Fig. 1: The workflow of the study.

Step 1: Saliva samples were collected from 312 CRC patients scheduled for surgical tumor resection. The median follow-up time was 21.5 months for patients with disease progression and 25.4 months for survival. Salivary microbiota profiling was performed using 16S rRNA full-length sequencing. Step 2: Species-level identification was conducted with inclusion criteria of prevalence ≥10% and relative abundance ≥0.01%, resulting in 98 species. A 1:1 cross-validation strategy was applied by randomly splitting the cohort into discovery (n = 156) and test (n = 156) sets, with 1000 subsampling iterations. Prognosis-related microbes were identified using univariate and multivariate Cox regression analyses, highlighting Campylobacter gracilis, Neisseria oralis, and Treponema medium as significant biomarkers. Step 3: A microbial risk score (MRS) was constructed based on the identified prognostic bacterial species. Clinical prognostic factors were also evaluated, and a comprehensive model integrating microbiota biomarkers (three species) and clinical factors (three variables) was developed. The performance of the comprehensive model was validated and compared with models based solely on the clinical factors, demonstrating superior prognostic accuracy.