Analyzing the effects of benzodiazepines on key virulence-related parameters of Pseudomonas aeruginosa

Sedation with benzodiazepines (BZs) has eventual side-effects proved to entail incremented risks for ventilator-associated pneumonia (VAP) (e.g. immunity alterations and nervous/mechanical responses increasing the aspiration of upper respiratory tract-colonizing bacteria), but some knowledge gaps on the topic exist. For instance, it has never been approached whether BZs could cause a modulation of intrinsic bacterial virulence, and/or influence the host-pathogen interaction in neglected contexts (e.g. respiratory epithelium features, or the lytic/opsonic activity of complement) to facilitate VAP development. Thus, to enrich the picture of factors contributing to the BZs-associated higher risk for VAP, we analyzed relevant in vitro, ex vivo and in vivo infection-related parameters to decipher whether they could be affected by these sedatives in the top VAP-causing pathogen Pseudomonas aeruginosa. While most variables were unaltered (including among others invertebrate infection assays and a rat model of VAP), a significantly attenuated pathogenic impact of infection on human respiratory A549 cells (invasion, cytotoxicity and inflammation reduced up to ≈50%) appeared upon BZ exposure at high therapeutic concentrations (diazepam/midazolam 10 µg/ml), potentially because of effects of the drugs mostly on the cultured cells. These facts could entail a BZs-associated stealth pathogen-like behavior of P. aeruginosa on the respiratory epithelium, consisting of a weak immune activation proportional to the mild damage caused, perhaps favoring VAP onset. Strikingly, BZs triggered a significantly increased biofilm formation (up to ≈2-fold > controls) on plastic plates and endotracheal tubes (supported by the upregulation of various biofilm-related genes and c-di-GMP accumulation), suggesting a groundbreaking idea: the BZ-dependent boosted formation of these P. aeruginosa sessile reservoirs, likely enabling bacterial release to deep airways and thus VAP progression. Animal models with prolonged sedation/ventilation periods should be conducted to confirm that BZs pose an added trigger for VAP through the surprising hitherto unknown phenomena revealed here. RNA-seq prfiling of Pseudomonas aeuginosa (from Denmark collection, DK) grown in planktonic (PL) or biofilm (BF) mode of life in response to midazolam 10 ug/ml treatment (M) vs untreated control (C)