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Background <p>Infections rank as the third leading cause of death among patients with type 2 diabetes (T2D), representing a critical clinical burden that cannot be ignored. The gut, as a major microbial reservoir, is strongly implicated in this heightened susceptibility. However, the specific enteric pathogens responsible remain poorly defined. This study, therefore, aims to identify specific enteric pathogens associated with T2D and translate these findings into an effective detection strategy by developing a robust molecular tool for clinical prevention.</p> Methods <p>We first conducted a systematic meta-analysis to identify T2D-associated gut pathogens. Then, targeted next-generation sequencing (tNGS) was performed on carefully matched stool samples to further characterize pathogen enrichment. Subsequently, findings from meta-analysis and tNGS were validated in independent clinical cohorts using qPCR analysis. Finally, we developed a dual-probe fluorescence-based PCR assay and conducted comparative evaluation between droplet digital PCR (ddPCR) and quantitative PCR (qPCR) platforms using clinical fecal samples.</p> Results <p>The meta-analysis revealed significantly increased abundance of <i>Klebsiella</i> and <i>Escherichia/Shigella</i> in T2D individuals, findings that were consistently replicated in independent validation cohorts. Subsequent tNGS analysis confirmed elevated abundance and detection rates of enteropathogenic <i>Escherichia coli</i> (EPEC) in the T2D group. Based on these findings, we developed a highly sensitive and specific dual-probe fluorescence-based PCR assay. Methodological comparisons demonstrated that ddPCR substantially outperformed qPCR in direct detection of target bacteria from fecal samples, exhibiting enhanced analytical sensitivity and superior resistance to fecal matrix inhibitors.</p> Conclusion <p>This study establishes a significant association between specific enteric pathogens and T2D, underscoring their putative role in diabetes-related complications. The demonstrated superiority of ddPCR enables sensitive, matrix-resistant enteric pathogen detection in T2D patients, providing a robust diagnostic tool that supports targeted diagnosis, risk stratification, and optimized clinical management of at-risk individuals.</p> Trial registration <p>National Medical Research Registration and Filing Information System MR4225044248, 13-Jun-2025.</p>

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Identification and optimized detection of two type 2 diabetes-associated enteric pathogens: Klebsiella and EPEC

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月7日

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  • Long Wang,
  • Xianglan Fang,
  • Liuliu Shi,
  • Peng Wang,
  • Wei Chen,
  • Jin Yang,
  • Yan Ding,
  • Zheng Cao,
  • Duoshuang Xie,
  • Kun Meng

摘要

Background

Infections rank as the third leading cause of death among patients with type 2 diabetes (T2D), representing a critical clinical burden that cannot be ignored. The gut, as a major microbial reservoir, is strongly implicated in this heightened susceptibility. However, the specific enteric pathogens responsible remain poorly defined. This study, therefore, aims to identify specific enteric pathogens associated with T2D and translate these findings into an effective detection strategy by developing a robust molecular tool for clinical prevention.

Methods

We first conducted a systematic meta-analysis to identify T2D-associated gut pathogens. Then, targeted next-generation sequencing (tNGS) was performed on carefully matched stool samples to further characterize pathogen enrichment. Subsequently, findings from meta-analysis and tNGS were validated in independent clinical cohorts using qPCR analysis. Finally, we developed a dual-probe fluorescence-based PCR assay and conducted comparative evaluation between droplet digital PCR (ddPCR) and quantitative PCR (qPCR) platforms using clinical fecal samples.

Results

The meta-analysis revealed significantly increased abundance of Klebsiella and Escherichia/Shigella in T2D individuals, findings that were consistently replicated in independent validation cohorts. Subsequent tNGS analysis confirmed elevated abundance and detection rates of enteropathogenic Escherichia coli (EPEC) in the T2D group. Based on these findings, we developed a highly sensitive and specific dual-probe fluorescence-based PCR assay. Methodological comparisons demonstrated that ddPCR substantially outperformed qPCR in direct detection of target bacteria from fecal samples, exhibiting enhanced analytical sensitivity and superior resistance to fecal matrix inhibitors.

Conclusion

This study establishes a significant association between specific enteric pathogens and T2D, underscoring their putative role in diabetes-related complications. The demonstrated superiority of ddPCR enables sensitive, matrix-resistant enteric pathogen detection in T2D patients, providing a robust diagnostic tool that supports targeted diagnosis, risk stratification, and optimized clinical management of at-risk individuals.

Trial registration

National Medical Research Registration and Filing Information System MR4225044248, 13-Jun-2025.