Epicatechin gallate disrupts methicillin resistant Staphylococcus aureus cell envelope integrity and enhances antibiotic activity

Abstract

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for alternative therapeutic strategies. Natural product-derived resistance-modifying compounds are gaining attention as antibiotic adjuvants to restore drug activity. Epicatechin gallate (EPCG) has antibacterial and anti-virulence properties. This study investigates the antibacterial activity of EPCG and its potential to enhance the efficacy of selected antibiotics against S. aureus and MRSA. Standard broth microdilution assays were used to determine the minimum inhibitory concentration (MIC) of EPCG against S. aureus and MRSA. Synergistic interactions with selected antibiotics were evaluated using checkerboard and isobologram assays. Furthermore, cytotoxicity was evaluated using Artemia franciscana nauplii (brine shrimp) lethality and human dermal fibroblast (HDF) assays. The impact of EPCG, cefotaxime, and their combinations on cells and biofilms was visualised through confocal and cryo-electron microscopy. EPCG demonstrated strong antibacterial activity (MIC = 208 µg/mL against S. aureus and 188 µg/mL against MRSA). Isobologram assays also revealed strong synergy for the EPCG–cefotaxime combination (∑ fractional inhibitory concentration = 0.15 for MRSA; 0.25 for S. aureus). EPCG exhibited moderate toxicity toward HDF cells (LC₅₀ = 100 µg/mL), whereas the optimised cefotaxime: EPCG (90:10 [v/v]) combination was non-toxic across all assays. Additionally, confocal microscopy revealed reduced bacterial aggregation and diminished biofilm-associated fluorescence following EPCG treatment. Cryo-electron microscopy revealed pronounced ultrastructural disruptions in MRSA cells treated with the combination. In conclusion, EPCG enhances the antibacterial activity of antibiotics such as cefotaxime against S. aureus and MRSA. Its synergistic action, lower cytotoxicity at optimal ratios, and confirmed damage to bacterial cells highlight EPCG’s potential as a natural antibiotic adjuvant.

Keywords

Methicillin-resistant Staphylococcus aureus / Epicatechin gallate / Minimum inhibitory concentration / Synergy / Confocal microscopy / Cryo-electron microscopy

References

[1] World Health Organization. Antimicrobial Resistance. World Health Organization ; 2024. Accessed April 9, 2026. https://www.who.int/europe/news-room/fact-sheets/item/antimicrobial-resistance

[2] World Health Organization. WHO Bacterial Priority Pathogens List, 2024: Bacterial Pathogens of Public Health Importance to Guide Research, Development and Strategies to Prevent and Control Antimicrobial Resistance. World Health Organization ; 2024. Accessed April 9, 2026. https://www.who.int/publications/i/item/9789240093461

[3] Centers for Disease Control and Prevention. Methicillin-Resistant Staphylococcus aureus (MRSA) Basics. Centers for Disease Control and Prevention; 2025. Accessed April 9, 2026. https://www.cdc.gov/mrsa/about/index.html

[4] Ventola CL . The antibiotic resistance crisis, part 1: Causes and threats. Pharm Ther. 2015; 40(4): 277-283.

[5] Uddin TM, Chakraborty AJ, Khusro A, et al. Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. J Infect Public Health. 2021; 14(12): 1750-1766. doi: 10.1016/j.jiph.2021.10.020

[6] Shlaes DM, Bradford PA . Antibiotics-From there to where?: How the antibiotic miracle is threatened by resistance and a broken market and what we can do about it. Pathog Immun. 2018; 3(1): 19-43. doi: 10.20411/pai.v3i1.231

[7] Hutchings MI, Truman AW, Wilkinson B . Antibiotics: Past, present and future. Curr Opin Microbiol. 2019; 51: 72-80. doi: 10.1016/j.mib.2019.10.008

[8] McCarthy H, Rudkin JK, Black NS, et al. Methicillin resistance and the biofilm phenotype in Staphylococcus aureus. Front Cell Infect Microbiol. 2015; 5: 1. doi: 10.3389/fcimb.2015.00001

