Towards understanding antimicrobial resistance

Project Outline

Antimicrobial resistance (AMR) is a major crisis for human medicine. Globally, untreatable bacterial infections are increasing, leaving limited treatment options. Gram-negative Enterobacteriaceae e.g. Klebsiella pneumoniae with carbapenem resistance are classified as critical priorities by the WHO. An important characteristic of bacteria is their ability to share genetic information, including AMR genes, via mobile-genetic elements such as plasmids. Plasmids can share multiple genes producing resistance to clinically important antibiotics such as b-lactams, (including carbapenems), and even drugs-of-last-resort such as colistin. In addition, plasmids can carry genes for increased virulence. Evidence indicates clinically-relevant AMR plasmids persist even in the absence of antibiotics (e.g. Buckner et al, mBio, 2018). Pathogens with plasmids carrying AMR genes are responsible for some of the most difficult to treat and often multi-drug resistant infections.

In many infection and in the natural environment, bacteria live in dense and intricate communities called biofilms. Biofilms form in, for example, hospital surfaces, deep wound infections, and indwelling devices. Evidence generated in part by the Buckner lab suggests that plasmids containing AMR and/or virulence genes can be shared between bacteria in these biofilms, often at a higher rate than in non-biofilm lifestyles (E.g. Element et al, mSphere 2023). Furthermore, biofilms are intrinsically more difficult to treat with antibiotics, and can continue to “release” bacteria, which may contain additional AMR/virulence plasmids, to infect/colonise other areas.

The Buckner lab have developed a flow-cytometry and microscopy-based assay to monitor plasmid transmission and persistence in bacterial populations, using clinically relevant multi-drug resistant bacteria. The assay includes K. pneumoniae with AMR plasmids tagged with the gfp gene, in conjunction with recipient bacteria labelled with the mcherry gene, to measure plasmid dynamics (conjugation and persistence) (Buckner et al, mBio, 2020). The Buckner lab have also been using biofilm models to study plasmid dynamics in clinical isolates of K. pneumoniae (Element et al, mSphere, 2023). The McNally lab are world-leaders in genomics and bioinformatics, and the bioinformatic components of this project will take place within the McNally group.

Unfortunately, plasmid dynamics in biofilms remain relatively understudied, with information from free-floating “planktonic” cells not translating to the biofilm lifestyle. Therefore, this project will aim to elucidate factors impacting plasmid dynamics within single- and multi-species biofilms using clinical K. pneumoniae isolates. This will be done by using and expanding the fluorescent plasmid system, long-read whole genome sequencing and genomic comparison of isolates, classical plasmid biology, flow cytometry and microscopy to characterise AMR plasmid dynamics in biofilms.

Objectives

1. Sequence and characterise clinical isolates of carbapenem resistant K. pneumoniae
2. Expand the fluorescent AMR plasmid system to these K. pneumoniae isolates.
3. Monitor plasmid dynamics in single species biofilms.
4. Compare plasmid dynamics in multi-species biofilm settings.

Methods

Techniques include, but are not limited to: whole genome sequencing, bioinformatic analysis/genomics, molecular biology, genetic manipulation, generation of single- and multi-species biofilm models, flow cytometry, FACS, confocal microscopy, plasmid conjugation and persistence assays.

Importance

This project will significantly increase our fundamental understanding of how AMR plasmids move within and evolve in biofilm settings. This is a significantly understudied area, and the understanding generated by this project will be instrumental in our ability to understand, and then develop strategies to prevent transmission and/or remove AMR plasmids from bacterial populations.

Techniques

This project will utilise a variety of techniques including but not limited to: whole genome sequencing, bioinformatic analysis/genomics, molecular biology, genetic manipulation, cloning, generation of single- and multi-species biofilm models, flow cytometry, FACS, confocal microscopy, plasmid conjugation and persistence assays.