Prospecting microbiota of Adriatic fish: Bacillus velezensis as a potential probiotic candidate

Abstract

Background: Aquaculture is one of the fastest growing sectors of food production and covers more than half of the market demand for fish and fishery products. However, aquaculture itself faces numerous challenges, such as infectious disease outbreaks, which are one of the limiting factors for the growth and environmental sustainability of modern aquaculture. Understanding the composition and diversity of the gut microbiota of fish is important to elucidate its role in host health and aquaculture management. In addition, the gut microbiota represents a valuable source of bacteria with probiotic potential for farmed fish.

Results: In this study, we analysed the intestinal microbiota of two economically important fish species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), using 16S rRNA gene amplicon sequencing. The taxonomic analysis identified 462 amplicon sequence variants at a similarity level of 99 and showed similar alpha diversity indices between seabass and gilthead seabream. Beta diversity analysis showed no significant differentiation in gut microbiota between fish species or aquaculture sites. Among the culturable isolates, a high proportion of Photobacterium damselae and Bacillus spp. was detected. We selected a single Bacillus velezensis isolate and further characterised its biosynthetic potential by performing whole genome sequencing. Its genome contains biosynthetic gene clusters for most of the common secondary metabolites typical of B. velezensis. Antibiotic susceptibility testing showed the sensitivity of the selected isolates to several antibiotics according to EFSA recommendations. Furthermore, stimulation of peripheral blood leukocytes (PBL) with B. velezensis resulted in a strong pro-inflammatory response, with a pronounced upregulation of cytokines il1b, il6, tnfa and il10 observed over time.

Conclusions: Overall, this study provides an insight into the composition of the intestinal microbiota and the diversity of culturable intestinal bacteria of two economically most important fish species from Adriatic cage culture and sheds light on the autochthonous intestinal B. velezensis as a promising probiotic candidate for Mediterranean aquaculture.

Keywords: Bacillus velezensis; Dicentrarchus labrax; Sparus aurata; Aquaculture; Probiotics; Whole genome sequencing.

Fig. 1

Map of Adriatic Sea with sampling locations. Shown are locations of farms from which European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) were sampled in 2019. Note that from farm G only European seabass sample was collected. (Map downloaded from https://d-maps.com/carte.php?num_car=5352&lang=en)

Fig. 2

Alpha and beta diversity plots. (A) Alpha diversity analyses of gut microbiomes in two aquaculture fish species: Dicentrarchus labrax (green) and Sparus aurata (purple): Observed number of ASVs, Pielou’s evenness index and Shannon’s diversity index. Kruskal–Wallis hypothesis test P values are indicated above horizontal bars. (B) Principal coordinate analysis (PcoA) based on weighted and unweighted Unifrac distances of fish gut samples according to species and the locations. Fish farm location (A – H) match the labels on Fig. 1

Fig. 3

(A) Venn diagram displays the number of shared and unique ASVs among two fish species. (B) Heatmap of core microbiome at Genus level. Scale represents prevalence (relative abundance)

Fig. 4

Taxonomical structure of bacterial assemblages with over 3% relative abundance. Taxa with < 3% are denoted as Other. Hypervariable V3-V4 regions of 16S rRNA were used to assign taxonomy at genus level in two aquaculture fish species – Dicentrarchus labrax and Sparus aurata at different locations (A – H) in the eastern Adriatic Sea. Fish farm location (A – H) match the labels on Fig. 1

Fig. 5

Abundance heatmap of discriminative gut microbiomes detected between two fish species – ESB (green) and GSB (purple). Taxonomic annotation was included at the family (Genus) level for labelling. A color-coded annotation bar was added above the heatmap to indicate host species identity, as well indicated in the fish farm location (A – H) that match the labels on Fig. 1

Fig. 6

Principal components analysis for gene expression profiles during in vitro stimulation of European seabass PBLs. Relative positions of treatment groups (time-points and stimuli) are shown. Values are log2-transformed and scaled. Numbers within symbols indicate the fish PBLs were isolated from

Fig. 7

Expression of target genes in European seabass PBLs stimulated with B. velezensis and lipoteichoic acid (LTA). (A) Distribution of target gene expression is shown according to state (control, B. velezensis-stimulated and LTA-stimulated) and time. Box plots represent distribution of log2-normalised expression; (B) Log2-transformed fold changes of target genes expression in respect to time-matched controls with respective adjusted p-values. Statistically significant fold changes are indicated in bold