Green mycosynthesis of a CuO/ZnO heterojunction nanocomposite using Aspergillus terreus and its antibacterial and anti-virulence activity against multidrug-resistant Escherichia coli

Abstract

The rapid spread of multidrug-resistant (MDR) Escherichia coli poses a severe global health risk and demands the development of new antibacterial strategies. Biologically manufactured metal oxide nanocomposites have gained popularity due to their superior antibacterial activity and environmentally sustainable synthesis. This study uniquely integrates fungal metabolite profiling, green synthesis of a CuO/ZnO heterojunction nanocomposite, and molecular evaluation of anti-virulence activity against clinically isolated MDR Escherichia coli. The CuO/ZnO system was selected due to its synergistic heterostructure, which enhances reactive oxygen species generation and improves antibacterial performance compared to single metal oxides. In this investigation, an MDR E. coli strain was isolated from wound infections and identified using 16 S rRNA sequencing. A soil-derived fungus capable of forming CuO/ZnO nanocomposites was isolated and identified as Aspergillus terreus using 18 S rRNA sequencing. The bioactive constituents in the fungal filtrate were identified using High performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GC-MS). UV-Vis spectroscopy, transmission electron microscope (TEM), Fourier transform infrared spectroscopy(FTIR), and X-ray diffraction (XRD) were used to characterize the biosynthesized CuO/ZnO nanocomposite. The antibacterial effect against MDR E. coli was examined using disc diffusion and minimum inhibitory concentration (MIC) assays, and the influence on virulence and quorum-sensing genes was measured using quantitative reverse transcription PCR (qRT-PCR). The MDR E. coli isolate was resistant to all antibiotics tested. A variety of phenolic acids, flavonoids, and aromatic compounds were detected in the fungal filtrate; however, the filtrate alone did not exhibit antibacterial activity. The CuO/ZnO nanocomposite showed strong antibacterial activity, with an inhibition zone diameter of 2.3 ± 0.4 mm and MIC of 62.5 ± 0.2 µg/mL. Gene expression analysis indicated considerable downregulation of critical virulence genes, including fimH, luxS, toxA, and papC, suggesting impairment of adhesion, toxin synthesis, and quorum sensing. The biosynthesized CuO/ZnO nanocomposite shows strong antibacterial and anti-virulence activity against MDR E. coli, indicating its potential as an alternative antibacterial agent.