Dataset Information

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

ABSTRACT: In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli.

SUBMITTER: Banjo M

PROVIDER: S-EPMC5971530 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

Banjo Masaya M Iguchi Atsushi A Seto Kazuko K Kikuchi Taisei T Harada Tetsuya T Scheutz Flemming F Iyoda Sunao S

Journal of clinical microbiology 20180525 6

In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and pra ...[more]

PMID: 29593058

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Project description:Easy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing of universal pair of primers and selection of the optimal restriction enzyme RsaI was preceded by in silico comparative analysis of the sequences of the genes coding for 53 different serotypes of H-antigen (E. coli flagellin). The target fragments of E. coli genomes for MLST method were selected on the basis of bioinformatics analysis of complete sequences of 16 genomes of E. coli. Initially, seven molecular targets were proposed (seven pairs of primers) and five of them were found useful for effective genotyping of E. coli strains. Both developed methods revealed high differentiation power, and a high genetic diversity of the strains tested was observed. Within the group of 71 strains tested, 29 and 47 clusters were revealed with fliC RFLP-PCR and MLST methods, respectively. Differentiation of the strains with the reference BOX-PCR method revealed 31 different genotypes. The in silico analysis revealed that the discriminatory power of the new MLST method is comparable to the Pasteur and Achtman schemes and is higher than the discriminatory power of the method developed by Clermont. From the epidemiology point of view, the outcomes of our investigation revealed that in most cases, the patients were infected with unique strains, probably from environmental sources. However, some strains isolated from different patients of the wards of pediatrics, internal medicine, and neurology were classified to the same genotype when the results of all three methods were taken into account. It could suggest that they were transferred between the patients.

Project description:BACKGROUND: Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC. METHODS: In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5'end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real-time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR. RESULTS: The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n?=?111, 64.9%) and EPEC (n?=?38, 22.2%), which were the dominating pathotypes of DEC strains. CONCLUSION: The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories.

Dataset Information

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

Publications

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

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