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Efflux detectives: Identifying and investigating novel efflux systems in important human pathogens

Primary Supervisor: Dr Jessica Blair, Institute of Microbiology and Infection

Secondary supervisor: Prof Alan McNally

PhD project title: Efflux detectives: Identifying and investigating novel efflux systems in important human pathogens

University of Registration: University of Birmingham

Project outline:

Antibiotic Resistance is a current global health crisis that kills 700,000 people every year. Bacterial infections are becoming harder to treat as they evolve resistance to the drugs used to treat them. In our team we try to understand the mechanisms bacteria use to become resistant to antibiotics. One way bacteria become resistant to antibiotics is using efflux pumps(1). These are protein machines in the bacterial membrane which pump antibiotic molecules directly out of the cell to keep internal concentrations below toxic levels. In Salmonella and other Gram-negative bacteria the most clinically important pump is AcrAB-TolC which confers resistance to multiple classes of antibiotics and underpins many other mechanisms of antibiotic resistance. RND efflux pumps are also fundamental to the biology of Gram-negative organisms and are critically important for virulence and biofilm formation.

Many Gram-negative bacteria encode multiple RND efflux pumps with different functions and which are expressed under different conditions. For example, Salmonella commonly has 5 RND pumps while E. coli has 6 and Pseudomonas aeruginosa has 13. The number of RND efflux pumps encoded differs between genera, between species of the same genera and even between strains of the same species (manuscript in preparation).

We have recently developed a method to search bacterial genomes for novel RND efflux pumps and used it to search available genomes. As part of this we have discovered multiple novel RND pumps including a novel E. coli RND efflux pump that is present throughout disease causing lineages of E. coli but has been lost from commensal and environmental lineages. The pump is highly conserved suggesting it is under strong selective pressure but the pump function is currently not clear.

Experimental plan:

  1. Investigate the function of the novel RND efflux pump in E. coli. This will include phenotypic characterisation of engineered E. coli that either lack or over-express the pump. We will investigate whether the pump exports antibiotics or is required for biofilm formation or to cause infection.
  2. Search available genomes of other important human pathogens to look for other new efflux systems. Our preliminary work shows that there are a significant number of undescribed efflux systems in clinically, industrially, and environmentally important organisms. Another element of this project will be to search for further novel pumps in selected organisms. This will allow us to understand the prevalence and variation in the number of RND efflux pumps in different bacterial genera/species/strains. Once identified we will select pumps of particular interest and study their prevalence and characterise the function.

Work in the team also covers many other aspects of efflux pump biology and I’m happy to discuss alternative projects in this area.

BBSRC Strategic Research Priority: Understanding the Rules of Life:Microbiology

    Techniques that will be undertaken during the project:

    • Bioinformatics/microbial genomics
    • Molecular microbiology
    • Measurement of aspects of bacterial physiology including bacterial growth, antimicrobial susceptibility, biofilm formation, virulence, motility etc.
    • Measurement of intracellular drug accumulation and efflux rate.
    • Flow cytometry.

    Contact: Dr Jessica Blair, University of Birmingham