Increased mutations in lipopolysaccharide biosynthetic genes cause time-dependent development of phage resistance in .

Understanding how bacteria evolve resistance to phages has implications for phage-based therapies and microbial evolution. In this study, the susceptibility of 335 isolates to the wide host range phage BPSELC-1 was tested. Potentially significant gene sets that could confer resistance were identified using bioinformatics approaches based on phage susceptibility phenotypes; more than 90 potential antiphage defense gene sets, including those involved in lipopolysaccharide (LPS) biosynthesis, DNA replication, secretion systems, and respiratory chain, were found. The evolutionary dynamics of resistance to phage were assessed through laboratory evolution experiments, which showed that phage-resistant mutants rapidly developed and exhibited genetic heterogeneity. Most representative hosts (58.1% of 62) rapidly developed phage resistance within 24 h. All phage-resistant mutant clones exhibited genetic heterogeneity and observed mutations in LPS-related genes ( and ) as well as other genes such as cellular respiration, transport, and cell replication-related genes. The study also identified potential trade-offs, indicating that bacteria tend to escape fitness trade-offs through multi-site mutations, all tested mutants increased sensitivity to polymyxin B, but this does not always affect their relative fitness or biofilm-forming capacity. Furthermore, complementing the mutant gene could partially restore the phage sensitivity of phage-resistant mutants. These results provide insight into the phage resistance mechanisms of and the complexity of bacterial evolution resulting from phage predation, which can inform future strategies for phage-based therapies and microbial evolution.