Study protocol on antimicrobial resistance burden, transmission dynamics, and therapeutic bacteriophages in livestock and exposed farming populations in Nagpur, India: An integrated One Health approach

The rise in antimicrobial resistance (AMR) is a severe public health threat worldwide. India bears a disproportionately heavy burden of this problem due to ample antimicrobial usage in both humans and animals and scarce integrated surveillance. Since humans, animals, and environmental reservoirs which can harbour resistant microorganisms interact very closely on farms livestock, these are considered critical hotspots for the emergence and dissemination of antimicrobial-resistant bacteria and resistance genes. This work presents a 36-month prospective longitudinal observational study protocol aimed at quantifying the burden and characterizing the transmission dynamics of a selected set of key bacteria, that are clinically significant and hence, pathogenic— Escherichia coli , Staphylococcus aureus , Klebsiella pneumoniae , Streptococcus pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa —alongside their AMRprofiles in livestock, farm-exposed human populations, and environmental reservoirs in Nagpur, India, within the framework of One Health. Seasonal sampling of milk, animal faeces, human stool, soil, wastewater, drinking water, and animal feed will be carried out on dairy farms located in urban, peri-urban, and rural areas. Pathogens will be isolated using standard microbiological techniques and characterized based on antimicrobial susceptibility by employing VITEK®2 and disc diffusion methods. At the same time, bacteriophages against multidrug-resistant isolates will be isolated, purified, and characterized through plaque assays, host-range analysis, electron microscopy, and whole-genome sequencing for their therapeutic potential evaluation. Additionally, metagenomic next-generation sequencing will be utilized on a select number of samples to comprehensively characterize the resistomes and diversity of phages. The research will provide detailed longitudinal data on the frequency and spread of AMR among human, animal, and environmental compartments, create a biobank of AMR isolates and lytic bacteriophages, and offer genomic clues to delineate phage-based treatments and well-informed mitigation strategies of AMR within the framework of One Health in India. The results will be made public through peer-reviewed articles, presentations at scientific meetings, and deposition of sequence data in open-access databases.