Vibrio is ubiquitous genus found in marine and brackish environments. Despite being commonly found in seawater, sediment and marine organisms, numerous Vibrio species are considered pathogenic for marine organisms and humans. Bivalves are highly susceptible to bacterial colonization due to their capacity to accumulate bacteria in their tissues through filtration. Focus of this study is Anguillarum clade, known for bivalve and fish pathogens. Additionally, one of the growing problems in marine environments is the emergence of antibiotic resistance due to the excessive use of antibiotics and anthropogenic discharges into the marine environment. However, antibiotic resistance in eastern Adriatic bivalve aquaculture has been poorly studied.
Focus of our research are two most important species in aquaculture in Croatia - European flat oyster, Ostrea edulis Linnaeus, 1758 and Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819. These species were sampled in two protected marine reserves – Lim Bay and Mali Ston Bay, locations which are known for bivalve aquaculture. We sampled seawater, sediment and bivalve tissue (hepatopancreas and gills) on both locations bimonthly over the course of one year. For bacteria isolation, Vibrio selective medium was used, while we identified Vibrio clades with MALDI-TOF mass spectrometry. As final step, we performed Multilocus Sequence Analysis (MLSA) with selected phylogenetic marker genes for species identification. Vibrio isolates belonging to Anguillarum clade, were tested for antibiotic resistance via disk diffusion on Mueller Hinton agar for 12 selected antibiotics which are frequently used in aquaculture and medicine.
MALDI-TOF MS results showed that only 23 isolates belonged to Anguillarum clade. All isolates were found during sampling during colder months (T ≥ 13⁰C). As for antibiotic resistance, all isolates were resistant to ampicillin. Also, we recorded high resistance to antibiotic imipenem (83%), commonly used in human medicine. To some extent, isolates were resistant to erythromycin (48%) while 52% of isolates were intermediately resistant. More than half of isolates (56%) were multi-resistant, meaning that they were resistant to two or more antibiotics.
This study shows first report on the diversity and antibiotic resistance of Anguillarum clade Vibrio species from bivalve aquaculture in Croatia and shows rising concern that on-going increasing anthropogenic pressures might negatively affect bivalve aquaculture both with bigger susceptibility of farmed bivalves to colonization by Vibrio pathogens and with more resistant Vibrio bacteria.