Proteus mirabilis is a Gram-negative uropathogenic bacterial species responsible for many catheter-associated urinary tract infections (CAUTIs). Due to the growing rates of antimicrobial resistance among CAUTI pathogens, novel antimicrobial solutions are urgently needed. Bacteriophage endolysins are an emerging class of antimicrobial agents, but no endolysins from P. mirabilis phages have been reported to date. Here, we describe two new Proteus phage endolysins, LysPM1 and LysPM2, containing Peptidase_M15_3 and CHAP domains, respectively. We experimentally confirmed their antibacterial activity against several Proteus spp. when used in combination with outer membrane permeabilizers, including EDTA, citric acid, and chloroform, as well as against frozen bacteria. Both enzymes have optimum activity at pH 7–8 and retain activity at temperatures up to 60 °C. Both endolysins completely lost their lytic activity upon treatment with EDTA, suggesting that divalent cations participate in the catalytic mechanism; the addition of Ca2⁺ and Zn2⁺ to LysPM1, and Ca2⁺ and Mn2⁺ to LysPM2 partially restored the activity. Our study confirms LysPM1 and LysPM2 as the first experimentally characterized Proteus phage endolysins, with LysPM2 also being the first experimentally characterized Gram-negative endolysin with a single CHAP domain. These findings could guide further development of phage-derived lytic enzymes for treatment of Proteus infections. • LysPM1 LysPM2 are the first experimentally characterized Proteus phage endolysins. • Both are single-domain endolysins: Peptidase_M15_3 in LysPM1 and CHAP in LysPM2. • Metal cofactors are required for their activity: Zn2⁺ for LysPM1, Ca2⁺ for LysPM2.

Motivation: Bacteria carry a large repertoire of antiviral defence systems, our knowledge of which is expanding rapidly. Several bioinformatics tools now exist to identify them. Though powerful, these tools can differ in the models they use and the nomenclature they return, thus a single tool could both miss an annotation and disagree with its peers. Results: Here we describe Ptolemaea, a pipeline for harmonising phage-defence annotations across multiple tools by reconciling PADLOC, DefenseFinder, and a bidirectional BLAST. Over a common predicted set of proteins, Ptolemaea provides a consensus annotation list per genome. The pipeline is not intended to outperform or replace its component tools; its purpose is to maximise the number of defence systems recovered from a genome and to make disagreements between tools explicit and resolvable. We demonstrate the pipeline on 700 complete genomes spanning the ESKAPE pathogens and Escherichia coli, recovering 32,509 defence annotations, of which 50.6% were supported by more than one annotation source. Availability: The Ptolemaea pipeline is freely available at https://github.com/ecampbell50/Ptolemaea. Supplementary information: Genome accessions used in this analysis can be found in S1, and script for genome retrieval in S2. Collated consensus annotation counts can be found in S3, while all raw tool outputs and curated decisions for each species can be found in S4-10

Artificial intelligence is revolutionizing structural bioinformatics, with AlphaFold arguably being the most impactful development to date. The structural atlases generated by these methods present significant opportunities for unraveling biological mysteries but also pose challenges in leveraging such massive datasets effectively. In this work, we explore the dynamic landscape of hundreds of thousands of AlphaFold-predicted structures using normal mode analysis. The resulting data serve to empirically define an alphabet summarizing relative protein flexibility, termed ProFlex. Leveraging ProFlex, we describe the flexibility information space occupied by this massive dataset. We believe leveraging the data compression offered by ProFlex-like approaches opens opportunities for understanding protein function, refining structural predictions, and rendering analyses computationally tractable.

