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Objectives: Escherichia coli sequence type (ST) 73 is a pandemic lineage of the ExPEC (Extraintestinal Pathogenic E. coli) family associated with conserved virulence. We report the complete genome of a genomically hypervirulent E. coli ST73 strain isolated from the oral cavity of a patient with a diagnosis of treatment resistant schizophrenia and receiving clozapine treatment., Methods: E. coli strain GABEEC132 underwent second and third generation sequencing with Illumina and Oxford-Nanopore-Technologies (ONT) platforms. Antibiotic Resistance Genes (ARGs) and Virulence Factors (VFs) were bioinformatically identified using the NCBI-AMR-Finder-Plus database and Virulence-Factors-database (VFDB), respectively. To contextualize the genome within a broader epidemiological framework, phylogenetic analysis was conducted using representative genomes of E. coli ST73 O6:H1 (n=55)., Results: E. coli strain GABEEC132 was identified as possessing the O6:H1 serotype and classified within the B2 phylogroup. The strain exhibited a high genomic virulence load, encoding for 194 VFs. Additionally, it encoded three ARGs, including an acquired blaTEM-1 located on a rep_cluster_2350 8 237 Kb mobilisable plasmid, presenting phenotypic resistance to ampicillin and piperacillin., Conclusion: This report provides novel insights into the oral prevalence of genotypically hypervirulent and drug-resistant E. coli ST73, a pandemic lineage., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Antimicrobial resistant bacteria can occur in the primary food production environment. The emergence and dissemination of antimicrobial resistance (AMR) in the environment can be influenced by several factors, including the presence of heavy metals. The aim of this study was to examine the presence and characteristics of antimicrobial resistant Enterobacterales in soils and spinach grown in soils with and without zinc amendment. A total of 160 samples (92 soil and 68 spinach) were collected from two locations, in which some plots had been amended with zinc. Samples were cultured on selective agars for detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL), carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales. Samples were also cultured for enumeration of total Enterobacterales. Isolates were identified by MALDI-TOF. Antimicrobial susceptibility testing was carried out in accordance with EUCAST and CLSI criteria. The whole genome sequence (WGS) of selected isolates was determined. Inductively coupled plasma atomic emission spectrometry was also performed on soil samples in order to measure the concentration of zinc. In total 20 antimicrobial resistant Enterobacterales were isolated from the soil (n = 8) and spinach samples (n = 12). In both sample types, Serratia fonticola (n = 16) was the dominant species, followed by Escherichia coli (n = 1), Citrobacter freundii (n = 1) and Morganella morganii (n = 1) detected in spinach samples, and Enterobacter cloacae (n = 1) detected in a soil sample. The WGS identified genes conferring resistance to different antimicrobials in agreement with the phenotypic results; 14 S. fonticola isolates were confirmed as ESBL producers and harboured the bla FONA gene. Genes that encoded for zinc resistance and multidrug efflux pumps, transporters that can target both antimicrobials and heavy metals, were also identified. Overall, the findings of this study suggest the presence of zinc did not influence the AMR Enterobacterales in soil or spinach samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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.

