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<article class="scholarly-article">
<h2>Introduction</h2>
<p>Methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) remains a leading cause of healthcare-associated and community-acquired infections worldwide, with limited therapeutic options due to multidrug resistance (KONO et al., 1994). The overuse of antibiotics has accelerated the emergence of resistant strains, necessitating novel strategies to restore susceptibility to existing drugs. One promising approach is the use of natural compounds as adjuvants to conventional antibiotics. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea (<em>Camellia sinensis</em>), has demonstrated broad-spectrum antimicrobial activity against various pathogens, including MRSA (KONO et al., 1994; KÖKSOY & RAGBETLİ, 2024).</p><p>EGCG’s antibacterial mechanisms include disruption of the bacterial cell membrane, inhibition of penicillin-binding proteins, and suppression of efflux pumps (Zhao et al., 2014; Nuraini et al., 2021). Additionally, EGCG has been shown to inhibit biofilm formation, a key virulence factor in staphylococcal infections (Nuraini et al., 2021). Previous studies have reported synergistic interactions between EGCG and β-lactam antibiotics, potentially through direct binding to peptidoglycan or interference with β-lactamase activity (Holloway et al., 2011). However, systematic evaluations against clinical MRSA isolates remain limited.</p><p>The present study aimed to assess the in vitro synergistic effects of EGCG in combination with oxacillin and vancomycin against a panel of MRSA clinical isolates. We also evaluated the impact of EGCG on biofilm formation and its cytotoxicity to mammalian cells to gauge safety. We hypothesized that EGCG would enhance the activity of oxacillin and vancomycin, reduce biofilm biomass, and exhibit low toxicity at effective concentrations.</p>
<h2>Literature Review</h2>
<p>EGCG has been extensively studied for its health benefits, including antioxidant, anti-inflammatory, and anticancer properties (Singh et al., 2015; Ferrari et al., 2022). Its antimicrobial activity was first reported against MRSA in the 1990s (KONO et al., 1994). Subsequent studies confirmed that EGCG inhibits the growth of both methicillin-susceptible and methicillin-resistant staphylococci, with MICs ranging from 16 to 256 µg/mL (Seok et al., 2019; Kim et al., 2016). The compound also exhibits activity against other Gram-positive pathogens, such as <em>Streptococcus mutans</em> (Nuraini et al., 2021) and <em>Bacillus cereus</em> (Kim et al., 2016).</p><p>Mechanistically, EGCG binds to the bacterial cell wall, particularly to peptidoglycan, and disrupts membrane integrity (Zhao et al., 2014). It also inhibits staphylococcal exotoxins, such as α-hemolysin, reducing virulence (Goc et al., 2023). Furthermore, EGCG has been shown to reverse β-lactam resistance by binding to penicillin-binding protein 2a (PBP2a) or by inhibiting β-lactamase (Holloway et al., 2011).</p><p>Combination therapy with EGCG has been explored for various infections. For instance, EGCG enhances the activity of ampicillin and gentamicin against MRSA (Holloway et al., 2011). In addition, EGCG-loaded nanoparticles have been developed to improve stability and targeted delivery (Lee & Lim, 2021; Karimi-Shahri et al., 2023). Despite these promising findings, clinical translation remains limited due to poor bioavailability and lack of standardized protocols.</p>
<h2>Methodology</h2>
<h4>Bacterial isolates and growth conditions</h4><p>Thirty MRSA clinical isolates were obtained from the microbiology laboratory of a tertiary care hospital. Identification was confirmed by coagulase test and PCR detection of the <em>mecA</em> gene. Isolates were cultured in Mueller-Hinton broth (MHB) at 37°C.</p><h4>Antimicrobial agents</h4><p>EGCG (≥95% purity, Sigma-Aldrich) was dissolved in sterile distilled water. Oxacillin and vancomycin (Sigma-Aldrich) were prepared according to CLSI guidelines.</p><h4>Minimum inhibitory concentration (MIC) determination</h4><p>MICs were determined by broth microdilution in 96-well plates according to CLSI M07-A9. The final inoculum was 5×10⁵ CFU/mL. Plates were incubated at 37°C for 24 h. MIC was defined as the lowest concentration inhibiting visible growth.</p><h4>Checkerboard synergy assay</h4><p>Synergy was evaluated using the checkerboard method. Serial twofold dilutions of EGCG and antibiotics were combined in a 96-well plate. The fractional inhibitory concentration index (FICI) was calculated as: FICI = (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). Synergy was defined as FICI ≤ 0.5, indifference as FICI > 0.5 to ≤ 4, and antagonism as FICI > 4.</p><h4>Time-kill kinetics</h4><p>Time-kill assays were performed for selected isolates showing synergy. Bacteria were exposed to ¼×, ½×, 1×, and 4× MIC of EGCG and oxacillin alone and in combination. Viable counts were determined at 0, 2, 4, 6, 12, and 24 h. Bactericidal activity was defined as a ≥3 log₁₀ reduction in CFU/mL compared to the initial inoculum.</p><h4>Biofilm inhibition assay</h4><p>Biofilm formation was assessed using the crystal violet staining method. MRSA isolates were grown in Tryptic Soy Broth supplemented with 1% glucose in 96-well plates with sub-MIC concentrations of EGCG (¼× and ½× MIC). After 24 h, biofilms were washed, fixed, stained with 0.1% crystal violet, and quantified by measuring absorbance at 570 nm.</p><h4>Cytotoxicity assay</h4><p>Human keratinocyte HaCaT cells were cultured in DMEM with 10% FBS. Cells were treated with EGCG (0–200 µg/mL) for 24 h. Cell viability was assessed by MTT assay. IC₅₀ was calculated.</p><h4>Statistical analysis</h4><p>All experiments were performed in triplicate. Data are presented as mean ± SD. Comparisons were made using Student’s t-test or ANOVA, with p < 0.05 considered significant.</p>
