Detection and distribution of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter jejuni isolates from chicken livers

Authors: Yeh, Hung-Yueh; Cox Jr, Nelson; Hinton Jr, Arthur; Berrang, Mark

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2024
Publication Date: 2/19/2024
Citation: Yeh, H., Cox Jr, N.A., Hinton Jr, A., Berrang, M.E. 2024. Detection and distribution of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter jejuni isolates from chicken livers. Journal of Food Protection. 87(4):100250. https://doi.org/10.1016/j.jfp.2024.100250.
DOI: https://doi.org/10.1016/j.jfp.2024.100250

Interpretive Summary: Campylobacter jejuni, the leading foodborne bacterial pathogen, causes human gastroenteritis worldwide, and disease outbreaks have been linked to consumption of undercooked broiler livers. Application of phages during poultry production is an alternative approach to mitigate the Campylobacter burden. To make this approach work, whether the presence of the phage genes in the C. jejuni genome (located at the CRISPR) is critical. Therefore, in this study, we explored the distribution of the CRISPR arrays from our C. jejuni isolates from chicken livers. A total of 181 C. jejuni isolates were identified from poultry livers and used for this CRISPR study. Genomic DNA of C. jejuni isolates were extracted, and the CRISPR type 1 sequences were amplified by PCR. The PCR products were purified and sequenced by the Sanger method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder program. Further, the spacer sequences were submitted to the CRISPRTarget to identify the potential bacteriophage types. A total of 155 (87%) C. jejuni isolates were detected that harbored the CRISPR sequences, and one type of DR was identified in all 155 isolates. The lengths of the CRISPR loci ranged from 97 to 431 nucleotides. The numbers of the spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates. Analysis of these spacer sequences indicated that the sequences could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases shows the most spacer sequences were homology to Campylobacter phage DA10. The results of our study provide important information for development of phage use in the study of Campylobacter mitigation during poultry production.

Technical Abstract: Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide; disease outbreaks have been linked to consumption of undercooked broiler livers. Application of bacteriophages during poultry production is an alternative approach to mitigate the Campylobacter burden. To make this approach effective, understanding the presence of the bacteriophage sequences in the CRISPR spacers in C. jejuni is critical as they may confer bacterial resistance to bacteriophage treatment. Therefore, in this study, we explored the distribution of the CRISPR arrays from 178 C. jejuni isolated from chicken livers. Genomic DNA of C. jejuni isolates was extracted, and CRISPR type 1 sequences were amplified by PCR. Amplicons were purified and sequenced by the Sanger dideoxy sequencing method. Direct repeats (DR) and spacers of CRISPR sequences were identified using the CRISPRFinder program. Further, spacer sequences were submitted to the CRISPRTarget to identify potential homology to bacteriophage types. A total of 155 (87%) C. jejuni isolates were found to harbor CRISPR sequences; one type of DR was identified in all 155 isolates. The lengths of the CRISPR loci ranged from 97 to 431 nucleotides. The numbers of spacers ranged from one to six. A total of 371 spacer sequences were identified in the 155 isolates. Analysis of these spacer sequences indicated that the sequences could be grouped into 51 distinctive individual sequences. Further comparison of these 51 spacer sequences with those in databases showed that most spacer sequences were homologous to Campylobacter bacteriophage DA10. The results of our study provide important information relative to development of an effective bacteriophage treatment to mitigate Campylobacter mitigation during poultry production.