Ferry Dwi Kurniawan, Dina Alia, Mamoru Shiraishi, Megumi Higo, Yoshiaki Inoue, Koichi Hagiwara
Over half of community-acquired pneumonia cases are caused by a few dozen bacterial species, and accurate identification of these pathogens is essential for effective treatment. In this study, we developed a reliable diagnostic method using 16S ribosomal RNA (16S rRNA) sequencing, considering intra-species variation, the need to differentiate Streptococcus pneumoniae from oral α-hemolytic streptococci, and applicability to the battlefield hypothesis, which helps distinguish true pathogens from commensal organisms that are not causative pathogens. We designed specific primers and a BLAST wrapper program, Cheryblast + ob, to classify 37 pneumonia-causing bacteria and 4 α-hemolytic streptococci. In simulation experiments involving a total of 20,309 copies of the 16S rRNA from 41 species of bacteria deposited in Genbank, the algorithm achieved a sensitivity greater than 0.996 and a specificity of 1.000. It was robust against sequencing errors and successfully distinguished S. pneumoniae from closely related species. In an experiment using next-generation sequencing on artificial mixtures containing genomic DNA from 10 bacterial species and human DNA at varying two-fold ratios, the species with the highest copy number was correctly identified in 8 out of 11 samples, and the top two species by copy number were identified in all 11 samples. This high-performance method offers a promising tool for accurate pneumonia diagnosis and could also be applied to other infections in which a limited number of bacterial species must be reliably identified. © The Author(s) 2025.
Comprehensive Medicine I, Saitama Medical Center, Jichi Medical University, 1-847 Amanuma-cho, Omiya-ku, Saitama-shi, Saitama, 330-8503, Japan; Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, University of Syiah Kuala, Aceh, Indonesia; Department of Otolaryngology-Head and Neck Surgery, Faculty of Medicine, University of Syiah Kuala, Aceh, Indonesia; Division of Pulmonary Medicine, Department of Internal Medicine, Jichi Medical University, Tochigi, Japan; General Thoracic Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Kawagoe, Japan; Clinical Laboratories for Cardiovascular Diseases, Saitama Medical Center, Jichi Medical University, Saitama, Japan
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