Integrating rapid pathogen identification and antimicrobial susceptibility testing through multiplex TaqMan qPCR assay

微生物学 鲍曼不动杆菌 支气管肺泡灌洗 塔克曼 多路复用 金黄色葡萄球菌 生物 肉汤微量稀释 细菌 抗菌剂 实时聚合酶链反应 医学 最小抑制浓度 铜绿假单胞菌 基因 生物信息学 内科学 生物化学 遗传学
作者
Libo Xie,Min Yang,Min Liu,Qianyuan Li,Chunhua Luo,Jun Luo
出处
期刊:Journal of Microbiological Methods [Elsevier BV]
卷期号:217-218: 106888-106888 被引量:2
标识
DOI:10.1016/j.mimet.2023.106888
摘要

Timely bacterial identification (ID) and antimicrobial susceptibility testing (AST) are of significance for therapy of bacteria-infected patients. In the present study, we developed a multiplex TaqMan qPCR assay for rapid and accurate ID and AST of three common hospital acquired pneumonia species, namely Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus. In this assay, DNA extraction and bacterial co-incubation with antibiotics are accomplished based on a common PCR instrument. ID of three bacteria is based on specific conserved DNA sequence fragment (gltA for A. baumannii, phoE for K. pneumoniae and nuc for S. aureus) detection through multiplex TaqMan qPCR assay within 80 min. AST of three bacteria could be acquired within 200 min based on genomic DNA fold change detection after 2 h of antibiotic exposure. Testing of 23 bronchoalveolar lavage fluid samples spiked by different A. baumannii isolates, 20 bronchoalveolar lavage fluid samples spiked by different K. pneumoniae isolates, and 14 bronchoalveolar lavage fluid samples spiked by different S. aureus isolates showed that the multiplex TaqMan qPCR assay had 100% (95% CI: 85.69–100), 100% (95% CI: 83.89–100) and 100% (95% CI:78.47–100) identification agreement with the initial spiked bacteria. Subsequent AST results compared with the standard broth microdilution method showed an overall agreement of 91.30% (95% CI: 73.20 to 97.58) for A. baumannii, 90% (95% CI: 69.90 to 97.21) for K. pneumoniae and 92.86% (95% CI: 68.53 to 98.73) for S. aureus based on the current multiplex TaqMan assay. Due to the high rapidity, good agreement, simplicity, and high throughput, this multiplex TaqMan assay could be helpful for ID and broad-spectrum AST in A. baumannii, K. pneumoniae and S. aureus, as well as potentially applicable for other clinical bacteria by changing the primers and probes.
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