Heavy metals occur naturally in the environment, and their concentration varies in soil across different regions. However, the presence of heavy metals may influence the antimicrobial resistance (AMR) in bacterial populations. Therefore, the objective of this study was to investigate and characterise the antimicrobial resistance profiles of Enterobacterales in soil and bovine milk filters from high and low zinc-containing regions in Ireland. In total, 50 soil samples and 29 milk filters were collected from two geographic locations with varying soil zinc concentrations. Samples were cultured for the enumeration and detection of Enterobacterales. Specifically, extended-spectrum beta-lactamase-producing Enterobacterales, carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales were isolated using selective media. Species identification was performed using MALDI-TOF. The phenotypic resistance profiles of selected Enterobacterales were determined by disk diffusion testing, following EUCAST and CLSI criteria; while, the genotypic resistance profiles of the same isolates were determined by whole genome sequencing (WGS). Heavy metal concentrations were also measured for all soil samples. A total of 40 antimicrobial resistant Enterobacterales were identified in soil (n = 31) and milk filters (n = 9). The predominant species detected in the high zinc-containing region was Escherichia coli in both sample types (soil n = 10, milk filters n = 2), while in the low zinc-containing region Serratia fonticola was predominant in soil samples (n = 8) and E. coli in milk filters (n = 4). Ten E. coli isolates identified from soil samples in the high zinc-containing region were multidrug resistant, showing resistance to all the antimicrobials tested, except for carbapenems. The WGS findings confirmed the phenotypic resistance results. Moreover, zinc resistance-associated genes and genes encoding for efflux pumps were identified. The current study revealed distinct phenotypic resistance profiles of Enterobacterales in low and high zinc-containing regions, and highlighted the benefit of utilising milk filters for AMR surveillance in dairy production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the nuclear genomes of 24 Mycena species, we find them to defy the expected patterns for fungi based on both their traditionally perceived saprotrophic ecology and substrate specializations. Mycena displayed massive genome expansions overall affecting all gene families, driven by novel gene family emergence, gene duplications, enlarged secretomes encoding polysaccharide degradation enzymes, transposable element (TE) proliferation, and horizontal gene transfers. Mainly due to TE proliferation, Arctic Mycena species display genomes of up to 502 Mbp (2-8× the temperate Mycena), the largest among mushroom-forming Agaricomycetes, indicating a possible evolutionary convergence to genomic expansions sometimes seen in Arctic plants. Overall, Mycena show highly unusual, varied mosaic-like genomic structures adaptable to multiple lifestyles, providing genomic illustration for the growing realization that fungal niche adaptations can be far more fluid than traditionally believed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inland saline ecosystems suffer multiple stresses (e.g., high radiation, salinity, water scarcity) that may compromise essential ecosystem functions such as organic matter decomposition. Here, we investigated the effects of drought on microbial colonization and decomposition of Sarcocornia fruticosa woody stems across different habitats in a saline watershed: on the dry floodplain, submerged in the stream channel and at the shoreline (first submerged, then emerged). Unexpectedly, weight loss was not enhanced in the submerged stems, while decomposition process differed between habitats. On the floodplain, it was dominated by fungi and high cellulolytic activity; in submerged conditions, a diverse community of bacteria and high ligninolytic activity dominated; and, on the shoreline, enzyme activities were like submerged conditions, but with a fungal community similar to the dry conditions. Results indicate distinct degradation paths being driven by different stress factors: strong water scarcity and photodegradation in dry conditions, and high salinity and reduced oxygen in wet conditions. This suggests that fungi are more resistant to drought, and bacteria to salinity. Overall, in saline watersheds, variations in multiple stress factors exert distinct environmental filters on bacteria and fungi and their role in the decomposition of plant material, affecting carbon cycling and microbial interactions., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Microbial necromass is increasingly recognized as an important fast-cycling component of the long-term carbon present in soils. To better understand how fungi and bacteria individually contribute to the decomposition of fungal necromass, three particle sizes (>500, 250-500, and <250 μm) of Hyaloscypha bicolor necromass were incubated in laboratory microcosms inoculated with individual strains of two fungi and two bacteria. Decomposition was assessed after 15 and 28 days via necromass loss, microbial respiration, and changes in necromass pH, water content, and chemistry. To examine how fungal-bacterial interactions impact microbial growth on necromass, single and paired cultures of bacteria and fungi were grown in microplates containing necromass-infused media. Microbial growth was measured after 5 days through quantitative PCR. Regardless of particle size, necromass colonized by fungi had higher mass loss and respiration than both bacteria and uninoculated controls. Fungal colonization increased necromass pH, water content, and altered chemistry, while necromass colonized by bacteria remained mostly unaltered. Bacteria grew significantly more when co-cultured with a fungus, while fungal growth was not significantly affected by bacteria. Collectively, our results suggest that fungi act as key early decomposers of fungal necromass and that bacteria may require the presence of fungi to actively participate in necromass decomposition., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

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-HS T , 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-HS T is most closely related to Virgibacillus halophilus 5B73C T , 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-HS T is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HS T = DSM 115946 T = NRRL B-65666 T ), and its closest neighbor, V. halophilus , is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73C T = DSM 21623 T = JCM 21758 T = KCTC 13935 T ). 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-6 T = DSM 22952 T = CCM 7714 T ). Within Amphibacillaceae ( n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known β-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., Competing Interests: The authors declare no conflict of interest.