Plasma-activated water (PAW) is a promising disinfection strategy that generates a complex mixture of reactive oxygen and nitrogen species (ROS/RNS), including hydrogen peroxide (H2O2), nitrate (NO3 -), and transient oxidants, in an acidic aqueous environment. These reactive species contribute to both immediate and extended antimicrobial activity. This study investigates how the addition of low concentrations (<100 μM) of potassium iodide (KI) enhances the bactericidal properties of spark-generated PAW by enabling the in-situ generation of reactive iodine species (RIS), particularly hypoiodous acid (HIO), under acidic conditions. KI addition (10-100 μM) led to a counterintuitive, dose-dependent increase in H2O2 concentrations, from ∼1.2 mM in PAW alone to ∼1.8 mM at 30 μM KI, possibly due to iodine-mediated catalytic effects or reduced H2O2 degradation. NO3 - levels also increased by ∼17 % with increasing KI. Equivalent concentrations of H2O2 + KI failed to replicate the rapid antimicrobial activity observed in PAW + KI, which achieved complete inactivation of Escherichia coli and Listeria monocytogenes planktonic cells within 3 min, compared to over 10 min for PAW alone, indicating the involvement of additional reactive species in KI-enhanced antimicrobial activity of PAW. However, Salmonella enterica planktonic cells exhibited only partial inactivation even with KI, indicating species-specific tolerance under these conditions. 24h biofilms of L. monocytogenes and E. coli were eradicated with PAW + KI in 10 min, whereas S. enterica showed only a 2-log reduction. Scavenger assays revealed that both longer-lived species (H2O2) and shorter-lived oxidants such as singlet oxygen are essential for this enhanced killing, while ozone and superoxide appeared dispensable. These findings support a multi-step antimicrobial mechanism: (1) plasma treatment creates a low pH, H2O2-rich solution; (2) iodide is oxidised to RIS such as I3 - and HIO; (3) additional PAW-derived oxidants potentiate RIS chemistry; and (4) unionised HIO diffuses across bacterial membranes to induce oxidative damage. PAW-KI remained stable for at least 14 days at 4 °C, with sustained RIS activity and minimal loss of H2O2 or NO3 -, suggesting preserved antimicrobial capacity over time. The antimicrobial mechanism likely proceeds through a four-step pathway: plasma-mediated generation of H2O2 and NO3 -; oxidation of I- to I2 and HIO; potentiation of RIS via PAW-derived ROS/RNS; and subsequent microbial inactivation via membrane damage. Together, these results demonstrate that PAW + KI forms a powerful, in situ RIS-generating system, offering a residue-minimising and environmentally sustainable disinfection platform. Its rapid action, scalability, and reliance on only air, water, electricity, and GRAS-listed KI make it an attractive intervention for food safety, clinical disinfection, and decentralised sanitation settings.

Viral proteins often evolve so rapidly that sequence similarity is lost even among conserved functional genes, complicating phylogenetic analysis and classification. This challenge is acute for Caudoviricetes , a highly diverse class of dsDNA tailed bacteriophages recently redefined by the International Committee on Taxonomy of Viruses (ICTV). To address the limitations of sequence-based taxonomy, we present a structure-based classification framework using whole-proteome structural clustering. From 4,082 exemplar genomes, we predicted 445,098 protein structures with ESMFold and clustered them using Foldseek. Genomes were encoded as binary profiles of structural fold presence, and phylogenetic distances were inferred and normalized using Relative Evolutionary Divergence (RED). This yielded a revised structural taxonomy comprising 159 orders, 267 families, 502 subfamilies, and 1,189 genera. We also introduce PhagePleats , a Python tool for classifying novel phage genomes based on structural similarity. Our approach highlights the utility of protein structure for resolving distant viral relationships. ### Competing Interest Statement The authors have declared no competing interest. Research Ireland Centre for Research Training in Genomics Data Science, 18/CRT/6214 Wellcome Trust Research Career Development Fellowship, 220646/Z/20/Z Research Ireland, 12/RC/2273-P2

Effective pathogen surveillance is critical for public health decision-making, with both individual and environmental monitoring playing essential roles. While wastewater (WW) and individual whole genome sequencing (WGS) have been used to monitor SARS-CoV-2 dynamics, their complementary potential for enhancing national-level genomic surveillance remains underexplored. This study aimed to evaluate the unique and combined contributions of WW and individual WGS to genomic surveillance. We conducted SARS-CoV-2 WGS on over 4000 WW samples and 23,000 individual samples across Northern Ireland (NI) between 2021 and 2023. SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 and Mini-XT protocols and sequenced on Illumina MiSeq. Variant compositions in WW data were analysed using Freyja and compared to individual data using time series analysis, correlation assessments, and volatility measurements via numerical derivatives, with mean absolute error (MAE) calculations used to assess concordance. Wastewater treatment plants (WWTPs) were ranked by concordance to individual WGS data. WW and individual WGS complementarity was quantified by mutation classification and overlap analysis. Temporal curve shifting was used to identify lags or leads in variant detection and to infer differences in geospatial spread between WW and individual sequencing data. We confirmed strong concordance between WW and individual variant compositions (mean MAE = 6.2 %). MAE was inversely correlated with sequencing rate (Pearson r=-0.37, p < 0.001) and increased during periods with more circulating variants, highlighting the value of increased sequencing efforts during volatile periods. The population size served by a WWTP was not a reliable indicator of how well its variant composition matched that of the national individual sequencing programme. Both individual and WW-based sequencing (WBS) detected unique, as well as common mutations. Patterns of variant spread within NI were consistent between both programmes (Pearson r = 0.63, p = 0.036), providing complementary insights into variant trends and geospatial spread. We demonstrate that integration of individual and WW WGS data offers more comprehensive SARS-CoV-2 genomic surveillance and improves confidence in predictions of variant composition and spread.