[9] Dilworth TJ, Ibrahim O, Hall P, et al. β-Lactams enhance vancomycin activity against methicillin-resistant Staphylococcus aureus bacteremia compared to vancomycin alone. Antimicrob Agents Chemother. 2014; 58(1): 102-109. doi: 10.1128/AAC.01204-13

[10] Worthington RJ, Melander C . Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 2013; 31(3): 177-184. doi: 10.1016/j.tibtech.2012.12.006

[11] Davis JS, Sud A, O’Sullivan MVN, et al. Combination of vancomycin and β-lactam therapy for methicillin-resistant Staphylococcus aureus bacteremia: A pilot multicenter randomized controlled trial. Clin Infect Dis. 2016; 62(2): 173-180. doi: 10.1093/cid/civ808

[12] Basavegowda N, Baek KH . Combination strategies of different antimicrobials: An efficient and alternative tool for pathogen inactivation. Biomedicines. 2022; 10(9): 2219. doi: 10.3390/biomedicines10092219

[13] Tong SYC, Lye DC, Yahav D, et al. Effect of vancomycin or daptomycin with vs without an antistaphylococcal β-lactam on mortality, bacteremia, relapse, or treatment failure in patients with MRSA bacteremia: A randomized clinical trial. JAMA. 2020; 323(6): 527-537. doi: 10.1001/jama.2020.0103

[14] Stapleton PD, Shah S, Ehlert K, et al. The β-lactam-resistance modifier (-)-epicatechin gallate alters the architecture of the cell wall of Staphylococcus aureus. Microbiology. 2007; 153(7): 2093-2103. doi: 10.1099/mic.0.2007/007807-0

[15] Bernal P, Lemaire S, Pinho MG, et al. Insertion of epicatechin gallate into the cytoplasmic membrane of methicillin-resistant Staphylococcus aureus disrupts penicillin-binding protein (PBP) 2a-mediated β-lactam resistance by delocalizing PBP2. J Biol Chem. 2010; 285(31): 24055-24065. doi: 10.1074/jbc.M110.114793

[16] Zhao Y, Qu Y, Tang J, et al. Tea catechin inhibits biofilm formation of methicillin-resistant S. aureus. J Food Qual. 2021; 2021: 1-7. doi: 10.1155/2021/8873091

[17] Teng F, Wang L, Wen J, et al. Epicatechin gallate and its analogues interact with sortase A and β-lactamase to suppress Staphylococcus aureus virulence. Front Cell Infect Microbiol. 2025; 15: 1537564. doi: 10.3389/fcimb.2025.1537564

[18] Carmine AA, Brogden RN, Heel RC, et al. Cefotaxime: A review of its antibacterial activity, pharmacological properties and therapeutic use. Drugs. 1983; 25(3): 223-289. doi: 10.2165/00003495-198325030-00001

[19] Hübsch Z, Van Zyl RL, Cock IE, et al. Interactive antimicrobial and toxicity profiles of conventional antimicrobials with Southern African medicinal plants. South African Journal of Botany. 2014; 93: 185-197. doi: 10.1016/j.sajb.2014.04.005

[20] Eloff JN . A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica. 1998; 64(8): 711-713. doi: 10.1055/s-2006-957563

[21] Ang ST, Kim TH, Cheesman MJ, et al. Antibacterial and synergistic effects of Terminalia citrina leaf extracts against gastrointestinal pathogens: Insights from metabolomic analysis. Antibiotics. 2025; 14(6). doi: 10.3390/antibiotics14060593

[22] Mastronarde DN . SerialEM: a program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position. Microsc Microanal. 2003; 9(Suppl 2): 1182-1183. doi: 10.1017/S1431927603445911

[23] Mastronarde DN . Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol. 2005; 152(1): 36-51. doi: 10.1016/j.jsb.2005.07.007

[24] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100. 30th ed. Clinical and Laboratory Standards Institute ; 2020. Accessed June 30, 2025. https://www.nih.org.pk/wp-content/uploads/2021/02/CLSI-2020.pdf

[25] Bucevičius J, Lukinavičius G, Gerasimaitė R . The use of Hoechst dyes for DNA staining and beyond. Chemosensors. 2018; 6(2). doi: 10.3390/chemosensors6020018

