Integrated molecular analysis of multidrug-resistant bacterial pathogens from healthcare and environmental settings

ABSTRACT: Antimicrobial resistance (AMR) poses an escalating global health threat, driven by the emergence and dissemination of multidrug-resistant (MDR) bacterial pathogens. This study investigates the molecular and genomic landscape of MDR bacteria isolated from both clinical and environmental sources, highlighting their resistance profiles, genetic determinants, and transmission mechanisms. A total of 60 bacterial isolates—30 from healthcare settings and 30 from environmental reservoirs such as hospital effluents, wastewater, and contaminated soil—were analyzed through a comprehensive pipeline encompassing bacterial culture, antibiotic susceptibility testing (AST), molecular identification via 16S rRNA sequencing, PCR-based detection of resistance genes, and whole-genome sequencing (WGS). Phenotypic testing revealed high resistance rates across multiple antibiotic classes, with notable differences between ecological sources: clinical isolates were predominantly resistant to β-lactams and carbapenems, while environmental strains showed elevated resistance to tetracyclines and sulfonamides. Genotypic analysis uncovered the widespread presence of resistance genes such as blaCTX-M, blaNDM-1, qnrS, sul1, and tetA in both groups, suggesting horizontal gene transfer (HGT) between human and environmental microbiomes. Mobile genetic elements, including plasmids (IncF, IncX3), class 1 integrons, and Tn3-family transposons, were implicated in the mobility and dissemination of these genes. Conjugation assays confirmed plasmid-mediated transfer of resistance traits from environmental isolates to laboratory strains. Phylogenomic comparisons and core genome analyses indicated high genetic relatedness among certain clinical and environmental strains, supporting the hypothesis of cross-ecosystem transmission. The findings affirm the One Health paradigm, emphasizing the ecological interconnectedness of AMR and the need for integrated surveillance encompassing both medical and environmental domains. This study not only broadens our understanding of resistance mechanisms and gene flow but also underscores the critical importance of environmental monitoring and molecular diagnostics in combating the AMR crisis.