Multidrug efflux pumps have been found to play a crucial role in drug resistance in bacteria and eukaryotes. In this study, we investigated the presence of functional multidrug and toxic compound extrusion (MATE) efflux pumps, inferred from whole genome sequencing, in the halophilic archaeon Halorubrum amylolyticum CSM52 using Hoechst 33342 dye accumulation and antimicrobial sensitivity tests in the presence and absence of efflux pump inhibitors (EPIs). The whole genome sequence of H. amylolyticum CSM52 contained two putative MATE-type efflux pump genes, which may contribute to the inherent resistance to conventional antimicrobial agents reported in archaea. Antimicrobial susceptibility of the wild-type H. amylolyticum CSM52 testing revealed a lack of sensitivity to a wide range of antimicrobials, including glycopeptides, aminoglycosides, macrolides, fluoroquinolones, tetracycline, and chloramphenicol. However, the presence of EPIs, such as thioridazine, fluoxetine, and chlorpromazine, significantly increased the susceptibility of H. amylolyticum CSM52 to a number of these antimicrobials, indicating the potential involvement of efflux pumps in the observed resistance. A molecular modelling study with EPIs and substrate antimicrobials provided important insights into the molecular interactions with the putative transporter. It suggests that the occupancy of the transporter channel by EPIs has the potential to impact the efflux of antimicrobials. Phylogenetic analysis of the amino acid sequences of both MATE pumps showed low similarity with bacterial representatives, suggesting the presence of novel and distinct MATE efflux pumps in archaea. Our findings provide the first experimental evidence of active antibiotic efflux mechanisms in archaea and their potential roles in antimicrobial resistance, broadening our understanding of mechanisms of archaeal antimicrobial resistance, an overlooked aspect of AMR research.

The ongoing antimicrobial resistance crisis has incentivised research into alternative antibacterial and antibiofilm agents. One of them is plasma-activated water (PAW), which is produced by exposing water to a cold plasma discharge. This process generates a diverse array of reactive oxygen and nitrogen species (ROS/RNS) with antimicrobial properties. Another intensively studied class of alternative antimicrobials are bacteriophages, attracting attention due to their specificity and strong antibacterial activity. As combinations of different types of antimicrobials are known to often exhibit synergistic interactions, in this study we investigated the combined use of cold atmospheric-pressure plasma-activated water and the bacteriophage vB_PmiS_PM-CJR against Proteus mirabilis biofilms as a potential option for treatment of catheter-associated urinary tract infections (CAUTIs).We compared the effect of two cold plasma discharge setups for PAW production on its antimicrobial efficacy against P. mirabilis planktonic and biofilm cultures. Next, we assessed the stability of the phage vB_PmiS_PM-CJR in PAW. Finally, we tested the antimicrobial activity of the phages and PAW against biofilms, both individually and in combinations.Our findings demonstrate that the combination of PAW with phage is more effective against biofilms compared to individual treatments, being able to reduce the number of biofilm-embedded cells by approximately 4 log. We were also able to show that the order of treatment plays an important role in the anti-biofilm activity of the phage-PAW combination, as the exposure of the biofilm to PAW prior to phage administration results in a stronger effect than the reverse order.This research underlines PAW's ability to potentiate phage activity, showcasing a considerable reduction in biofilm viability and biomass. Additionally, it contributes to the growing body of evidence supporting the use of phage-based combinatorial treatments. Overall, this sequential treatment strategy demonstrates the potential of leveraging multiple approaches to address the mounting challenge of antibiotic resistance and offers a promising avenue for enhancing the efficacy of CAUTI management.

Benzylisoquinoline alkaloids are valuable active ingredients in medicines that are typically extracted from plants and subsequently derivatized. Buprenorphine is a member of this family of compounds and is an effective analgesic and is also used for the treatment of opioid use disorder. The commercial route of synthesis for buprenorphine starts from thebaine and uses toxic reagents and harsh reaction conditions for the N- and O-demethylation steps. Here, we propose an alternative approach for buprenorphine synthesis via enzymatic N- and O-demethylation reactions. Utilizing rational enzyme design and directed evolution, we identified and engineered two oxygenase enzymes. For the N-demethylation reaction, the best variant achieved a cumulative improvement in conversion of 567-fold, while for the O-demethylation, the best variant achieved 22-fold cumulative improvement in conversion. A separate variant was able to efficiently catalyze both the N-demethylation and the O-demethylation reactions.

