Article

European Journal of Clinical Microbiology & Infectious Diseases

, Volume 27, Issue 7, pp 587-596

Pyrosequencing™ analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases

  • X.-L. HouAffiliated withInstitute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Key Laboratory of Infectious Diseases, Ministry of Public Health of ChinaInstitute of Biochemistry and Molecular Biology, Medical College, Zhejiang University
  • , Q.-Y. CaoAffiliated withInstitute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Key Laboratory of Infectious Diseases, Ministry of Public Health of China
  • , H.-Y. JiaAffiliated withInstitute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Key Laboratory of Infectious Diseases, Ministry of Public Health of China
  • , Z. ChenAffiliated withInstitute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Key Laboratory of Infectious Diseases, Ministry of Public Health of China Email author 

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Abstract

Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing™ was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long (~1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing™. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 104 CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing™ approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.