Viruses in the North Sea: viromics and prophage genomics

Despite their small size, viruses have an enormous influence on microbial population dynamics, due to lysis and horizontal gene transfer. Due the high abundance of their hosts, bacteriophages or phages comprise the majority of viruses and provide the largest reservoirs of unexplored genetic diversity in marine environments. The rise of Next Generation Sequencing (NGS) techniques brings new opportunities to investigate the marine virus community. However, there is no current statutory pipeline applied in marine phage ecology. Therefore, this thesis proposes a virus-specific pipeline based on the integration of existing tools and state of the art techniques. The developed pipeline was applied to accomplish the two research aims of this thesis: (1) to analyze the virus community in the North Sea with viromics(virus metagenomics), and (2) to characterize lysogenic phages from potentially pathogenic Vibrio species. The results of the first part of this thesis show that the virus community is dominated by phages and they are not evenly distributed throughout the North Sea. In general, the coastal virus community was genetically more diverse than the open sea community. The influence of riverine inflow and currents affects the genetic virus diversity with the community carrying genes from a variety of metabolic pathways and other functions. In the second part, lysogenic Vibrio phages from the North Sea were induced (ca. 40 % of tested isolates) and four phage genomes were characterized. The phages from V. parahaemolyticus (2 tailed phages, 1 filamentous phage) and V. cholerae (1 tailed phage) can integrate into their host genome and might have a role in pathogenicity. This thesis represents an exemplary study of the virus community in the North Sea, with special emphasis on the marine phages. The settled virus-specific pipeline the obtained insights will contribute to extend the study of the virus diversity dynamics in other marine areas to characterize novel phage groups

Genomic characterization of Filamentous Phage vB_VpaI_VP-3218, an Inducible Prophage of Vibrio parahaemolyticus

The seawater temperature rise promotes the growth of potentially human pathogenic Vibrio species. In the North Sea, V. parahaemolyticus strains have been isolated and characterized. These strains contain prophages that may contribute to the emergence of pathogenic strains in the marine environment. Here, we present the genome structure and possible biological functions of the inducible phage vB_VpaI_VP-3218, a novel filamentous phage carried by the V. parahaemolyticus strain VN-3218. Prophages of the strain VN-3218 were induced with mitomycin C and the DNA from the phage induction was sequenced. Two incomplete prophages were identified, only one complete phage genome with length of 11,082 bp was characterized. The phage vB_VpaI_VP-3218 belongs to the Inoviridae family and shows close homology to the Saetivirus genus. This phage can integrate into the chromosomal host genome and carries host-related regions absent in similar phage genomes, suggesting that this phage integrates within other host genomes. Furthermore, this phage might have a role in pathogenicity due to potential zonula occludens toxin genes. Based on genomic similarity, this phage is an episomally reversible integrated lysogenic phage. This study complements prophage induction and bioinformatic studies applied to non-model species of potentially pathogenic Vibrio species. The characterization of this phage provides new insights with respect to the presence of filamentous phages in environmental V. parahaemolyticus strains, which might have a role in the emergence of new pathogenic strains in the North Sea

Temperate Phages of potentially pathogenic Vibrio species from North Sea

Temperate phages are important players in genetic diversification of their bacterial host by transforming their genomes by transduction. During this process, the phage forms a stable interaction with its host, either by integrating into the bacterial chromosome or as plasmid. The integrated phage genome replicates along with the host until the lytic cycle is induced. Temperate phages carry mobile genetic elements which contribute to emergence of disease-causing strains from environmental bacterial populations, e.g. temperate Vibriophages can turn its host Vibrio cholera into a fatal disease upon successful infection and transduction. However, the potential of transferring pathogenic genes due in the marine environments to temperate phages is far less understood. We present results of potential pathogenic Vibrio strains isolated from the North Sea which were screened for lysogenic phages. Three potentially pathogenic Vibrio species V. vulnificus, V. parahaemolyticus and V. cholera were induced using Mitomycin C. New Vibriophages were isolated to investigate their role in pathogenicity. The present work shows the first insights of temperate phages presence on potential pathogenic Vibrio strains from Northern European seawaters

