Characterization and determination of the immunomodulatory activity of intestinal microbiota microorganisms isolated from free-range Cornu aspersum snails in olive groves before and after aestivation.

The present study investigated the impact of aestivation of free-range snails on the intestinal microbiota and its potential to protect the host from infection by pathogenic bacteria. Bacterial strains were isolated just before and at the end of aestivation from the gut of free-range Cornu aspersum aspersum snails from the island of Crete (Greece). Α total of 31 BA (Before Aestivation) and 25 EA (End Aestivation) isolates, were tested for their in vitro probiotic properties. Principal component analysis (PCA) and a machine learning model showed that BA isolates exhibited a different pattern of in vitro properties from that of EA isolates, and that all the EA isolates, except one, were presumptive probiotics, while among BA only isolates BA2Ae and BA5An were presumptive probiotics. 16SrRNA sequencing analysis at species-level revealed that most BA isolates belonged to potentially snail pathogens, while EA isolates belonged to the genera of Citrobacter, Lactiplantibacillus and Enterococcus, that belong to normal intestinal microbiota and presumptive probiotics. Injection of selected isolates in snails resulted in increased immune activity such as chemotaxis and phagocytosis. Food administration of the strains, E. malodoratus BA5An and L. plantarum EA2An, exhibiting the highest immunomodulatory activity, showed further probiotic properties since symbiosis achieved by enhancing some immune humoral responses, as well as restoration of intestinal homeostasis by inhibiting the dysbiosis induced by the snail pathogenic strain Listeria monocytogenes SN3. Each presumptive probiotic strain exhibited a different pattern of immunomodulatory activity against the pathogen.