Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3'-digallate

Abstract

Theaflavin-3,3'-digallate (TFDG), a polyphenol derived from the leaves of Camellia sinensis, is known to have many health benefits. In this study, the antibacterial effect of TFDG against nine bacteria and the sporicidal activities on spore-forming Bacillus spp. have been investigated. Microplate assay, colony-forming unit, BacTiter-Glo^TM, and Live/Dead Assays showed that 250 µg/mL TFDG was able to inhibit bacterial growth up to 99.97%, while 625 µg/mL TFDG was able to inhibit up to 99.92% of the spores from germinating after a one-hour treatment. Binding analysis revealed the favorable binding affinity of two germination-associated proteins, GPR and Lgt (GerF), to TFDG, ranging from -7.6 to -10.3 kcal/mol. Semi-quantitative RT-PCR showed that TFDG treatment lowered the expression of gpr, ranging from 0.20 to 0.39 compared to the control in both Bacillus spp. The results suggest that TFDG not only inhibits the growth of vegetative cells but also prevents the germination of bacterial spores. This report indicates that TFDG is a promising broad-spectrum antibacterial and anti-spore agent against Gram-positive, Gram-negative, acid-fast bacteria, and endospores. The potential anti-germination mechanism has also been elucidated.

Keywords: anti-germination; antibacterial; binding analysis; black tea polyphenol; natural product; sporicidal; theaflavin.

Figure 1

The (A) 2D and (B) 3D chemical structures of TFDG.

Figure 2

Live/Dead assay of Gram-negative species with various concentrations of TFDG at 6-h incubation. The samples were visualized using Olympus confocal microscope. The green indicates live bacteria, while the red indicates dead bacteria. (A) K. aerogenes control; (B) K. aerogenes with 62.5 µg/mL TFDG; (C) K. aerogenes with 250 µg/mL TFDG; (D) E. coli control; (E) E. coli with 62.5 µg/mL TFDG; (F) E. coli with 250 µg/mL TFDG; (G) P. aeruginosa control; (H) P. aeruginosa with 62.5 µg/mL TFDG; (I) P. aeruginosa with 250 µg/mL TFDG; (J) P. mirabilis control; (K) P. mirabilis with 62.5 µg/mL TFDG; and (L) P. mirabilis with 250 µg/mL TFDG.

Figure 3

Live/Dead assay of Gram-positive species with various concentrations of TFDG at 6-h incubation. The samples were visualized using Olympus confocal microscope. The green indicates live bacteria, while the red indicates dead bacteria. (A) B. cereus control; (B) B. cereus with 62.5 µg/mL TFDG; (C) B. cereus with 250 µg/mL TFDG; (D) B. subtilis control; (E) B. subtilis with 62.5 µg/mL TFDG; (F) B. subtilis with 250 µg/mL TFDG; (G) S. aureus control; (H) S. aureus with 62.5 µg/mL TFDG; (I) S. aureus with 250 µg/mL TFDG; (J) S. pyogenes control; (K) S. pyogenes with 62.5 µg/mL TFDG; and (L) S. pyogenes with 250 µg/mL TFDG.

Figure 4

Live/Dead assay of M. smegmatis with various concentrations of TFDG at 6-h incubation. The samples were visualized using Olympus confocal microscope. The green indicates live bacteria, while the red indicates dead bacteria. (A) M. smegmatis control; (B) M. smegmatis with 62.5 µg/mL TFDG; and (C) M. smegmatis with 250 µg/mL TFDG.

Figure 5

Live/Dead assay of Bacillus spp. spores with TFDG at 60-min incubation. The green indicates viable spores, while the red indicates non-viable spores. (A) B. cereus spores control; (B) B. cereus spores with 312.5 µg/mL TFDG; (C) B. cereus spores with 625 µg/mL TFDG; (D) B. subtilis spores control; (E) B. subtilis spores with 312.5 µg/mL TFDG; and (F) B. subtilis spores with 625 µg/mL TFDG.

Figure 6

Docking visualization of TFDG with the germination protein using BIOVIA Discovery Studio Visualizer. (A–C) B. cereus GPR with TFDG; (D–F) B. cereus Lgt with TFDG; (G–I) B. subtilis GPR with TFDG; (J–L) B. subtilis Lgt with TFDG. For (C,F,I,L) the bright green line shows conventional hydrogen bond; bright pink shows the pi-pi interaction; light pink shows pi-alkyl interaction; orange shows pi cation, pi carbon, and pi-anion interaction; the red line shows unfavorable bond interaction; light green shows pi-donor hydrogen interaction and carbon-hydrogen interaction; and bright purple shows pi sigma interaction.

Figure 7

Semi-quantitative RT-PCR lgt and gpr gene expression analysis of Bacillus spore when treated with TFDG. (A) B. cereus spores; (B) B. subtilis spores.