Ugokwe, N. I. | bioRxiv Science

bioRxiv2025

Characterisation of novel bacteriophages against the cattle pathogen Moraxella bovis

BackgroundInfectious bovine keratoconjunctivitis is the most important cattle ocular disease worldwide. The infection is primarily caused by Moraxella bovis and is a highly contagious disease that significantly affects cattle welfare. Currently, antibiotic medication is the primary treatment for infectious bovine keratoconjunctivitis. However, with rising concerns over antibiotic resistance, we propose developing a more targeted therapeutic strategy using bacteriophages (phages). Materials and MethodsWe have isolated the first known Moraxella bovis phages, characterised them according to their genome sequence, local virulence index and with transmission electron microscopy. The host ranges were assessed using 41 clinical M. bovis strains isolated from infected cows. ResultsFour phages were isolated and characterised. Comparative analysis identified a high degree of genomic similarity between the phages MB15, MB16, MB26 and MB43. MB43 was the most distinct, with the smallest host range phenotype. ConclusionsThe isolated phages show therapeutic potential for further development against Moraxella infections.

microbiology↗

bioRxiv2025

Rolling out plaque-2-sequence: a single plaque sequencing approach enabling rapid, low-cost sequencing of phages directly from plaques

Rapid, accurate, and scalable sequencing of bacteriophage genomes is critical to advance phage therapy, build phage biobanks and understand phage genomic diversity. Current methods are based on sequencing and assembling complete bacteriophage genomes using short- or long-read technologies. However, current protocols require large DNA input and are cost prohibitive which limits their application to phage collections that typically are large and have low-biomass. In order to address this we have developed plaque-2-sequence, a robust and cost-effective workflow for high-throughput phage genome sequencing that will transform the speed and cost of attaining phage genomes. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We applied the method to phages isolated on seven genetically diverse bacterial hosts; Escherichia, Pseudomonas, Synechococcus, Enterococcus, Klebsiella , Serratia and Enterobacter. High quality genome assemblies were validated using CheckV and benchmarking against previously sequenced phage isolates. Compared to standard Illumina sequencing, plaque-2-sequence offers [~]10-fold savings in sequencing price for individual labs. Furthermore, it substantially decreases the time required to produce a phage genome, once a plaque is obtained. Offering the ability to routinely obtain hundreds of phage genome sequences a week, with minimal hands-on time. Plaque-2-sequence enables systematic genomic characterisation of phage isolates, facilitating taxonomic classification, for the development of large scale phage biobanks. Impact StatementHere we have optimised a method for high-throughput sequencing of bacteriophage genomes from single plaques (plaque-2-sequence). We present a robust, high-throughput and cost-effective workflow. Plaque-2-sequence combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimised assembly steps tailored to phage genomes. We demonstrate the scalability of this approach by sequencing over 100 phages from multiple bacterial hosts. This marks a step-change for the field, allowing phage genome sequencing to keep pace with phage isolation rates, and transforming how rapidly we can explore and understand phage genomic diversity.

microbiology↗