Exploring the Inhibitory Effects of Plant Phytoalexins on Escherichia coli: A Genetic Study of Bacterial Resistance Interactions and Gene Expression Associated with Defense Response

Abstract

Background: The emergence of multidrug-resistant Escherichia coli (MDR) is a major public health threat, necessitating the search for novel antibiotic alternatives capable of inhibiting bacterial growth and biofilm formation, as well as modifying resistance mechanisms. The growing interest in plant phytoalexins stems from their diverse spectrum of biological activities and antimicrobial properties. Aim: This study aimed to evaluate the anti-biofilm and antibacterial activity of resveratrol and naringenin against MDR E. coli isolates, their effect on the gene expression levels of marA and acrB, and the relationship between gene expression, biofilm formation, and antibiotic resistance. Materials and Methods: Twenty clinical MDR E. coli isolates were identified using conventional microbiological and biochemical methods, with molecular confirmation where required. Antibacterial activity was assessed using a broth microdilution assay and growth inhibition analysis, while biofilm formation was measured using a crystal violet microplate method. Relative gene expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) according to the 2ΔΔCt method, with recA used as a reference gene. Statistical analyses were performed using one-way analysis of variance (ANOVA), the chi- square test, and Pearson's correlation test, with p < 0.05 considered statistically significant. Results: Over 90% of the strains exhibited resistance to ampicillin and cefotaxime, while resistance rates to ciprofloxacin and tetracycline exceeded 70%. Resveratrol exhibited greater antibacterial activity than naringin, with a significant reduction in bacterial growth (OD₆₀₀ = 0.48 ± 0.04 vs. 0.61 ± 0.03) and more potent, concentration-dependent inhibition of biofilm formation (68% vs. 54%). Gene expression analysis: All compounds affected the gene expression of two resistance-associated genes, marA and acrB. A general correlation (χ² = 24.7, p = 0.002) was found between resistance genes and apparent resistance. Correlation analysis also indicated a positive relationship between gene expression and biofilm formation (r = 0.78, p < 0.001) and minimum inhibitory concentration (MIC) values ​​(r = 0.41, p < 0.05). Results: qPCR results showed significant differences in buvirdin biosynthesis genes between non-biofilm-forming and biofilm-forming samples. The expression of two genes was significantly higher in all six bioflux strains compared to non-biofilm-forming samples (p ≤ 0.0388). Conclusion: The results show that both resveratrol and naringenin possess antibacterial and anti-biofilm activity against multidrug-resistant E. coli (MDR-EC), with resveratrol exhibiting superior overall activity. The modulation of resistance-associated gene expression and the emerging correlations between gene expression, biofilm formation, and antibiotic resistance suggest that certain plant phytoalexins, particularly resveratrol, may be promising candidates for treating MDR-EC infections.