Detection and Distribution of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in Campylobacter jejuni Isolates from Chicken Livers

Abstract

Campylobacter jejuni is the leading foodborne bacterial pathogen that causes human gastroenteritis worldwide linked to the consumption of undercooked broiler livers. Application of bacteriophages during poultry production has been used as an alternative approach to reduce contamination of poultry meat by Campylobacter. 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 between January and July 2018. 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 (DRs) 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. Even though CRISPR-Cas is reportedly not an active system in Campylobacter, 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 CRISPR loci lengths 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 that 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 the development of an effective bacteriophage treatment to mitigate Campylobacter during poultry production.

Keywords

• Bacteriophages
• Campylobacter jejuni
• Chicken liver
• Clustered regularly interspaced short palindromic repeats
• CRISPR