Efficacy of Chlorogenic Acid Combined With Cefazolin Against Methicillin-Resistant Staphylococcus aureus Biofilms

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA), has the ability to cause biofilm associated chronic infections with a high mortality rate. This creates a demand for the improved antibiofilm therapies. The aim of this study was to evaluate the anti-MRSA and antibiofilm activity of a natural product, chlorogenic acid, in synergy with an antibiotic, cefazolin. Material and Methods: The synergistic effect was measured by the checkerboard method. The antibiofilm activity was analysed by crystal violet staining, MTT assay, and atomic force microscopy (AFM). The cytotoxic effect on Human Embryonic Kidney (HEK 293) cells was determined using the MTT assay. Assays were performed in triplicate, and compared using the one-way ANOVA test. Results: When chlorogenic acid and cefazolin were combined at low concentrations, a strong biofilm inhibition in terms of biofilm biomass (88%), and metabolic activity (82%) was observed, as compared to the results obtained for each compound alone. AFM images of biofilms, treated with chlorogenic acid combined with cefazolin, revealed a high destruction of biofilms and extracellular polymeric substances, as compared to each drug alone. A nontoxic effect on HEK 293 cells was observed for the combination of chlorogenic acid and cefazolin. Conclusion: Chlorogenic acid can be used as an adjuvant with currently used antibiotic in the development of combinatory therapies to treat biofilm-associated bacterial infections.

Figure 1

Effect of individual and combinatorial treatments of chlorogenic acid and cefazolin against biofilm formation and mature biofilms in MRSA. (a) Effect of treatments on the inhibition of biofilm formation. (b) Effect of treatments on the eradication of mature biofilm. The concentrations of drug that shows synergistic effect were represented in red color.

Figure 2

AFM analyses of the synergistic antibiofilm effect of chlorogenic acid and cefazolin against MRSA. (a) AFM images of the combination of chlorogenic acid and cefazolin against biofilm inhibition and (b) AFM images of the combination of chlorogenic acid and cefazolin against eradication of mature biofilm: (A–D; I–L) represents the AFM 2D topography images and (E–H; M–P) represents the AFM 3D topography images.

Figure 3

Cytotoxicity effect of chlorogenic acid and cefazolin alone, and their combination on HEK 293 cells. ^∗p < 0.05: statistically significant difference, as compared to the untreated control.