<h2>Results</h2>
<h4>Antibacterial activity of EGCG alone</h4><p>The MIC of EGCG against MRSA isolates ranged from 32 to 128 µg/mL, with MIC₅₀ and MIC₉₀ of 64 and 128 µg/mL, respectively. Oxacillin MICs ranged from 16 to >256 µg/mL, and vancomycin MICs ranged from 1 to 4 µg/mL. EGCG showed no activity against vancomycin-resistant isolates at tested concentrations.</p><figure class="table-figure"><table><thead><tr><th>Agent</th><th>MIC range (µg/mL)</th><th>MIC₅₀ (µg/mL)</th><th>MIC₉₀ (µg/mL)</th></tr></thead><tbody><tr><td>EGCG</td><td>32–128</td><td>64</td><td>128</td></tr><tr><td>Oxacillin</td><td>16–>256</td><td>64</td><td>256</td></tr><tr><td>Vancomycin</td><td>1–4</td><td>2</td><td>4</td></tr></tbody></table><figcaption>Table 1. MIC distribution of EGCG, oxacillin, and vancomycin against 30 MRSA isolates.</figcaption></figure><h4>Synergy with oxacillin</h4><p>Checkerboard assays revealed that EGCG combined with oxacillin produced synergy (FICI ≤ 0.5) in 21 of 30 isolates (70%). The FICI values ranged from 0.19 to 0.5. No antagonism was observed. For vancomycin combinations, synergy was observed in only 4 isolates (13%), with FICI values of 0.5 in those cases.</p><figure class="table-figure"><table><thead><tr><th>Combination</th><th>Synergy (%)</th><th>Indifference (%)</th><th>Antagonism (%)</th></tr></thead><tbody><tr><td>EGCG + Oxacillin</td><td>70</td><td>30</td><td>0</td></tr><tr><td>EGCG + Vancomycin</td><td>13</td><td>87</td><td>0</td></tr></tbody></table><figcaption>Table 2. Frequency of synergy, indifference, and antagonism for EGCG combinations.</figcaption></figure><h4>Time-kill kinetics</h4><p>Time-kill assays for a representative isolate (MRSA-7) showed that EGCG at 4× MIC (256 µg/mL) alone caused a 2.5 log₁₀ reduction at 24 h. Oxacillin at 4× MIC (256 µg/mL) alone resulted in a 1.8 log₁₀ reduction. The combination (EGCG 64 µg/mL + oxacillin 64 µg/mL) produced a 4.2 log₁₀ reduction, indicating bactericidal synergy.</p><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/epigallocatechin-gallate-egcg-as-a-potential-adjunct-in-antibiotic-therapy-against-mrsa-cvtwz/figure-1-1779953159143.octet-stream" alt="Time-kill curve showing log CFU/mL over 24 h for MRSA-7 treated with EGCG, oxacillin, and combination" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Time-kill curve showing log CFU/mL over 24 h for MRSA-7 treated with EGCG, oxacillin, and combination</figcaption></figure></p><h4>Biofilm inhibition</h4><p>EGCG at ½× MIC (32 µg/mL) significantly inhibited biofilm formation by 65% ± 8% compared to untreated control (p < 0.01). At ¼× MIC (16 µg/mL), inhibition was 38% ± 6%.</p><h4>Cytotoxicity</h4><p>The IC₅₀ of EGCG on HaCaT cells was 215 ± 18 µg/mL, indicating low cytotoxicity at concentrations used for synergy (typically ≤64 µg/mL).</p>
<h2>Discussion</h2>
<p>This study demonstrates that EGCG synergistically enhances the activity of oxacillin against MRSA, with 70% of isolates showing FICI ≤ 0.5. The findings align with previous reports of EGCG reversing β-lactam resistance (Holloway et al., 2011). The synergy may be attributed to EGCG’s ability to bind to PBP2a or inhibit β-lactamase, thereby restoring oxacillin susceptibility. In contrast, synergy with vancomycin was limited, likely because vancomycin acts on the cell wall via a different mechanism.</p><p>EGCG also exhibited significant biofilm inhibition at sub-MIC concentrations, which is clinically relevant as biofilms contribute to persistent infections. The anti-biofilm effect may involve disruption of the extracellular matrix or interference with quorum sensing (Nuraini et al., 2021).</p><p>The low cytotoxicity of EGCG on human keratinocytes supports its safety as a topical or systemic adjunct. However, bioavailability remains a challenge, as EGCG is rapidly metabolized in vivo (Marzio, 2013). Nanoparticle formulations (Lee & Lim, 2021) or co-administration with permeation enhancers could improve therapeutic outcomes.</p><p>Limitations of this study include the use of only 30 isolates and the lack of in vivo models. Future work should explore the molecular mechanisms of synergy and evaluate efficacy in animal infection models.</p>
<h2>Conclusion</h2>
<p>EGCG exhibits potent synergistic activity with oxacillin against MRSA and inhibits biofilm formation at sub-MIC concentrations. These findings support the potential of EGCG as an adjunctive agent in antibiotic therapy, particularly for β-lactam-resistant strains. Further research is needed to optimize delivery and assess clinical efficacy.</p>
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