Background: Robust methods to track pathogens support public health surveillance. Both wastewater (WW) and individual whole genome sequencing (WGS) are used to assess viral variant diversity and spread. However, their relative performance and the information provided by each approach have not been sufficiently quantified. Therefore, we conducted a comparative evaluation using extensive individual and wastewater longitudinal SARS-CoV-2 WGS datasets in Northern Ireland (NI). Methods: WGS of SARS-CoV-2 was performed on >4k WW samples and >23k individuals across NI from 14th November 2021 to 11th March 2023. SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 protocol and sequenced on an Illumina MiSeq. Wastewater data were analysed using Freyja to determine variant compositions, which were compared to individual data through time series and correlation analyses. Inter-programme agreements were evaluated by mean absolute error (MAE) calculations. WW treatment plant (WWTP) performances were ranked by mean MAE. Volatile periods were identified using numerical derivative analyses. Geospatial spreading patterns were determined by horizontal curve shifting. Findings: Strong concordance was observed between wastewater and individual variant compositions and distributions, influenced by sequencing rate and variant diversity. Overall variant compositions derived from individual sequences and each WWTP were regionally clustered rather than dominated by local population size. Both individual and WW sequencing detected common nucleotide substitutions across many variants and complementary additional substitutions. Conserved spreading patterns were identified using both approaches. Interpretation: Both individual and wastewater WGS effectively monitor SARS-CoV-2 variant dynamics. Combining these approaches enhances confidence in predicting the composition and spread of major variants, particularly with higher sequencing rates. Each method detects unique mutations, and their integration improves overall genome surveillance. ### Competing Interest Statement JWM and DFG are directors of BioSeer Ltd (NI701440), a UK company that has the potential to offer wastewater testing services. ### Funding Statement Funding: Individual sequencing was funded via the Belfast Health and Social Care Trust (Department of Health for Northern Ireland) and the COVID-19 Genomics UK (COG-UK) consortium, which was supported by the Medical Research Council (MRC), UK Research and Innovation (UKRI), the National Institute for Health Research (NIHR), the Department of Health and Social Care (DHSC), and the Wellcome Sanger Institute. The NI Wastewater Surveillance Programme was funded by the Department of Health for Northern Ireland. EPT was supported through the COG-UK Early Career Funding Scheme. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The COG-UK study protocol was approved by the Public Health England Research Ethics Governance Group. (Reference: R&D NR0195) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

AIMS:This study aimed to develop a new bioinformatic approach for the identification of novel antimicrobial peptides (AMPs), which did not depend on sequence similarity to known AMPs held within databases, but on structural mimicry of another antimicrobial compound, in this case an ultrashort, synthetic, cationic lipopeptide (C12-OOWW-NH2). METHODS AND RESULTS:When applied to a collection of metagenomic datasets, our outlined bioinformatic method successfully identified several short (8-10aa) functional AMPs, the activity of which was verified via disk diffusion and minimum inhibitory concentration assays against a panel of 12 bacterial strains. Some peptides had activity comparable to, or in some cases, greater than, those from published studies that identified AMPs using more conventional methods. We also explored the effects of modifications, including extension of the peptides, observing an activity peak at 9-12aa. Additionally, the inclusion of a C-terminal amide enhanced activity in most cases. Our most promising candidate (named PB2-10aa-NH2) was thermally stable, lipid-soluble, and possessed synergistic activity with ethanol but not with a conventional antibiotic (streptomycin). CONCLUSIONS:While several bioinformatic methods exist to predict AMPs, the approach outlined here is much simpler and can be used to quickly scan huge datasets. Searching for peptide sequences bearing structural similarity to other antimicrobial compounds may present a further opportunity to identify novel AMPs with clinical relevance, and provide a meaningful contribution to the pressing global issue of AMR.

