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). b-lactamases are a major mechanism for AMR within the Enterobacteriaeae. The most problematic b-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 lplasmidsr. 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 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 ISCR1 and either IS26 or ISCR27 were involved with blaNDM insertion into different locations of the antibiotic resistance island ARI-A on IncA/C2 plasmids. Tn3-derived Inverted-repeat Transposable Elements (TIMEs) and an IS903-like element were identified for IncFIIY plasmids. This thesis collectively identified eight different transposable elements associated with blaNDM: ISCR27 and/or IS26 on type 1 IncA/C2; ISCR1 on IncN2, IncA/C2 and IncFII2; ISCR6-like, IS903-like and TIMEs on IncFIIY; IS26 and/or IS3000 on IncX3; and an IS26 composite transposon on IncH1B. Thirdly, to investigate the relationship between plasmid types and bacterial species, in silico plasmid typing (via plasmid typing database, PlasmidFinder) and Principal Component Analysis (PCA) was performed to survey the plasmid content across 1683 Enterobacteriaceae isolates. These whole genome sequenced isolates comprised of K. pneumoniae (n=494), Shigella sonnei (n=223), Yersinia spp. (n=214), Shigella flexneri (n=171), E. coli (n=355), E. cloacae (n=133) and Salmonella enteria serovar Typhimurium (n=95). Twelve main plasmid types were identified distributed into three levels of occurence: common, IncF (~65% of strains); intermediate, IncHI, IncI, IncR (8-10%); and rare, IncA/C, B/O/K/Z, L/M N, O, P, Q, X, and Y (0.5-3%). PCA of isolates and their shared plasmid content identified specific plasmid sub-types to represent possible routes of gene exchange between different genera. Furthermore, two primary clusters of species were identified based on their shared plasmid sub-type content, Group 1: K. pneumoniae and E. cloacae, and Group 2: E. coli, S. sonnei and S. flexneri. Species within each group were seen to be phyogenetically similar. Collectively the analysis presented in this thesis, proposes an underlying network of interactions between AMR genes, transposable elements, plasmids types and the bacterial host, where each interaction may involve a degree of compatibility depending on the genera of the strain. The blaNDM genes appear to have transmitted through this proposed network, from Acinetobacter spp. to disseminate amongst the Enterobacteriaceae family, following its interactions, compatibilities and limitations. Further surveillance of the Enterobacteriaceae family, including environment and community samples, will be required to define the extent plasmid-mediated AMR genes have spread within the Enterobacteriaceae family. The combined molecular/genetic approach and subsequent whole plasmid sequence analysis would be recommended for this survelliance. This PhD thesis provides insights into the acquisition and spread of the blaNDM gene and emphasizes the capability of Enterobacteriaceae to transmit plasmid-mediated AMR genes amongst themselves to adapt to their environment, especially where antimicrobial pressure is present.