Insights into novel diagnostic assay development, antimicrobial resistance, and pathogenicity in <i>Proteus mirabilis</i> through pan-genome analysis

<i>Proteus mirabilis</i>, a significant pathogen associated with human urinary tract infections (UTIs), demonstrates escalating multidrug resistance (MDR) that complicates clinical management. Accurate identification and in-depth genomic analysis are essential for monitoring and controlling this pathogen. This study aimed to identify the species-specific gene repertoire, antimicrobial resistance (AMR), and virulence genetic profiles through pan-genome analysis to develop novel detection methods and better understand emerging public health threats. The genus <i>Proteus</i> exhibits an open pan-genome, with <i>P. mirabilis</i> harboring a distinct species-specific gene repertoire. Two species-specific core genes, <i>PMI3020</i> and <i>PMI3598</i>, were identified as molecular targets. We developed conventional PCR and TaqMan probe-based real-time PCR assays, which demonstrated high specificity when tested against <i>P. mirabilis</i> and non-<i>P</i>. <i>mirabilis</i> isolates. The TaqMan probe-based real-time PCR demonstrated a sensitivity of 3.43 × 10² CFU/mL using serial dilutions of <i>P. mirabilis</i> DNA. Comparative genomic analysis revealed significant differences in AMR and pathogenicity-related gene repertoires between <i>P. mirabilis</i> and other <i>Proteus</i> spp. The higher prevalence of AMR phenotypes in <i>P. mirabilis</i> correlated with its greater abundance of AMR genes. Emerging AMR genes acquired through horizontal gene transfer (HGT) have increased MDR risks, particularly to carbapenems and cephalosporins. Additionally, <i>P. mirabilis</i> genomes contain more virulence genes mainly related to adherence and iron acquisition. Our findings establish pan-genome analysis as an effective tool for identifying specific genetic markers to detect pathogens accurately and provide a comprehensive genomic framework illuminating AMR dynamics and virulence in <i>P. mirabilis</i>, thereby providing a valuable foundation for future public health risk assessments.IMPORTANCE<i>P. mirabilis</i> is a major uropathogen with increasing AMR prevalence. The dissemination of AMR genes across healthcare and community settings poses critical challenges to infection control. This study conducted pan-genome analysis of <i>Proteus</i> to identify <i>P. mirabilis</i>-specific gene repertoire, of which species-specific core genes were used as molecular targets to develop highly sensitive PCR assays for accurate detection of this pathogen. Compared with other <i>Proteus</i> spp., <i>P. mirabilis</i> possesses a greater abundance of AMR genes, resulting in a higher prevalence of AMR phenotypes, including significant resistance to carbapenems and cephalosporins. This study establishes pan-genome analysis as an effective strategy for mining species-specific genetic markers, enabling the development of novel PCR assays for accurate pathogen detection. The comprehensive genomic framework enhances understanding of AMR dynamics and virulence mechanisms essential for public health risk assessment.