Land degradation by deforestation adversely impacts soil properties, and long-term restoration practices have been reported to potentially reverse these effects, particularly on soil microorganisms. However, there is limited knowledge regarding the short-term effects of restoration on the soil bacterial community in semiarid areas. This study evaluates the bacterial community in soils experiencing degradation (due to slash-and-burn deforestation) and restoration (utilizing stone cordons and revegetation), in comparison to a native soil in the Brazilian semiarid region. Three areas were selected: (a) under degradation; (b) undergoing short-term restoration; and (c) a native area, and the bacterial community was assessed using 16S rRNA sequencing on soil samples collected during both dry and rainy seasons. The dry and rainy seasons exhibited distinct bacterial patterns, and native sites differed from degraded and restoration sites. Chloroflexi and Proteobacteria phyla exhibited higher prevalence in degraded and restoration sites, respectively, while Acidobacteria and Actinobacteria were more abundant in sites undergoing restoration compared to degraded sites. Microbial connections varied across sites and seasons, with an increase in nodes observed in the native site during the dry season, more edges and positive connections in the restoration site, and a higher occurrence of negative connections in the degradation site during the rainy season. Niche occupancy analysis revealed that degradation favored specialists over generalists, whereas restoration exhibited a higher prevalence of generalists compared to native sites. Specifically, degraded sites showed a higher abundance of specialists in contrast to restoration sites. This study reveals that land degradation impacts the soil bacterial community, leading to differences between native and degraded sites. Restoring the soil over a short period alters the status of the bacterial community in degraded soil, fostering an increase in generalist microbes that contribute to enhanced soil stability., (© 2024. The Author(s).)

The remediation of mixed contaminated soil is challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. Here, the effectiveness of different bioremediation strategies, namely, rhizoremediation with native plant species, mycoremediation with Pleurotus ostreatus spent mushroom substrate, and biostimulation with organic by-products (i.e., composted sewage sludge and spent mushroom substrate), for the recovery of a mixed contaminated soil from an abandoned gravel pit was studied. The combination of biostimulation and rhizoremediation led to the most significant increase in soil health, according to microbial indicator values. The application of composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. None of the strategies managed to decrease contamination levels below regulatory limits, but they did enhance soil health. It was concluded that the biological remediation treatments improved soil functioning in a short time, before the concentration of soil contaminants was significantly reduced., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 bla IMI-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., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Objectives: Escherichia coli sequence type (ST) 127 is a pandemic lineage that belongs to the extraintestial pathogenic (ExPEC) family, mainly associated with urinary tract infections and bloodstream infections. Here, we report the complete genome of an E. coli ST 127 isolate which was identified in the saliva of a patient with treatment-resistant schizophrenia (TRS) exhibiting no signs of infection. The objective of this work is to determine the mobile genetic elements (MGEs), antibiotic resistance genes (ARGs), and virulence factors (VFs) that contribute to the pathogenicity of such ST127 isolates., Methods: Whole-genome sequencing (WGS) of isolate GABEEC10 was performed using DNABseq and Nanopore MinION platforms. Hybrid assembly of GABEEC10 was conducted with Unicycler v. 0.5.0. and annotated using PROKKA v1.14.5. Comparative genomics and phylogenomics were conducted using average nucleotide identity (ANI) and approximately-maximum-likelihood phylogenetic inference. ARGs, VFs, and serotyping were identified with Abricate v1.0.0 using CARD, vfdb, and EcOH databases, respectively., Results: Escherichia coli salivary isolate GABEEC10 was identified to belong to phylogroup B2 and have a serotype of O6 H31 with a total genome length of 4,940,530 bp and a mean guanine-cytosine (GC) content of 50.40 %. GABEEC10 was identified to have a highly virulent genotype with the presence of 84 VFs in addition to 44 ARGs, including an acquired blaTEM-30. The strain was identified to additionally carry four mobilisable plasmids., Conclusion: We report the complete genome of E. coli GABAEEC10 that can be used for gaining insights into the pathogenicity, drug resistance mechanisms, and dissemination patterns of the emerging pandemic lineage ST 127., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency., (© 2023. The Author(s).)

