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Gallichan, Sarah (2024) Bias in the laboratory detection of ESBL-producing Escherichia coli and the consequences for disease transmission modelling, Thesis (Doctoral), Liverpool School of Tropical Medicine.
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S Gallichan_Thesis_FINAL_updated_07.02.2025.pdf - Accepted Version Restricted to Repository staff only until 29 July 2025. Download (14MB) |
Abstract
Understanding transmission pathways of clinically relevant, drug-resistant pathogens, such as extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC), is essential to developing and implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To capture the transmission of ESBL-EC between two sources, all the ESBL-EC strains present in each of the two samples need to be recovered and compared at a single nucleotide variant (SNV) level. However, the microbiological and bioinformatic methods to best analyse ESBL-EC are unclear. In Chapter One of this thesis, I highlighted the lack of consensus between studies on the impact that different microbiological laboratory methods and sequence analysis have on investigating ESBL-EC diversity within a complex microbial sample. Recognising this gap in the literature, I conducted a controlled comparison of different steps in the microbiological workflow. In Chapter Three and Four I determined the impact different pre-enrichment broths, pre-enrichment incubation times, antibiotic selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high quality DNA for sequencing and genomic
epidemiology. I demonstrated that using a 4-hour pre-enrichment in Buffered Peptone Water (BPW), plating on cefotaxime supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole genome sequencing. An alternative approach to investigating within-host E. coli diversity in healthy human stool was investigated in Chapter 5 by coupling pre-enrichment with shotgun metagenomics. Using the bioinformatics tool StrainGE I was able to determine that pre-enrichment was highly effective in amplifying E. coli while maintaining the ratios of multiple strains. However, this approach was not sufficient for accurate strain calls and SNV analysis. Utra-deep sequencing may be able to improve accuracy but currently costs are prohibitive. As a result, I investigated the feasibility of SNV calling of a plate sweep using the mSWEEP/ mGEMS pipeline by directly comparing it to the SNV analysis of multiple single colony picks from the rectal swabs of six TRACS-Liverpool study participants. The plate sweep method offered a workable alternative to both multiple single colony picks and shotgun metagenomics for describing within-host ESBL-EC diversity. In this thesis I have described the successful optimisation of a workflow for the effective recovery and SNV analysis of ESBL-EC from human stool/ rectal swabs that is applicable at scale to investigate ESBL-EC transmission in healthcare settings
| Item Type: | Thesis (Doctoral) | ||||
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| Subjects: | QW Microbiology and Immunology > Bacteria > QW 138 Enterobacteriaceae QW Microbiology and Immunology > QW 50 Bacteria (General). Bacteriology. Archaea WC Communicable Diseases > WC 20 Research (General) WC Communicable Diseases > Infection. Bacterial Infections > Enteric Infections > WC 290 Escherichia coli infections |
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| Faculty: Department: | Clinical Sciences & International Health > Clinical Sciences Department | ||||
| Depositing User: | Lynn Roberts-Maloney | ||||
| Date Deposited: | 29 Apr 2025 12:05 | ||||
| Last Modified: | 29 Apr 2025 12:10 | ||||
| URI: | https://archive.lstmed.ac.uk/id/eprint/26644 |
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