Antibiotic residues in the aquatic environment - current perspective and risk considerations.

Antimicrobial resistance is a major concern for human and animal health, projected to deteriorate with time and given current trends of antimicrobial usage. Antimicrobial use, particularly in healthcare and agriculture, can result in the release of antimicrobials into surface waters, promoting the development of antibiotic resistance in the environment, and potentially leading to human health risks. This study reviews relevant literature, and investigates current European and Irish antimicrobial usage trends in humans and animals, as well as potential pathways that antibiotics can take into surface waters following use. Reported levels in the aquatic environment are summarized, with particular focus on Ireland. There are relatively few studies examining Irish water bodies or sewage effluent for antibiotic residues, however, five antibiotics, namely azithromycin, ciprofloxacin, clarithromycin, metronidazole, and trimethoprim, have been measured in Irish waters, in concentrations predicted to select for resistance. Numerous isolates of multi-drug resistant bacteria have also been found in water bodies throughout Ireland and Europe. The value of risk assessment methodologies in understanding risks posed by antibiotic residues is reviewed including the advantages and disadvantages of specific approaches. Hazard quotient and bespoke Monte Carlo approaches are predominant risk assessment tools used to examine antimicrobial release and their complex pathways. This study highlights the need for monitoring of antimicrobial releases and the potential for resistance development, persistence and transmission while highlighting the role of risk assessment methodologies in assessing potential human and environmental health impacts.

An MLST approach to support tracking of plasmids carrying OXA-48-like carbapenemase.

OBJECTIVES: The prevalence of infections caused by OXA-48-like carbapenemase-producing organisms in Ireland has increased dramatically since 2011 and is an urgent public health issue. Genome-based high-resolution genotyping was used to analyse clinical isolates submitted to the Irish Carbapenemase-Producing Enterobacteriaceae Reference Laboratory Service for a 13 month period (2016-17). METHODS: A total of 109 OXA-48-producing non-duplicate clinical isolates from 16 submitting centres were sequenced. Using a gene-by-gene approach, isolate genomes were characterized by MLST and core genome MLST, and the presence of antimicrobial resistance determinants was determined. Reference mapping and a novel plasmid MLST-type approach was applied to determine plasmid background. RESULTS: The OXA-48-like-producing isolates were Escherichia coli (n = 56), Klebsiella spp. (n = 46) and Enterobacter cloacae (n = 7). Amongst the E. coli isolates there were 37 different STs and amongst the Klebsiella spp. isolates there were 27 different STs. blaOXA-48 was present in 105/109 (96.3%) of isolates. Based on mapping analysis and detection of the pOXA-48 IncL-type plasmid replicon and backbone genes, a pOXA-48-like plasmid was identified in 93/109 isolates (85.3%). The remaining isolates (n = 16; 14.7%) harboured blaOXA-48-like genes in unknown environments. Using a gene-by-gene approach two pOXA-48-like plasmid groups with 2/71 pOXA-48-like locus differences between them were identified. CONCLUSIONS: In Ireland we found a diversity of genotypes associated with OXA-48-like-producing clinical isolates with the IncL pOXA-48 plasmid type predominating as the blaOXA-48 genetic environment. A plasmid MLST approach can rapidly identify plasmids associated with outbreaks and monitor spread of types temporally and geographically.

Economic Assessment of Waterborne Outbreak of Cryptosporidiosis.

In 2007, a waterborne outbreak of Cryptosporidium hominis infection occurred in western Ireland, resulting in 242 laboratory-confirmed cases and an uncertain number of unconfirmed cases. A boil water notice was in place for 158 days that affected 120,432 persons residing in the area, businesses, visitors, and commuters. This outbreak represented the largest outbreak of cryptosporidiosis in Ireland. The purpose of this study was to evaluate the cost of this outbreak. We adopted a societal perspective in estimating costs associated with the outbreak. Economic cost estimated was based on totaling direct and indirect costs incurred by public and private agencies. The cost of the outbreak was estimated based on 2007 figures. We estimate that the cost of the outbreak was >€19 million (≈€120,000/day of the outbreak). The US dollar equivalent based on today's exchange rates would be $22.44 million (≈$142,000/day of the outbreak). This study highlights the economic need for a safe drinking water supply.

