title: Pyrosequencing analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases.
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 ( approximately1.2 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 10(4) 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.
authors.affiliation: Institute of Infectious Diseases, First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou, Zhejiang, 310003, People's Republic of China.
authors.forename: X-L
authors.initials: XL
authors.lastname: Hou
authors.forename: Q-Y
authors.initials: QY
authors.lastname: Cao
authors.forename: H-Y
authors.initials: HY
authors.lastname: Jia
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000328:Adult; D001426:Bacterial Proteins / Q000235:genetics*; D002681:China; D027081:DNA Gyrase / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology*; D004755:Enterobacteriaceae / Q000145:classification* / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology*; D006801:Humans; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014737:Vibrionaceae / Q000145:classification* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D027081:DNA Gyrase