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

Abstract

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.