Comprehensive Exploration of Antimicrobial Potential of Artemisia brevifolia Wall

Abstract

The development of resistance and treatment challenges against the bacterial infections has become a major concern due to which medicinal plants with therapeutic properties have been screened and used as anti-bacterials. Artemisia brevifolia Wall. belongs to the family Asteraceae. It is locally known as “Tarkha” or “Mori”. Traditionally, this species is used as antiseptic, anthelminthic, antidiabetic, carminative, blood purifier, relieving stress, diuresis, reducing pain, antitussive, stomachic, febrifuge and antidot for scorpion sting. Owing to the ethnomedicinal importance of the subject plant, it was selected in the present study for the estimation of antibacterial potential. N-hexane (NH), ethyl acetate (EA), methanol (M), and aqueous extracts (Aq) of aerial parts of the plant were obtained by successive solvent extraction. Preliminary resistance profiling against antibiotics from chief antibiotic classes against different gram-positive and gram-negative bacteria was performed by the disc diffusion method. Antibacterial susceptibility testing against the determined resistant strains was executed using standard disc diffusion assay whereas microbroth dilution method was used for minimum inhibitory concentration determination. For the evaluation of synergistic interaction of plant extract with cefixime, checker board microdilution method and time kill curve studies were used. Cell wall integrity of bacterial cells was also evaluated by determining extracellular bacterial protein leakage. Finally, the biocompatibility of extracts was determined by hemolytic analysis. The percent extract retrieval was recorded highest when water was used as extraction solvent (6.1% w/w). Preliminary resistance profiling of antibiotics showed that resistant clinical isolates of two gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two gram-positive (Staphylococcus haemolyticus and MRSA) bacteria were resistant to cefixime. Therefore, cefixime and the resistant clinical isolates were chosen to proceed for evaluation of synergistic efficiency of A. brevifolia standardized extracts. Antibacterial susceptibility testing of A. brevifolia standardized extracts showed moderate antibacterial activity against all resistant bacterial isolates with significant bacterial inhibition shown by EA extract with ZOI of 10±1.4 mm and 12±0.7 mm against S. haemolyticus and MRSA respectively, while NH extract exhibit noteworthy inhibition i.e., 12±0.7 mm ZOI against E. coli and M extract was most active extracts against P. aeruginosa with ZOI of 11±0.7 mm. Minimum inhibitory concentration (MIC) of 60 μg/ml was recorded in EA (S. ix haemolyticus and MRSA) and M (E. coli and P. aeruginosa) extracts . EA extract exhibited a total synergy when given with cefixime against all bacterial isolates with Fractional inhibitory concentration index (FICI) 0.5 and a 4-fold reduction in minimum inhibitory concentration (MIC) of extract and cefixime against S. haemolyticus likewise FICI value of 0.375 and a reduction (4 and 8) in MIC of extract and cefixime was observed against P. aeruginosa. FICI of E.A extract was calculated 0.3125 with a 4 and 10 fold reduction in MICI against MRSA and E. coli. Kinetic studies of antibacterial activity were determined by plotting time kill curve graphs. Results depicted that all extracts showed same pattern against all bacterial isolates when tested at MICI and 2MICI i.e., bacteria remained in log phase from 0-3 hour and in decline phase from 3-6 hour followed by a log phase from 6-9 hour. However, when the extracts were given in synergy the bacteria remained in stationary phase from 0-6 hour while log phase started at 6 hours. Doubling the concentration of synergistic dose caused bacteria to remain in stationary phase throughout the reaction time. Disintegration of cell envelop was measured by quantification of cellular protein leakage as a function of cell death. Analysis revealed that all extracts showed same results against all bacterial isolates i.e., they do not disrupt cell membrane of bacteria when given alone but in combination a reduction in protein content was observed. A negligible hemolysis established extracts biocompatibility. In conclusion, the results of the current study shows that EA extract exhibit significant synergistic effects with cefixime and may be used in combination to combat multidrug resistance. However, mechanistic insight for the observed therapeutic potential is recommended.