Indistinguishable NDM-producing Escherichia coli isolated from recreational waters, sewage, and a clinical specimen in Ireland, 2016 to 2017.

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.

Colonisation with ESBL-producing and carbapenemase-producing Enterobacteriaceae, vancomycin-resistant enterococci, and meticillin-resistant Staphylococcus aureus in a long-term care facility over one year.

BACKGROUND: This study examined colonisation with and characteristics of antimicrobial-resistant organisms among residents of a long-term care facility (LTCF) over one year, including strain persistence and molecular diversity among isolates of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. METHODS: Sixty-four residents of a LTCF were recruited (51 at baseline, 13 during the year). Data on dependency levels, hospitalisations, and antimicrobial prescribing were collected. Nasal and rectal swabs and catheter urine specimens were examined quarterly, using chromogenic agars, for ESBL-producing Enterobacteriaceae, carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and meticillin-resistant S. aureus (MRSA). All ESBL-producing E. coli (ESBL-EC) were characterised by pulsed-field gel electrophoresis (PFGE) and PCR to assess for sequence type (ST) ST131, its resistance-associated H30 and H30-Rx subclones, and blaCTX-M, blaTEM, blaSHV, and blaOXA-1. RESULTS: The overall number of residents colonised, by organism, was as follows: ESBL-EC, 35 (55%); MRSA, 17 (27%); ESBL-producing K. pneumoniae (ESBL-KP), 5 (8%); VRE, 2 (3%) and CPE, 0 (0%). All 98 ESBL-EC isolates were H30-Rx ST131, with bla CTX-M-group 1. By PFGE, a group of 91 ESBL-EC (from 33 participants) had ≥85% similar profiles and resembled UK epidemic strain A/ international pulsotype PFGE812. Sequential ESBL-EC from individual residents were closely related. Six ESBL-KP isolates, from five participants, had bla CTX-M-group 1 and by PFGE were closely related. Colonisation with ESBL and MRSA was associated with location within the LTCF and previous exposure to antimicrobials. CONCLUSIONS: Among LTCF residents, colonisation with ESBL-EC and MRSA was common. All ESBL-EC were H30-Rx ST131, consistent with clonal dissemination.

Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

A comprehensive microbial surveillance was conducted at NASA's Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known ß-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE: The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Simulation model to predict the fate of ciprofloxacin in the environment after wastewater treatment.

The extent hospital effluent contributes to antimicrobial presence in the environment and its impact on resistance dissemination remains unknown. To investigate the fate of the antimicrobial ciprofloxacin in hospital effluent a Monte Carlo simulation model was developed to model levels from hospital use to wastewater treatment plant (WWTP) effluent release, in addition to modeling resistance formation potential, hazard quotient (HQ) and swimmer exposure. The mean predicted concentration (PC) of ciprofloxacin in hospital effluent, urban effluent, WWTP effluent, sludge, soil and sea water was 579, 6.06, 2.59, 3.48, 0.006 and 0.15 mg/m(3), respectively. A parallel surveillance study confirmed levels of ciprofloxacin above or below the limit of detection. The model predicted levels would never exceed the ECOSAR toxicity value. The model predicted a 98% probability of ciprofloxacin exhibiting a HQ > 1 (low toxicity concern). The mean ciprofloxacin PC in WWTP effluent was less than the minimum inhibitory concentration (MIC). The probability of conditions in WWTP effluent being favorable for resistance at 20% and 80% of the MIC was 3% and 72%, respectively. In all instances, when the MIC was bound, the probability for resistance formation within soil and sea water was < 1%. The probability of a swimmer being exposed to a level of ciprofloxacin greater than the acceptable daily intake was negligible. The study concluded that release of hospital effluent into the environment may lead to concentrations of ciprofloxacin which are of low toxicity concern but may be conducive to resistance formation and allow for the dissemination of resistance.

Risk ranking of macrolide antibiotics - Release levels, resistance formation potential and ecological risk.

