Emergence of phage-resistant Pseudomonas aeruginosa biofilm cells in response to the pressure exerted by bacteriophage treatment

Antibiotic resistance constitutes one of the most serious threats to the global public health and it urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to the traditional antibiotic therapies. In the present study, the efficacy of phages against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. In this work, the emergence of phage-resistant variants was tracked during phage infection experiments, in which the phages were targeting different cell receptors. Resistant bacterial variants were isolated as early as 6-h post-phage biofilm treatment, depending on the phage used. In most cases, the emergence of phage-resistant variants occurred at a later time point in planktonic cultures than in biofilms. To further investigate the genetic makeup of these phage-resistant variants and possible mutations accumulated, some of these bacteria were selected for further phenotypic and genotypic characterization. Whole genome sequencing was performed on five of the P. aeruginosa PAO1-derived phage-resistant variants and all of them carried mutations affecting the galU gene, which is involved in lipopolysaccharide core biosynthesis, as well as in one of pil genes, which are involved in the synthesis of type IV pilus. These results were corroborated by results from the LPS analysis of the five variants, which revealed defects in LPS biosynthesis as reflected by the presence of truncated core oligosaccharide and the absence of long-chain O-antigen bands. Examination of these variants by transmission electron microscopy, revealed that in contrast to the wild-type bacteria, none of the variants produces pili . Genomic sequencing further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (> 200 kbp) in their genomes. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develops phage-resistant variants that are well adapted to the biofilm mode of growth.