Application of bacterial genomics and culture-free microbiota diagnostics to respiratory infections

Bettridge, Aimee 2022. Application of bacterial genomics and culture-free microbiota diagnostics to respiratory infections. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Polymicrobial infections drive the progression of chronic lung diseases and can dramatically affect clinical outcomes. Clinical laboratories routinely employ culture-based tools to diagnose lung infection. Nevertheless, culture cannot capture the full extent of bacterial diversity and is prone to misidentification. The suitability of 16S rRNA gene sequencing and ribosomal intergenic spacer analysis (RISA) was explored in context of respiratory infection diagnosis in the Public Health Wales (PHW) microbiology service. Optimised RISA and PCR protocols were used to analyse mock communities simulating respiratory bacterial pathogen assemblages. Sequence-based community abundance and diversity was distinct between each mock community, although community composition substantially deviated from expected proportions. RISA proved useful in predicting species identification. A genomic DNA extraction protocol was then validated for microbiota analysis using silica-based gDNA extraction employing bead-beating. These optimised protocols were applied to profile bronchiectasis, cystic fibrosis, and chronic obstructive pulmonary disease sputa, and the results compared to PHW routine culture-based analyses. Respiratory microbiota was highly individualised, and bacterial diversity did not statistically correlate with reduced lung function. Combining both culture-free and culture-dependent methodologies proved beneficial in the identification of potentially pathogenic microorganisms missed in culture and for resolving poor sequence identification in a minority of samples. Whole genome sequencing (WGS) was deployed for the accurate characterisation of the emerging cystic fibrosis pathogens Achromobacter spp., and was compared to conventional MALDI-TOF. WGS was instrumental in determining sources of Achromobacter insuavis acquisition and the multidrug-resistant nature of these species, suggesting the presence of previously unreported metallo-beta-lactamases. Phenotypic traits suggest that Achromobacter xylosoxidans’ competitive fitness was demonstrably similar to traditional CF Gram-negative species. These results also suggested these species interacted as an intransitive hierarchy. Overall, these DNA-based methods show promise for routinisation in the PHW however WGS is still largely a research tool in context of lung infection.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Funders:	KESS
Date of First Compliant Deposit:	6 September 2022
Date of Acceptance:	6 September 2022
Last Modified:	06 Sep 2023 08:23
URI:	https://orca.cardiff.ac.uk/id/eprint/152392

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