Green Tea Catechin Epigallocatechin Gallate Inhibits Vegetative Cell Outgrowth and Expression of Beta-Lactamase Genes in Penicillin-Resistant Bacillus anthracis Strain PCr

Abstract

The green tea catechin epigallocatechin gallate (EGCg) has antimicrobial effects on many bacteria. In this study, we investigated the inhibitory effects of EGCg on Bacillus anthracis spores and vegetative cells. The B. anthracis spores were insensitive to EGCg, but the growth of vegetative cells derived from germinated spores was inhibited by EGCg. Moreover, EGCg decreased the minimum inhibitory concentration of penicillin and meropenem for penicillin-resistant B. anthracis. In the penicillin-resistant B. anthracis strain, the transcription levels of the beta-lactamase genes (bla1 and bla2) decreased significantly following the treatment with 50 µg/mL EGCg. These results suggest that the appropriate application of EGCg may effectively control the penicillin-resistant B. anthracis growth and beta-lactamase production.

Keywords: Bacillus anthracis; beta-lactamase; green tea catechin; penicillin.

Figure 1

Schematic overview of experimental design for investigating EGCg effects on B. anthracis of MIC determination, spore germination studies, time-kill assays, and beta-lactamase activity and gene expression analysis.

Figure 2

(a) Growth curve from spores of penicillin-resistant Bacillus anthracis strain PCr treated without EGCg (no-treatment control) or with 100 μg/mL EGCg. The optical density at 600 nm (OD₆₀₀) was calculated (relative to the initial OD₆₀₀). Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001. The graph inset presents the decrease in OD₆₀₀ due to spore germination. (b) Growth curve of vegetative cells of penicillin-resistant B. anthracis strain PCr treated with EGCg (25, 50, and 100 μg/mL). Colony-forming unit (CFU)/mL was calculated after 3, 6, 9, 12, and 27 h incubations. Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001. (c) Growth curve of vegetative cells of wild-type B. anthracis strain BA103 treated with EGCg (25, 50, and 100 μg/mL). Colony-forming unit (CFU)/mL was calculated after 3, 6, 9, 12, and 27 h incubations. Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001.

Figure 2

(a) Growth curve from spores of penicillin-resistant Bacillus anthracis strain PCr treated without EGCg (no-treatment control) or with 100 μg/mL EGCg. The optical density at 600 nm (OD₆₀₀) was calculated (relative to the initial OD₆₀₀). Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001. The graph inset presents the decrease in OD₆₀₀ due to spore germination. (b) Growth curve of vegetative cells of penicillin-resistant B. anthracis strain PCr treated with EGCg (25, 50, and 100 μg/mL). Colony-forming unit (CFU)/mL was calculated after 3, 6, 9, 12, and 27 h incubations. Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001. (c) Growth curve of vegetative cells of wild-type B. anthracis strain BA103 treated with EGCg (25, 50, and 100 μg/mL). Colony-forming unit (CFU)/mL was calculated after 3, 6, 9, 12, and 27 h incubations. Significant differences from the no-treatment control are indicated by asterisks: * p < 0.05 and **** p < 0.0001.

Figure 3

Production of β-lactamase in penicillin-resistant and wild-type Bacillus anthracis. Overnight cultures of penicillin-resistant B. anthracis (PCr) (a) and wild-type B. anthracis (BA103) (b) in MH broth supplemented with EGCg (0, 10, 50, 100, and 500 μg/mL) were added to Cefinase discs. The production of β-lactamase was indicated by a change in disc color (yellow to red). The intensity of the color change reflected the extent of β-lactamase production.

Figure 4

Analysis of β-lactamase gene (bla1 and bla2) expression in Bacillus anthracis PCr grown in MH broth supplemented with EGCg (25 and 50 µg/mL) and in B. anthracis BA103 with EGCg 50 µg/mL. The bla1 (a) and bla2 (b) mRNA levels after 1.5, 3, and 6 h incubations were determined by qRT-PCR and normalized against gapA mRNA levels. The mean and standard deviation were calculated from two independent experiments. Significant differences between EGCg treatments and the no-treatment control are indicated.