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Quorum sensing in the genus Hafnia - in vitro and in silico investigations
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Date
2024
Authors
O'Mahony, Amy Kate
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Publisher
University College Cork
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Abstract
Food production systems are subject to compounding anthropogenic pressures and understanding and managing their microbial components is more critical than ever. Food quality and safety are strongly influenced by the microbiota present in the raw materials from primary production as well as those that adapt over time to food processing environments. This doctoral thesis investigates the role of quorum sensing as a specific microbial adaptive mechanism in the context of food microbiology, and further interrogates quorum quenching or microbial interference with the quorum sensing strategies of other microbes for competitive advantage with a focus on the genus Hafnia within the order "Enterobacterales".
A comprehensive review on bacterial quorum sensing (QS) and quorum sensing inhibition (QSI) mechanisms was undertaken in Chapter 1, with particular focus on microbiomes associated with foods and the human gut. This was complemented with a general review of the genus Hafnia with a particular focus on food/gut microbiomes in Chapter 2, as Hafnia spp. emerged as promising hits in the screening work documented in Chapter 3. Here, in vitro high-throughput screening was undertaken to identify food-grade bacteria with potential applications in bioprotection as quorum quenching strains. In Chapter 4, the nature of the presumptive QSI activity of Hafnia spp. isolates towards acylated homoserine lactone (AHL) signalling was explored using a variety of in vitro approaches. Long-read sequencing of Hafnia spp. isolates was performed and a variety of basecalling and assembly tools were benchmarked in Chapter with the resulting assemblies being used to augment the pangenomic and phylogenomic investigation of the genus Hafnia undertaken in Chapter 6. Finally, Chapter 7 uses a combined vitro/in silico approach to interrogate the landscape of prophages within the genus Hafnia.
The review of literature regarding QS highlighted the need to disambiguate which strains merely possess QS genes from those that express them, and moreover to understand the environmental contexts influencing their expression. This guided the in vitro screening performed in Chapter 3 and the combination of bioinformatic and biochemical analysis in Chapter 4, resulting in the identification of QSI activity in dairy isolates across diverse genera, including several potentially food-grade isolates.
The focus was placed on Hafnia spp., reviewing the prevalence and diversity of the genus in Chapter 2. It was determined that QS activity was remarkably conserved in the expanded panel of strains assayed, but also that loss of QS activity has arisen at least once in the genus. Novel and improved assemblies of several Hafnia spp. isolates were generated using long-read sequencing, with a demonstration of the impact of choices in basecalling and assembly parameters and tools on the final assembly. High quality assemblies were fundamental for deciphering in vitro phenotypes in terms of QSI in Chapter 4 and prophages in Chapter 7 and also augment the quality of pangenomic analysis in Chapter 6. The curation and incorporation of MAGs complements the closed genomes by contributing important information on and insight into emerging and poorly understood clades, which are identified in this work.
This thesis demonstrates the ubiquity of quorum sensing in food-grade bacteria and provides evidence for its importance in governing microbial interactions with relevance to food production and human nutrition. Proposed taxonomic revisions are being prepared for submission to the International Committee on Systematics of Prokaryotes and SeqCode, providing clarification to taxonomy within the genus Hafnia. Outputs from the phylogenomic and pangenomic investigations provide a framework for future surveillance and risk analysis of Hafnia spp. in foods, while the pipelines developed may be deployed to conduct similar investigations on other microbial species of interest. Overall, it also demonstrates the potential for hybrid approaches which incorporate both wet lab and computational approaches to gain novel biological insights into microbiomes and validates the potential for the hybrid role of "damp-lab" microbiology in which researchers are trained in tenets of classical microbiology but also gain proficiency in the discipline of bioinformatics, while maintaining the sanctity of each discipline in its own right.
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Controlled Access
Keywords
Hafnia , Quorum sensing , Quorum quenching , Food microbiology , Bacteriology , Phylogenomics
Citation
O'Mahony, A. K. 2024. Quorum sensing in the genus Hafnia - in vitro and in silico investigations. PhD Thesis, University College Cork.