Investigation of interspecies crosstalk between probiotic Bacillus subtilis BR4 and Pseudomonas aeruginosa using metabolomics analysis

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes high mortality in cystic fibrosis patients. Treatment failures often occur due to the emergence of antibiotic resistance. Inhibition of virulence factors production without suppressing the growth of the pathogens is a potential alternative strategy to control the antibiotic resistance. In order to accomplish, three different interaction studies were performed using Bacillus subtilis BR4, PA and their extracellular contents. Firstly, co-cultivation was performed with different cell density of BR4 or PA. In co-culture setup (F), high cell density of BR4 significantly inhibits the biofilm formation of PA in a growth-independent manner (p < 0.01). To substantiate the biofilm inhibition, LC-MS/MS was performed and metabolic profile of monocultures and cocultures were compared. Multivariate analysis corroborated that metabolic profile of coculture setup (F) is drastically different from other coculture and monoculture setups. To check the effect of extracellular content of PA on BR4, supernatant of PA was extracted with ethyl acetate and different concentration of that extract (PA-EXT) was supplemented with BR4 culture. Exogenous supplementation PA-EXT (40 μg/mL) led to increased biofilm inhibitory activity (p < 0.01) in BR4. Further, to check the effect of extracellular content of BR4, PA was grown in the supernatant of BR4. PA survives in the spent media of BR4 without biofilm formation. Though 50% spent media of BR4 was replaced with fresh media, PA could not produce biofilm. In support of this, LC-MS/MS analysis has revealed that abundance of quorum sensing (QS) signals was reduced in the spent media grown PA than control. Furthermore, BR4 protects zebrafish larvae (Danio rerio) against PA infection and increases their survival rate (p < 0.05). We found that PA-induced oxidative stress and apoptosis were also significantly reduced in the BR4-pretreated larval group than control group. These results clearly indicate that BR4 exerts growth-independent QS inhibition in PA, suggesting that it could be used as a probiotic for future therapeutic interventions.

Keywords: Apoptosis; Biofilm; LC-MS/MS; Quorum sensing; ROS; Zebrafish.