Public-domain availability for bioinformatics software resources is a key requirement that ensures long-term permanence and methodological reproducibility for research and development across the life sciences. These issues are particularly critical for widely used, efficient, and well-proven methods, especially those developed in research settings that often face funding discontinuities. We re-launch a range of established software components for computational genomics, as legacy version 1.0.1, suitable for sequence matching, masking, searching, clustering and visualization for protein family discovery, annotation and functional characterization on a genome scale. These applications are made available online as open source and include MagicMatch, GeneCAST, support scripts for CoGenT-like sequence collections, GeneRAGE and DifFuse, supported by centrally administered bioinformatics infrastructure funding. The toolkit may also be conceived as a flexible genome comparison software pipeline that supports research in this domain. We illustrate basic use by examples and pictorial representations of the registered tools, which are further described with appropriate documentation files in the corresponding GitHub release., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Vasileiou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited < 93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average nucleotide identity and digital DNA-DNA hybridization values, when compared to any known bacterial species, were < 94% and <50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including β-lactone and type III polyketide synthase (T3PKS) clusters. Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10 T (= NRRL B-65660 T = DSM 115933 T ), Leifsonia virtsii F6&#95;8S&#95;P&#95;1A T (= NRRL B-65661 T = DSM 115931 T ), Leifsonia williamsii F6&#95;8S&#95;P&#95;1B T (= NRRL B-65662 T = DSM 115932 T ), Paenibacillus vandeheii F6&#95;3S&#95;P&#95;1C T (= NRRL B-65663 T = DSM 115940 T ), and Sporosarcina highlanderae F6&#95;3S&#95;P&#95;2 T (= NRRL B-65664 T = DSM 115943 T ). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions., (© 2023. The Author(s).)

Natural ecosystems can become significant reservoirs and/or pathways for antimicrobial resistance (AMR) dissemination, with the potential to affect nearby microbiological, animal, and ultimately human communities. This is further accentuated in environments that provide direct human exposure, such as drinking water. To date, however, few studies have investigated AMR dissemination potential and the presence of co-selective stressors (e.g., metals/metalloids) in groundwater environments of human health significance. Accordingly, the present study analysed samples from rural (drinking) groundwater supplies (i.e., private wells) in the Republic of Ireland, where land use is dominated by livestock grazing activities. In total, 48 Escherichia coli isolates tested phenotypically for antimicrobial susceptibility in an earlier study were further subject to whole genome sequencing (WGS) and corresponding water samples were further analysed for trace metal/metalloid concentrations. Eight isolates (i.e., 16.7%) were genotypically resistant to antimicrobials, confirming prior phenotypic results through the identification of ten antimicrobial resistance genes (ARGs); namely: aph(3″)-lb (strA; n=7), aph(6)-Id (strA; n = 6), blaTEM (n = 6), sul2 (n = 6), tetA (n = 4), floR (n = 2), dfrA5 (n = 1), tetB (n = 1), and tetY (n = 1). Additional bioinformatic analysis revealed that all ARGs were plasmid-borne, except for two of the six sul2 genes, and that 31.2% of all tested isolates (n = 15) and 37.5% of resistant ones (n = 3) carried virulence genes. Study results also found no significant relationships between metal concentrations and ARG abundance. Additionally, just one genetic linkage was identified between ARGs and a metal resistance gene (MRG), namely merA, a mercury-resistant gene found on the same plasmid as blaTEM, dfrA5, strA, strB, and sul2 in the only isolate of inferred porcine (as opposed to bovine) origin. Overall, findings suggest that ARG (and MRG) acquisition may be occurring prior to groundwater ingress, and are likely a legacy issue arising from agricultural practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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, χ 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis .•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis .•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Despite growing interest in fungal necromass decomposition due to its importance in soil carbon retention, whether a consistent group of microorganisms is associated with decomposing necromass remains unresolved. Here, we synthesize knowledge on the composition of the bacterial and fungal communities present on decomposing fungal necromass from a variety of fungal species, geographic locations, habitats, and incubation times. We found that there is a core group of both bacterial and fungal genera (i.e. a core fungal necrobiome), although the specific size of the core depended on definition. Based on a metric that included both microbial frequency and abundance, we demonstrate that the core is taxonomically and functionally diverse, including bacterial copiotrophs and oligotrophs as well as fungal saprotrophs, ectomycorrhizal fungi, and both fungal and animal parasites. We also show that the composition of the core necrobiome is notably dynamic over time, with many core bacterial and fungal genera having specific associations with the early, middle, or late stages of necromass decomposition. While this study establishes the existence of a core fungal necrobiome, we advocate that profiling the composition of fungal necromass decomposer communities in tropical environments and other terrestrial biomes beyond forests is needed to fill key knowledge gaps regarding the global nature of the fungal necrobiome., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Guidelines identifying appropriate DNA extraction methods for both museum and modern biological samples are scarce or non-existent for mammalian species. Yet, obtaining large-scale genetic material collections are vital for conservation and management purposes. In this study, we evaluated five protocols making use of either spin-column, organic solvents, or magnetic bead-based methods for DNA extraction on skin samples from both modern, traffic-killed (n = 10) and museum (n = 10) samples of European hedgehogs, Ericaneus europaeus. We showed that phenol-chloroform or silica column (NucleoSpin Tissue) protocols yielded the highest amount of DNA with satisfactory purity compared with magnetic bead-based protocols, especially for museum samples. Furthermore, extractions using the silica column protocol appeared to produce longer DNA fragments on average than the other methods tested. Our investigation demonstrates that both commercial extraction kits and phenol-chloroform protocol retrieve acceptable DNA concentrations for downstream processes, from degraded remnants of traffic-killed and museum samples of mammalian specimens. Although all the tested methods could be applied depending on the research questions and laboratory conditions, commercial extraction kits may be preferred due to their effectiveness, safety and the higher quality of the DNA extractions., (© 2023. Springer Nature Limited.)

