Introduction
Bivalves, which filter seawater as part of their feeding process, are particularly prone to bacterial colonization of their tissues. Among the bacteria commonly found in marine environments, Vibrio species are predominant and include commensals, opportunistic pathogens, and significant pathogens of both marine organisms and humans. Human infections are most frequently associated with the consumption of contaminated seafood, especially bivalves, which are often eaten raw or undercooked. In addition to the threat they pose to human health, Vibrio species can cause serious diseases in bivalves, impacting aquaculture production. Moreover, studies indicate that rising of seawater temperatures associated with global warming will significantly affect the dynamics of Vibrio genus species. Therefore, understanding the structure of natural Vibrio communities, including the presence of potential pathogens, is crucial for the management of bivalve aquaculture. Species belonging to the Mediterranei and Orientalis clades, such as V. mediterranei, V. bivalvicida and V. tubiashii, have frequently been associated with mortality events in marine organisms, but are under investigated in the context of eastern Adriatic bivalve aquaculture. In addition, the increasing problem of antibiotic resistance, driven by the overuse of antibiotics and anthropogenic pollution in marine environments, represents a growing challenge. Despite its importance, antibiotic resistance among Vibrio species in the eastern Adriatic bivalve aquaculture remains poorly investigated when it comes to Mediterranei and Orientalis clade species.
Materials and methods
Our study is focused in two protected marine reserves – Lim Bay and Mali Ston Bay, locations which are known for aquaculture of Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819 and Ostrea edulis Linnaeus, 1758. On both location we sampled seawater, sediment and bivalve tissue bimonthly over the course of one year. All bacteria isolated on Vibrio selective media were screened with MALDI-TOF mass spectrometry to confirm the genus and clade. More precise species identification was carried out by sequencing pyrH gene for improved taxonomic resolution. All isolates identified as members of Mediterranei and Orientalis 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.
Results
MALDI-TOF MS results showed that 58 isolates belonged to Mediterranei clade while only 21 Orientalis clade isolates were found. Isolates of both clades were found during warmer periods especially in Lim Bay at seawater temperatures above 17⁰C. Furthermore, Orientalis clade isolates didn’t show a high incidence of antibiotic resistance except for erythromycin (42.9%). For Mediterranei clade, higher occurrence of resistance to erythromycin (62.1%) was found. Also, while only 15.5% of isolates were resistant to oxytetracycline, 58.6% were intermediately resistant. Almost 71% of isolates were multi-resistant, meaning that they were resistant to two or more antibiotics.
This study provides the first report on the diversity and antibiotic resistance of Vibrio species from the Mediterranei and Orientalis clades associated with bivalve aquaculture in the eastern Adriatic. The findings highlight the need to focus on a broader range of species that make up the Vibrio community, in order to improve the current knowledge of the risk for infections and diseases outbreaks in bivalve aquaculture.
This will be crucial in the near future, considering the predictions that increasing anthropogenic pressure and climate change will enhance the susceptibility of farmed bivalves to potentially pathogenic Vibrio species and promote the spread of antibiotic-resistant strains, posing a growing threat to the sustainability of bivalve aquaculture.
Acknowledgements
This research was funded by the Croatian Science Foundation programmes: “Research Project” [grant number IP-2019-04-1956] and Career Development of Young Researchers – Training New Doctoral Students [grant number DOK 2021-02-7567].