Facile synthesis of CuONPs using Citrus limon juice for enhancing antibacterial activity against methicillin-resistant Staphylococcus aureus, beta-lactamase and tetracycline-resistant Escherichia coli

Abstract

Antimicrobial resistance (AMR) resulting from indiscriminate use of antibiotics in various fields of agriculture such as livestock farming, aquaculture, and croup fields become an emerging catatroph for the health (human, animal) and environment. Among those, poultry farming has been considered as one of the major contributors of multidrug-resistant (MDR) bacteria. Focusing this, the present research is designed for green synthesis of copper oxide nanoparticles (CuONPs) with the aim of their application in antibiotic-free poultry farming for curving use of antibiotics in that sector. For that, antibacterial CuONPs were nanoformulated to decrease the required doses of bulk CuSO₄. We used a CuSO₄·5H₂O solution as a Cu²⁺ source and Citrus limon juice as a reducing agent as well as capping agent. Particle yield was initially confirmed by the λ_max specific to CuONPs (295 nm) using UV-Vis spectroscopy. The presence of the Cu–O group during particle formation and crystallinity with the purity of yielded NPs was confirmed with Fourier-transform infrared spectroscopy and X-ray diffractometry. The round to spherical CuONPs of 92–155 nm average size was confirmed with atomic force, scanning electron, and transmission electron microscopy. The concentration of yielded NPs was calculated with the dynamic light scattering. The physical characterization tools indicated a maximum CuONPs yield with a 0.001 M ion source with 15% reducing agents after 12 h reduction. Antibacterial effectivity was tested against methicillin-resistant Staphylococcus aureus and tetracycline- and beta-lactamase-resistant Escherichia coli, confirmed by PCR amplicon band at 163 bp, 643 bp, and 577 bp for the mecA, blaTEM-1 and tetA genes, respectively. An antibiogram assay of CuONPs showed a maximum zone of inhibition of 26 ± 0.5 mm for the synthesized particles. The minimum inhibitory and bactericidal concentrations were 1.6 μg ml⁻¹ and 3.1 μg ml⁻¹, respectively, for broad-spectrum application. Finally, the biocompatibility of CuONPs was determined by demonstrating a nonsignificant decrease of BHK-21 cell viability at <2 MIC doses for complying their future in vivo applicability.