Antibiotic resistance (AR) development in natural water bodies is a significant source of concern. Macrolide antibiotics in particular have been identified as pollutants of concern for AR development throughout the literature, as well as by state and international authorities. This study utilises a probabilistic model to examine the risk of AR development arising from human-use macrolide residues, utilising administration rates from Ireland as a case study. Stages modelled included level of administration, excretion, degradation in wastewater, removal in wastewater treatment, assuming conventional activated sludge (CAS) treatment, and dilution. Release estimates per day, as well as risk quotient values for antibiotic resistance development and ecological impact, are generated for erythromycin, clarithromycin, and azithromycin. In the modelled scenario in which conventional activated sludge treatment is utilised in wastewater treatment, this model ranks risk of resistance development for each antibiotic in the order clarithromycin > azithromycin > erythromycin, with mean risk quotient values of 0.50, 0.34 and 0.12, respectively. A membrane bioreactor scenario was also modelled, which reduced risk quotient values for all three macrolides by at least 50 %. Risk of ecological impact for each antibiotic was also examined, by comparing environmental concentrations predicted to safety limits based on toxicity data for cyanobacteria and other organisms from the literature, with azithromycin being identified as the macrolide of highest risk. This study compares and quantifies the risk of resistance development and ecological impact for a high-risk antibiotic group in the Irish context, and demonstrates the potential for risk reduction achieved by adoption of alternative (e.g. membrane bioreactor) technology.

Evaluating the impact of heavy metals on antimicrobial resistance in the primary food production environment: A scoping review.

Heavy metals are naturally occurring environmental compounds, which can influence antimicrobial resistance (AMR) dissemination. However, there is limited information on how heavy metals may act as a selective pressure on AMR in the primary food production environment. This review aims to examine the literature on this topic in order to identify knowledge gaps. A total of 73 studies, which met pre-established criteria, were included. These investigations were undertaken between 2008 and 2021, with a significant increase in the last three years. The majority of studies included were undertaken in China. Soil, water and manure were the most common samples analysed, and the sampling locations varied from areas with a natural presence of heavy metals, areas intentionally amended with heavy metals or manure, to areas close to industrial activity or mines. Fifty-four per cent of the investigations focused on the analysis of four or more heavy metals, and copper and zinc were the metals most frequently analysed (n = 59, n = 49, respectively). The findings of this review highlight a link between heavy metals and AMR in the primary food production environment. Heavy metals impacted the abundance and dissemination of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), with MGEs also observed as playing a key role in the spread of ARGs and metal resistance genes (MRGs). Harmonization of methodologies used in future studies would increase the opportunity for comparison between studies. Further research is also required to broaden the availability of data at a global level.

Genomic analysis reveals the presence of emerging pathogenic Klebsiella lineages aboard the International Space Station.

IMPORTANCE: The International Space Station (ISS) is a unique, hermetically sealed environment, subject to environmental pressures not encountered on Earth, including microgravity and radiation (cosmic ionising/UV). While bacteria's adaptability during spaceflight remains elusive, recent research suggests that it may be species and even clone-specific. Considering the documented spaceflight-induced suppression of the human immune system, a deper understanding of the genomics of potential human pathogens in space could shed light on species and lineages of medical astromicrobiological significance. In this study, we used hybrid assembly methods and comparative genomics to deliver a comprehensive genomic characterization of 10 Klebsiella isolates retrieved from the ISS. Our analysis unveiled that Klebsiella quasipneumoniae ST138 demonstrates both spatial and temporal persistence aboard the ISS, showing evidence of genomic divergence from its Earth-based ST138 lineage. Moreover, we characterized plasmids from Klebsiella species of ISS origin, which harbored genes for disinfectant resistance and enhanced thermotolerance, suggestin possible adaptive advantages. Furthermore, we identified a mobile genetic element containing a hypervirulence-associated locus belonging to a Klebsiella pneumoniae isolate of the "high-risk" ST101 clone. Our work provides insights into the adaptability and persistence of Klebsiella species during spaceflight, highlighting the importance of understanding the dynamics of potential pathogenic bacteria in such environments.