Microbial electrosynthesis (MES) is a promising carbon utilization technology, but the low-value products (i.e., acetate or methane) and the high electric power demand hinder its industrial adoption. In this study, electrically efficient MES cells with a low ohmic resistance of 15.7 mΩ m 2 were operated galvanostatically in fed-batch mode, alternating periods of high CO 2 and H 2 availability. This promoted acetic acid and ethanol production, ultimately triggering selective (78% on a carbon basis) butyric acid production via chain elongation. An average production rate of 14.5 g m -2  d -1 was obtained at an applied current of 1.0 or 1.5 mA cm -2 , being Megasphaera sp. the key chain elongating player. Inoculating a second cell with the catholyte containing the enriched community resulted in butyric acid production at the same rate as the previous cell, but the lag phase was reduced by 82%. Furthermore, interrupting the CO 2 feeding and setting a constant pH 2 of 1.7-1.8 atm in the cathode compartment triggered solventogenic butanol production at a pH below 4.8. The efficient cell design resulted in average cell voltages of 2.6-2.8 V and a remarkably low electric energy requirement of 34.6 kWh el kg -1 of butyric acid produced, despite coulombic efficiencies being restricted to 45% due to the cross-over of O 2 and H 2 through the membrane. In conclusion, this study revealed the optimal operating conditions to achieve energy-efficient butyric acid production from CO 2 and suggested a strategy to further upgrade it to valuable butanol., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance., (© 2023. The Author(s).)

