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Background <p>Enterobacteria harboring the polyketide synthase (<i>pks</i>) island (<i>clbA</i>–<i>clbS</i>), which encodes colibactin (<i>pks⁺</i> 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 <i>pks⁺</i> enterobacteria remain unstandardized. This study aimed to evaluate how accurately SMS can reproduce PCR-defined <i>pks⁺</i> status and identify practical cutoffs by analyzing fecal samples from 223 healthy adults in a Japanese cohort.</p> Results <p>PCR identified 60/223 (26.9%) participants as <i>pks⁺</i>. SMS detected at least one <i>clb</i> gene in 85/223 (38.1%) at ≥ 90% identity; 49 of these agreed with PCR positives. Among the single-gene rules, <i>clbB</i> yielded the best discrimination (area under the curve (AUC) = 0.826) across 70–90% identity. The total-read rule across 19 <i>clb</i> genes performed best overall (AUC = 0.838), with higher AUCs above the 60% threshold (all <i>p</i> ≤ 0.045). The rule requiring detection of at least eight <i>clb</i> genes was inferior (AUC = 0.734–0.773). Detection performance depended on gene length; longer <i>clb</i> genes (&gt; 1,500 amino acids) achieved higher accuracy (AUC ≥ 0.800).</p> Conclusions <p>Optimized SMS can reproduce PCR-based detection of <i>pks⁺</i> enterobacteria in feces with high accuracy. <i>clbB</i> and a cluster-level rule aggregating reads across <i>clbA</i>–<i>clbS</i> 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 <i>pks⁺</i> enterobacteria and its clinical translation for colorectal cancer prevention.</p>

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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

  • 期刊论文

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  • 发表日期：

    2026年8月1日

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  • Yusei Tataka,
  • Hitoshi Kawashima,
  • Hinako Nanri,
  • Daiki Watanabe,
  • Kenji Watanabe,
  • Masaya Yamaguchi,
  • Jun Kunisawa,
  • Motohiko Miyachi

摘要

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.