Rapid detection of viable Acinetobacter baumannii and antibiotic susceptibility testing based on a phage amplification-Taqman qPCR assay

Abstract

BackgroundTimely and accurate identification of pathogenic bacteria and antibiotic susceptibility could decrease misuse and overuse of antibiotics. In this study, we developed a phage amplification-Taqman qPCR assay for simultaneous viable Acinetobacter baumannii (A. baumannii) detection and determination of broad-spectrum antimicrobial susceptibility based on quantification of phage DNA replication in one single tube. ResultsThe two-phage cocktail used in the current phage amplification-Taqman qPCR assay consists of phage p53 and phage pB3074. Once a lytic phage infects its host, phages could specifically recognize the viable cells and replicate themselves within the cells to produce hundreds of progeny phages. With dual phage cocktail concentration (5.00 × 103 PFU/mL to 5.00 × 104PFU/mL) used, down to 50 CFU /mL of viable A. baumannii could be detected within 3 h from bronchoalveolar lavage fluid spiked with A. baumannii in an incubation volume of 2 mL. We further illustrated that antibiotic susceptibility testing results could be obtained within 2 h based on current phage amplification-Taqman qPCR assay to measure the phenotypic response of A. baumannii from spiked bronchoalveolar lavage fluid after only 30 min of antibiotic exposure. Testing of 12 bronchoalveolar lavage fluid samples spiked by different strains showed that the phage amplification-Taqman qPCR assay had 100 % (95 %CI: 74.12 to 100) identification agreement with the initial spiked concentrations of the bacteria. Subsequent antibiotic susceptibility testing results of 9 bronchoalveolar lavage fluid samples spiked by different strains also had shown that the phage amplification-Taqman qPCR assay had good agreements with the standard broth microdilution method, with an overall agreement of 100 % (95 % CI, 70.09 to 100) for ceftazidime and aztreonam. Significance and NoveltyDue to the high rapidity, high sensitivity, good specificity, low cost, and the ability to distinguish between live and dead bacteria, the phage amplification-Taqman qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria and determination of broad-spectrum antimicrobial susceptibility.