Fluctuating Bacteriophage-induced galU Deficiency Region is Involved in Trade-off Effects on the Phage and Fluoroquinolone Sensitivity in Pseudomonas aeruginosa

• Phage-resistant Pseudomonas aeruginosa brown mutants (brmts) with galU and hmgA deletions were isolated. • Whole-genome sequencing of the brmts revealed that upstream and downstream of galU were largely deleted from the parental chromosome. • These deleted sequences commonly harbored multi-drug efflux system genes ( mexXY ). • These phage-resistant variants exhibited increased fluoroquinolones sensitivity compared with the parental strain. • Our findings may provide insights into part of the potential strategies for controlling the phage-resistant variants during phage therapy. Pseudomonas aeruginosa , which causes chronic infections, has demonstrated rapid acquisition of antimicrobial resistance (AMR). Therefore, bacteriophages have received significant attention as promising antimicrobial agents; however, previous trials have reported the occurrence of phage-resistant variants. P. aeruginosa has lost large chromosomal fragments via evolutionary selection by MutL. Mutants lacking galU and hmgA, located in close proximity, exhibit phage resistance and brown color phenotype since hmgA encodes a homogentisic acid metabolic enzyme and deletion of galU results in a lack of O -antigen polysaccharide and absence of the phage receptor. In the present study, we evaluated this mechanism for controlling phage resistance in P. aeruginosa veterinary isolate Pa12. Phage-resistant Pa12 brown mutants (brmts) with galU and hmgA deletions were isolated. Whole-genome sequencing of the brmts revealed that regions 148-27 kbp upstream and 261-110 kbp downstream of galU were largely deleted from the Pa12 parental chromosome. Furthermore, all of these fluctuating deleted sequences in Pa12 brmts, tentatively designated bacteriophage-induced galU deficiency (BigD) regions, harbor multi-drug efflux system genes ( mexXY ). Minimum inhibitory concentration (MIC) assays demonstrated that brmts altered sensitivity to antibiotics and exhibited increased levofloxacin sensitivity compared with the Pa12 parent. Orbifloxacin and enrofloxacin also effectively suppressed growth of the Pa12 brmts, suggesting that MexXY, which mediates quinolone efflux and is located in the BigD region, might be associated with restoration of fluoroquinolone sensitivity. Our findings indicate that AMR-related genes in the BigD region could produce trade-off effects between phages and drug sensitivity and thereby contribute to a potential strategy to control and prevent phage-resistant variants in phage therapy.