Carbapenem resistance and hypervirulence in enterobacterales: A genomic and phylogenetic analysis of clinical and environmental isolates in Ireland

Antimicrobial resistance is one of the most important global public health challenges of the 21st century. Carbapenemase-producing Enterobacterales (CPE) in particular pose a significant threat due to their rapid spread and limited treatment options. Infections caused by CPE are associated with very high mortality rates, especially among hospitalised patients. However, understanding of the mobile genetic elements (MGE) responsible for transmission of these genes and the convergence of multiple resistance genes and virulence determinants on single plasmids remains poorly understood, especially in an Irish context. This thesis investigates the genomic characteristics and dissemination pathways of CPE in Ireland, with a specific focus on the MGE involved in the spread of carbapenem and aminoglycoside resistance, as well as the emerging convergence with hypervirulent Klebsiella pneumoniae. To achieve this, whole genome sequencing (WGS) incorporating both short and long read technologies was employed to characterise isolates from human samples, the hospital environment and natural water sources between 2018 and 2023. The first study examined the relationship and dissemination pathways of CPE within a defined geographical region. To identify and compare the MGE carrying carbapenemase genes across different ecological niches and bacterial genera, a total of thirty-nine isolates were analysed. These isolates were collected from hospital patients, the hospital environment and local natural water sources (river and seawater) between 2018 and 2020. These isolates carried blaKPC-2 (n = 6), blaNDM-5 (n = 12) and blaOXA-48 (n = 21). Carbapenemase genes were plasmid-borne in 95% of isolates with blaOXA-48 almost exclusively associated with a highly conserved, ~63 kb IncL plasmid. The blaKPC-2 and blaNDM-5 genes were typically located on large multi-replicon IncF plasmids. Conserved genomic regions were observed surrounding the carbapenemase genes and were similar across different plasmids and the chromosome. These findings highlight the role of MGE, such as plasmids and transposons, in the transmission of carbapenemase genes across different ecological niches and genera. The second study investigated the molecular epidemiology of isolates co-carrying the carbapenemase gene blaNDM alongside 16S rRNA methyltransferases (16S-RMTases), which confer a pan-aminoglycoside resistant phenotype. Fifty-eight isolates collected between 2019 and 2023, co-carrying blaNDM and 16S-RMTase genes were analysed. Short read sequencing was carried out on all isolates and long-read sequencing was carried out on a subset (n = 27). MOB-recon was used to predict plasmid derived contigs from draft assemblies. A strong association between blaNDM-1 and armA and blaNDM-5 and rmtB1 was observed. Several clusters of closely related plasmids carrying both blaNDM and 16S-RMTase genes were identified across multiple healthcare facilities and species, suggesting plasmid mediated dissemination. The final study investigated the emergence of hypervirulence in Ireland and its convergence with carbapenemase resistance. Ninety K. pneumoniae isolates obtained from screening swabs (n = 59), invasive infections (n = 18), non-invasive sites (n = 12) and the hospital environment (n = 1), were sequenced. All isolates were genomically hypervirulent and 82% of isolates carried a carbapenemase gene (blaOXA-48/blaOXA-181/blaNDM-1). While all isolates were closely related with similar resistance and virulence profiles, two distinct clusters were detected. In vivo murine systemic infection models demonstrated equivalent virulence in isolates from infection and from asymptomatic carriage, suggesting that colonisation may serve as a reservoir for invasive disease. Collectively, these studies highlight the diversity of plasmids carrying carbapenemase genes, often in combination with other clinically relevant resistance genes. While carriage of blaOXA-48 was largely associated with a single conserved 63 kb plasmid carrying blaOXA-48 alone, blaKPC and blaNDM were found on a more diverse array of IncF, IncM and IncC plasmids. The identification of conserved plasmid backbones across species and in multiple ecological niches highlights the adaptability and persistence of these MGE. Furthermore, the convergence of AMR and hypervirulence determinants on the same plasmids represents a serious emerging threat, particularly in the absence of standardised screening for hypervirulent K. pneumoniae. These findings reinforce the importance of a coordinated One Health approach utilising enhanced genomic surveillance across environmental, clinical and public health sectors to monitor and control the ongoing threat of antimicrobial resistance.

Funder

Research Ireland

Publisher

University of Galway

Rights

CC BY-NC-ND

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University of Galway Theses (PhD Theses)