Incorporation of V. vulnificus into marine snow for oyster uptake and in vivo bacterial competition assays

Vibrio vulnificus is a significant human pathogen found in high numbers in oysters. Despite the environmental prevalence of V. vulnificus, clinical cases are uncommon. We hypothesised that in vivo competition between V. vulnificus and other strains/species resulted in the killing of hypervirulent strains, reducing clinical incidence. To assess this, we have developed an oyster model into which we can ensure ingestion of high quantities of V. vulnificus using a defined ‘marine snow’. Marine snow describes the aggregation of naturally occurring phytoplankton, bacteria, debris and other organic materials. Our marine snow substrate is comprised solely of the diatom, Thalassiosira pseudonana. Bottles containing artificial seawater, 109 T. pseudonana, V. vulnificus culture and hyaluronic acid were rotated at 16 r.p.m. for 24 h to generate aggregates. V. vulnificus-containing marine snow was added to beakers holding individual oysters. Following 24 h uptake, oyster stomachs were excised and homogenised in PBS. The resulting suspension was serially diluted for plate-counts and DNA extracted for downstream qPCR analysis. Diatom-based aggregates present a controllable and reproducible model for incorporating V. vulnificus into marine snow. Using this methodology, we demonstrate greater uptake of V. vulnificus by oysters than any current study. This has potential applications for future in vivo work studying a range of microorganisms in oysters, such as other human pathogens or those of interest to aquaculture. Future work will undertake in vivobacterial competition assays to determine the role that intra-/inter-species competition has on the ecology of V. vulnificus and whether this impacts the clinical incidence.