Intestinal Bacillus velezensis successfully simulates European seabass peripheral blood leukocytes

Introduction: In an era of rising antimicrobial resistance and a limited number of curative measures for disease outbreaks, aquaculture struggles to find environmentally friendly and cost-effective alternatives to various chemotherapeutics. One such alternative is the use of health-promoting agents, such as probiotics. They are known to have a beneficial effect on fish health at local and systemic levels, by improving health status, disease resistance and host and environmental balance.

Methodology: We isolated a Bacillus spp. strain from Adriatic aquaculture fish and characterized it by whole genome sequencing (WGS), combining Illumina and Nanopore sequencing data and assessing its biosynthetic potential using the antiSMASH web interface. The same isolate was tested for its immunostimulatory efficiency on European seabass peripheral blood leukocytes (PBLs). The PBLs were isolated by hypotonic lysis and stimulated with live bacteria for 3h, 6h and 18h. In addition, a batch of PBLs was treated with lipoteichoic acid (LTA) from B. subtilis as a positive control. Finally, expression of selected immune-related genes (Il1-β, Il6, Il10, Tnf-α and Tlr2) in stimulated PBLs was quantified by real-time PCR.

Results: Based on Genome Taxonomy Database, the isolated strain was identified as Bacillus velezensis. antiSMASH analysis identified biosynthetic gene clusters (BCGs) for most of the usual B. velezensis specific secondary metabolites, including surfactin, bacillaene, macrolactin, fengycin, difficilin, bacillibactin and bacilysin. However, antiSMASH could not separate between two possible BCGs in “region 8” due to overlapping genes, thus possibly suggesting that biosynthetic potential for iturin is also present. Gene expression analysis showed that the most perturbed genes were those coding for canonical pro-inflammatory leukocytes, indicating a positive effect on innate immunity.

Conclusion: In conclusion, B. velezensis and/or its identified secondary metabolites positively stimulate European seabass peripheral blood leukocytes indicating its potential use as a probiotic in seabass aquaculture.

This work was funded by Interreg Italy-Croatia "AdriAquaNet" project and the Danish National Research Foundation (DNRF137) for the Center for Microbial Secondary Metabollites.