Antibacterial Potential and Synergistic Interaction between Alkanna tinctoria (L.) Tausch and Cefixime against Resistant Bacterial Strains

Abstract

Antimicrobial resistance makes the antibiotic treatment difficult. Alkanna tinctoria (L.) Tausch (A. tinctoria) is effective against bacterial infections. Current study evaluated the synergistic antimicrobial interactions of A. tinctoria extracts along with cefixime against resistant pathogenic bacterial strains (MRSA, E. coli, and R. A. baumanniii) to counteract microbial resistance. A. tinctoria extracts (ethyl acetate, methanol, and aqueous) were prepared by using sonication aided maceration. Extracts were subjected to phytochemical evaluation i.e. total phenolic content (TPC), total flavonoid content (TFC) and standardized by RP-HPLC. Antioxidant colorimetric assays i.e. total antioxidant capacity (TAC), free radical scavenging assay (FRSA), and ferric reducing antioxidant power (FRAP) were employed. Initial susceptibility profiling of antibiotics and extracts were done by disc diffusion and microbroth dilution assays. Synergistic evaluation of extracts and cefixime were assessed by checkerboard method. Time kill kinetics studies were done to study concentration and time dependent effects of extracts and cefixime. Protein estimation studies were employed to study mechanistic impact of combination on bacterial growth inhibition. Hemocompatibility was evaluated by hemolytic assay. Ethyl acetate extract showed maximum percent extract recovery (4.25%). Phytochemical evaluation showed that methanol extract had significant amount of phenols and flavonoids (23.7 ± 0.02 µg QE/mg of extract and 19.5 ± 0.18 μg GAE/mg of extract) in it. Rutin, catechin, and myricetin were major polyphenols detected by RP-HPLC. Methanol extract showed remarkable antioxidant potential, TAC (130.67 ± 0.08 g AAE/mg of extract), FRSA (17.3 ± 0.08%) and FRAP (121.46 ± 0.04 g AAE/mg of extract). All the extracts showed antibacterial activity with MIC value of 500 g/ml. Ethyl acetate extract in combination exhibited total synergism with 8 and 16 fold reduction in extract and cefixime MIC values against all resistant strains used. The time kill kinetics revealed bacteriostatic effect when extract and cefixime was used in combination. The synergism exhibited mechanistic inhibition of bacterial proteins specifically, the ethyl acetate extract in combination yielded a significant percentage inhibition (84.6 ± 0.09%) against resistant strains. All the extracts alone and in combination were safe to use at the maximum concentration of 500 g/ml (3.66 ± 0.36% hemolysis). Thus, A. tinctoria extracts have significant antimicrobial activity and also lowered the MIC of cefixime against resistant strains might be considered a useful combination to boost the cefixime antibacterial activity