Phages infecting marine Vibrios: prevalence, diversity and role in the dissemination of antibiotic resistance genes

Antimicrobial resistance (AMR) is a major public health issue. Recent studies indicate that antimicrobial resistance genes (ARGs) can be readily identified in both bacteriophages and bacteria found within the marine environment. Furthermore, experiments show that bacteria can obtain AMR when transduced with phage genetic material, suggesting that bacteriophages might be responsible for the transfer and spread of antimicrobial resistance in these systems. This project sought to determine the role of phages in mediating the transfer of ARGs within the marine environment, focusing on Vibrio spp. as model organisms due to their high abundance and impact caused on both public health and the seafood industry. First, a framework for the analysis of marine phages was established (Chapter 3). Genomic, p,hylogenetic, proteomic, host range and infection parameters of a range of novel marine coliphage isolates were determined and their ability to transduce ARGs was assessed. This suggested that host DNA encapsidation rate should be determined as a proxy for the transduction rate, due to the inability of stopping secondary phage infections during the transduction assay. The established framework was used for the analysis of marine Vibrio phages (Chapter 4). Experiments showed a high prevalence of Inoviridae prophages derived from the host in the obtained phage lysates, necessitating further studies to determine their exact composition. Nevertheless, the study resulted in the isolation and exhaustive characterisation of two novel Vibrio phages, including the second largest Vibrio phage isolated to date. Encapsidation of host DNA by the analysed phage was not observed, indicating this process may be below the experimental detection thresholds, which is not unexpected for generalised transducing phages. Bioinformatics analysis of all publicly available Vibrio genomes was performed to identify prophages encoding ARGs (Chapter 5) leading to the creation of the largest Vibrio prophage database to date. Analyses showed a high prevalence of the Inoviridae family of filamentous prophages in the Vibrio genomes and indicated that ~25% of the detected Vibrio prophages represent novel sequences not related to any known phages. Subsequent analysis of prophage-encoded ARGs indicated the presence of a range of ARGs, with genes encoding β-lactamases such as TEM-1, CTX-M and OXA being the most common. Selected ARGs were subsequently synthesised and their functionality was determined by heterologous expression in E. coli (Chapter 6). This not only showed that detected ARGs are often functional, but also led to the discovery of a novel cat ARG encoded on an intact Vibrio prophage conferring resistance to chloramphenicol. Finally, metagenomics and functional metagenomics analyses were performed to determine the prevalence of ARGs in marine phageomes (Chapter 7). This led to the reconstruction of a plethora of novel phage genomes, including a representative of megaphages, the largest known bacterial viruses. Surprisingly, genomics and functional genomics analyses failed to detect known or novel ARGs, respectively, suggesting that their distribution across the marine environment might not be uniform. This study represents a major step in our knowledge of lytic and temperate phages and their ability to encode functional ARGs and transfer them within the marine environment. Furthermore, it pinpoints the areas which require further investigation to fully understand the importance of generalized and specialized transduction in the dissemination of the ARGs in the environment