Polymyxin B (PMB) is increasingly used as a last-line antibiotic; however, the emergence of PMB resistance is a serious threat to global health. Here, a total of 40 clinical isolates were collected to screen for PMB-resistant strains. Several clinical isolates including NCCP 16007 were far more resistant to PMB (MIC: 128-256 μg/ml) than the ATCC 17978 strain (MIC: 2 μg/ml) and appeared to possess resistance to broad-spectrum antibiotics including meropenem and 12 others. Four highly PMB-resistant strains possessed point mutations in the histidine kinase PmrB, leading to an increased expression of encoding a phosphoethanolamine transferase. Whole-genome analyses revealed that the NCCP 16007 stain had acquired two additional copies of the gene with phage integrase and 13 antibiotic resistance genes (ARGs) from other pathogens, including and . The GC ratios of the ARGs (50-60%) were higher than that of the chromosomal backbone (39.06%), further supporting the horizontal gene transfer of ARGs. Comparative genomics with other multidrug-resistant strains revealed that the NCCP 16007 strain has many additional ARGs and has lost several virulence factors including Csu pili and heme oxygenase but exhibited high pathogenicity in -infection models. The observation of condensed biofilm through confocal and scanning electron microscopy suggested that the NCCP 16007 strain may possess high adhesion capacity during urinary tract infection. Therefore, our genomic and phenotypic analyses suggested that the multidrug-resistant NCCP 16007 strain possesses high genome plasticity, natural transformation ability, and pathogenicity.