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.

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.

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.

A Point Prevalence Survey of Antibiotic Resistance in the Irish Environment, 2018-2019.

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 role of the natural aquatic environment in the dissemination of extended spectrum beta-lactamase and carbapenemase encoding genes: A scoping review.

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.

Antibiotic resistomes of healthy pig faecal metagenomes.

Antibiotic resistance reservoirs within food-producing animals are thought to be a risk to animal and human health. This study describes the minimum natural resistome of pig faeces as the bacteria are under no direct antibiotic selective pressure. The faecal resistome of 257 different genes comprised 56 core and 201 accessory resistance genes. The genes present at the highest relative abundances across all samples were tetW, tetQ, tet44, tet37, tet40, mefA, aadE, ant(9)-1, ermB and cfxA2. This study characterized the baseline resistome, the microbiome composition and the metabolic components described by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in healthy pig faeces, without antibiotic selective pressures. The microbiome hierarchical analysis resulted in a cluster tree with a highly similar pattern to that of the accessory resistome cluster tree. Functional capacity profiling identified genes associated with horizontal gene transfer. We identified a statistically significant positive correlation between the total antibiotic resistome and suggested indicator genes, which agree with using these genes as indicators of the total resistomes. The correlation between total resistome and total microbiome in this study was positive and statistically significant. Therefore, the microbiome composition influenced the resistome composition. This study identified a core and accessory resistome present in a cohort of healthy pigs, in the same conditions without antibiotics. It highlights the presence of antibiotic resistance in the absence of antibiotic selective pressure and the variability between animals even under the same housing, food and living conditions. Antibiotic resistance will remain in the healthy pig gut even when antibiotics are not used. Therefore, the risk of antibiotic resistance transfer from animal faeces to human pathogens or the environment will remain in the absence of antibiotics.

A robust, cost-effective method for DNA, RNA and protein co-extraction from soil, other complex microbiomes and pure cultures.

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co-recovered from the same biological samples. Commercial kits are currently available for the co-extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol-chloroform-based methods for nucleic acids co-extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost-effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high-throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co-extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram-positive and Gram-negative pure cultures.

Linking Microbial Community Structure and Function During the Acidified Anaerobic Digestion of Grass.

Harvesting valuable bioproducts from various renewable feedstocks is necessary for the critical development of a sustainable bioeconomy. Anaerobic digestion is a well-established technology for the conversion of wastewater and solid feedstocks to energy with the additional potential for production of process intermediates of high market values (e.g., carboxylates). In recent years, first-generation biofuels typically derived from food crops have been widely utilized as a renewable source of energy. The environmental and socioeconomic limitations of such strategy, however, have led to the development of second-generation biofuels utilizing, amongst other feedstocks, lignocellulosic biomass. In this context, the anaerobic digestion of perennial grass holds great promise for the conversion of sustainable renewable feedstock to energy and other process intermediates. The advancement of this technology however, and its implementation for industrial applications, relies on a greater understanding of the microbiome underpinning the process. To this end, microbial communities recovered from replicated anaerobic bioreactors digesting grass were analyzed. The bioreactors leachates were not buffered and acidic pH (between 5.5 and 6.3) prevailed at the time of sampling as a result of microbial activities. Community composition and transcriptionally active taxa were examined using 16S rRNA sequencing and microbial functions were investigated using metaproteomics. Bioreactor fraction, i.e., grass or leachate, was found to be the main discriminator of community analysis across the three molecular level of investigation (DNA, RNA, and proteins). Six taxa, namely Bacteroidia, Betaproteobacteria, Clostridia, Gammaproteobacteria, Methanomicrobia, and Negativicutes accounted for the large majority of the three datasets. The initial stages of grass hydrolysis were carried out by Bacteroidia, Gammaproteobacteria, and Negativicutes in the grass biofilms, in addition to Clostridia in the bioreactor leachates. Numerous glycolytic enzymes and carbohydrate transporters were detected throughout the bioreactors in addition to proteins involved in butanol and lactate production. Finally, evidence of the prevalence of stressful conditions within the bioreactors and particularly impacting Clostridia was observed in the metaproteomes. Taken together, this study highlights the functional importance of Clostridia during the anaerobic digestion of grass and thus research avenues allowing members of this taxon to thrive should be explored.