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

Antimicrobial resistance (AMR) is global health crisis. Klebsiella pneumoniae, a Gram-negative bacteria, that are resistant to carbapenems (part of the last-line drugs for difficult-to-treat infections) are on the World Health Organization's list of critically resistant pathogens. They contribute to many healthcare-associated infections and are associated with increased mortality and morbidity. This is partly due to their ability to rapidly become systemic from the initial infection site, but also because they can share resistance genes on mobile genetic elements, such as plasmids, leading them to rapidly spread AMR to other Gram-negative species. This can lead to failed antimicrobial therapy and negative patient outcomes, especially in cases where rapid administration of the correct antibiotics is crucial, such as sepsis. These plasmids can also carry genes that aid in environmental survival, regulate virulence factors and confer multidrug resistance. Plasmid acquisition can, therefore, change the bacterial phenotype and alter disease severity and patient outcomes. Thus, it is important to understand the mechanisms of plasmid evolution to be able to predict changes in virulence and risk of plasmid spread. In this project, we will employ long-read sequencing to analyse the genomes of clinical isolates of K. pneumoniae where it caused sepsis in patients. We aim to understand why some strains caused fatal infections while others did not. Bioinformatic analyses will be employed to identify both chromosomal and plasmid-borne AMR genes, virulence factors and error-prone polymerases that may lead to mutations. This will be complemented by phenotypic assessments such as survival within phagocytic cells and antimicrobial susceptibility to understand the differences in patient outcomes. Environmental survival in the presence of disinfectants, desiccated conditions and adherence to surfaces will be investigated to assess environmental survival and spread as fomites. We will also set up long-term cultures in the presence of sub-inhibitory concentrations of antibiotics to evaluate the role of error-prone polymerases in carbapenemase evolution, enzymes that inactivate Beta-lactam antibiotics. This will be supported by frequent sequencing and phenotypic characterisation to identify genetic mutations and changes in phenotypic expression. We will also conduct inter- and intra-species plasmid transfer and associated changes in phenotypes to plasmid-free strains in gut bacteria. These investigations will result in an understanding how whole-genome sequencing can influence patient treatment outcomes, manage pathogen and plasmid spread and inform infection control practices and surveillance of resistant pathogens in healthcare settings. This should aid in the development of new strategies to combat the spread of plasmids and resistant pathogens.