Grant: Discovery Grants Program - Individual

Antimicrobial resistance (AMR) attributed to disinfectant cationic biocides (CB) is a growing problem as it reduces the effectiveness of CBs we rely on in agriculture, health, and pipeline industries. Even before the pandemic, our reliance on disinfectant CBs has increases annually; CBs are a common environmental pollutant and driver of AMR. CBs kill bacteria by disrupting membrane lipids and are relied upon to eradicate bacterial biofilms. Efflux pumps are the most well-recognized CB resistance mechanism, where efflux membrane proteins actively expel CBs as they enter the cell rendering them ineffective. However, CB mechanisms cannot explain how these membrane protein pumps resist the lipid-damaging actions of CBs. We have recently discovered that lipid-modifying CB-resistance mechanisms are equally, if not more important than efflux. Our previous studies of E. coli K-12 adapted to the most common CBs revealed an assortment of lipid biosynthetic genes and lipid transport system mutations that confer CB resistance. Many of the lipid mutants we discovered also enhanced the formation of outer membrane vesicles (OMV), which may represent a potent physiological CB/ disinfectant resistance mechanism. The long-term objective of my program is to understand how bacterial lipid alterations influence efflux-mediated CB resistance mechanisms. We hypothesize that CB-resistant mechanisms in bacteria rely on efflux pump activity during biofilm growth, but during planktonic growth, cells rely on lipid alterations that enhance OMV release to remove CB-damaged membranes. To assess this, we will expand our analysis of E. coli to include other commonly identified Gram-negative environmental contaminants, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We propose 3-short term objectives to explore this: 1. Generate CB-adapted species in vitro to identify and characterize CB-resistant genes using antimicrobial susceptibility testing and multiomics analyses. This aim determines if CB-resistant lipid gene variants detected in previous E. coli studies are similarly identified in other adapted species. 2. Characterize CB-resistant lipid genes for their impacts on biofilm and OMV formation. By determining how various CB-resistant lipid mutants influence biofilm and OMV formation, we can determine the extent of each mechanism's contributions toward different CB-resistant cell physiologies. 3. Identify phospholipids directly associated with CB-selective efflux pump function using Peptidiscs and mass spectrometry-based approaches. Since CB adaptation alters lipid content, these approaches identify what types of lipids may directly regulate efflux pump activity. This program offers much-needed insights into how lipid- and efflux-mediated resistance mechanisms in bacteria contribute to CB as well as AMR. These findings will lead to innovations in CB-resistance detection tools and methodologies, as well as improvements in the design of more effective CBs.