Standardization of shotgun metagenomics parameters for pks-positive enterobacteria detection in human fecal samples: Receiver operating characteristic analysis using polymerase chain reaction as reference standard
13 affiliations
- 1Graduate School of Sport Sciences, Waseda University, Tokorozawa, 359-1192 Japan
- 2Center for Clinical Nutrition, National Institutes of Biomedical Innovation, Health and Nutrition, Settsu, 566-0002 Japan
- 3Microbial Research Center for Health and Medicine, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, 567-0085 Japan
- 4Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, 359-1192 Japan
- 5Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 422-8526 Japan
- 6Graduate School of Medicine, The University of Osaka, Suita, 565-0871 Japan
- 7Graduate School of Pharmaceutical Sciences, The University of Osaka, Suita, 565-0871 Japan
- 8Graduate School of Dentistry, The University of Osaka, Suita, 565-0871 Japan
- 9Graduate School of Science, The University of Osaka, Toyonaka, 560-0043 Japan
- 10International Vaccine Design Center, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
- 11Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Kobe, 650-0017 Japan
- 12Research Organization for Nano and Life Innovation, Waseda University, Tokyo, 162-8480 Japan
- 13Department of Physical Activity Research, National Institutes of Biomedical Innovation, Health and Nutrition, Settsu, 566-0002 Japan
Abstract
Background: Enterobacteria harboring the polyketide synthase (pks) island (clbA–clbS), which encodes colibactin (pks⁺ enterobacteria), is a putative risk factor for colorectal cancer. Polymerase chain reaction (PCR) is the current standard for detection. Primer-independent shotgun metagenomic sequencing (SMS) is increasingly used; however, thresholds and analysis parameters for identifying pks⁺ enterobacteria remain unstandardized. This study aimed to evaluate how accurately SMS can reproduce PCR-defined pks⁺ status and identify practical cutoffs by analyzing fecal samples from 223 healthy adults in a Japanese cohort.
Results: PCR identified 60/223 (26.9%) participants as pks⁺. SMS detected at least one clb gene in 85/223 (38.1%) at ≥ 90% identity; 49 of these agreed with PCR positives. Among the single-gene rules, clbB yielded the best discrimination (area under the curve (AUC) = 0.826) across 70–90% identity. The total-read rule across 19 clb genes performed best overall (AUC = 0.838), with higher AUCs above the 60% threshold (all p ≤ 0.045). The rule requiring detection of at least eight clb genes was inferior (AUC = 0.734–0.773). Detection performance depended on gene length; longer clb genes (> 1,500 amino acids) achieved higher accuracy (AUC ≥ 0.800).
Conclusions: Optimized SMS can reproduce PCR-based detection of pks⁺ enterobacteria in feces with high accuracy. clbB and a cluster-level rule aggregating reads across clbA–clbS are effective, although tightening the sequence-identity threshold did not consistently improve performance. The standardized criteria proposed here enable cross-study comparability and may accelerate epidemiologic assessment of pks⁺ enterobacteria and its clinical translation for colorectal cancer prevention.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s12866-026-05460-3.
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BibTeX
@article{tataka2026standardization,
title = {{Standardization of shotgun metagenomics parameters for pks-positive enterobacteria detection in human fecal samples: Receiver operating characteristic analysis using polymerase chain reaction as reference standard}},
author = {Yusei Tataka and Hitoshi Kawashima and Hinako Nanri and Daiki Watanabe and Kenji Watanabe and Masaya Yamaguchi and Jun Kunisawa and Motohiko Miyachi},
journal = {BMC Microbiology},
year = {2026},
month = aug,
doi = {10.1186/s12866-026-05460-3},
url = {https://doi.org/10.1186/s12866-026-05460-3},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Standardization of shotgun metagenomics parameters for pks-positive enterobacteria detection in human fecal samples: Receiver operating characteristic analysis using polymerase chain reaction as reference standard
AU - Yusei Tataka
AU - Hitoshi Kawashima
AU - Hinako Nanri
AU - Daiki Watanabe
AU - Kenji Watanabe
AU - Masaya Yamaguchi
AU - Jun Kunisawa
AU - Motohiko Miyachi
PY - 2026
DA - 2026/08/01
JO - BMC Microbiology
DO - 10.1186/s12866-026-05460-3
UR - https://doi.org/10.1186/s12866-026-05460-3
AB - Background: Enterobacteria harboring the polyketide synthase (pks) island (clbA–clbS), which encodes colibactin (pks⁺ enterobacteria), is a putative risk factor for colorectal cancer. Polymerase chain reaction (PCR) is the current standard for detection. Primer-independent shotgun metagenomic sequencing (SMS) is increasingly used; however, thresholds and analysis parameters for identifying pks⁺ enterobacteria remain unstandardized. This study aimed to evaluate how accurately SMS can reproduce PCR-defined pks⁺ status and identify practical cutoffs by analyzing fecal samples from 223 healthy adults in a Japanese cohort. Results: PCR identified 60/223 (26.9%) participants as pks⁺. SMS detected at least one clb gene in 85/223 (38.1%) at ≥ 90% identity; 49 of these agreed with PCR positives. Among the single-gene rules, clbB yielded the best discrimination (area under the curve (AUC) = 0.826) across 70–90% identity. The total-read rule across 19 clb genes performed best overall (AUC = 0.838), with higher AUCs above the 60% threshold (all p ≤ 0.045). The rule requiring detection of at least eight clb genes was inferior (AUC = 0.734–0.773). Detection performance depended on gene length; longer clb genes (> 1,500 amino acids) achieved higher accuracy (AUC ≥ 0.800). Conclusions: Optimized SMS can reproduce PCR-based detection of pks⁺ enterobacteria in feces with high accuracy. clbB and a cluster-level rule aggregating reads across clbA–clbS are effective, although tightening the sequence-identity threshold did not consistently improve performance. The standardized criteria proposed here enable cross-study comparability and may accelerate epidemiologic assessment of pks⁺ enterobacteria and its clinical translation for colorectal cancer prevention. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s12866-026-05460-3.
SN - 1471-2180
ER -