Development of an efficient antimicrobial susceptibility testing method with species identification by Nanopore sequencing of 16S rRNA amplicons

While amplicon sequencing of 16S rRNA is a common method for studying microbial community, it has been difficult to identify genera and species using next-generation sequencers to examine some regions (e.g., V3-V4 of 16S rRNA) because of the short read lengths. However, the advent of third-generation sequencers has made it possible to analyze the full length of the 16S rRNA gene, which allowed for species level identification at low cost. In this study, we evaluated the accuracy of the identification with a third-generation sequencer, MinION from Oxford Nanopore Technologies, using nine indigenous bacteria that can pose problems with food poisoning and opportunistic infections as an example. We demonstrated that Enterococcus faecalis and Enterococcus hirae could be identified at the species level with an accuracy of 96.4% to 97.5%. We also demonstrated that the absolute counts of various bacteria could be determined by spiking the sample with a bacterium as an internal standard. Then, we tested whether this convenient bacterial identification method could evaluate the antibiotic sensitivities of multiple bacteria simultaneously. In order to evaluate antimicrobial susceptibility, a mock community, an artificial mixture of the nine bacterial strains, was prepared and cultured in the presence of the antibiotics ofloxacin or chloramphenicol, and the 16S rRNAs were analyzed by using Nanopore sequencer. We confirmed that antibiotic-induced cell count reductions could be measured simultaneously by quantifying the abundances of various bacteria in the mock community before and after culture. It was thus shown that the antibiotic sensitivities of multiple bacteria could be evaluated simultaneously, with distinction made between bactericidal action and bacteriostatic action. This methodology would allow rapid evaluation of antibiotic activity spectrum at the species level containing a wide variety of bacteria, such as biofilm bacteria and gut microbiota.