Pseudomonas aeruginosa, an opportunistic pathogen, has been found to cause several chronic and acute infections in human. Moreover, it often shows drug-tolerance and poses a severe threat to public healthcare through biofilm formation. In this scenario, two molecules, namely, cuminaldehyde and tobramycin, were used separately and in combination for the efficient management of biofilm challenge. The minimum inhibitory concentration (MIC) of cuminaldehyde and tobramycin was found to be 150 μg/mL and 1 μg/mL, respectively, against Pseudomonas aeruginosa. The checkerboard assay revealed that the fractional inhibitory concentration (FIC) index of cuminaldehyde and tobramycin was 0.36 suggesting a synergistic association between them. The sub-MIC dose of cuminaldehyde (60 μg/mL) or tobramycin (0.06 μg/mL) individually did not show any effect on the microbial growth curve. However, the same combinations could affect microbial growth curve of Pseudomonas aeruginosa efficiently. In connection to biofilm management, it was observed that the synergistic interaction between cuminaldehyde and tobramycin could inhibit biofilm formation more efficiently than their single use (p < 0.01). Further investigation revealed that the combinations of cuminaldehyde and tobramycin could generate reactive oxygen species (ROS) that resulted in the increase of membrane permeability of bacterial cells leading to the efficient inhibition of microbial biofilm formation. Besides, the synergistic interaction between cuminaldehyde (20 μg/mL) and tobramycin (0.03 μg/mL) also showed significant biofilm dispersal of the test microorganism (p < 0.01). Hence, the results suggested that synergistic action of cuminaldehyde and tobramycin could be applied for the efficient management of microbial biofilm.
Microbial biofilm indicates a cluster of microorganisms having the capability to display drug resistance property, thereby increasing its proficiency in spreading diseases. In the present study, the antibiofilm potential of thymoquinone, a black seed-producing natural molecule, was contemplated against the biofilm formation by Pseudomonas aeruginosa. Substantial antimicrobial activity was exhibited by thymoquinone against the test organism wherein the minimum inhibitory concentration of the compound was found to be 20 μg/mL. Thereafter, an array of experiments (crystal violet staining, protein count, and microscopic observation, etc.) were carried out by considering the sub-MIC doses of thymoquinone (5 and 10 μg/mL), each of which confirmed the biofilm attenuating capacity of thymoquinone. However, these concentrations did not show any antimicrobial activity. Further explorations on understanding the underlying mechanism of the same revealed that thymoquinone accumulated reactive oxygen species (ROS) and also inhibited the expression of the quorum sensing gene (lasI) in Pseudomonas aeruginosa. Furthermore, by taking up a combinatorial approach with two other reported antibiofilm agents (tetrazine-capped silver nanoparticles and tryptophan), the antibiofilm efficiency of thymoquinone was expanded. In this regard, the highest antibiofilm activity was observed when thymoquinone, tryptophan, and tetrazine-capped silver nanoparticles were applied together against Pseudomonas aeruginosa. These combinatorial applications of antibiofilm molecules were found to accumulate ROS in cells that resulted in the inhibition of biofilm formation. Thus, the combinatorial study of these antibiofilm molecules could be applied to control biofilm threats as the tested antibiofilm molecules alone or in combinations showed negligible or very little cytotoxicity.
