Genomic pathogen surveillance is a powerful tool for public health and research, but is costly and unachievable in low-resource settings. Most sub-genomic typing methods sacrifice resolution whilst remaining costly. We developed "Phylo-Plex", a novel approach that identifies information-rich genomic regions to maximise phylogenetic information whilst minimising the number of regions. Applied to Treponema pallidum and Neisseria gonorrhoeae, we designed a high-resolution multiplex PCR sequencing scheme for lineage tracking pathogens with different extremes of genome variation. For Treponema pallidum, we also designed and evaluated the Phylo-Plex scheme in the laboratory and field settings by sequencing 72 clinical samples using MinION Flongle cells. Our T. pallidum scheme comprising 59 multiplex amplicons achieved high discrimination of fine-scale sublineages comparable to those defined using whole genomes, and demonstrating a qPCR detection limit ≤Ct 32. Variant calls from MinION amplicon sequencing were highly correlated with Illumina whole genome sequencing. We successfully deployed the method in a low-resource laboratory in Zimbabwe, costed at <£300/24 samples (£12.47/sample). Phylo-Plex enables low-cost tracking of priority pathogenic lineages in low resource settings and at scale.

Background Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease, particularly in low—and middle-income countries, including Zambia. ETEC pathogenesis is driven by colonisation factors (CF) and enterotoxins, and increasing antimicrobial resistance compounds the global health burden. Despite its impact, limited genomic data exists for ETEC strains in Sub-Saharan Africa. Methods This study conducted whole-genome sequencing of 62 ETEC isolates collected from children under five years old presenting with moderate-to-severe diarrhoea in Lusaka, Zambia. Genomic DNA was extracted, sequenced using the Illumina MiSeq platform, and analysed for phylogenetic relationships, toxin profiles, CF combinations, AMR genes, and plasmid incompatibility groups. Sequences were processed using bioinformatics tools, including SPAdes for genome assembly and Abricate for virulence and AMR profiling. Results The isolates displayed diverse phylogenetic groupings, predominantly within phylogroups A (22 isolates) and B1 (29 isolates). Forty-five serotypes and 39 sequence types were identified, with ST155, ST4, and ST847 being most prevalent. 35% of isolates lacked a known CF, but CS6-only stains were the most common (8%). The most frequent toxin profile was LTh (31%); AMR analysis revealed 350 resistance genes, with Sul2 (14%) and blaTEM-1 (10%) being predominant. ∼89% of isolates exhibited genomic multidrug resistance. Plasmid analysis identified IncFII as the most prevalent incompatibility group (19%). Conclusion This study highlights the genomic diversity of ETEC in Zambia, revealing concerns about multidrug resistance and identifying virulence profiles. These findings reiterate the urgent need for enhanced surveillance and targeted interventions, including vaccine development, to combat ETEC-related diarrhoea effectively. Authors Summary Diarrhoea caused by a bacteria called Escherichia coli is a significant health problem for young children in Zambia and many similar countries. Despite its profound impact, not much is known about the types of ETEC found in Zambia. In this study, we collected this type of bacteria from children under five years old who had diarrhoea and studied them in detail using advanced genetic tools. We discovered that the bacteria are very diverse, with different groups, toxin types, and ways of attaching to the host. Worryingly, most bacteria were resistant to several antibiotics, potentially making treatment challenging. This research helps us understand how ETEC spreads and causes illness in Zambia, showing the urgent need for tracking, improved treatments, and vaccines to protect children from this serious disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement AVM was supported by The Swedish Research Council (grant no. 2022-01449) and the Sahlgrenska Academy International Starting Grant. RC was awarded funding for this project as part of the European and Developing Countries Clinical Trials Partnership (EDCTP) program, supported by the European Union (grant number RIA 2018V-2309—ETEC, ETVAX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript ### 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 study was conducted in line with ethical recommendations and requirements for the protection of human participants in research (HSP). The study obtained ethics approval from the University of Zambia Biomedical Research Ethics Committee (UNZABREC Ref: 1091-2020) and the National Health Research Authority (NHRA). 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 Data cannot be shared publicly to protect the confidentiality of study participants. However, researchers can inquire on accessing the metadata by requesting from the Centre for Infectious Disease Research in Zambia (CIDRZ) Institutional Data Access/Ethics Committee by contacting [info@cidrz.org][1]. All sequence data has been submitted to the NCBI database. [1]: https://info@cidrz.org

The parasitic nematode Teladorsagia circumcincta is one of the most important pathogens of sheep and goats in temperate climates worldwide and can rapidly evolve resistance to drugs used to control it. To understand the genetics of drug resistance, we have generated a highly contiguous genome assembly for the UK T. circumcincta isolate, MTci2. Assembly using PacBio long-reads and Hi-C long-molecule scaffolding together with manual curation resulted in a 573 Mb assembly (N50 = 84 Mb, total scaffolds = 1,286) with five autosomal and one sex-linked chromosomal-scale scaffolds consistent with its karyotype. The genome resource was further improved via annotation of 22,948 genes, with manual curation of over 3,200 of these, resulting in a robust and near complete resource (96.3% complete protein BUSCOs) to support basic and applied research on this important veterinary pathogen. Genome-wide analyses of drug resistance, combining evidence from three distinct experiments, identified selection around known candidate genes for benzimidazole, levamisole and ivermectin resistance, as well as novel regions associated with ivermectin and moxidectin resistance. These insights into contemporary and historic genetic selection further emphasise the importance of contiguous genome assemblies in interpreting genome-wide genetic variation associated with drug resistance and identifying key loci to prioritise in developing diagnostic markers of anthelmintic resistance to support parasite control.

BACKGROUND:The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180). METHODS:A genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain's evolution. RESULTS:GPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage ϕOXC141. Prior to the introduction of PCV13, this clade's composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in ~30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation. CONCLUSION:This interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.