Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms.

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Anti-oxidant, antibacterial, anti-biofilm, and anti-quorum sensing activities of four essential oils against multidrug-resistant bacterial clinical isolates.

PURPOSE OF THE STUDY: Outbreaks of multidrug-resistant bacteria are increasingly reported at the clinical setting. The antimicrobial, anti-biofilm, anti-quorum sensing, and anti-oxidant activities of four essential oils extracted from Cinnamomum verum, Origanum majorana, Thymus vulgaris, and Eugenia caryophyllata against Gram-positive and Gram-negative multidrug-resistant bacteria were evaluated in vitro. MATERIALS AND METHODS: This study was conducted on 105 multidrug resistant clinical strains. Inhibition diameter zone, minimum inhibitory concentration, and minimum bactericide concentration of the oils were determined using agar disc diffusion method and microdilution. The ability of the 4 essential oils to inhibit the production of bacterial biofilms was tested on polystyrene plates, as well as their inhibitory effect on the production of violacein by Chromobacterium violaceum CV026. The anti-oxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl scavenging method. RESULTS: Essential oils of Cinnamomum verum, Thymus vulgaris and Eugenia caryophyllata showed an important antibacterial activity. The inhibition diameter zone was higher than 20 mm for 90.24 %, 85.71 % and 60.95 % of strains respectively. These essential oils have a remarkable anti-biofilm and anti-quorum sensing activities against almost all the species studied. Clove extract revealed the highest anti-oxidant activity (Pourcentage of inhibtion of DPPH = 90.3 %). CONCLUSION: These results supported the use of the 4 essential oils as alternative or complementary agents to treat infections caused by multidrug-resistant bacteria, and to prevent biofilm formation and quorum sensing signaling. They might be used as a safe anti-oxidants instead of harmful artificial ones.