Impact of gas-mediated respiratory inhibition on antibiotic tolerance in Escherichia coli

Carbon monoxide (CO) and nitric oxide (NO) are potent respiratory inhibitors that can be present at the site of bacterial infections. It is well-known that bacterial respiratory metabolism can module susceptibility to antibiotics, so the overarching goal of this PhD project was to analyse the effect of NO and CO mediated respiratory inhibition upon antibiotic susceptibility.

To develop tools for this project, an E. coli strain capable of endogenous CO production was created via expression of recombinant Human haem oxygenase HO-1 protein encoded on a plasmid vector. This strain was shown to accumulate CO via haemoglobin-binding assays, along with the green pigment biliverdin, the second product of haem oxygenase activity. However, recombinant HO-1 expression had a negligible effect upon the growth of E. coli and elicited a modest decrease in oxygen consumption. In addition, bubbling growth medium with CO gas did not impair respiratory oxygen consumption, which prompted a switch to focus on NO in subsequent chapters.

The involvement of reactive oxygen species and respiratory metabolism in antibiotic susceptibility has been contentious for many years, yet antibiotics are effective under anaerobic conditions and the respiratory inhibitor NO is produced by the host in response to bacterial infection. Recent work has supported a prominent role for the proton motive force (PMF) in aminoglycoside susceptibility, so it was of interest to focus upon the interplay between anaerobic conditions, NO exposure, and the efficacy of aminoglycoside antibiotics. Under anaerobic conditions, respiratory inhibition by NO or dissipation of the PMF by the uncoupler 2,4-DNP were both shown to significantly decrease the susceptibility of E. coli cultures against gentamicin.

A logical extension of the E. coli antibiotic susceptibility work was focussing on nitrofurantoin, an antibiotic that can accumulate in the urine where pathogenic E. coli may also be exposed to NO. Surprisingly, the presence of NO increased the susceptibility of E. coli to nitrofurantoin. Proteomic analyses were performed to identify perturbations in protein abundances to provide mechanistic insights into this interaction, and systems involved in acid stress and potential routes for enzymatic reduction of nitrofurantoin were implicated in this study.

Through these investigations we identified interactions between antibiotic susceptibility of E. coli and nitric oxide exposure. These findings suggest potential combination therapies could be developed as antibiotic resistance continues to grow as a global threat.