Precision Medicine DTP - Genomic characterisation of multidrug-resistant septicaemia-causing Klebsiella pneumoniae to inform patient care and infection control

Additional Supervisor: Dr Surabhi Taori [NHS Lothian]

Background

Antimicrobial resistance (AMR) is a global health crisis. Resistance to carbapenems, one of the last-resort treatments for severe infections, is of particular concern in Gram-negative organisms. Klebsiella pneumoniae is a clinically important pathogen that causes hospital-acquired pneumonia and life-threatening invasive septicaemia, and is most commonly associated with carbapenemases, enzymes that can render carbapenems inactive. Carbapenem resistance is often a consequence of mutation and gene acquisition via plasmids that can carry additional resistance genes and virulence factors. In Klebsiella pneumoniae, plasmid acquisition has led to the emergence of high-risk multidrug-resistance clones with enhanced pathogenicity and fitness that are associated with increased morbidity and mortality. 

Plasmid transfer between Klebsiella pneumoniae and Gram-negative organisms like Escherichia coli and Enterobacter species can lead to more than one type of carbapenemase being present in a clinical isolate that can directly impede treatment. Plasmids can also carry genes that confer resistance to other antibiotics, siderophores that enhance systemic survival, genes that regulate prominent virulence factors like the capsule, anti-restriction factors that facilitate inter- and intra-species transfer and error-prone polymerases. Plasmid acquisition can, therefore, change the bacterial phenotype and alter disease severity. Bacterial exposure to antibiotics of either the wrong class or at suboptimal levels can induce stress responses that increase plasmid persistence and transfer. Stress also induces the production of error-prone polymerases that can introduce new genetic mutations, for example, the proliferation of new carbapenemases, which is an increasing threat to human health. Genome analysis of clinical isolates and understanding the mechanisms of plasmid evolution are essential to predict changes in virulence, to determine strain-relatedness and to predict the risk of plasmid spread to ultimately inform patient treatment and management and infection control practices. 

Aims

In this project, long-read sequencing will be employed to analyse the genomes of septicemia-causing clinical isolates of Klebsiella pneumoniaeto understand why some strains caused fatal infections while others didn’t. This will be complemented by phenotypic analyses of virulence and persistence in the environment. Further, the creation of new carbapenemases and plasmid transfer will be investigated to assess the effects on virulence, treatment options and infection control practices. 

1. Perform long-read sequencing of clinical isolates of Klebsiella pneumoniae

MinION sequencing will be performed to generate whole-genome sequences for K. pneumoniae isolates that did and did not cause fatal septicaemia. Bioinformatics analyses will be performed to identify chromosomal and plasmid-borne AMR genes, virulence factors, anti-restriction factors and error-prone polymerases. Sequences will be compared to those in publicly available databases to understand strain distribution and plasmid evolution. Data will be analysed in the context of clinical information on patient outcomes. 

2. Assess clinically relevant phenotypes of lethal and non-lethal strains

Phenotypic analyses including adherence to and invasion of epithelial cells, survival within phagocytic cells and antimicrobial susceptibility will be performed to understand differences in patient outcomes. Adherence to surfaces and resistance to desiccation and disinfectants will be investigated to assess environmental persistence.

3. Characterise chromosomal and plasmid-borne carbapenemases

Focused comparisons of carbapenemase-encoding gene sequences will be performed to identify novel members and evolution of gene families. Long-term cultures of strains in the presence of sub-inhibitory levels of antibiotics including carbapenems and fluoroquinolones will be carried out to assess the role of error-prone polymerases in carbapenemase evolution. Sequencing to identify genetic mutations and phenotypic characterisation (as in Aim 2) will be performed at regular intervals.

4. Assess inter-and intra-species plasmid transfer and associated changes in phenotypes

Transfer of plasmids from clinical isolates to plasmid-free strains of K. pneumoniae and E. coli will be assessed in co-culture experiments with and without antibiotic selection. Phenotypic changes in recipient strains will be investigated (as in Aim 2). 

Training outcomes

This project will provide training in laboratory skills required to culture bacterial pathogens and assess their phenotypes alongside extensive training in bioinformatics, genomics and bacterial genetics in the context of clinical information, resulting in an understanding of how whole-genome sequencing can influence patient treatment and clinical outcomes, and manage pathogen spread in clinical settings. It will also identify specific genes that would serve as markers for strains with the potential to become lethal. These could be employed to screen patients admitted to or moving between intensive care settings to inform patient management at the individual and group levels. Moreover, the information on strain persistence in the environment and resistance to disinfection will directly inform infection control management.

Q&A Session

If you have any questions regarding this project, you are invited to attend a Q&A  session hosted by the Supervisor(s) on 5th December at 3pm via Microsoft Teams. Click here to join the meeting. Meeting ID: 395 314 382 637. Passcode: zR6Dsy

About the Programme

 This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

 All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the following link: 

https://www.ed.ac.uk/usher/precision-medicine/app-process-eligibility-criteria  

For more information about Precision Medicine visit:

http://www.ed.ac.uk/usher/precision-medicine