Medical Astro-Microbiology: Current Role and Future Challenges.

The second and third decades of the twenty-first century are marked by a flourishing of space technology which may soon realise human aspirations of a permanent multiplanetary presence. The prevention, control and management of infection with microbial pathogens is likely to play a key role in how successful human space aspirations will become. This review considers the emerging field of medical astro-microbiology. It examines the current evidence regarding the risk of infection during spaceflight via host susceptibility, alterations to the host's microbiome as well as exposure to other crew members and spacecraft's microbiomes. It also considers the relevance of the hygiene hypothesis in this regard. It then reviews the current evidence related to infection risk associated with microbial adaptability in spaceflight conditions. There is a particular focus on the International Space Station (ISS), as one of the only two  crewed objects in low Earth orbit. It discusses the effects of spaceflight related stressors on viruses and the infection risks associated with latent viral reactivation and increased viral shedding during spaceflight. It then examines the effects of the same stressors on bacteria, particularly in relation to changes in virulence and drug resistance. It also considers our current understanding of fungal adaptability in spaceflight. The global public health and environmental risks associated with a possible re-introduction to Earth of invasive species are also briefly discussed. Finally, this review examines the largely unknown microbiology and infection implications of celestial body habitation with an emphasis placed on Mars. Overall, this review summarises much of our current understanding of medical astro-microbiology and identifies significant knowledge gaps.

Characterization of cephalosporin and fluoroquinolone resistant Enterobacterales from Irish farm waste by whole genome sequencing.

Background: The Enterobacterales are a group of Gram-negative bacteria frequently exhibiting extended antimicrobial resistance (AMR) and involved in the transmission of resistance genes to other bacterial species present in the same environment. Due to their impact on human health and the paucity of new antibiotics, the World Health Organization (WHO) categorized carbapenem resistant and ESBL-producing as critical. Enterobacterales are ubiquitous and the role of the environment in the transmission of AMR organisms or antimicrobial resistance genes (ARGs) must be examined in tackling AMR in both humans and animals under the one health approach. Animal manure is recognized as an important source of AMR bacteria entering the environment, in which resistant genes can accumulate. Methods: To gain a better understanding of the dissemination of third generation cephalosporin and fluoroquinolone resistance genes between isolates in the environment, we applied whole genome sequencing (WGS) to Enterobacterales (79 E. coli, 1 Enterobacter cloacae, 1 Klebsiella pneumoniae, and 1 Citrobacter gillenii) isolated from farm effluents in Ireland before (n = 72) and after (n = 10) treatment by integrated constructed wetlands (ICWs). DNA was extracted using the MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) followed by WGS on a MiSeq platform (Illumina, Eindhoven, Netherlands) using v3 chemistry as 300-cycle paired-end runs. AMR genes and point mutations were identified and compared to the phenotypic results for better understanding of the mechanisms of resistance and resistance transmission. Results: A wide variety of cephalosporin and fluoroquinolone resistance genes (mobile genetic elements (MGEs) and chromosomal mutations) were identified among isolates that mostly explained the phenotypic AMR patterns. A total of 31 plasmid replicon types were identified among the 82 isolates, with a subset of them (n = 24), identified in E. coli isolates. Five plasmid replicons were confined to the Enterobacter cloacae isolate and two were confined to the Klebsiella pneumoniae isolate. Virulence genes associated with functions including stress, survival, regulation, iron uptake secretion systems, invasion, adherence and toxin production were identified. Conclusion: Our study showed that antimicrobial resistant organisms (AROs) can persist even following wastewater treatment and could transmit AMR of clinical relevance to the environment and ultimately pose a risk to human or animal health.

Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples.

