Multiplexed and Rapid AST for Escherichia coli Infection by Simultaneously Pyrosequencing Multiple Barcodes Each Specific to an Antibiotic Exposed to a Sample.

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.