Investigations of novel bacterial drug efflux systems
The antimicrobial resistance (AMR) crisis represents one of the greatest challenges to modern medicine this century and threatens a return to the pre-antimicrobial era. It is evident that novel therapeutic options are required. Multidrug efflux pumps are ancient integral membrane transporters that provide a direct avenue for the polyspecific export of noxious compounds out of the cell, including antimicrobial therapeutics, thus promoting cell survival under antimicrobial challenge. Advances in genome sequencing and genomic techniques have allowed researchers to sequence, screen, identify and characterise drug efflux systems of novel bacterial isolates at exceptional rates. Furthermore, advances in single cell technology have provided a unique opportunity to study subtleties in heterogeneous efflux function at the single-cell level. Acinetobacter baumannii is a Gram-negative coccobacillus that is classified as a global top priority for the development of novel antimicrobials. A. baumannii infections are opportunistic and primarily affect the most critically ill patients. The emergence of multidrug resistant A. baumannii isolates is a significant unsolved public health problem.
The work presented in this thesis explores a range of high-throughput genomic techniques to further understand antimicrobial resistance in this pathogen. Transposon directed insertion sequencing was used to investigate the core fitness genes that confer biocide tolerance in A. baumannii. Transcriptomics explored the global gene response to subinhibitory concentration exposure of tetracycline and ciprofloxacin on planktonic and biofilm cultures of A. baumannii.
The high-throughput genomic analyses identified various hypothetical membrane proteins of unknown function that were important for antimicrobial fitness that could potentially represent new drug efflux pumps. Investigations in this thesis focused on three novel membrane proteins that were important for fitness under subinhibitory silver nitrate exposure in A. baumannii. The ABC-2 transporter, YadGH, the ArAE transport protein, ABUW_0700 and the DUF817 family protein, ABUW_1191 were found to be highly conserved in regions related to cell envelope homeostasis, redox and virulence respectively. Heterologous expression in Escherichia coli provided evidence for multidrug resistance phenotype and acriflavine efflux activity in comparison to the empty vector controls. Microscopy, flow cytometry and lipid composition analyses suggested that YadGH conferred various pleiotropic effects on the cell envelope. Further experimental work will be required to clarify whether these membrane proteins are multidrug efflux pumps or confer drug resistance via indirect effects on the cell envelope.
Advances in single-cell technology provide a unique opportunity to study heterogeneity of efflux activity at the individual cell level. In this thesis, we provide a proof-of-concept study where we designed a microfluidic chip that allows for the direct visualisation and quantitation of heterogeneous efflux rates of individual cells in an isogenic bacterial population. To validate this, we studied real-time R6G efflux heterogeneity in a wild-type population of E. coli cells and discover a broad distribution of different efflux rates in individual cells. Furthermore, our technique enabled visualisation of heterogeneous R6G efflux cycles at the single-molecule level.
