Cross-kingdom antimicrobial resistance profiling: A unified multiplex PCR assay for simultaneous bacterial and fungal detection

Antimicrobial-resistant (AMR) pathogens have emerged as a critical global health concern, necessitating the development of rapid and reliable screening tools. Conventional culture-based identification is labor-intensive and often delays appropriate antimicrobial therapy. This study aimed to develop and validate a multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of methicillin-resistant Staphylococcus aureus , carbapenem-resistant Acinetobacter baumannii , and fluconazole-resistant Candida albicans . Initially, monoplex PCR was performed to evaluate the specificity and sensitivity of each primer set targeting 16S rRNA (bacterial identification), mecA (methicillin resistance), bla OXA-23 (carbapenem resistance), ITS (fungal identification), and MDR1 (azole resistance). Based on these results, multiplex PCR was developed to simultaneously detect multiple target pathogens within a single reaction. The assay successfully amplified all target genes, and no cross-reactivity was observed. Furthermore, multiplex PCR enabled the rapid, simultaneous detection of multiple AMR pathogens within a single reaction. A prototype multiplex PCR kit was also developed based on this study. Collectively, these findings indicate that the developed multiplex PCR assay has potential as a feasible approach for the simultaneous detection of AMR pathogens.