Mutation of Eamy_RS26425, <i>hldE</i> , or <i>pgm</i> renders <i>Erwinia amylovora</i> CFBP 1430 multi-phage resistant and avirulent

With the rise of antibiotic-resistant pathogens, bacterial infections have become an increasingly significant threat. Bacteriophages have the potential to complement the current treatment options. To ensure that phage biocontrol is efficient and long-lasting, the risk of resistance development must be minimized, and phage resistance mechanisms should be thoroughly investigated. In this study, a Tn5 transposon mutagenesis library of the fire blight pathogen <i>Erwinia amylovora</i> was screened to identify multi-phage-resistant mutants. These mutants had transposon insertions in the genes Eamy_RS26425 (formerly Eamy1960, putative NAD-dependent epimerase), <i>hldE</i> (putative ADP-heptose synthase), or <i>pgm</i> (putative phosphoglucomutase) and were able to survive attacks of phages that require different receptors for host infection. The generated knockout mutants were analyzed for modification of outer membrane structures and for potential fitness loss. Indeed, multiple alterations that could explain phage resistance were observed. Lipopolysaccharide structures were altered in all three deletion mutants. Amylovoran production was diminished, or almost absent, and cellulose production was affected in the deletion mutants. These findings strongly indicate that the affected enzymes are essential for the correct synthesis of polysaccharides that are targeted by phages as receptors. However, modification of these genes as a resistance strategy against phage attack does not pose a risk to phage treatment, since deletion mutants demonstrated reduced virulence on detached blossoms.IMPORTANCEThe understanding of bacteriophage host interactions is essentially needed if bacteriophages are considered as an alternative treatment option for bacterial infections.