Introduction
Infectious bacterial fish diseases remain a major challenge for aquaculture development. Although antibiotics are an important tool for disease treatment, their damaging effects on the environment and public health have led to increased restrictions to their use in aquaculture. Quorum-Sensing (QS) is a regulatory mechanism closely related to the expression of virulence factors contributing to the pathogenicity of fish diseases. Hence, disruption of bacterial QS, known as Quorum-Quenching (QQ) is being proposed as a possibility to fight aquaculture diseases dissemination [1]. Bacillus spp. are sporeforming bacteria with recognizable biotechnology applications including as a source of natural antimicrobial molecules, such as QQ molecules. Indeed, we recently isolated from the gut of three marine fish species, Bacillus spp. capable of inhibiting the growth and biofilm formation of 14 different fish pathogens and with in vitro QQ capacity [2]. Alongside with other probiotic health-promoting characteristics, Bacillus spp. are also known for their immunostimulatory effects enhancing the host’s innate and adaptive immunity against fish pathogens [3].
Here, we screened the QQ capacity of the extracellular components of our collection of Bacillus spp., previously isolated from the gut of different marine fish species and tested their ability to degrade AHLs QS-signals produced by important fish pathogens, their protective effects in an in vivo model when challenged with E. tarda and their putative modulation of the fish immune system.
Materials and Methods
We took advantage of 200 Fish isolates (FI) isolated from the gut of different fish species (Sparus aurata, Dicentrarchus labrax, and Diplodus sargus) [2, 4] to explore their QQ potential, by testing the ability to degrade AHLs QS molecules using CV026 biosensor. FI with extracellular QQ activity were tested for AHL enzymatic degradation of 30 µM of 3-Oxo-C6-HSL and evaluated for their QQ capacity against AHLs QS-signals produced by Aeromonas spp., Vibrio spp., Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei using a well-diffusion method and CV026 biosensor. The fish isolates’ protection against E. tarda infection was performed using zebrafish larvae (Danio rerio). Larvae were treated once with the lyophilized extracts of FI314, FI436 and FI464 after mouth’s opening (7dpf), for 2h at 28°C. The treated 10 dpf larvae were challenged by immersion with E. tarda at 1x108 CFU mL-1, and cumulative mortalities were registered between 16-24hours post-infection.
To elucidate the immune-modulatory properties of the QQ strains, head–kidney leucocytes from gilthead seabream were isolated and treated for 24h with extracts of FI314, FI436 and FI464 before stimulation with E. tarda (5x106CFU/mL). Different innate immune parameters – viability (propidium iodide), respiratory burst activity and phagocytosis – were evaluated using flow cytometry. Simultaneously, expression of immune-relevant genes (Hsp70, Cox-2, IL-1β, IL-6, IL-10 and Tnfα) were measured using real-time PCR.
Results
We found out that ~12% of our fish-gut Bacillus spp. were able to interfere with synthetic AHLs signalling molecules. We further selected 10 isolates as producers of extracellular putative AHL-lactonase enzymes. Conjugating the QQ genomic profile, QQ bioactivities and molecular identification FI314, FI436 and FI464 were tested for their QQ capacity against natural AHLs produced by fish pathogens. We observed that A. veronii and E. tarda produce AHLs molecules detectable by Chr. violaceum biosensor, that were degraded when subjected to the extracellular extracts of fish-gut isolates FI314, FI436 and FI464. Moreover, when compared to the control (non-treated zebrafish larvae infected with E. tarda), FI extracellular compounds from FI314, increased the average survival rate of challenged larvae by 43% (p<0.01), and strains FI436 and FI464 increased the survival rate upon challenge by 50% (p<0.001).
Although, Bacillus spp. extracellular extracts did not stimulate the respiratory burst activity and cell viability, they remarkably increased pathogens’ phagocytosis when the seabream leukocytes were exposed to E. tarda. Statistical analysis revealed that all extracts significantly increased (p<0.001) the engulfment of E. tarda 1h post-infection. In cells treated with the extracellular extracts, we observed an up-regulation of the immune genes associated with inflammation, including IL-1β, IL-6 and Cox-2, indicating a stimulus of the immune system. FI314 extracellular extract significantly increased the expression of IL-1β (p=0.04), IL-6 (p=0.005) and Cox-2 (p=0.021) and, FI436 and FI464 significantly increased IL-6 expression (p=0.047 and p=0.009, respectively).
Conclusions
Taken together our experiments revealed three promising fish-gut Bacillus spp. that produce extracellular molecules capable of quenching aquaculture pathogens’ communication, protect fish from Edwarsiella tarda infection in an in vivo model and improve the immune system of gilthead seabream. Thus, the extracellular molecules from FI314 (B. subtilis), FI436 (B. vezelensis) and FI464 (B. pumilus) may be promising tools for disease control in aquaculture.
Funding: RAS, NP and NM are recipients of a PhD grant (SFRH/BD/131069/2017, SFRH/BD/138187/2018, SFRH/BD/137919/2018, respectively) from FCT; CRS has a scientific employment contract supported by national funds through FCT. This research was partially supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020, through national funds provided by FCT and ERDF.
References
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2. Santos, R.A., et al., Isolation and Characterization of Fish-Gut Bacillus spp. as Source of Natural Antimicrobial Compounds to Fight Aquaculture Bacterial Diseases. Mar Biotechnol (NY), 2021.
3. Kuebutornye, F.K.A., E.D. Abarike, and Y. Lu, A review on the application of Bacillus as probiotics in aquaculture. Fish & Shellfish Immunology, 2019. 87: p. 820-828.
4. Serra, C.R., et al., Selection of carbohydrate-active probiotics from the gut of carnivorous fish fed plant-based diets. Scientific Reports, 2019. 9(1): p. 6384.