Temperate Phages of potentially pathogenic Vibrio species from the North Sea

Question: Temperate phages play a major role in bacterial genomic diversity, by transferring mobile genetic elements. Phages may contribute to emergence of disease-causing strains from environmental Vibrio species. Thus, potentially pathogenic Vibrio species from the North Sea were subjected to phage induction to gather information on the vibriophage related genepool. Methods: Screening for lysogenic phages from potential pathogenic Vibrio strains isolated from North Sea in 2014 was performed. V. vulnificus, V. parahaemolyticus and V. cholera strains were induced using Mitomycin C. After phage induction, each sample was checked for phage production using epifluorescence microscopy. Positive phage induced samples were used for a host screening assay using 154 potentially pathogenic Vibrios of the respective species Results: During the research cruise 39 potentially pathogenic Vibrio strains from the coastal waters were isolated from stations near the coastline. Temperate phages were successfully induced from 18 potentially pathogenic Vibrio isolates, in detail 14 correspond to V. parahaemolyticus, 3 to V. vulnificus and 1 to V. cholerae. All inducible phages were found in Vibrio strains from coastal stations of Germany and the Netherlands. The induced phage samples presented inhibitory activity against half of the tested Vibrio strains, mostly environmental isolates from North and Baltic Sea. Conclusion: About 46% of Vibrio isolates possess inducible temperate phages, all of them isolated from coastal waters. These phages might play a role in transference of pathogenic genes into the environment. This will be subject of further studies. The present work shows the first insights of temperate phages presence on potential pathogenic Vibrio strains from Northern European seawaters

Temperate Phages of potentially pathogenic Vibrio species from the North Sea

Temperate phages are important players in genetic diversification of their bacterial host by transforming their genomes by transduction. During this process, the phage forms a stable interaction with its host. The integrated phage genome replicates along with the host until the lytic cycle is induced. Phages may contribute to emergence of disease-causing strains from environmental Vibrio species by transference of mobile genetic elements. However, the potential of transferring pathogenic genes due in the marine environments to temperate phages is far less understood. Thus, potentially pathogenic Vibrio species from the North Sea were subjected to phage induction to gather information on the vibriophage related genepool. During the research cruise 39 potentially pathogenic Vibrio strains from the coastal waters were isolated from stations near the coastline. Temperate phages were successfully induced from 18 potentially pathogenic Vibrio isolates. In detail, 14 correspond to V. parahaemolyticus, 3 to V. vulnificus and 1 to V. cholerae. All inducible prophages were found in Vibrio strains from coastal stations of Germany and the Netherlands. The induced phage samples presented inhibitory activity against half of the tested Vibrio strains, mostly environmental isolates from North and Baltic Sea. As conclusion, about 46% of Vibrio isolates possess inducible temperate phages, all of them isolated from coastal waters. These phages might play a role in transference of pathogenic genes into the environment. This will be subject of further studies. The present work shows the first insights of temperate phages presence on potential pathogenic Vibrio strains from Northern European seawaters

The North Sea goes viral: Occurrence and distribution of North Sea bacteriophages

Marine viruses are dominated by phages and have an enormous influence on microbial population dynamics, due to lysis and horizontal gene transfer. The aim of this study is to analyze the occurrence and diversity of phages in the North Sea, considering the virus-host interactions and biogeographic factors. The virus community of four sampling stations were described using virus metagenomics (viromes). The results show that the virus community was not evenly distributed throughout the North Sea. The dominant phage members were identified as unclassified phage group, followed by Caudovirales order. Myoviridae was the dominant phage family in the North Sea, which occurrence decreased from the coast to the open sea. In contrast, the occurrence of Podoviridae increased and the occurrence of Siphoviridae was low throughout the North Sea. The occurrence of other groups such as Phycodnaviridae decreased from the coast to the open sea. The coastal virus community was genetically more diverse than the open sea community. The influence of riverine inflow and currents, for instance the English Channel flow affects the genetic virus diversity with the community carrying genes from a variety of metabolic pathways and other functions. The present study offers the first insights in the virus community in the North Sea using viromes and shows the variation in virus diversity and the genetic information moved from coastal to open sea areas

DANGEROUS HITCHHIKERS? EVIDENCE FOR POTENTIALLY PATHOGENIC VIBRIO SPP. ON MICROPLASTIC PARTICLES

The qualitative and quantitative composition of biofilms on microplastic surfaces is widely unknown. A previous study (Zettler et al., 2013; EST) reports the presence of potentially pathogenic bacteria (Vibrio spp.) on floating microplastic particles. Hence microplastics could function as vectors for the dispersal of microorganisms to new habitats. Several Vibrio species are serious human pathogens. Contact with contaminated water and consumption of raw seafood are the main infection factors for Vibrio associated diseases. On research cruises to the North- and Baltic Sea, microplastic particles were collected and subjected to APW (alkaline peptone water) enrichment. Growth on selective CHROMagar™ Vibrio and further identification of isolates by MALDI-TOF (matrix assisted laser desorption/ionization time-of-flight) clearly indicate the presence of potentially pathogenic Vibrio spp. on microplastics. Our results highlight the urgent need for detailed microbiological analyses of floating microplastic particles in the future