However, particular safety concerns based on antibiotic resistances and virulence factors were dominant within E. faecalis (100%) and E. faecium (79%), and acquired antibiotic resistance genes were not commonly found (7.5%; erythromycin and clindamycin) amongst the non-enterococcal
isolates of aquatic origin. To our knowledge, this is the first large-scale study selleck compound describing the antimicrobial activity against fish pathogens and the safety assessment beyond the QPS approach of LAB isolated from aquatic animals. The in vitro subtractive screening presented herein, which allowed the selection of 33 strains (8 E. faecium, 11 P. pentosaceus, 1 Lb. carnosus, 1 Lb. curvatus, 3 L. cremoris, 3 Lc. cremoris and 6 W. cibaria) out of 99 LAB isolates of aquatic origin, constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture and to avoid the spreading of bacterial cultures with harmful traits into the aquatic environment. Nevertheless, a comprehensive in vivo assessment of their lack of toxicity and undesirable effects must be also carried out using cell
lines, live food and, ultimately, aquatic animals before their unequivocal consideration as safe probiotics for a sustainable aquaculture. Methods Bacterial strains and growth conditions A total of 99 LAB (59 enterococci and 40 non-enterococci) of aquatic origin with antimicrobial activity against spoilage and food-borne pathogenic bacteria of concern for the fish industry, previously isolated
and identified by our group from RG7420 cell line Selleck Crenigacestat fish, seafood and fish products , were used in this study (Table 1). The LAB strains were isolated on non-supplemented MRS (Oxoid, Ltd., Basingstoke, United Kingdom) or KAA (Oxoid) agar (1,5%, w/v) at 25°C, and taxonomically identified  by sequencing of the genes encoding 16S rRNA (16S rDNA)  and/or superoxide dismutase (sodA) . Unless otherwise stated, LAB were grown aerobically in MRS broth at 32°C. Direct antimicrobial activity assay The antimicrobial activity of the 99 LAB against the main Gram-positive and Gram-negative fish pathogens was assayed by a qualitative stab-on-agar test (SOAT) as previously described by Cintas et al. . Briefly, pure cultures were stabbed onto MRS or Tryptone Soya Agar (TSA) (Oxoid) plates supplemented with glucose (2%, w/v) and incubated at 32°C for 5 h, and then 40 ml of the corresponding soft agar (0.8%, w/v) this website medium containing about 1 × 105 CFU/ml of the indicator strain was poured over the plates. After incubation at 28-37°C for 16–24 h depending on the indicator strain, the plates were checked for inhibition zones (absence of visible microbial growth around the stabbed cultures), and only inhibition halos with diameters >3 mm were considered positive. L.