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Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

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Abstract

Campylobacter jejuni is the leading bacterial foodborne pathogen that causes human acute gastrointestinal illness worldwide. Due to its genetic diversity, fastidious growth and sophisticated biochemical requirements, classification of Campylobacter by traditional techniques is problematic. Several molecular typing methods have been explored in this bacterium. One such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). These CRISPRs consist of a direct repeat interspaced with nonrepetitive spacer sequences. In this study, we applied this genotyping method to explore the genetic diversity of C. jejuni isolated from poultry sources. Ninety-nine C. jejuni isolates from poultry environments in four different US states were used. Genomic DNA of the isolates were extracted from cultures using a commercial kit. PCR primers and conditions for CRISPR type 1 amplification were described previously. The amplicons were purified and sequenced by the Sanger dideoxy sequencing method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder. The results show there were 21% isolates no detectable, 30% isolates questionable, and 49% isolates confirmed CRISPR, respectively. The lengths of CRISPR range from 100 to 695 nucleotides. One type of DR was found in CRISPR of these isolates. The number of spacers in CRISPR ranges from 1 to 10 with various sequences. A total of 55 distinctive spacer sequences were identified in 78 isolates. Among them, 33 sequences were found unique in this study. In addition, the CRISPR genotyping had higher the Simpson’s index of diversity value than that from flaA nucleotide typing. The results of our study show the CRISPR genotyping on C. jejuni may be complementary to the other genotyping methods.

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Acknowledgements

We thank Susan Q. Brooks and Manju Amin of Poultry Microbiological Safety and Processing Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA, USA for the technical supports. Amal Awad, an Egyptian Junior Scientist, was supported by the U.S.—Egypt Science and Technology Joint Fund, Cairo, Egypt. This study was supported by the USDA Agricultural Research Service CRIS Project No. 6040-32000-071-00D. Mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture, which is an equal opportunity provider and employer.

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Authors and Affiliations

  1. Poultry Microbiological Safety and Processing Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, 950 College Station Road, Athens, GA, 30605-2720, USA

    Hung-Yueh Yeh & Amal Awad

  2. Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt

    Amal Awad

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  1. Hung-Yueh Yeh
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  2. Amal Awad
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HY and AA conceived, designed and performed the experiments, analyzed the data, and wrote the manuscript.

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Yeh, HY., Awad, A. Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Curr Microbiol 77, 1647–1652 (2020). https://doi.org/10.1007/s00284-020-01965-w

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