Cytotoxicity and antibacterial BioEffects of CuO-Zn nanoparticles synthesized from Lactobacillus helveticus yogurt isolates targeting RsbA gene expression

Proteus mirabilis is an opportunistic urinary tract pathogen distinguished by its swarming motility and dimorphic behavior. This study examined the antibacterial potential of native Bulgarian dairy Lactobacillus strains against clinically significant P. mirabilis isolates. By developing and characterizing copper oxide (CuONPs) and zinc oxide (ZnONPs) nanoparticles as antimicrobial agents targeting P. mirabilis, this work contributes to addressing the global challenge of antimicrobial resistance, aligning with the United Nations Sustainable Development Goals (UNSDGs) that aim to reduce infectious disease mortality through innovative therapeutic approaches. Two multidrug-resistant P. mirabilis isolates (M1 and M2) displaying swarming motility were selected for analysis. The antimicrobial activity of Lactobacillus helveticus, a heterofermentative lactic acid bacterium commonly found in dairy products, was evaluated. This strain produces helveticin, a bacteriocin-like inhibitory substance (BLIS) with notable bactericidal activity. CuONPs and ZnONPs were biosynthesized using L. helveticus isolated from yogurt, with strain identity confirmed via 16 S rRNA gene sequencing. Nanoparticles were subsequently extracted and partially purified through 70% ammonium sulfate precipitation. Characterization revealed surface plasmon resonance peaks at 250 nm (CuONPs) and 300 nm (ZnONPs). AFM analysis indicated particle sizes ranging from 15 to 26 nm, while TEM imaging showed predominantly spherical to irregular morphologies between 9 and 20 nm. Cytotoxicity evaluation using the MTT assay demonstrated dose-dependent reductions in the viability of WRL68 human hepatic cells, with up to 60% inhibition at higher concentrations. Notably, both nanoparticle types effectively inhibited P. mirabilis swarming motility, and qPCR analysis confirmed significant downregulation of the rsbA gene, a crucial regulator of this behavior. Overall, green-synthesized CuONPs and ZnONPs derived from L. helveticus displayed potent anti-swarming and antivirulence effects against P. mirabilis. Their ability to suppress virulence-associated gene expression underscores their promise as eco-friendly antimicrobial agents for managing opportunistic pathogens and mitigating multidrug resistance.