Molecular Basis of Tobacco Induced Bacterial Biofilms

Abstract

Objective: Tobacco smoke exposure has been correlated to recalcitrant chronic rhinosinustis. Prior work has demonstrated tobacco smoke mediated sinonasal biofilm formation. Thus our objectives were: 1) Construct a series of reporter constructs for pseudomonas biofilm forming genes. 2) Determine which biofilm forming genes are regulated by tobacco smoke. Method: Promoter regions of 6 genes involved in pseudomonas biofilm formation (LasI, LasR, RhlA, PilF, FlgK, and AlgC) were subcloned into the luciferase reporter plasmid pMini-CTX-Lux. Subcloning was confirmed with PCR with subsequent conjugation into Pseudomonas (PAO1). Each subclone was then challenged with serial tobacco smoke exposure and luciferase activity quantified. Results: Elevated levels of luminescence, indicating specific promoter activation was identified after daily tobacco-smoke exposure in the LasI, PilF, FlgK, and AlgC genes. The promoter activation of these genes was significantly elevated ( P < .05) after 2 consecutive exposures with no additional activation following a third exposure (Kruskall-Wallis non-parametric test). The level of promoter activation of the LasB gene was not affected by tobacco smoke exposure, while the RhlA gene decreased progressively by daily exposure. Conclusion: Utilizing the luciferase reporter construct mLuxI we demonstrate activation of several key biofilm forming genes by exposure to tobacco smoke. This confirms our prior finding that tobacco smoke induces biofilms in sinonasal microbes. Interestingly, not all biofilm pathways interrogated demonstrated activation, suggesting unique tobacco mediated biofilm forming pathways in pseudomonas.