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, bla OXA-48 (n = 2) and bla NDM-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mycoremediation with mushroom growth substrates can be used for the recovery of mixed contaminated soils due to the benefits derived from the physicochemical characteristics of the substrates, the activity of extracellular enzymes secreted by the fungi, and the presence of the fungal mycelia. The objective of this work was to assess the potential of Agaricus bisporus and Pleurotus ostreatus growth substrates (inoculated mushroom substrates vs. spent mushroom substrates) for the mycoremediation of soils co-contaminated with lead and lindane (γ-HCH). We compared the efficiency of these mycoremediation strategies with the phytoremediation with Brassica spp. Or Festuca rubra plants, in terms of both reduction in contaminant levels and enhancement of soil health. An enhanced soil health was achieved as a result of the application of mycoremediation treatments, compared to phytoremediation and control (untreated) treatments. The application of P. ostreatus inoculated substrate led to the most significant reduction in γ-HCH concentration (up to 88.9% compared to corresponding controls). In the presence of inoculated mushroom substrate, P. ostreatus fruiting bodies extracted more Pb than Brassica spp. Or F. rubra plants. Mycoremediation with P. ostreatus growth substrates appears a promising strategy for the recovery of the health of soils co-contaminated with Pb and γ-HCH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Plants modulate the soil microbiota and select a specific microbial community in the rhizosphere. However, plant domestication reduces genetic diversity, changes plant physiology, and could have an impact on the associated microbiome assembly. Here, we used 16S rRNA gene sequencing to assess the microbial community in the bulk soil and rhizosphere of wild, semi-domesticated, and domesticated genotypes of lima bean (Phaseolus lunatus), to investigate the effect of plant domestication on microbial community assembly. In general, rhizosphere communities were more diverse than bulk soil, but no differences were found among genotypes. Our results showed that the microbial community's structure was different from wild and semi-domesticated as compared to domesticated genotypes. The community similarity decreased 57.67% from wild to domesticated genotypes. In general, the most abundant phyla were Actinobacteria (21.9%), Proteobacteria (20.7%), Acidobacteria (14%), and Firmicutes (9.7%). Comparing the different genotypes, the analysis showed that Firmicutes (Bacillus) was abundant in the rhizosphere of the wild genotypes, while Acidobacteria dominated semi-domesticated plants, and Proteobacteria (including rhizobia) was enriched in domesticated P. lunatus rhizosphere. The domestication process also affected the microbial community network, in which the complexity of connections decreased from wild to domesticated genotypes in the rhizosphere. Together, our work showed that the domestication of P. lunatus shaped rhizosphere microbial communities from taxonomic to a functional level, changing the abundance of specific microbial groups and decreasing the complexity of interactions among them., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., Competing Interests: Conflict of InterestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2023.)

The physiological performance of organisms depends on their environmental context, resulting in performance-response curves along environmental gradients. Parasite performance-response curves are generally expected to be broader than those of their hosts due to shorter generation times and hence faster adaptation. However, certain environmental conditions may limit parasite performance more than that of the host, thereby providing an environmental refuge from disease. Thermal disease refuges have been extensively studied in response to climate warming, but other environmental factors may also provide environmental disease refuges which, in turn, respond to global change. Here, we (1) showcase laboratory and natural examples of refuges from parasites along various environmental gradients, and (2) provide hypotheses on how global environmental change may affect these refuges. We strive to synthesize knowledge on potential environmental disease refuges along different environmental gradients including salinity and nutrients, in both natural and food-production systems. Although scaling up from single host-parasite relationships along one environmental gradient to their interaction outcome in the full complexity of natural environments remains difficult, integrating host and parasite performance-response can serve to formulate testable hypotheses about the variability in parasitism outcomes and the occurrence of environmental disease refuges under current and future environmental conditions., (© 2023 The Authors. Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Prendergast, Slowey, Burgess, Murphy, Johnston, Morris, O’ Doherty, Moriarty and Gutierrez.)

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 β-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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species ( Klebsiella pneumoniae , Escherichia coli , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter hormaechei and Citrobacter freundii ) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli .

Objectives: Kalamiella piersonii is a newly identified bacterial species, first isolated from surfaces of the International Space Station (ISS). It also appears as a novel human pathogen reported to be implicated in bacteremia and kidney stone disease. Here, we report the first complete genome of a multidrug-resistant strain of K. piersonii (GABEKP28), isolated from the saliva of a patient with treatment-resistant schizophrenia (TRS), to determine the mobile genetic elements (MGEs), antibiotic resistance genes (ARGs), and virulence factors (VFs) harboured by such a strain of this novel species., Methods: Whole-genome sequencing was performed using DNABSEQ (PE150) and Nanopore MinION platforms. Hybrid assembly was conducted using Unicycler v0.5.0. Genome assembly quality was verified using QUAST v5.0.2. The assembly was annotated using PROKKA v1.14.5. ARGs and VFs were identified using Abricate v1.0.0., Results: K. piersonii strain GABEKP28 was classified as multidrug-resistant while also carrying plasmidic genetic determinants associated with a hypervirulent phenotype. The complete genome size is 3 881 479 bp and has a guanine-cytosine content of 57.76% while it encodes for 3 525 chromosome coding sequences. The strain was also identified to carry three plasmids of 513 647 bp, 261 771 bp, and 106 029 bp, respectively., Conclusions: K.piersonii GABEKP28 is the first complete genome of this species to be submitted to GenBank and only the second to be sequenced from a human host. The whole-genome sequencing data with multiple plasmids, ARGs, and VFs will aid in understanding the pathogenicity, evolution, and phylogeny of this novel opportunistic pathogen., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 bla CTX-M-1 and bla CTX-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 bla CTX-M-1 and bla CTX-M-15 . Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for bla CTX-M-1 ( n  = 18), bla CTX-M-15 ( n  = 35), and other closely related bla CTX-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 bla CTX-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 bla CTX-M-1 and bla CTX-M-15 , respectively, and E. coli bla CTX-M-1 was identified in all bla CTX-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 bla CTX-M-1 and bla CTX-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.

