Vitamin D3 potentiates antimicrobial and antibiofilm activities of streptomycin and thymoquinone against Pseudomonas aeruginosa

Biofilm formed by Pseudomonas aeruginosa is a three dimensional microbial matrix that confers multidrug resistance properties along with the proficiency to evade the host immune system. The present study aims to determine the combinatorial effects of vitamin D3 (cholecalciferol) with two already reported antibiofilm agents: streptomycin and thymoquinone separately against P. aeruginosa biofilms. The minimum inhibitory concentration of streptomycin, thymoquinone and D3 was found to be 20, 10 and 100 mu g/mL respectively. The inhibition of biofilm formation and pre-formed biofilm disintegration properties of streptomycin and thymoquinone alone or in combination with D3 at their sub-MIC concentration was determined by crystal violet staining and confocal laser scanning microscopy. A significant inhibition of metabolic activities like oxygen consumption rate and reduction in quorum sensing related cellular activities like swarming motilities, pyocyanin production and extracellular protease secretion by P. aeruginosa were also observed as a result of this combinatorial effect. Both of these combinatorial applications were found to accumulate ROS in bacterial cells, which has been proved to be one of the main causes of their antibiofilm activity. Effect of these two drug combinations on bacterial lettuce leaf infection was also evaluated. Molecular docking analysis indicated that thymoquinone combined D3 can interact more efficiently with the quorum sensing proteins LasI and LasR. The host cell cytotoxicity of these two combinations was found to be negligible on the murine macrophage cell line. These findings suggest that D3 potentiates the antimicrobial and antibiofilm efficacy of both streptomycin and thymoquinone against P. aeruginosa. Although both combinations have shown significant antibiofilm and antimicrobial potential, combinatorial performances of D3 combined thymoquinone were found to be more promising.