Photodynamic antimicrobial efficacy and mode of action of polypyridyl‐based Ru(II) complexes against multidrug‐resistant Klebsiella pneumoniae

Background and Purpose The rise of multidrug‐resistant (MDR) bacteria poses a significant threat to global health. MDR Klebsiella pneumoniae is of particular concern because of its increasing antibiotic resistance. With limited therapeutic options, antimicrobial photodynamic therapy (aPDT), which employs light‐activated photosensitizers to produce reactive oxygen species (ROS), represents a promising alternative. Experimental Approach We assessed the antimicrobial activity of two ruthenium‐based photosensitisers, PSRu‐379 and PSRu‐381, against both susceptible and MDR K. pneumoniae strains and explored their mode of action. Bacterial survival was measured after activation with 61.2 J·cm −2 of blue light. ROS production was analysed using fluorescent probes; transcriptional responses were examined by RT‐qPCR and RNA sequencing; cytotoxicity was tested in human cell lines; and interactions with cefotaxime were evaluated using fractional inhibitory concentration (FIC) analysis. Key Results The minimum effective concentration was 4 μg·ml −1 for the susceptible strain and 8 μg·ml −1 for MDR strains. ROS analysis indicated predominant singlet oxygen generation, with minimal contribution from hydrogen peroxide, supporting a predominantly Type II photodynamic mechanism. Transcriptomic analysis revealed upregulation of sulphur metabolism and oxidative stress response pathways, along with downregulation of aminoacid biosynthesis, capsule formation and metal ion transport. The photosensitisers exhibited synergistic interactions with cefotaxime, reducing MIC values and yielding an FIC index of 0.08. Low cytotoxicity was observed in human cell lines under the tested conditions. Conclusions and Implications Ruthenium‐based photosensitisers appear to act predominantly through singlet oxygen‐mediated oxidative stress, enhancing cefotaxime efficacy while exhibiting low cytotoxicity, and therefore represent promising adjunct therapies against MDR K. pneumoniae .