Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae

ABSTRACT The increasing prevalence of hypervirulent (hv) and carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) highlights the importance of timely and accurate differential diagnosis for epidemiological investigation and clinical management. A multiplex quantitative real-time PCR (qRT-PCR) assay for the simultaneous identification of hvKp and CR-Kp was developed and validated with excellent performance in sensitivity and specificity. Generally, the gltA gene for Kp, the iucA, rmpA and rmpA2 genes for hvKp, and the Klebsiella pneumoniae carbapenemases (KPC) gene for CR-Kp were included in the qRT-PCR assay. The detection limits for classic Kp (cKp), CR-cKp, hvKp, and CR-hvKp strains could all reach 50 genome equivalent copies and 20 CFUs per reaction with high accuracy (R 2 > 0.99) and reliability (CV values < 3%). Detection results from 84 Kp positive clinical samples showed 31 hvKp with 8 CR-hvKp and 53 cKp with 1 CR-cKp strains. The presence of virulence-associated factors for the identified hvKp and KPC genes for CR-Kp was confirmed by previously developed conventional PCR and antimicrobial susceptibility tests, respectively. Furthermore, the qRT-PCR identified hvKp strains showed mortality rates of ≥40% in the outbred murine infection model, while no death for the identified cKp strains. These results indicated that our multiplex qRT-PCR assay could accurately identify hvKp and CR-Kp strains, which will be of great use for the rapid and accurate diagnosis in a clinical setting and the surveillance of the circulating Kp. IMPORTANCE Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.