Defining the role of efflux in bacterial biofilm formation and antimicrobial resistance to develop new treatments for infection

Molecular efflux systems remove toxic substances from cells via a pump-like mechanism, with presence in bacterial species associated with the development of antimicrobial resistance (AMR). They are also involved in biofilm formation, a surface associated bacterial community with intrinsic AMR, the development of which occurs in up to 80% of infections. Recent studies have identified upregulation of efflux systems in many biofilm-forming pathogens, and an association with resistance to many of the biocides used in hospitals. A greater understanding of the role of efflux in biofilm formation and AMR would therefore support the development of new antimicrobial agents.

Aims and objectives:
The overall aim of this project is to investigate the hypothesis that efflux systems have a waste management and regulatory role in biofilm formation, and that drugs already used in human medicine can be repurposed as efflux-inhibitors and anti-biofilm agents. To achieve this, Proteus mirabilis will be used as a clinically relevant model organism. This is a Gram negative organism frequently associated with catheter associated urinary tract infections (CAUTI) due to its ability to form extensive crystalline biofilm networks that block urinary catheters, leading to severe complications such as septicaemia.

Bioinformatic approaches will be used to identify efflux systems and associated regulatory genes encoded by clinical isolates of P. mirabilis. Further to this, mutants lacking key efflux systems and/or regulatory genes will be constructed, and biofilm formation compared to parenteral strains using in vitro models of biofilm formation, including the use of a clinically relevant CAUTI model. This will increase understanding of how efflux systems support the formation of biofilms, and their integration with gene networks that govern this process.
As well as the role of the identified efflux systems in biofilm formation, their role in biocide tolerance will be examined via susceptibility testing in strains with upregulated and downregulated efflux systems. In addition, Galleria mellonella larvae will be used to examine how these mutations affect the virulence of the strains in vivo.
In order to understand the role that the identified efflux systems play in the modulation of wider gene networks controlling biofilm formation, global gene expression profiles will be obtained from specific efflux mutants, as well as from cells which have undergone non-specific chemical efflux inhibition. Target systems will be identified, and in silico modelling approaches will then be used to assess interactions with potential inhibitors in order to identify existing compounds with the potential for use as efflux-inhibitors and anti-biofilm agents. Interactions will be further characterised using biological assays.

Applications and benefits:
The MRC has identified AMR as a priority research area therefore this project supports this. The main benefits will be novel insight into the role of efflux systems in biofilm formation and AMR, and identification of key systems as pharmacological targets. This will aid the identification of anti-biofilm agents from a pool of existing compounds, supporting development of antimicrobial agents.