A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water.

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A Longitudinal Survey of Antibiotic-Resistant Enterobacterales in the Irish Environment, 2019-2020.

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 blaCTX-M, blaVIM, blaIMP, blaOXA-48, blaNDM, and blaKPC 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 blaOXA-48 (n = 18), blaNDM (n = 14), blaKPC (n = 4) and blaOXA-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.

A comparative risk ranking of antibiotic pollution from human and veterinary antibiotic usage - An Irish case study.

Antibiotic use in the healthcare and agriculture sectors has resulted in levels being found in environmental compartments including surface waters. This can create a selective pressure toward antibiotic resistance development, representing a potential risk to human health. Examining the Irish scenario, this screening paper develops a novel risk ranking model to comparatively assess, on a national scale, the predicted amount of antibiotics entering water bodies as a result of their use in healthcare and agricultural sectors, and the subsequent risk of antibiotic resistance development. Probabilistic modelling approaches, based on data sourced from published literature on antibiotics, are used to account for inherent uncertainty and variability in the input factors; usage, metabolism, degradation and wastewater removal rates, estimating the mass of six antibiotic classes released daily from both sectors. These mass estimates are used to generate predicted concentrations and risk quotient values for each drug class, utilising estimated minimum inhibitory concentration values sourced from the literature. Modelled results predict higher risk quotient (RQ) values in the healthcare compared to agriculture sector, with macrolides and penicillins ranking highest in terms of RQ value. A lower RQ is also predicted from human-use tetracyclines, trimethoprim, and quinolones. Avenues for runoff reduction for each antibiotic class, in particular the higher-risk classes, in both usage sectors are discussed. For validation, predicted levels are compared to observed levels of antibiotic residues in Ireland. Key knowledge gaps to assist prediction and modelling of antibiotic pollution in future studies are also discussed. This research paper establishes a protocol and model structure, applicable to other regions, to compare the contributions of healthcare and agriculture to antibiotic pollution, and identifies highest-ranked antibiotic classes in terms of potential resistance development for prioritisation in the Irish situation.

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods.

The Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples-same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)-suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Natural recreational waters and the risk that exposure to antibiotic resistant bacteria poses to human health.

Antimicrobial resistance (AMR) is widely recognised as a considerable threat to human health, wellbeing and prosperity. Many clinically important antibiotic resistance genes are understood to have originated in the natural environment. However, the complex interactions between humans, animals and the environment makes the health implications of environmental AMR difficult to quantify. This narrative review focuses on the current state of knowledge regarding antibiotic resistant bacteria (ARB) in natural bathing waters and implications for human health. It considers the latest research focusing on the transmission of ARB from bathing waters to humans. The limitations of existing evidence are discussed, as well as research priorities. The authors are of the opinion that future studies should include faecally contaminated bathing waters and people exposed to these environments to accurately parameterise environment-to-human transmission.

Critically important antimicrobial resistant Enterobacteriaceae in Irish farm effluent and their removal in integrated constructed wetlands.

This study investigated the ability of Integrated Constructed Wetlands (ICWs) to remove critically important antimicrobial resistant organisms (AROs) from farm wastewater. Influent samples from the untreated farm waste and effluent samples taken at the end of the ICW system were collected monthly from four ICWs, serving four different farm types (suckler, dairy, dairy & poultry and pig). Using selective media to screen for the presence of carbapenemase resistant organisms, plasmid mediated and AmpC ß-Lactamase producing organisms (ESBL/pAmpC) and fluoroquinolone resistant organisms, a total of 82 AROs were obtained with the majority being E. coli (n = 79). Statistically significant were the differences on the number of AROs isolated from influent (higher) compared to effluent, as well as a seasonal effect, with less AROs recovered during winter in comparison to other seasons (P < 0.05). On the other hand, there was no significant differences in the recovery of AROs on different farms. The majority of isolates from each of the farms (99%) were multi drug resistant, with 65% resistant to seven or more antimicrobials. A high incidence of tetracycline, trimethoprim/sulfamethoxazole, and ampicillin resistance was common to the isolates from all four farms but there were differences in ESBL levels with 63% of the isolates recovered from Farm 4 (piggery) being ESBLs compared to 18%, 36% and 4.5% recovered from Farms 1 (suckler), 2 (dairy) and 3 (dairy & poultry), respectively. No carbapenemase producing organisms were isolated. Our results showed that ICWs are effective in removing critically important AROs from farm wastewater on all four farm types.

Evaluating the potential for exposure to organisms of public health concern in naturally occurring bathing waters in Europe: A scoping review.

Globally, water-based bathing pastimes are important for both mental and physical health. However, exposure to waterborne organisms could present a substantial public health issue. Bathing waters are shown to contribute to the transmission of illness and disease and represent a reservoir and pathway for the dissemination of antimicrobial resistant (AMR) organisms. Current bathing water quality regulations focus on enumeration of faecal indicator organisms and are not designed for detection of specific waterborne organisms of public health concern (WOPHC), such as antimicrobial resistant (AMR)/pathogenic bacteria, or viruses. This investigation presents the first scoping review of the occurrence of waterborne organisms of public health concern (WOPHC) in identified natural bathing waters across the European Union (EU), which aimed to critically evaluate the potential risk of human exposure and to assess the appropriateness of the current EU bathing water regulations for the protection of public health. Accordingly, this review sought to identify and synthesise all literature pertaining to a selection of bacterial (Campylobacter spp., Escherichia coli, Salmonella spp., Shigella spp., Vibrio spp., Pseudomonas spp., AMR bacteria), viral (Hepatitis spp., enteroviruses, rotavirus, adenovirus, norovirus), and protozoan (Giardia spp., and Cryptosporidium spp.) contaminants in EU bathing waters. Sixty investigations were identified as eligible for inclusion and data was extracted. Peer-reviewed investigations included were from 18 countries across the EU, totalling 87 investigations across a period of 35 years, with 30% published between 2011 and 2015. A variety of water bodies were identified, with 27 investigations exclusively assessing coastal waters. Waterborne organisms were classified into three categories; bacteria, viruses, and protozoa; amounting to 58%, 36% and 17% of the total investigations, respectively. The total number of samples across all investigations was 8,118, with detection of one or more organisms in 2,449 (30%) of these. Viruses were detected in 1281 (52%) of all samples where WOPHC were found, followed by bacteria (865(35%)) and protozoa (303(12%)). Where assessed (442 samples), AMR bacteria had a 47% detection rate, emphasising their widespread occurrence in bathing waters. Results of this scoping review highlight the potential public health risk of exposure to WOPHC in bathing waters that normally remain undetected within the current monitoring parameters.