Synergistic effect of Ru(II)-based type II photodynamic therapy with cefotaxime on clinical isolates of ESBL-producing Klebsiella pneumoniae

Multidrug-resistant bacteria, such as ESBL producing-Klebsiella pneumoniae, have increased substantially, encouraging the development of complementary therapies such as photodynamic inactivation (PDI). PDI uses photosensitizer (PS) compounds that kill bacteria using light to produce reactive oxygen species. We test Ru-based PS to inhibit K. pneumoniae and advance in the characterization of the mode of action. The PDI activity of PSRu-L2, and PSRu-L3, was determined by serial micro dilutions exposing K. pneumoniae to 0.612 J/cm 2 of light dose. PS interaction with cefotaxime was determined on a collection of 118 clinical isolates of K. pneumoniae. To characterize the mode of action of PDI, the bacterial response to oxidative stress was measured by RT-qPCR. Also, the cytotoxicity on mammalian cells was assessed by trypan blue exclusion. Over clinical isolates, the compounds are bactericidal, at doses of 8 µg/mL PSRu-L2 and 4 µg/mL PSRu-L3, inhibit bacterial growth by 3 log10 (>99.9%) with a lethality of 30 min. A remarkable synergistic effect of the PSRu-L2 and PSRu-L3 compounds with cefotaxime increased the bactericidal effect in a subpopulation of 66 ESBL-clinical isolates to > 6 log10 with an FIC-value of 0.16 and 0.17, respectively. The bacterial transcription response suggests that the mode of action occurs through Type II oxidative stress. The upregulation of the extracytoplasmic virulence factors mrkD, magA, and rmpA accompanied this response. Also, the compounds show little or no toxicity in vitro on HEp-2 and HEK293T cells. Through the type II effect, PSs compounds are bactericidal, synergistic on K. pneumoniae, and have low cytotoxicity in mammals.