Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes

<b>Introduction.</b> <i>Klebsiella pneumoniae</i> (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.<b>Gap Statement.</b> While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.<b>Aim.</b> To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.<b>Methodology.</b> In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (<i>rmpA</i>, <i>rmpA2</i>, <i>iucA</i>, <i>peg344</i> and <i>iroB</i>), carbapenem-resistant (<i>bla</i> _NDM, <i>bla</i> _OXA-48-like and <i>bla</i> _KPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.<b>Results.</b> The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl^-1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.<b>Conclusion.</b> This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.