Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity. Author summary There is a poor correlation between the activity of antibiotics in the laboratory and in patients, including in several infectious diseases of the respiratory tract. What may help explaining differences between antibiotic activity in vitro and in vivo is that current antibiotic susceptibility tests do not consider the in vivo lung environment. The lung environment contains many factors that may influence bacterial susceptibility to antibiotics. This includes lung epithelial cells, which have been shown to improve the activity of aminoglycoside antibiotics. Yet, how lung epithelial cells increase aminoglycoside activity is currently unknown. Here, we cultured lung epithelial cells in an in vivo-like model and found that they secrete metabolites that enhance the activity of aminoglycoside antibiotics. We found that host cell secretions increased antibiotic uptake through stimulation of bacterial metabolism, which in turn resulted in enhanced activity. Our findings highlight that cross-talk between host and bacterial metabolisms contributes to the efficacy of antibiotic treatment. Understanding how the host metabolism influences antibiotic activity may open up therapeutic avenues to exploit host metabolism for improving antibiotic activity and help explaining discrepancies between antibiotic efficacy in vitro and in vivo.