Evolutionary selection of trimethoprim-resistant <i>dfrA</i> genes in lytic phages affects phage and host fitness during infection

Temperate phages and prophages are well-known carriers of antibiotic resistance genes (ARGs) facilitating their transmission. In contrast, lytic phages rarely harbor functional ARGs. However, by a lenient threshold search strategy combined with a machine learning approach based on structural similarity, here we identified 9419 potential ARGs within lytic phages. We showed that potential trimethoprim-resistance dihydrofolate reductase (<i>dfrA</i>) genes enriched in lytic phages could confer trimethoprim resistance to <i>Escherichia coli</i>. Sequence analysis revealed that lytic phages rarely transfer these potential <i>dfrA</i> genes into their bacterial hosts. Functional studies showed that these <i>dfrA</i> genes not only enhance phage reproduction in the presence of trimethoprim but also promote bacterial growth during phage infection. These results highlight that abundant functional ARGs were selected in evolution to improve lytic phage reproduction by promoting bacterial growth during infection, suggesting that <i>dfrA</i> genes play important roles in evolutionary mutualism between lytic phages and their hosts.