Environmental water is considered one of the main vehicles for the transmission of antimicrobial resistance (AMR), posing an increasing threat to humans and animals health. Continuous efforts are being made to eliminate AMR; however, the detection of AMR pathogens from water samples often requires at least one culture step, which is time-consuming and can limit sensitivity. In this study, we employed comparative genomics to identify the prevalence of AMR genes within among: Escherichia coli, Klebsiella, Salmonella enterica and Acinetobacter, using publicly available genomes. The mcr-1, blaKPC (KPC-1 to KPC-4 alleles), blaOXA-48, blaOXA-23 and blaVIM (VIM-1 and VIM-2 alleles) genes are of great medical and veterinary significance, thus were selected as targets for the development of isothermal loop-mediated amplification (LAMP) detection assays. We also developed a rapid and sensitive sample preparation method for an integrated culture-independent LAMP-based detection from water samples. The developed assays successfully detected the five AMR gene markers from pond water within 1 h and were 100% sensitive and specific with a detection limit of 0.0625 µg/mL and 10 cfu/mL for genomic DNA and spiked bacterial cells, respectively. The integrated detection can be easily implemented in resource-limited areas to enhance One Health AMR surveillances and improve diagnostics.

Characterization of Shiga toxin-producing Escherichia coli presence, serogroups and risk factors from private groundwater sources in western Ireland.

Over recent years, Ireland has reported the highest crude incidence rates of Shiga toxin-producing Escherichia coli (STEC) enteritis in Europe. Unregulated private groundwater sources have emerged as an important potential transmission route for STEC, with up to 750,000 Irish residents reliant on these sources for domestic waters. This study aimed to investigate the prevalence and serogroup profile of STEC contamination from domestic private wells in western Ireland. Fifty-two groundwater sources were analysed during two sampling campaigns in the autumn (September/October) of 2019 (n = 21) and 2021 (n = 31). Untreated groundwater samples (30 L) were collected and analysed using the "CapE" (capture, amplify, extract) method. Extracted DNA was tested using multiplex real-time PCR for Shiga toxin stx1 and/or stx2 and eae genes. STEC positive DNA samples were tested for clinically relevant serogroups by real-time PCR. Data relating to 27 potential groundwater contamination risk factors were geospatially linked to each well and assessed for association with E. coli, stx1 and/or stx2 and eae presence/absence. Overall, 20/52 wells (38.4 %) were positive for E. coli (median concentration 8.5 MPN/100 mL as assessed by Colilert-18 method). Stx1 and/or stx2 was detected in 10/52 (19.2 %) wells overall and 8/20 E. coli positive wells, equating to a STEC to "generic" E. coli detection ratio of 40 %. Six of these wells (30 %) were also positive for eae. One or more serogroup-specific gene targets were identified in all but one stx1 and/or stx2 positive sample, with O145 (n = 6), O157 (n = 5) and O103 (n = 4) most prevalent. STEC presence was significantly associated with decreasing well depth (U = -2.243; p = 0.024) and increasing 30-day mean antecedent rainfall (U = 2.126; p = 0.034). Serogroup O104 was associated with increased sheep density (U = 2.089; p = 0.044) and detection of stx1 and/or stx2 + eae with increased septic tank density (U = 2.246 p = 0.023). Findings indicate high detection rates of clinically relevant STEC in E. coli contaminated groundwater sources in Ireland.

Longitudinal carriage of antimicrobial resistant Enterobacterales in healthy individuals in Ireland - Assessing the impact of recreational water use on duration of carriage.

The increasing prevalence of extended-spectrum beta-lactamase (ESBL) producing Enterobacterales (ESBL-PE) and carbapenemase-producing Enterobacterales (CPE) is a major public health concern worldwide. Despite the associated risk of infection from gut colonisation with a resistant Enterobacterales, the incidence and duration of carriage in healthy individuals is poorly studied. This "persistence study" is the first in Ireland to assess the longitudinal carriage of ESBL-PE and CPE in healthy individuals. A cohort of 45 participants, 22 of whom were colonised with ESBL-PE, was recruited from a recently completed point prevalence study that investigated colonisation in recreational water users (WU) versus controls. Six bi-monthly faecal samples per participant were analysed for CPE and ESBL-PE over one year and the relationship between persistent colonisation and exposure to natural waters was investigated. For 11 of 45 participants (24.4 %) ESBL-E. coli (ESBL-EC) was detected in at least one sample. Genomic analysis revealed that six participants harboured the same ESBL-EC strains as identified in the preceding study. ESBL-EC persisted in the gut for a median duration of 10.3 months (range 4-23 months), consistent with previous research. Five participants (11.1 %) carried ESBL-EC for the entire study year. The carbapenemase gene blaIMI-2 was detected once. Colonisation was higher in water users during the non-bathing season (n = 10, November 2021-April 2022), than during the bathing season (n = 5, May 2022-September 2022) [relative risk 1.99 (95 % CI 0.34-11.71)]. However, overall WU were less likely to be colonised with ESBL-EC than controls (19 % vs 25 % respectively, RR 0.76, CI 0.24-2.34). Further research is warranted to better understand the factors influencing the persistence of gut colonisation with ESBL-EC and CPE and to what extent bathing water quality impacts colonisation for those regularly exposed.

