Nano-microbial based technology employing polyvalent phage conjugate: A next generation weapon for antimicrobial resistance lurking behind wastewater

Abstract

Worldwide, due to a dearth of innovative interventions, new forms of antimicrobial resistance (AMR) are being discovered every day in clinical and environmental settings. Therefore, it is necessary to remove these contaminants directly or indirectly from the environment. Nanomicrobial-based technology employing nanomaterials with microbes is a new paradigm that finds a place in the antimicrobial crisis. Microbial entities such as phages can be used to treat antimicrobial resistance, but phage resistance is challenging and limits its applicability. Similarly, nanotechnology will not be able to selectively remove resistant strains from the environment individually. Therefore, we employ nanomicrobial-based technology that aims to fill these gaps. In the present study, polyvalent phages were isolated from wastewater with an easy-to-use modified multi-host sequential approach, characterized and conjugated with magnetite (Fe₃O₄) nanoparticles with the modified formulation to form nanomicrobial conjugates (NMCs). These NMCs were subjected to characterization and in vitro antibacterial studies. The results indicated a significant polyvalency of phages in the order of Caudovirales. Transmission electron microscopy (TEM) analysis of Fe₃O₄ nanoparticles formed by the co-precipitation method showed a particle size of 30 ± 5 nm and the selected area electron diffraction (SAED) pattern indicates a single-phase crystalline structure. To form NMCs, isolated phages (10⁵ PFU/mL) were immobilized onto Fe₃O₄ nanoparticles. Further, surface modification of Fe₃O₄ nanoparticles enables the covalent association of phages. Biosurfactant-functionalized Fe₃O₄ nanoparticles (FNMCs) were found to have higher phage loading capacity, with a significant value of p < 0.0127 and a zeta potential of -22.2 mV. TEM studies and in vitro biofilm assay showed that NMCs exhibit promising antibacterial activity against various resistant bacterial strains. Pilot studies showed that NMCs can selectively eliminate up to 98.3% of AMR in wastewater. Thus, these findings indicate a synergistic effect of both phage and nanomaterial and this technology is expected to be a new lead in wastewater management.

Keywords: Biofilm; Biosurfactant: multi-drug resistance; Confocal; Electron microscopy; Escherichia coli; Klebsiella pneumoniae; Live/dead bacteria; Magnetite (Fe(3)O(4)) nanoparticle; Multi-valent bacteriophage isolation; Phage therapy; Plaque.