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., Competing Interests: Declaration of competing interest This work was funded by the Ireland's Environmental Protection Agency and the Health Service Executive [grant number 2017-HS-LS-1]. The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microalgae that form phytoplankton live and die in a complex microbial consortium in which they co-exist with bacteria and other microorganisms. The dynamics of species succession in the plankton depends on the interplay of these partners. Bacteria utilize substrates produced by the phototrophic algae, while algal growth can be supported by bacterial exudates. Bacteria might also use chemical mediators with algicidal properties to attack algae. To elucidate whether specific bacteria play universal or context-specific roles in the interaction with phytoplankton, we investigated the effect of cocultured bacteria on the growth of 8 microalgae. An interaction matrix revealed that the function of a given bacterium is highly dependent on the cocultured partner. We observed no universally algicidal or universally growth-promoting bacteria. The activity of bacteria can even change during the aging of an algal culture from inhibitory to stimulatory or vice versa. We further established a synthetic phytoplankton/bacteria community with the centric diatom, Coscinodiscus radiatus, and 4 phylogenetically distinctive bacterial isolates, Mameliella sp., Roseovarius sp., Croceibacter sp., and Marinobacter sp. Supported by a Lotka-Volterra model, we show that interactions within the consortium are specific and that the sum of the pairwise interactions can explain algal and bacterial growth in the community. No synergistic effects between bacteria in the presence of the diatom was observed. Our survey documents highly species-specific interactions that are dependent on algal fitness, bacterial metabolism, and community composition. This species specificity may underly the high complexity of the multi-species plankton communities observed in nature. IMPORTANCE The marine food web is fueled by phototrophic phytoplankton. These algae are central primary producers responsible for the fixation of ca. 40% of the global CO 2 . Phytoplankton always co-occur with a diverse bacterial community in nature. This diversity suggests the existence of ecological niches for the associated bacteria. We show that the interaction between algae and bacteria is highly species-specific. Furthermore, both, the fitness stage of the algae and the community composition are relevant in determining the effect of bacteria on algal growth. We conclude that bacteria should not be sorted into algicidal or growth supporting categories; instead, a context-specific function of the bacteria in the plankton must be considered. This functional diversity of single players within a consortium may underly the observed diversity in the plankton.

Mushroom-forming fungi ( Agaricomycetes ) employ enzymatic and nonenzymatic cellulose degradation mechanisms, the latter presumably relying on Fenton-generated radicals. The effects of the two mechanisms on the cellulose microfibrils structure remain poorly understood. We examined cellulose degradation caused by litter decomposers and wood decomposers, including brown-rot and white-rot fungi and one fungus with uncertain wood decay type, by combining small- and wide-angle X-ray scattering. We also examined the effects of commercial enzymes and Fenton-generated radicals on cellulose using the same method. We detected two main degradation or modification mechanisms. The first characterized the mechanism used by most fungi and resembled enzymatic cellulose degradation, causing simultaneous microfibril thinning and decreased crystalline cellulose. The second mechanism was detected in one brown-rot fungus and one litter decomposer and was characterized by patchy amorphogenesis of crystalline cellulose without substantial thinning of the fibers. This pattern did not resemble the effect of Fenton-generated radicals, suggesting a more complex mechanism is involved in the destruction of cellulose crystallinity by fungi. Furthermore, our results showed a mismatch between decay classifications and cellulose degradation patterns and that even within litter decomposers two degradation mechanisms were found, suggesting higher functional diversity under current ecological classifications of fungi. IMPORTANCE Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose. Our data revealed two mechanisms involved in crystalline cellulose degradation by fungi: one that results in the thinning of the cellulose fibers, resembling the enzymatic degradation of cellulose, and one that involves amorphogenesis of crystalline cellulose by yet-unknown pathways, resulting in a patchy-like degradation pattern. These results pave the way to a deeper understanding of cellulose degradation and the development of novel ways to utilize crystalline cellulose.

