Osaka, T. | Search bioRxiv

bioRxiv2025

Novel Escherichia coli Phages Representing a Distinct Genus within Stephanstirmvirinae: Genome and Host Range Characteristics

Bacteriophages play crucial roles in microbial ecosystems and have potential biotechnological applications. However, our understanding of culturable phages remains limited. This study characterized six novel Escherichia coli phages isolated from pig farm wastewater and urban sewage using comprehensive genomic, morphological, and host-range analyses. Using multiple comparative approaches, including gene-sharing network analysis, average nucleotide identity (ANI), and nucleotide intergenomic similarity (NIS), we demonstrated that five of these phages form a distinct group within the subfamily Stephanstirmvirinae, potentially representing a novel genus provisionally named "Wecvirus". These phages were further classified into two distinct species within the proposed genus, each of which exhibits a unique host range pattern. This host specificity is reflected in the species-specific differences in the amino acid sequences of tail fibers, which are crucial for infection. The remaining phage, which was not classified as Wecvirus exhibited characteristics that challenged the current classification criteria, highlighting the need for more flexible taxonomic approaches. Our findings expand the understanding of phage diversity within Stephanstirmvirinae and contribute to the evolving phage taxonomy framework. ImportanceThe rapid emergence of antibiotic-resistant pathogens necessitates the development of alternative antimicrobial strategies, with phage therapy showing promising potential. However, the successful implementation of phage-based treatments requires a comprehensive understanding of phage diversity and host-pathogen interactions. Our study expands the known diversity within Stephanstirmvirinae by characterizing novel E. coli phages that constitute a distinct genus. The identification of species-specific host recognition mechanisms within this genus provides insights into phage-host adaptation. These findings contribute to our understanding of phage taxonomy and evolution, while also offering practical implications for the development of phage-based therapeutics against pathogenic E. coli strains. This research highlights the importance of continued exploration of novel phages for both fundamental understanding and therapeutic applications.

evolutionary biology↗

bioRxiv2024

From phenotype to receptor: validating physiological clustering of Escherichia coli phages through comprehensive receptor analysis

Understanding the relationship between bacteriophage (phage) classification and target receptors is crucial for phage ecology and applied research. In this study, we compared 13 previously isolated Escherichia coli phages based on physiological characteristics, whole-genome sequences, and tail fiber protein phylogenetics. We improved our previously proposed physiological clustering method by optimizing the bacterial panel for host range assessment, implementing appropriate distance metrics for mixed data types, and applying silhouette coefficient analysis for objective determination of optimal cluster numbers. We combined genomic analysis and lipopolysaccharide (LPS) structural analysis of phage-resistant E. coli strains to identify target receptors of the phages. Complementation experiments further confirmed the direct involvement of identified genes in phage reception. The results revealed that phylogenetically distinct E. coli phages target different sites in the LPS R-core region (modified by WaaV, WaaW, WaaT, and WaaY), membrane proteins (NfrB, TolA, YhaH), or flagella. Our analysis revealed that, subtle chemical modifications of LPS (such as Heptose phosphorylation) were shown to be important for E. coli phage recognition. Furthermore, physiological characteristics, tail fiber phylogenetics, and whole genome analysis independently classified the phages with high correlation to target receptor specificity. The addition of three phages with known receptors further validated our approach. Our results suggest that grouping based on physiological characteristics (such as lysis dynamics and host range) and genotypes (tail fiber phylogenetics or whole genome analysis) independently classified phages with high correlation to target receptor specificity. Here, we elucidated the diversity and specificity of E. coli phage target receptors, providing new insights into the classification of E. coli phages and phage-host interactions. ImportancePhage therapy is gaining attention as an alternative treatment for antibiotic-resistant bacteria. Developing effective phage cocktails requires combining phages with different target receptors, but traditional methods for identifying target receptors are labor-intensive. This study demonstrates that E. coli phages targeting the TK001 strain with different target receptors can be grouped based on their physiological characteristics, tail fiber sequences, or whole genomes. Our approach was enhanced through systematic bacterial panel selection for host range assessment, optimized distance metrics for physiological characteristics, and objective cluster determination using silhouette coefficient analysis for all three classifications. This insight can be used to more efficiently create diverse phage cocktails. Additionally, we identified phages targeting diverse sites, including different regions of LPS, membrane proteins, and flagella. These findings deepen our understanding of phage host recognition mechanisms, enabling the rapid preparation of effective phage cocktails and contributing to new advancements in bacterial infection treatment.

microbiology↗