Microbiota Profiling of Cerebrospinal Fluid in Bacterial Meningitis Using 16S rRNA Metagenomic Sequencing

Bacterial meningitis is a life-threatening central nervous system infection in which rapid and accurate pathogen identification is essential for effective treatment; however, conven-tional culture methods often show limited sensitivity due to prior antibiotic exposure, low microbial load, or fastidious organisms. Background: This study aimed to investigate the cerebrospinal fluid (CSF) microbiota in patients with bacterial meningitis and to evaluate the diagnostic performance of 16S rRNA gene-based metagenomic next-generation se-quencing (mNGS), particularly in culture-negative cases. Methods: CSF samples from 26 patients collected between September 2023 and July 2025 were analyzed. Standard aerobic culture and PCR were performed. DNA was extracted using the ZymoBIOMICS kit and sequenced on the Oxford Nanopore MinION platform using the ONT 16S Barcoding Kit. Sequencing data were processed using Dorado basecalling, FastQC quality control, and taxonomic classification against NCBI and proprietary databases. Results: Conventional culture identified pathogens in 3/26 samples (Klebsiella pneumoniae, Enterobacter aerogenes, Enterococcus spp.), and one sample was PCR-positive for Mycobacterium tuberculosis. In contrast, mNGS detected bacterial pathogens in five samples, confirming all cul-ture-positive organisms and additionally identifying Mycobacterium spp. and Neisseria meningitidis operational taxonomic units in culture-negative cases. Conclusions: 16S rRNA-based mNGS demonstrated higher diagnostic yield than conventional culture and provided complementary value in pathogen detection, particularly in culture-negative meningitis, and may improve clinical diagnostic workflows.