Objectives: Using available whole genome data, the objective of this in silico study was to identify genetic mechanisms that could explain the antimicrobial resistance profile of three multi-drug resistant (MDR) strains (CA17, CA51, CA39) of the skin bacterium Cutibacterium acnes previously recovered from patients with acne. In particular, we were interested in detecting novel genetic determinants associated with resistance to fluoroquinolone and macrolide antibiotics that could then be confirmed experimentally., Methods: A range of open source bioinformatics tools were used to 'mine' genetic determinants of antimicrobial resistance and plasmid borne contigs, and to characterise the phylogenetic diversity of the MDR strains., Results: As probable mechanisms of resistance to fluoroquinolones, we identified a previously described resistance associated allelic variant of the gyrA gene with a 'deleterious' S101L mutation in type IA 1 strains CA51 (ST1) and CA39 (ST1), as well as a novel E761R 'deleterious' mutation in the type II strain CA17 (ST153). A distinct genomic sequence of the efflux protein YfmO which is potentially associated with resistance to MLSB antibiotics was also present in CA17; homologues in CA51, CA39, and other strains of Cutibacterium acnes , were also found but differed in amino acid content. Strikingly, in CA17 we also identified a circular 2.7 kb non-conjugative plasmid (designated pCA17) that closely resembled a 4.8 kb plasmid (pYU39) from the MDR Salmonella enterica strain YU39., Conclusions: This study has provided a detailed explanation of potential genetic determinants for MDR in the Cutibacterium acnes strains CA17, CA39 and CA51. Further laboratory investigations will be required to validate these in silico results, especially in relation to pCA17., Competing Interests: The author(s) declared that there are no conflicts of interest, (© 2022 The Authors.)

Boreal forests have a large impact on the global greenhouse gas balance and their soils constitute an important carbon (C) reservoir. Mature boreal forests are typically a net CO 2 sink, but there are also examples of boreal forests that are persistent CO 2 sources. The reasons remain often unknown, presumably due to a lack of understanding of how biotic and abiotic drivers interact to determine the microbial respiration of soil organic matter (SOM). This study aimed at identifying the main drivers of microbial SOM respiration and CO 2 and CH 4 soil chamber-fluxes within dry and wet sampling areas at the mature boreal forest of Norunda, Sweden, a persistent net CO 2 source. The spatial heterogeneity of the drivers was assessed with a geostatistical approach combined with stepwise multiple regression. We found that heterotrophic soil respiration increased with SOM content and nitrogen (N) availability, while the SOM reactivity, i.e., SOM specific respiration, was determined by soil moisture and N availability. The latter suggests that microbial activity was N rather than C limited and that microbial N mining might be driving old-SOM decomposition, which was observed through a positive correlation between soil respiration and its δ 13 C values. SOM specific heterotrophic respiration was lower in wet than in dry areas, while no such dependencies were found for chamber-based soil CO 2 fluxes, implying that oxygen depletion resulted in lower SOM reactivity. The chamber-based soil CH 4 flux differed significantly between the wet and dry areas. In the wet area, we observed net CH 4 emission that was positively related to soil moisture and NH 4 + -N content. Taken together, our findings suggest that N availability has a strong regulatory effect on soil CO 2 emissions at Norunda, and that microbial decomposition of old-SOM to release bioavailable N might be partly responsible for the net CO 4 emissions at Norunda, and that microbial decomposition of old-SOM to release bioavailable N might be partly responsible for the net CO 2 emission at the site., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere and is not under consideration by another journal. We have read and understood STOTEN's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)