The interplay between antibiotic efflux and plasmid-mediated antibiotic resistance

Secondary Supervisor(s): Dr Michelle Buckner

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology)

Project Outline

Antimicrobial resistance (AMR) is global health crisis that is associated with ~5 million deaths a year. Understanding the mechanisms that confer AMR is key to being to able to design and develop novel therapeutics to tackle this (1).

Bacterial efflux pumps are an intrinsic mechanism of AMR (2). These are molecular machines that sit in bacterial membranes and can pump a broad range of antibiotic molecules out of bacterial cells. This underpins all other mechanisms of antibiotic resistance. In addition, many important AMR mechanisms are encoded on extrachromosomal pieces of DNA, called plasmids, which can be shared between bacteria. This enables rapid dissemination of AMR genes through and between species. For example, carbapenemase genes, which confer resistance to crucial last-line of defence antibiotics, are regularly carried on and transmitted between bacteria, including K. pneumoniae and E. coli (e.g. 3)

There is some evidence in the literature that efflux alters plasmid transfer under specific circumstances (e.g. 4) and we have preliminary evidence suggesting this is a more general effect. Understanding this link could have significant implications for designing novel therapeutics that target efflux function and/or prevent plasmid conjugation.

This project will explore the extent of the impact of efflux status on plasmid conjugation and AMR and elucidate the molecular mechanisms underpinning the effect.

Objective one: Measure the impact of efflux rate on conjugation of AMR plasmids in Gram-negative bacteria. Here we will measure how presence, absence or over-expression of different bacterial efflux pumps alters conjugation of different AMR plasmids in the presence of clinically relevant antibiotics. This will be done using a combination of traditional conjugation assays and flow cytometry (using fluorescently tagged AMR plasmids) (5,6).

Objective two: Molecules capable of inhibiting efflux pumps have been discovered, as have molecules able to reduce the level of plasmid conjugation, although neither have reached the clinic. Next, we will investigate whether chemically or genetically inhibiting either process alters the impact on AMR and whether combining inhibitors with different mechanisms has an additive effect.

Objective three: Based on results from objective one and two, and existing preliminary data, we will explore the mechanism by which efflux and conjugation rate are connected. Initially, this will involve knocking out and over-expressing genes, and exploring gene expression profiles. These findings will guide further molecular assays.

This project will be a collaboration across two research groups working on AMR, headed by Professor Jessica Blair who has expertise in bacterial efflux pumps and Dr Michelle Bucker who is an expert in plasmid biology and conjugation. Both teams are welcoming, inclusive environments that focus on student development and learning while working on important and exciting problems connected to AMR.

This project will involve mastering the measurement of many of aspects of microbial physiology including antimicrobial susceptibility, plasmid conjugation, efflux rates and intracellular antimicrobial accumulation. This project will involve training in a range of microbiology and molecular biology skills, likely to include flow cytometry, FACS, bacterial culture, genetic engineering, plasmid conjugation and persistence assays, sequencing and analysis.

References

1. Darby, E.M., Trampari, E., Siasat, P. et al. Molecular mechanisms of antibiotic resistance revisited. Nat Rev Microbiol 21, 280–295 (2023). DOI:10.1038/s41579-022-00820-y.

2. Siasat and Blair, 2024. Microbial Primer: Multidrug efflux pumps. Microbiology. doi:10.1099/mic.0.001370.

3. Element SJ, Moran RA, Beattie E, Hall RJ, van Schaik W, Buckner MM. 2023. Growth in a biofilm promotes conjugation of a blaNDM-1-bearing plasmid between Klebsiella pneumoniae strains. mSphere, doi:10.1128/msphere.00170-23.

4. Nolivos et al.

How do bacteria prevent intracellular accumulation of antibiotics?

Secondary Supervisor(s): Dr Tim Overton

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology)

Project Outline

All living cells must control the internal concentration of harmful molecules. This can include toxic by-products of metabolism, or harmful exogenous molecules encountered in the cell’s environment. For bacteria, a major threat is antibiotics, most of which have intracellular targets and require accumulation to a critical concentration for activity (1). The more a cell can limit intracellular concentration of an antibiotic, the more likely it will survive.

Cells adopt two fundamental strategies to prevent accumulation of antibiotics: limiting influx of toxic molecules into cells; and active export (efflux) to reduce intracellular concentration (2). However, we have shown that the relative importance of each process for bacteria changes dramatically in different physiological conditions (3). However, why this is the case, and how cells have evolved regulatory and effector mechanisms to allow them to control drug accumulation in diverse conditions is poorly understood.

This project aims to:

Objective one: Use established assays to measure how intracellular accumulation of antibiotics is altered in different physiologically and environmentally relevant conditions and when cells are in a biofilm. The conditions will include aspects of conditions encountered during infection or in the environment.

Objective two: Establish the total cellular impact of selected conditions using techniques including RNAseq. We will also study the physical changes in membrane composition in selected conditions.

Objective three: Understand how the mechanisms that control intracellular accumulation of antibiotics change in different conditions to impact bacterial susceptibility to drugs. We will use molecular microbiology and genetic engineering to inactivate or over-express genes that we hypothesise to be involved in controlling intracellular antibiotic concentration and measure the impact of this.

This project will be part of an existing collaboration between two research groups working on AMR, headed by Professor Jessica Blair and Dr Tim Overton. Both teams are welcoming, inclusive environments that focus on student development and learning while working on important and exciting problems connected to AMR.

This project will involve learning multiple techniques to measure the intracellular accumulation of molecules inside bacterial cells, many of which use flow cytometry and have been developed by us (e.g. 4). The student will also master the measurement of many of aspects of microbial physiology including antimicrobial susceptibility, growth and efflux rates. They will also learn how to perform and analyse RNAseq data and will get a thorough training in molecular microbiology.

References

1. Darby, E.M., Trampari, E., Siasat, P. et al. Molecular mechanisms of antibiotic resistance revisited. Nat Rev Microbiol 21, 280–295 (2023). DOI:10.1038/s41579-022-00820-y.

2. Siasat and Blair, 2024. Microbial Primer: Multidrug efflux pumps. Microbiology.

doi:10.1099/mic.0.001370.

3. Whittle EE, McNeil HE, Trampari E, Webber M, Overton TW, Blair JMA. 2021. Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells. mBio 12:10.1128/mbio.02608-21.

4. Emily E Whittle, Simon Legood, Ilyas Alav, Tim Overton and Jessica M A Blair. Flow Cytometric Analysis of Efflux by Dye Accumulation. Frontiers in Microbiology. 2019 10, 2319.