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

作者：Zhixiang Xu, Baisheng Li, Yushan Jiang, Jia Huang, Lebin Su, Weibo Wu, Qi-Lin Pang, Zhuolin Li, Jiaqi Zhang, Xiaohe Li, Jun Wang, Fulan Cen, Ling Peng, Jinhu Liang, Fuxiang Wang, Chang Liu, Chenguang Shen, Yingxia Liu, Yang Yang · 发表于：Microbiology Spectrum · 年份：2023 · DOI：10.1128/spectrum.00719-23 · 被引用次数：16 · 研究领域：Antibiotic Resistance in Bacteria、Bacterial Identification and Susceptibility Testing、Antibiotics Pharmacokinetics and Efficacy

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 ...