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

Abstract

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.