Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens

J Clin Microbiol. 2016 Feb;54(2):368-75. doi: 10.1128/JCM.01965-15. Epub 2015 Dec 4.


The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for Clostridium difficile identification in diarrhea stool samples. Twenty-two C. difficile-positive diarrhea samples identified by PCR and qPCR and five C. difficile-negative diarrhea controls were studied. C. difficile was detected in 90.9% of C. difficile-positive samples using 16S rRNA gene sequencing, and C. difficile was detected in 86.3% of C. difficile-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of C. difficile from 16S rRNA gene sequencing (r(2) = -0.60) and MSS (r(2) = -0.55). C. difficile was codetected with Clostridium perfringens, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of Candida spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without a priori knowledge.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adolescent
  • Child
  • Child, Preschool
  • Cluster Analysis
  • Diarrhea / diagnosis*
  • Diarrhea / microbiology*
  • Drug Resistance, Microbial
  • Feces / microbiology*
  • Feces / virology
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Infant
  • Infant, Newborn
  • Metagenome*
  • Metagenomics* / methods
  • Microbiota*
  • RNA, Ribosomal, 16S / genetics
  • Real-Time Polymerase Chain Reaction
  • Reproducibility of Results


  • RNA, Ribosomal, 16S