The genetic analysis of Enterobacteriaceae plasmids to provide insights into the acquisition and spread of the blaNDM gene

Abstract

Treatment options for infections caused by pathogenic Gram-negative bacteria, especially those of the Enterobacteriaceae family, are becoming limited with the increase of antimicrobial resistance (AMR). β-lactamases are a major mechanism for AMR within the Enterobacteriaeae. The most problematic β-lactamases are carbapenemases, which confer resistance to carbapenems, the major last-line antimicrobial. A recently emerged carbapenemase that has globally disseminated is the NDM carbapenemase, provided by blaNDM genes. These blaNDM genes (and other AMR genes) are able to transmit between strains when inserted on extrachromosomal self-replicating DNA molecules known as 'plasmids'. AMR genes within Enterobacteriacae are frequently associated with specific bacterial species, clonal lineage, plasmid Incompatibility (Inc) types or transposable elements. The blaNDM genes however do not have this association when oberserved in the Enterobacteriaceae family. To address and characterise this new paradigm presented by blaNDM genes, this thesis presents the bioinformatic analysis of plasmids associated with the Enterobacteriaceae to provide insights into the acquisition and spread of the blaNDM gene and an epidemiological approach to assess its plasmid-mediated dissemination between genetically unrelated species.Specifically these aims were achieved by; firstly, the establishment of a recent account of the blaNDM gene from an epidemiological perspective using a novel genetic/molecular approach. This would identify the spread of individual plasmids carrying blaNDM across multiple species and patients, both within a single facility and across multiple national facilities. The approach combined in-depth bioinformatic analysis of blaNDM genetic contexts (NGCs) with common molecular epidemiology techniques. IncN2 (n=4) and IncA/C (n=3) were identified as the most common plasmids types carrying blaNDM across four patients within a Pakistani military hospital. These patients harboured between two and four NDM-1 producing Gram-negative bacilli of different ii species coresident in their stool samples. IncFII-types (n=7) and IncX3 (n=4) were the most common plasmid types carrying blaNDM amongst 12 Enterobacteriaceae isolates, each from different patients across multiple Australian healthcare facilities. These isolates each carried one plasmid harbouring blaNDM but only five different blaNDM genetic contexts were identified, indicating five particular plasmids with a specific NGC had disseminated amongst these 12 isolates.Secondly, to investigate transposable elements involved for insertion of the blaNDM gene into different plasmid types, the complete sequence of four plasmids carrying blaNDM (two IncA/C2 and two IncFIIY) was bioinformatically analysed. These plasmids were from four different clinical samples of four patients, comprised of Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli. Each plasmid was observed to acquire blaNDM by different mechanisms on very similar plasmid backbones. Transposable elements ...