Antimicrobial and biofilm inhibiting potential of an amide derivative [N-(2', 4'-dinitrophenyl)-3β-hydroxyurs-12-en-28-carbonamide] of ursolic acid by modulating membrane potential and quorum sensing against colistin resistant Acinetobacter baumannii.

Acinetobacter baumannii is Gram-negative, an opportunistic pathogen responsible for life-threatening ventilator-associated pneumonia. World Health Organization (WHO) enlisted it as a priority pathogen for which therapeutic options need speculations. Biofilm further benefits this pathogen and aids 100-1000 folds more resistant against antimicrobials and the host immune system. In this study, ursolic acid (1) and its amide derivatives (2-4) explored for their antimicrobial and antibiofilm potential against colistin-resistant A. baumannii (CRAB) reference and clinical strains. Viability, crystal violet, microscopic, and gene expression assays further detailed the active compounds' antimicrobial and biofilm inhibition potential. Compound 4 [N-(2',4'-dinitrophenyl)-3β-hydroxyurs-12-en-28-carbonamide)], a synthetic amide derivate of ursolic acid significantly inhibits bacterial growth with MIC in the range of 78-156 μg/mL against CRAB isolates. This compound failed to completely kill the CRAB isolates even at 500 μg/mL concentration, suggesting the compound's anti-virulence and bacteriostatic nature. Short and prolonged exposure of 4 inhibited or delayed the bacterial growth at sub MIC, MIC, and 2× MIC, as evident in time-kill and post-antibacterial assay. It significantly inhibited and eradicated >70% of biofilm formation at MIC and sub MIC levels compared to colistin required in high concentrations. Microscopic analysis showed disintegrated biofilm after treatment with the 4 further strengthened its antibiofilm potential. Atomic force microscopy (AFM) hinted the membrane disrupting effect of 4 at MIC's. Further it was confirmed by DiBAC using fluorescence-activating cells sorting (FACS), suggesting a depolarized membrane at MIC. Gene expression analysis also supported our data as it showed reduced expression of biofilm-forming (bap) and quorum sensing (abaR) genes after treatment with sub MIC of 4. The results suggest that 4 significantly inhibit bacterial growth and biofilm mode of colistin-resistant A. baumannii. Thus, further studies are required to decipher the complete mechanism of action to develop 4 as a new pharmacophore against A. baumannii.