[26] Hermanson GT . Fluorescent Probes. In: Bioconjugate Techniques. Elsevier; 2013: 395-463. doi: 10.1016/b978-0-12-382239-0.00010-8

[27] Lichtman JW, Conchello J-A. Fluorescence microscopy. Nat Methods. 2005; 2(12): 910-919. doi: 10.1038/nmeth817

[28] Shaner NC, Steinbach PA, Tsien RY . A guide to choosing fluorescent proteins. Nat Methods. 2005; 2(12): 905-909. doi: 10.1038/nmeth819

[29] Gao T, Wang Y, Zhu J, et al. Antibacterial activity of a plant natural polyphenol against zoonotic Streptococcus suis. Microb Pathog. 2025; 205: 107655. doi: 10.1016/j.micpath.2025.107655

[30] Dhanda G, Acharya Y, Haldar J . Antibiotic adjuvants: A versatile approach to combat antibiotic resistance. ACS Omega. 2023; 8(12): 10757-10783. doi: 10.1021/acsomega.3c00312

[31] Caturla N, Vera-Samper E, Villalain J, et al. The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes. Free Radic Biol Med. 2003; 34(6): 648-662. doi: 10.1016/s0891-5849(02)01366-7

[32] Yoda Y, Hu ZQ, Zhao WH, et al. Different susceptibilities of Staphylococcus and Gram-negative rods to epigallocatechin gallate. J Infect Chemother. 2004; 10(1): 55-58. doi: 10.1007/s10156-003-0284-0

[33] Kitichalermkiat A, Katsuki M, Sato J, et al. Effect of epigallocatechin gallate on gene expression of Staphylococcus aureus. J Glob Antimicrob Resist. 2020; 22: 854-859. doi: 10.1016/j.jgar.2020.06.006

[34] Zhao WH, Hu ZQ, Okubo S, et al. Mechanism of synergy between epigallocatechin gallate and β-lactams against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2001; 45(6): 1737-1742. doi: 10.1128/AAC.45.6.1737-1742.2001

[35] Shah S, Stapleton P, Taylor PW . The polyphenol (−)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus. Lett Appl Microbiol. 2008; 46(2): 181-185. doi: 10.1111/j.1472-765X.2007.02296.x

[36] Friedman M, Henika PR, Levin CE, et al. Antimicrobial activities of tea catechins and theaflavins and tea extracts against Bacillus cereus. J Food Prot. 2006; 69(2): 354-361. doi: 10.4315/0362-028x-69.2.354

[37] Cui Y, Oh YJ, Lim J, et al. AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria. Food Microbiol. 2012; 29(1): 80-87. doi: 10.1016/j.fm.2011.08.019

[38] Stapleton PD, Taylor PW . Methicillin resistance in Staphylococcus aureus: mechanisms and modulation. Sci Prog. 2002; 85(1): 57-72. doi: 10.3184/003685002783238870

[39] Dmitriev BA, Toukach FV, Holst O, et al. Tertiary structure of Staphylococcus aureus cell wall murein. J Bacteriol. 2004; 186(21): 7141-7148. doi: 10.1128/JB.186.21.7141-7148.2004

[40] Rodriguez-Lazaro D, Alonso-Calleja C, Oniciuc EA, et al. Characterization of biofilms formed by foodborne methicillin-resistant Staphylococcus aureus. Front Microbiol. 2018; 9: 3004. doi: 10.3389/fmicb.2018.03004

[41] Romaniuk JA, Cegelski L . Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR. Philos Trans R Soc Lond B Biol Sci. 2015; 370(1679): 20150024. doi: 10.1098/rstb.2015.0024

[42] Brand C, Newton-Foot M, Grobbelaar M, et al. Antibiotic-induced stress responses in Gram-negative bacteria and their role in antibiotic resistance. J Antimicrob Chemother. 2025; 80(5): 1165-1184. doi: 10.1093/jac/dkaf068

[43] Sethuvel DPM, Bakthavatchalam YD, Karthik M, et al. β-Lactam resistance in ESKAPE pathogens mediated through modifications in penicillin-binding proteins: an overview. Infect Dis Ther. 2023; 12(3): 829-841. doi: 10.1007/s40121-023-00771-8