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Goodman, Richard (2022) Investigating the impact of intercellular and intracellular mobile genetic element transfer on the evolution of antimicrobial resistance in Enterobacterales, Thesis (Doctoral), Liverpool School of Tropical Medicine.
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R Goodman PhD - Final Thesis for LSTM Archive 1.pdf - Accepted Version Restricted to Repository staff only until 16 August 2024. Download (149MB) |
Abstract
Antimicrobial resistance (AMR) is a major threat to human health. AMR genes are often associated with mobile genetic elements (MGEs) which enables the movement of AMR genes between bacterial cells (intercellularly) and between and within replicons inside bacterial cells (intracellularly). In this study we aimed to understand the intercellular and intracellular movement of AMR genes and the MGEs associated with them and determine the effect this movement has on the evolution of Escherichia coli and Klebsiella pneumoniae, both Gram-negative organisms of the Enterobacterales order.
Using filter-mating experiments to study intercellular transfer, and the pBACpAK entrapment vector to study intracellular transfer, we were able to build a clear picture of the IncI1 plasmid mediated movement of the clinically important colistin resistant gene mcr-1 between cells and on a novel transposon, Tn7511, between replicons in the same cell. The use of whole genome sequencing, both long and short read, provided new insights into the mechanism of the ISApl1 mediated transposition
of mcr-1.
Building on these techniques, we went on to address the issue of increasing chloramphenicol susceptibility at a time of increasing β-lactam resistance in E. coli and K. pneumoniae from Malawi.
We aimed to understand the molecular mechanisms underpinning this phenomenon. We demonstrated the intercellular movement of the IncFIB(pQil) plasmid carrying a catA2 gene between K. pneumoniae and E. coli. Comparative growth rate assays showed that the acquisition of this plasmid led to a fitness cost on all transconjugants, but to different degrees. Comparative genomics revealed how the IncFIB(pQil) plasmid was related to the well characterised pKpQil plasmid but with a reshuffled set of AMR genes. Following the experiments described above, new entrapment vectors were designed and constructed to determine intracellular transfer in these chloramphenicol resistant transconjugants and they were shown to capture Tn2 (containing blaTEM-1B) and ISKpn25 from the conjugative IncFIB(pQil) plasmid.
To further address the questions around the re-emergence of chloramphenicol sensitivity bioinformatics analysis was carried out on short-read genomic datasets from K. pneumoniae and E. coli in Malawi. The co-occurrences of AMR genes were assessed across all isolates to build up a picture of the genomic interactions underpinning the loss of chloramphenicol resistance. We developed a hypothesis to explain one of the molecular mechanisms underlying the re-emergence of chloramphenicol susceptibility. Enterobacterales were shown to acquire plasmids with both ESBL genes and IS26 elements. In the proposed mechanism, the incoming ESBL genes provide resistance against third generation cephalosporins and the IS26 inactivate and silence the chloramphenicol resistant catB3 gene.
The work presented in this thesis shows how clinically important AMR genes are mobilised in a variety of contexts and how the promiscuity of MGEs has an impact on the evolution of resistance by moving genes and disabling others. We have developed a unique and adaptable system to
understand intracellular dynamics which can reveal the molecular mechanisms of phenotypic resistance and susceptibility to important antibiotics. Understanding the intercellular and intracellular movement of AMR genes associated with MGEs is key for the surveillance of AMR in local regions and to develop new therapies or inform current treatment regimens to counteract the movement of these genes.
| Item Type: | Thesis (Doctoral) | ||||||||||
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| Subjects: | QU Biochemistry > Genetics > QU 470 Genetic structures QW Microbiology and Immunology > Bacteria > QW 138 Enterobacteriaceae QW Microbiology and Immunology > QW 4 General works. Classify here works on microbiology as a whole. QW Microbiology and Immunology > QW 45 Microbial drug resistance. General or not elsewhere classified. |
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| Faculty: Department: | Biological Sciences > Department of Tropical Disease Biology | ||||||||||
| Depositing User: | Lynn Roberts-Maloney | ||||||||||
| Date Deposited: | 03 Oct 2023 13:08 | ||||||||||
| Last Modified: | 03 Oct 2023 13:08 | ||||||||||
| URI: | https://archive.lstmed.ac.uk/id/eprint/23258 |
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