Effective therapeutic monitoring of wound infections is crucial for wound care. We explore a novel approach using a previously described 3D-printed, colourimetric indicator to non-invasively monitor antimicrobial interventions in wound infection. The indicator contains a pH-sensitive dye which detects carbon dioxide production from wound pathogens by changing colour from blue to yellow. The sensor accurately reported the minimum inhibitory concentration of antibiotic against Pseudomonas aeruginosa in a sealed 96-well plate. Subinhibitory antibiotic concentrations resulted in the sensor changing colour, whilst remaining blue for inhibitory concentrations. The sensor was applied to a porcine skin wound model with Pseudomonas aeruginosa biofilms, testing several antimicrobial treatments: antibiotic monotherapy, phage monotherapy, and a combination of both. After treatment, the sensor, sealed with the wound, enabled visual monitoring of antimicrobial efficacy. Through comparing post-treatment bioburden and sensor response, greater log-fold reductions in P. aeruginosa following antimicrobial treatment correlated with longer sensor response times. Sensors monitoring unsuccessfully treated biofilms changed colour, while those treated at or above the minimum biofilm eradication concentration elicited no colour change, indicating P. aeruginosa eradication. This sensor holds promise for monitoring wound infection treatment efficacy, improving patient outcomes, and promoting undisturbed wound healing as a non-invasive point-of-care tool.

Abstract Background Ireland’s COVID-19 response combined extensive SARS-CoV-2 testing to estimate incidence, with whole genome sequencing (WGS) for genome surveillance. As an island with two political jurisdictions―Northern Ireland (NI) and Republic of Ireland (RoI)―and access to detailed passenger travel data, Ireland provides a unique setting to study virus introductions and evaluate public health measures. Using a substantial Irish genomic dataset alongside global data from GISAID, this study aimed to trace the introduction and spread of SARS-CoV-2 across the island. Methods We recursively searched for 29,518 SARS-CoV-2 genome sequences collected in Ireland from March 2020 to June 2022 within the global SARS-CoV-2 phylogenetic tree and identified clusters based on shared last common non-Irish ancestors. A maximum parsimony approach was used to assign a likely country of origin to each cluster. The geographic locations and collection dates of the samples in each introduction cluster were used to map the spread of the virus across Ireland. Downsampling was used to model the impact of varying levels of sequencing and normalisation for population permitted comparison between jurisdictions. Results Six periods spanning the early introductions and the emergence of Alpha, Delta, and Omicron variants were studied in detail. Among 4439 SARS-CoV-2 introductions to Ireland, 2535 originated in England, with additional cases largely from the rest of Great Britain, United States of America, and Northwestern Europe. Introduction clusters ranged in size from a single to thousands of cases. Introductions were concentrated in the densely populated Dublin and Belfast areas, with many clusters spreading islandwide. Genetic phylogeny was able to effectively trace localised transmission patterns. Introduction rates were similar in NI and RoI for most variants, except for Delta, which was more frequently introduced to NI. Conclusions Tracking individual introduction events enables detailed modelling of virus spread patterns and clearer assessment of the effectiveness of control measures. Stricter travel restrictions in RoI likely reduced Delta introductions but not infection rates, which were similar across jurisdictions. Local and global sequencing levels influence the information available from phylogenomic analyses and we describe an approach to assess the ability of a chosen WGS level to detect virus introductions.

