Potential Antimicrobial Properties of Coffee Beans and Coffee By-Products Against Drug-Resistant Vibrio cholerae

Abstract

Vibrio cholerae is the causative organism of the cholera epidemic, and it remains a serious global health problem, particularly the multidrug-resistant strain, despite the development of several generic drugs and vaccines over time. Natural products have long been exploited for the treatment of various diseases, and this study aimed to evaluate the in vitro antibacterial activity of coffee beans and coffee by-products against V. cholerae antimicrobial resistant strains. A total of 9 aqueous extracts were investigated, including light coffee (LC), medium coffee (MC), dark coffee (DC), dried green coffee (DGC), dried red coffee (DRC), fresh red coffee (FRC), Arabica leaf (AL), Robusta leaf (RL), and coffee pulp (CP). The influential coffee phytochemicals, i.e., chlorogenic acid (CGA), caffeic acid (CA), and caffeine, were determined using HPLC. The antibacterial properties were tested by agar well-diffusion techniques, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were further determined against 20 V. cholerae isolates. The results revealed that all tested strains were sensitive to coffee extracts, with MIC and MBC values in the range of 3.125-25.0 mg/mL and 12.5-50.0 mg/mL, respectively. With a MIC of 6.25 mg/mL, DGC, DRC, and CP appeared to be the most effective compounds against 65, 60, and 55% of clinical strains, respectively. The checkerboard assay revealed that the combination of coffee extract and tetracycline was greater than either treatment alone, with the fractional inhibitory concentration index (FICI) ranging from 0.005 to 0.258. It is important to note that CP had the lowest FICI (0.005) when combined with tetracycline at 60 ng/mL, which is the most effective dose against V. cholerae six-drug resistance strains (azithromycin, colistin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim), with a MIC of 47.5 μg/mL (MIC alone = 12.5 mg/mL). Time killing kinetics analysis suggested that CA might be the most effective treatment for drug-resistant V. cholerae as it reduced bacterial growth by 3 log₁₀ CFU/mL at a concentration of 8 mg/mL within 1 h, via disrupting membrane permeability, as confirmed by scanning electron microscopy (SEM). This is the first report showing that coffee beans and coffee by-product extracts are an alternative for multidrug-resistant V. cholerae treatment.

Keywords: Vibrio cholerae; antimicrobial activity; coffee by-products; coffee extract; drug-resistant.

Figure 1

Effect of coffee phytochemical on the viability of V. cholerae O1 El Tor N16961. Time-kill kinetics of CGA (A), CA (B), and caffeine (C) at concentrations of 1–8 MIC against V. cholerae were investigated over a 24 h incubation period at 37°C. The MIC for CGA was 0.5 mg/mL, while the MICs for CA and caffeine were 1 mg/mL. MHB was used as the control instead of compound. Samples were taken at 1, 2, 4, 8, 16, and 24 h to determine viable bacterial numbers. The bactericidal level is indicated by the dashed lines.

Figure 2

Effect of CA on membrane permeability. V. cholerae O1 El Tor N16961 was treated with CA at a concentration of 1–8 mg/mL for 1 h at 37°C. The intracellular leakage of nucleotides (A) and proteins (B) were measured, and 0.1% Triton X-100 (TX) was used as a positive control. The outer membrane disruption and membrane potential dissipation were investigated by the percentages of NPN uptake (C) and Rh123 relative fluorescence intensity (D), respectively. Significant differences compared to untreated controls are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 3

Effect of CA on bacterial cell morphology. V. cholerae O1 El Tor N16961 was treated with CA at a concentration of 8 mg/mL for 2 h at 37°C. SEM images at x10,000 and x20,000 magnifications were demonstrated. (A,B) Negative controls and (C,D) effective treatments. The cell membrane disruption is represented by the red circles.