Bacterial resistance to cationic biocides, including quaternary ammonium compound (QAC) disinfectants, is primarily conferred by modifications that increase membrane-bound efflux pump activity. In Gram-negative bacteria, numerous unrelated single-component efflux pump families contribute to efflux-mediated antimicrobial resistance; these pumps have preference for cationic antimicrobial expulsion. The single-component efflux pump family known as Small Multidrug Resistance (SMR) pumps, are frequently transmitted by class I integrons on multidrug-resistant plasmids in Enterobacteria. These SMR genes are renowned for ability to confer resistance to QACs/antibiotics in Enterobacteria growing planktonically, but their role during biofilm formation is unclear. The first aim of this thesis (Chapters 3-4) was to better understand substrate selectivity and regulation of plasmid-transmitted SMR members when grown as biofilms, which represents a clinically relevant physiological growth state. The most frequent plasmid-transmitted SMR members representing two subclasses, Gdx and Qac, were examined separately to determine phylogenetic relatedness and sequence diversity. Antimicrobial susceptibility testing (AST) was used to characterize Gdx and Qac substrate selectivity in E. coli when grown as planktonic and biofilm cultures. Results indicated that Gdx and Qac sequence diversity was low, suggesting these sequences are rapidly diverging from similar genes. Acquisition of either Gdx or Qac genes in E. coli resulted in broader QAC/antibiotic substrate recognition and resistance only when cells grew as biofilms.
The final thesis aim compared cation-selective single-component efflux members representing four families, MdtK, MdfA, EmrE, AceI, to explore if lipid-co-associations between these transporters influences their ability to recognize cationic antimicrobials. Cloned and affinity-tagged versions of each pump were overexpressed for AST in E. coli strains lacking key phospholipid synthesis genes for major phospholipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). Analysis revealed that most pumps increased antimicrobial susceptibility when negatively charged PG or CL levels increased. Isolation of annular phospholipids co-associated with each affinity-tagged transporter using Peptidisc methods revealed significant bias for CL co-association by all pumps, with each having differing ratios of PE and PG.
This thesis reveals the importance of monitoring biofilm growth physiology when examining the substrate selectivity of QAC selective efflux pumps and reveals the importance of anionic phospholipids (PG and CL) in regulating efflux activity.