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Biofilm production plays a clinically significant role in the pathogenicity of many bacteria, limiting our ability to apply antimicrobial agents and contributing in particular to the pathogenesis of chronic infections. Bacteriophage depolymerases, leveraged by these viruses to circumvent biofilm mediated resistance, represent a potentially powerful weapon in the fight against antibiotic resistant bacteria. Such enzymes are able to degrade the extracellular matrix that is integral to the formation of all biofilms and as such would allow complementary therapies or disinfection procedures to be successfully applied. In this manuscript, we describe the development and application of a machine learning based approach towards the identification of phage depolymerases. We demonstrate that on the basis of a relatively limited number of experimentally proven enzymes and using an amino acid derived feature vector that the development of a powerful model with an accuracy on the order of 90% is possible, showing the value of such approaches in the discovery of novel therapeutic agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had an unprecedented impact on the people of Ireland as waves of infection spread across the island during the global coronavirus disease 2019 (COVID-19) pandemic. Viral whole-genome sequencing (WGS) has provided insights into SARS-CoV-2 molecular mechanisms of pathogenicity and evolution and contributed to the development of anti-virals and vaccines. High levels of WGS have enabled effective SARS-CoV-2 genomic surveillance on the island of Ireland, leading to the generation of a sizeable data set with potential to provide additional insights into viral epidemiology. Because Ireland is an island, accurate documentation of travel rates to and from other regions, both by air and by sea, are available. Furthermore, the two distinct political jurisdictions on the island allow comparison of the impact of varying public health responses on viral dynamics, including SARS-CoV-2 introduction events. Using phylogenomic analysis incorporating sample collection date and location metadata, we identify multiple introduction and spreading events for all major viral lineages to the island of Ireland during the period studied (March 2020-June 2022). The majority of SARS-CoV-2 introductions originated from England, with frequent introductions from USA and northwestern Europe. The clusters of sequences predicted to derive from discrete introduction events ("introduction clusters") vary greatly in size, with some involving only one or two cases and others comprising thousands of samples. When introduction cluster samples are mapped sequentially by collection date, they appear predominantly in previously affected or adjacent areas. This mirroring of the phylogenetic relationships by the geospatiotemporal propagation of SARS-CoV-2 validates our analytic approach. By downsampling, we estimate the power to detect introductions to Ireland as a function of sequencing levels. Per capita normalisation of both sequencing levels and detected introductions accounts for biases due to differing sequencing efforts and total populations. This approach showed similar rates of introductions for all major lineages into Northern Ireland (NI) and Republic of Ireland (RoI) with the exception of Delta, which was higher in NI which is likely attributable to higher travel per capita. However, there were similar rates of Delta infection within NI and RoI, suggesting that although travel restrictions will reduce risk of introducing novel variants to the region, they may not substantially decrease total incidence. Our generalisable methodology to study introduction dynamics and optimal sequencing levels will assist public health authorities to select the most appropriate control measures and viral sequencing strategy.

Biofilm production plays a clinically significant role in the pathogenicity of many bacteria, limiting our ability to apply antimicrobial agents and contributing in particular to the pathogenesis of chronic infections. Bacteriophage depolymerases, leveraged by these viruses to circumvent biofilm mediated resistance, represent a potentially powerful weapon in the fight against antibiotic resistant bacteria. Such enzymes are able to degrade the extracellular matrix that is integral to the formation of all biofilms and as such would allow complementary therapies or disinfection procedures to be successfully applied. In this manuscript, we describe the development and application of a machine learning based approach towards the identification of phage depolymerases. We demonstrate that on the basis of a relatively limited number of experimentally proven enzymes and using an amino acid derived feature vector that the development of a powerful model with an accuracy on the order of 90% is possible, showing the value of such approaches in protein functional annotation and the discovery of novel therapeutic agents.

Abstract Background Gut microbiomes are crucial for host nutrition due to their feed energy-harvesting capacity. In the rumen microbiome Butyrivibrio and Pseudobutyrivibrio dominate and play a key role in harvesting dietary energy. Within these genera, five rumen species have been classified (Butyrivibrio fibrisolvens, B. hungatei, B. proteoclasticus, Pseudobutyrivibrio ruminis and P. xylanivorans) and more recently an additional sixth Butyrivibrio sp. group was added. Nonetheless, in recent years the explosion in available metagenomically assembled genomes (MAGs) offer a new insight into their taxonomy and function. Consequently, in this study we analysed the pangenome and function of 64 MAGs and 71 culture representatives of Butyrivibrio and Pseudobutyrivibrio. Results Using MASH and ANI we demonstrate that the 135 Butyrivibrio and Pseudobutyrivibrio genomes from MAGs and pure culture cluster into 59 genera and 75 species. Pangenome analysis of 8 groups containing > 4 strains showed average core genome percentages of between 38.49–76.67%. In turn, the accessory genome percentages for the 8 groups were between 23.33% and 65.39%. The most abundant GH families found in the 8 groups were GH13, GH3, GH43, GH2, GH25, GH31, GH36, and GH5 in descending order. Dendograms of the GH families show extensive variation, and expression of 20.45–47.11% of the genes is observed in metatranscriptome datasets. Conclusions Our findings demonstrate that Butyrivibrio and Pseudobutyrivibrio genomes cluster into 59 genera and 75 species. The 8 groups containing 4 or more genomes that were carried forward possess open genomes with extensive genomic diversity. The MAGs, alongside genomes for cultured isolates, contained an expansive repertoire of glycosyl hydrolase isoforms, which likely facilitate the symbiotic breakdown of plant matter under dietary perturbations allowing a competitive advantage and driving niche specialisation. This study has enabled a substantial enhancement in our understanding of the functional capacity and taxonomy of the dominant rumen isolates, Butyrivibrio and Pseudobutyrivibrio by utilising all recently published rumen MAGs.

Background The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. Results We present the ‘Mini-XT’ miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. Conclusions The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.