A Novel Untargeted Molecular Detection Technique for Rapid Fecal Microbiota Profiling in Very Preterm Infants: Optimization, Genus‐Level Comparison, and Application

Gut microbiota profiling shows potential for improving care in the neonatal intensive care unit (NICU). However, common techniques, including 16S rRNA gene and metagenomic sequencing, have limited bedside applicability. The IS-pro microbiota assay provides species-level abundances within 5 h. We aimed to optimize the taxa annotation for preterm infants (phase 1), compare its findings to 16S sequencing on the genus level (phase 2), and apply the assay in a preterm cohort (phase 3). 1445 fecal samples from 479 preterm infants (24-30 weeks gestation) across 10 NICUs were analyzed with IS-pro. For phase 1 (optimization), IS-pro amplicons of 32 fecal samples were additionally analyzed with nanopore sequencing to expand the IS-pro matching database. For phase 2 (comparison), 41 samples were compared to 16S sequencing. In phase 3 (application), the optimized IS-pro assay was applied to the total cohort. Following phase 1, a mean relative abundance of 82.5% was successfully annotated. In phase 2, IS-pro showed high concordance with 16S sequencing, with a strong positive correlation between the two techniques (Pearson's correlation coefficient: 0.77, SD 0.24). In phase 3, IS-pro analysis of the full cohort revealed Staphylococcus, Klebsiella, Enterococcus, Escherichia-Shigella, and Streptococcus as the predominant genera in the first 4 weeks of life. Our findings demonstrate that the IS-pro microbiota assay effectively detects and quantifies key bacterial taxa in fecal samples of preterm infants, with outcomes highly concordant with 16S sequencing. Unlike traditional techniques, IS-pro is a rapid tool, illustrating its potential for clinical practice. Future studies should explore its applications in the NICU.