Comparative Evaluation of Berberine and Berberis lycium Extracts to Combat Resistance in Bacteria

Abstract

Antimicrobial resistance in bacterial pathogens is a worldwide issue with high rates of morbidity and mortality. Antibiotics resistance influences as a persistent worldwide health danger are highlighted, as are efforts to address this complex issue. Due to the large diversity of secondary metabolites found in crude extracts of medicinal plants, they could be used as an alternative source of resistance modifying agents. Therefore, the current study was designed to appraise the antibacterial potential of Berberis lycium and its metabolite berberine with contemporary antibiotic against antibiotic resistant clinical isolates. Exhaustive sonication-assisted extraction was used to obtain extracts using four solvents (aqueous, methanol, ethyl acetate and n-Hexane). Extracts were phytochemically characterized and significant alkaloids were quantified therapeutically by using high resolution mass spectrometry (HRMS). Preliminary resistance profiling against antibiotics from chief antibiotic classes against different gram-positive (Staphylococcus haemolyticus and MRSA) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was performed by the disc diffusion method. Micro broth dilution method was used for minimum inhibitory concentration determination. Synergistic interaction of berberine and extracts was observed by using checkerboard method and time kill curve studies. Cell wall integrity of bacterial cell was checked by determining extracellular bacterial protein leakage. Finally, the hemocompatibility of berberine and extracts was determined by hemolytic assay. Toxicity of berberine and extracts was checked by brine shrimp lethality assay. The percent extract retrieval was recorded highest when water was used as extraction solvent (5.308% w/w). Preliminary resistance profiling of antibiotics showed that resistant clinical isolates of two gram-negative and two gram-positive bacteria were resistant to cefixime. Therefore, cefixime and the resistant clinical isolates were chosen to proceed for comparative evaluation of synergistic efficiency of Berberine and B. lycium extracts. Berberine and MeOH extract showed MIC value of 100 and 150 μg/ml against all four strains respectively. MeOH and Aq extract exhibited a total synergy except MeOH extract against R.P. aeruginosa when given in combination with cefixime against all resistant bacterial isolates. 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 MIC and 2MIC and berberine also showed same pattern except in case vii of R.S. haemolyticus. Quantification of cellular protein leakage as a function of cell death was used to assess cell envelop disintegration. Analysis revealed that berberine showed slightly less protein inhibition as compared to the Berberis lycium extracts that contain a variety of alkaloids and polyphenols 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. All extracts and berberine showing negligible hemolysis confirmed their hemocompatibility. For the cytotoxic analysis, brine shrimp lethality assay was performed in which berberine showed greater cytotoxicity as compared to the MeOH and Aq extract. In conclusion, the result of the current study shows that MeOH and Aq extracts of Berberis lycium exhibit significant results in terms of synergistic effects as compared to berberine with cefixime and can be used in combination to combat multidrug resistance. However, mechanistic research is recommended due to the observed therapeutic potential.