Work

Defense mechanisms of Pseudomonas aeruginosa and Klebsiella pneumoniae clinical strains against lytic phages and counter-defense

Year: 2026

Type: article

Abstract: Pseudomonas aeruginosa and Klebsiella pneumoniae are major Gram-negative pathogens in which the emergence of multidrug-resistant strains has increased the need for alternative antimicrobial strategies, including phage therapy. However, bacterial antiphage defense systems and the corresponding phage-encoded counter-defense mechanisms can critically influence phage infectivity and therapeutic potential. Here, we characterized bacterial defense systems in 75 clinical P. aeruginosa and 47 K. pneumoniae isolates and analyzed anti-defense repertoires in collections of 13 and 11 corresponding lytic phages, respectively. Abortive infection (Abi) systems were the predominant characterized defense category in P. aeruginosa , whereas systems of unknown function were the most abundant in K. pneumoniae . Among P. aeruginosa phages, DEDDh_I_II_III_IV_V_VI_1, a DnaQ-like exonuclease associated with anti-CRISPR activity, was the most frequently detected anti-defense determinant (76.92%). In K. pneumoniae phages, stp (anti-restriction) was detected in 54.55% and acb1/acb2 (anti-CBASS) in 27.27%. Phage–host interaction analysis revealed substantial heterogeneity in infectivity and showed that the presence or number of anti-defense systems alone did not consistently predict infection outcome. To investigate the functional contribution of specific anti-defense mechanisms, time-kill assays were performed against P. aeruginosa PA14 and CECT-110 and K. pneumoniae ATCC 700721, and bacterial growth inhibition was quantified from the area under the growth curve (AUC). Notably, anti-CBASS-positive P. aeruginosa phages showed significantly greater growth inhibition than anti-CBASS-negative phages (65.28% vs. 15.77%; difference, 49.51 percentage points; P = 0.0007), whereas no significant association was observed in K. pneumoniae . No significant association with growth inhibition was identified for the other anti-defense categories evaluated. Based on this genotype–phenotype association, we designed a LAMP–CRISPR-Cas13a assay targeting queC (CBASS_associated defense) as a proof of concept for rapid detection of CBASS-associated determinants in P. aeruginosa . Although validation in larger bacterial and phage collections is required, our findings support the integration of bacterial defense profiling with complementary phage anti-defense characterization as a potential strategy for prioritizing candidate phages for subsequent phenotypic evaluation and rational phage therapy.

Source: Scientific Reports

DOI: 10.1038/s41598-026-69902-8