Multiplex PCR assay for the Rapid detection of Klebsiella pneumoniae pathotypes

IntroductionKlebsiella pneumoniae is a major cause of nosocomial infections, with its evolving pathotypes--including multidrug-resistant (MDR), hypervirulent (hvKp), and convergent strains--posing significant diagnostic and treatment challenges. The convergence of antimicrobial resistance and hypervirulence in clinical settings complicates timely identification, leading to difficult-to-treat infections with limited therapeutic options. Materials and MethodsIn this study, we designed and optimized a multiplex polymerase chain reaction (m-PCR) assay for the simultaneous detection of key biomarkers/genes associated with hypervirulent (rmpA, rmpA2, iucA, peg344, iroB), carbapenem-resistant (bla-NDM, bla-OXA-48-like), and convergent K. pneumoniae (hv-CRKp) pathotypes in clinical isolates. Specific primers were designed for these targets in a five-pair combination, with rigorous optimization of reaction conditions, including annealing temperatures, specificity, and sensitivity. Results and ConclusionThe developed m-PCR assay exhibited 100% specificity when compared to whole genome sequencing 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/{micro}L. Additionally, validation through sequencing confirmed the accuracy of detected amplicons. This m-PCR assay offers a rapid, sensitive, and specific diagnostic tool for differentiating K. pneumoniae pathotypes in clinical settings, aiding in timely intervention and improved infection control measures. Impact StatementThis study presents a multiplex PCR assay for the rapid and accurate detection of Klebsiella pneumoniae pathotypes, addressing the challenge of defining hypervirulence due to the diverse and evolving set of markers. By analyzing genomic data from public databases, the most well-established virulence and resistance markers were carefully selected to enable comprehensive strain differentiation. The assay simultaneously detects hypervirulent, carbapenem-resistant, and classical strains, offering a faster and more targeted alternative to conventional methods. Additionally, its adaptable design allows for modifications based on regional strain variations, making it a valuable tool for both clinical and epidemiological applications. This approach enhances infection control efforts and antimicrobial stewardship by enabling timely and precise pathogen identification, particularly in resource-limited settings. By bridging genomic insights with practical diagnostics, this assay provides a cost-effective, scalable solution to monitor and manage high-risk K. pneumoniae infections more effectively.