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

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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.

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Acknowledgments

This work was supported by grants 2003C13015, 021103128 from the Science and Technology Department of Zhejiang Province, China. We appreciate Barbara J. Chang for critical discussion and Robert Wohlhueter for his help in preparing the manuscript.

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Authors and Affiliations

  1. Institute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Key Laboratory of Infectious Diseases, Ministry of Public Health of China, Qingchun Road 79, Hangzhou, Zhejiang, 310003, People’s Republic of China

    X.-L. Hou, Q.-Y. Cao, H.-Y. Jia & Z. Chen

  2. Institute of Biochemistry and Molecular Biology, Medical College, Zhejiang University, Yuhang tang Road 88, Hangzhou, Zhejiang, 310058, People’s Republic of China

    X.-L. Hou

Authors
  1. X.-L. Hou
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  2. Q.-Y. Cao
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  3. H.-Y. Jia
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  4. Z. Chen
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Corresponding author

Correspondence to Z. Chen.

Additional information

Hou and Cao contributed equally to this work. Pyrosequencing™ is a trademark of Biotage, Uppsala, Sweden and is functionally equivalent to the generic “sequencing by synthesis”; we use it in the text in this sense without ™ designation.

Electronic supplementary material

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Supplementary Table 1

The analysis of positional differences in the gyrB sequences of 1.2 kba (DOC 55.0 KB)

Supplementary Table 2

Power of the assay with respect to differentiating Salmonella serotypes. Discrimination among serotypes depends on uniqueness within the 262 gyrB sequences considered in designing the assay. In addition to the six serotypes of central interest to this report, another 53 serotypes of S. enterica subsp. enterica were also analyzed (1–9 replicates of each). Non-discriminated clusters are shown here. (DOC 77.5 KB)

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Hou, XL., Cao, QY., Jia, HY. et al. Pyrosequencing™ analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases. Eur J Clin Microbiol Infect Dis 27, 587–596 (2008). https://doi.org/10.1007/s10096-008-0477-7

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  • DOI: https://doi.org/10.1007/s10096-008-0477-7

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