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Investigations of novel bacterial drug efflux systems

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posted on 2023-11-01, 04:39 authored by Stephanie Sabina Nagy

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.

History

Table of Contents

Chapter 1: General Introduction -- Chapter 2: Genomic fitness profiling of Acinetobacter baumannii reveals modes of action for common biocides and mechanisms of biocide-antibiotic antagonism -- Chapter 3: The ABC-2 transporter, YadGH from Acinetobacter baumannii has pleiotropic effects on membrane homeostasis and drug resistance -- Chapter 4: Identification and characterisation of an Aromatic Acid Exporter (ArAE) family protein from Acinetobacter baumannii -- Chapter 5: Characterisation of the Acinetobacter baumannii DUF817 protein as a multidrug resistance protein -- Chapter 6: A tool for the direct visualisation of rapid drug efflux out of individual bacterial cells -- Chapter 7: Identification of a novel ciprofloxacin tolerance gene, aciT, which contributes to filamentation in Acinetobacter baumannii -- Chapter 8: Rapid microevolution of biofilm cells in response to antibiotics -- Chapter 9: Discussion and future directions -- Appendices

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

Doctor of Philosophy

Department, Centre or School

School of Natural Sciences

Year of Award

2023

Principal Supervisor

Ian Paulsen

Additional Supervisor 1

Bhumika Shah

Rights

Copyright: The Author Copyright disclaimer: https://www.mq.edu.au/copyright-disclaimer

Language

English

Extent

318 pages

Former Identifiers

AMIS ID: 258595