Comparison of 16S rRNA Gene Sequencing, API 20E, and RT-PCR for Identification of Autopsy-derived Escherichia coli and Shigella spp.

Abstract

Practical techniques to differentiate Escherichia coli from Shigella spp. remains underexplored in the field of forensic microbiology. We aimed to compare the performance of 16S rRNA gene sequencing, and two commercially available API 20E and RT-PCR systems to distinguish between E. coli and Shigella spp. on the basis of analysis of 24 isolates collected from 12 forensic autopsies to evaluate the level of species identification. The accuracy of 16S rRNA gene sequencing at the species level was ranged from 16.7% to 33.3%. However, 79.2% (19/24) were unidentified, even though analyzed using three distinct databases (MicroSEQ 500 library, BLAST, EzTaxon). Phylogenetic analysis confirmed that 16S rRNA gene sequencing could not differentiate Shigella spp. and E. coli. Of the 19 isolates, the API 20E showed complete identification of 78.9% (15/19) at the species level, whereas RT-PCR analysis showed complete identification of 19 isolates (100%) at the species level. Our comparison analyses revealed that only 16S rRNA gene sequencing is not enough to differentiate E. coli and Shigella spp., and the accuracy and rapidity order was as follows: RT-PCR >API 20E>standard analysis. These results suggest that the polyphasic strategy with the standard analysis and RT-PCR or API 20E techniques are ideal for differentiation of these microbes.