial use and the subsequent emergence of resistant organisms, including carbapenemase-producing Enterobacterales (CPE). CPE isolate co-carrying bla_NDM-1 and mcr-9.1 have been scarcely reported internationally. The identification of 20 such isolates, including 16 of one species, within a group of three hospitals in one region indicated potential dissemination within and between healthcare facilities. Twenty isolates were pseudo-anonymised and identified via MALDI-ToF MS. Antimicrobial susceptibility testing was performed by disc diffusion, and Minimal Inhibition Concentration for colistin was carried out using the UMIC system. Short-read sequencing was conducted using the Illumina MiSeq platform, and genomic analysis identified antimicrobial resistance genes, virulence factors and plasmid contigs. Taxonomic classification of draft genomes was bioinformatically assessed using Kraken2. This collection comprised of Enterobacter hormaechei (n = 16), Citrobacter freundii (n = 3) and Enterobacter cloacae (n = 1) sourced from patient rectal swabs collected during routine screening (n = 13) or from healthcare-associated environmental sites (n = 7). The E. hormaechei isolates included four different ST types with one unassigned ST. Contig-based plasmid analysis identified 17 plasmid replicon types among the isolates. IncHI2A, IncHI2, and pKPCCAV1321_1 were detected in all isolates. Linked bla_NDM-1 and mcr-9.1 gene spread in hospitals likely occurred via plasmid-mediated transfer rather than spread of E. hormaechei. This study represents the first documented instance of bla_NDM-1/mcr-9.1 co-occurrence in Europe to date. It highlights the increasing public health threat posed by antimicrobial resistance and underscores the importance of genomic surveillance and clinical screening.

Evidence shows persistence of non-spore-forming Acinetobacter johnsonii in high-stakes controlled and nutrient-limited environments. This study aims to explore the mechanisms underpinning such adaptability through a comprehensive genomic analysis of 22 isolates of A. johnsonii from NASA’s Payload Hazardous Servicing Facility (PHSF) and one carbapenem-resistant strain (E154408A) from patient colonization in Ireland. Core-genome phylogeny revealed clustering of PHSF-originating isolates in a monophyletic clade divergent from the main species lineage. Species-wide virulence-associated genes and metabolic profiling indicated the unique presence in PHSF-originating isolates of two complete efflux pumps and of a conserved allantoin racemase, suggesting adaptability for multiple environmental stresses. Observed ubiquity of bla _OXA in investigated genomes (n=112) and phenotypically-validated multidrug-resistant profile of E154408A strain highlight A. johnsonii ’s potential as antimicrobial resistance (AMR) reservoir. Plasmidome analysis suggested gain/loss events across the monophyletic population and potential AMR acquisition pathways. Genome-to-metagenome mapping identified genomic signatures of A. johnsonii in PHSF >10 years post initial isolation. Importance: Acinetobacter johnsonii is increasingly recognized as an emerging human pathogen, with growing evidence of its ability to persist in controlled, high-stakes environments, posing risks as both persisting environmental contaminant and antimicrobial resistance (AMR) reservoir. Yet, gaps remain in our understanding of its AMR profile and the mechanisms that enable its enhanced environmental adaptability. This knowledge is necessary in contexts where biological cleanliness is a priority such as clinical settings and spacecraft assembly facilities cleanrooms, where contamination of hardware with terrestrial microorganisms is concerning. In this study, we aim to address some of key knowledge gaps by providing genomic insights into a rare multi-drug resistant clinical isolate and 22 NASA cleanroom isolates that persisted for over a decade in extremely clean conditions. Our findings will help evaluate the contamination risk of A. johnsonii in high-stakes environments and ultimately strengthen our ability to manage this microbial contaminant across terrestrial and extraterrestrial settings. Highlights: Cleanrooms-derived A. johnsonii genomes show favorable traits for increased adaptability Genomic signatures of A. johnsonii persisted in the cleanrooms for >10 years bla _OXA is ubiquitously found in the genome of all A. johnsonii E154408A is the first patient colonization by carbapenem-resistant A. johnsonii in Europe