Genomic investigation of bacterial pathogens with anti-microbial resistance genes and their lytic bacteriophage

Abstract

As the antibiotic resistance (AMR) of pathogenic bacteria continued to rise, it became one of the most critical concerns in public health, necessitating urgent and comprehensive strategies to unravel the complexity of AMR genes and establish effective countermeasures. The diversity of resistant bacterial pathogens, the varied characteristics of resistance mechanisms, and the rapid spread of resistance genes across multiple bacterial species highlighted the complexity of this issue, making it essential to develop tools and methodologies that could advance our understanding of AMR at the genetic and structural levels. To gain a comprehensive understanding of this crucial task and devise effective control strategies, three approaches were proposed in these studies. In the monitoring studies of antibiotic resistance, a comprehensive antibiotic resistance gene database, named BOARDS (accessible at sbml.unist.ac.kr), was constructed. This database, which encompassed extensive data on 3,943 AMR genes and structural information derived from whole genome sequencing (WGS) data, could serve as a pivotal resource for in-depth AMR analysis. The identification of resistance genes was facilitated by BOARDS, while insights into their structural basis were provided. Concurrently, the Rapid Analysis and Detection tool of Antimicrobial-Resistance (RADAR), a one-stop analysis pipeline for the detection of AMR genes from WGS data, was developed. These integrated analyses led to a better understanding of the AMR landscape and uncovered trends. Additionally, the use of structural information confirmed that insights could be gained into the impact of single nucleotide polymorphisms (SNPs). In the method development phase, the emphasis of this study was on advancing AMR detection capabilities through the creation of a new diagnostic kit utilizing the SYBR Green Real-Time PCR assay. The kit was crafted to target genes that confer resistance to vancomycin, methicillin, erythromycin, and tetracycline. It was designed to cover over 99% of targets selected based on WGS analysis, enabling the distinction between genes with very high sequence homology that are differentiated by SNPs. This provided a rapid, reliable, and efficient method for AMR detection. The diagnostic tool played a crucial role in the timely monitoring and management of AMR spread, ensuring appropriate therapeutic intervention. Lastly, to counteract the issue of antibiotic resistance, a novel lytic bacteriophage targeting Klebsiella pneumoniae, named KPP105, was extensively characterized to understand its properties and potential as an antimicrobial agent. The potential of bacteriophage-derived lytic proteins, endolysins, was explored further. The stability of this bacteriophage across various pH levels and temperatures was demonstrated, with its host specificity and classification within the Demerecviridae family being confirmed. Specifically, in silico mutagenesis screening was conducted for the endolysin derived from KPP105, proposing specific amino acid substitutions to enhance its binding affinity with its ligand, peptidoglycan. In conclusion, this comprehensive study highlighted the significance of a multifaceted approach, including databases, diagnostic tools, and bacteriophage biology, in addressing the complex and urgent issue of antibiotic resistance. The study provides valuable insights and tools for the in-depth AMR analysis of multidrug-resistant bacterial pathogens and potential countermeasures. Key word: Antimicrobial resistance, multifaceted approach, multiple bacterial pathogens, database construction, predicted protein structure, one-stop analysis pipeline development, SYBR Green, Real-Time PCR assay, ready-to-use kit, novel bacteriophage, Klebsiella pneumoniae, lysis-associated protein.