Nanopore 16S amplicon sequencing enhances the understanding of pathogens in medically intractable diabetic foot infections

Diabetic foot infections (DFIs) cause substantial morbidity and mortality. The mainstay of the treatment is empiric antibiotic treatment and surgical debridement in severe cases. In this study, we performed Nanopore 16S ribosomal DNA (rDNA) sequencing from the debridement specimens of DFIs. Fifty-four surgical debridement specimens obtained from 45 patients of medically intractable DFI were included. The 16S rDNA PCR was performed from the specimen and Nanopore sequencing was performed for up to 3 hours. The reads were aligned to the BLAST database, and the results were compared to conventional culture studies. The 16S sequencing results revealed that majority of the DFIs (44 of 54, 81.5%) were polymicrobial infections. All bacteria isolated by conventional culture studies were detected by the 16S sequencing. Several anaerobes (Prevotella, Finegoldia, Anaerococcus, Bacteroides) were commonly identified by the 16S sequencing but were frequently missed by culture studies. In many cases, certain bacteria only revealed by the 16S sequencing were more abundant than the bacteria isolated by the culture studies. In conclusion, Nanopore 16S sequencing was capable of pathogen identification in DFIs and has many advantages over conventional culture studies. Nanopore 16S sequencing enables a comprehensive understanding of the bacteria involved in DFIs.