Fighting phage-resistant variants of Pseudomonas kielensis by synergistic interaction between bacteriophages and...

Pseudomonas kielensis is one of the key psychrophilic bacteria that is known to cause food spoilage. Bacteriophages have been demonstrated to be a promising alternative to suppress the growth of spoilage bacteria in the food industry. This work isolated and characterized a novel lytic phage phiPKYZU08 targeting P. kielensis PK-YZU08. The Autographiviridae family phage phiPKYZU08 has a short tail (16.05 nm length) and an icosahedral head (49.85 nm diameter), and exhibited a burst size of 226 PFU/cell, 10-min latent period, and 30-min burst period. This phage had a 43,378 bp dsDNA genome containing 52 open reading frames (ORFs), which showed 81.18% homology with that of Pseudomonas agarici phage phiNV3 but without virulence or antibiotic resistance genes. However, phage-resistant strains were observed after a period of phage treatment, and phage-resistant variants showed higher biofilm-forming abilities. RNA-sequencing results revealed 12 KEGG pathways associated with energy metabolism were upregulated, while the pentose phosphate pathway, sulfur metabolism, and ABC transporter were significantly downregulated. To overcome phage resistance, a combined treatment of Lactiplantibacillus plantarum LP03 extract (across a concentration from 12.5% to 100%) with phage phiPKYZU08 (10⁶ PFU/mL) showed synergistic interaction on the regrowth of P. kielensis PK-YZU08, with complete inhibition (100%) on bacterial growth after 12 h, 24 h, and 48 h treatments. Even the lowest L. plantarum LP03 extract concentration (6.25%) with phage phiPKYZU08 treatment maintained an antibacterial efficacy of 79.06% at 48 h, 71.63% at 24 h, and 56.60% at 12 h. The synergistic mechanism is mediated by three primary pathways, including (1) direct bacteriostatic activity to suppress bacterial growth, (2) inhibition of biofilm formation to compromise bacterial community integrity, and (3) suppression of phage-insensitive mutants to mitigate resistance evolution. Overall, this multi-target strategy offers an innovative and effective approach for controlling Pseudomonas in the food industry.