Shotgun metagenomic sequencing of serum extracellular vesicle-derived DNA enables microbial profiling in urothelial carcinoma

Shotgun metagenomic sequencing provides more detailed microbial information than 16 S rRNA sequencing but requires higher cost and larger sample volumes. We evaluated whether shotgun metagenomic sequencing could detect bacterial DNA in serum extracellular vesicles. Serum samples from 72 patients with urothelial carcinoma (surgery, n = 45; immune checkpoint inhibitor therapy, n = 27) and 20 healthy controls were analyzed; all had been previously examined using 16 S rRNA sequencing. Microbial DNA fragments from multiple bacterial species were detected in serum EV fractions. In our previous 16 S study, bacterial diversity differed significantly between patients and healthy controls, whereas no significant difference was observed in the present shotgun analysis. In contrast, among patients receiving immune checkpoint inhibitors, both 16 S and shotgun analyses showed significant differences in bacterial diversity between responders ( n = 7) and nonresponders ( n = 20). These results indicate that bacterial DNA in serum extracellular vesicles can be analyzed using shotgun metagenomic sequencing.