Assessing the impact of recreational water use on carriage of antimicrobial resistant organisms.

Understanding the role of exposure to natural recreational waters in the acquisition and transmission of antimicrobial resistance (AMR) is an area of increasing interest. A point prevalence study was carried out in the island of Ireland to determine the prevalence of colonisation with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) in recreational water users (WU) and matched controls. A total of 411 adult participants (199 WU, 212 controls) submitted at least one faecal sample between September 2020 - October 2021. In total, 80 Enterobacterales were isolated from 73 participants. ESBL-PE were detected in 29 (7.1 %) participants (7 WU, 22 controls), and CRE were detected in nine (2.2 %) participants (4 WU, 5 controls). No carbapenemase-producing Enterobacterales (CPE) were detected. WU were significantly less likely to harbour ESBL-PE than controls (risk ratio = 0.34, 95 % CI 0.148 to 0.776, χ2 7.37, p = 0.007). This study demonstrates the occurrence of ESBL-PE and CRE in healthy participants in Ireland. Recreational exposure to bathing water in Ireland was associated with a decreased prevalence of colonisation with ESBL-PE and CRE.

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. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

First reported detection of the mobile colistin resistance genes, mcr-8 and mcr-9, in the Irish environment.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n = 2), healthcare facility wastewater (n = 2), wastewater treatment plant influent (n = 1) and integrated constructed wetland influent (piggery farm waste) (n = 1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, blaOXA-48 (n = 2) and blaNDM-1 (n = 1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

Inter-hospital outbreak of Klebsiella pneumoniae producing KPC-2 carbapenemase in Ireland.

OBJECTIVES: To describe an outbreak of KPC-2-producing Klebsiella pneumoniae with inter-hospital spread and measures taken to control transmission. METHODS: Between January and March 2011, 13 K. pneumoniae isolates were collected from nine patients at hospital A and two patients at hospital B. Meropenem, imipenem and ertapenem MICs were determined by Etest, carbapenemase production was confirmed by the modified Hodge method and by a disc synergy test, and confirmed carbapenemase producers were tested for the presence of carbapenemase-encoding genes by PCR. PFGE, plasmid analysis, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST) analysis were performed on all or a subset of isolates. RESULTS: Meropenem, imipenem and ertapenem MICs were 4 to >32, 8-32 and >16 mg/L, respectively. PCR and sequencing confirmed the presence of bla(KPC-2). PFGE identified four distinguishable (≥88%) pulsed-field profiles (PFPs). Isolates distinguishable by PFGE had identical MLVA profiles, and MLST analysis indicated all isolates belonged to the ST258 clone. Stringent infection prevention and control measures were implemented. Over a period of almost 8 months no further carbapenemase-producing Enterobacteriaceae (CPE) were isolated. However, KPC-2-producing K. pneumoniae was detected in two further patients in hospital A in August (PFP indistinguishable from previous isolates) and October 2011 (PFP similar to but distinguishable from previous isolates). CONCLUSIONS: Stringent infection prevention and control measures help contain CPE in the healthcare setting; however, in the case of hospital A, where CPE appears to be established in the population served, it may be virtually impossible to achieve eradication or avoid reintroduction into the hospital.