Introduction
Bivalves filter water as part of feeding process and accumulate large number of various particles and microorganisms, including the potential bacterial pathogens. One of the most relevant genera found in bivalve tissue is genus Vibrio, due to the fact that they cause mass mortality and huge economic losses in aquaculture. Among Vibrio pathogens, Splendidus clade is of particular concern due to numerus bivalve pathogens such as V. splendidus, V. crassostreae, V. tasmaniensis etc. (Destoumieux-Garzón et al., 2020). 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 (Baralla et al, 2021).
Materials and methods
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. For bacteria isolation, Vibrio selective medium was used (Tiosulphate Citrate Bile Salt Sucrose - TCBS). Subsequently, we identified Vibrio clades with MALDI-TOF mass spectrometry (Culot et al, 2021). Due to large number of isolates, we applied BOX-PCR genomic fingerprinting method as intermediate step for determination of differences between species and identification of clones (Canellas et al, 2021; Culot et al, 2021). As final step, we preformed Multilocus Sequence Analysis (MLSA) with three selected phylogenetic marker genes (gyrB, mreB and rpoD) for species identification (Pérez-Cataluña et al, 2016). Vibrio isolates belonging to Splendidus clade, were tested for antibiotic resistance via disk diffusion on Mueller Hinton agar for 13 selected antibiotics which are frequently used in aquaculture and medicine.
Results
MALDI-TOF MS results showed that more than half of all culturable Vibrio bacteria belonged to Splendidus clade. As we isolated large amount of Splendidus clade samples (n=688), we applied BOX-PCR genomic fingerprinting method as intermediate step for identification of clones which eliminated numerous isolates from further molecular analysis. Our final step of identification to species-level we preformed MLSA which resulted in phylogenetic tree, displaying clusters of two or three species, namely, V. splendidus-hemicentroti, V. tasmaniensis-atlanticus, V. crassostrea-gigantis-celticus, V. kanaloae-toranzoniae and V. chagasii-pomeroyi. Results of antibiotic resistance showed that more than 94% of all isolated bacteria belonging to Splendidus were resistant to one or more antibiotics. Furthermore, almost all isolates were resistant to vancomycin (96%). However, there is rising concern for antibiotics erythromycin, oxytetracycline and oxytetracycline due to intermediate resistance for around half of isolates. This study shows first report on the diversity and antibiotic resistance of Splendidus 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.
Acknowledgements
This work was supported by the Croatian Science Foundation - project IP-2019-04-1956
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