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.

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.