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

Authors: Yeh, Hung-Yueh; AMAD, AMAL - Mansoura University

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/21/2020
Publication Date: 7/5/2020
Citation: Yeh, H., Amad, A. 2020. Genotyping of Campylobacter jejuni Isolates from Poultry by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). Current Microbiology. 77:1647–1652. https://doi.org/10.1007/s00284-020-01965-w.
DOI: https://doi.org/10.1007/s00284-020-01965-w

Interpretive Summary: 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 Campylobacter jejuni isolated from poultry sources. Ninety-nine Campylobacter jejuni isolates from poultry environments in four different U.S. 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 one 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. The results of our study show the CRISPR genotyping on Campylobacter jejuni may be complementary to the other genotyping methods.

Technical 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 Campylobacter jejuni isolated from poultry sources. Ninety-nine Campylobacter jejuni isolates from poultry environments in four different U.S. 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 one 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. The results of our study show the CRISPR genotyping on Campylobacter jejuni may be complementary to the other genotyping methods.