CRISPR analysis in Salmonella infantis strains of poultry origin

Salmonella species are widespread in the world while remaining one of the top reasons for food sourced infections. Therefore, fast and accurate diagnosis becomes crucial. With this thesis study, CRISPR region analysis of S. Infantis strains was done to provide information on usability of these sequences by pointing out differences according to regional and breeding type factors. For the study, a total of 100 S. Infantis strains were used consisting of 60 sourced from broilers, 30 sourced from laying hens and 10 sourced from turkeys that were isolated and identified between 2018 and 2020 from Marmara, Aegean and Eastern Black Sea based corporations belonging to the Culture Collection of Ankara University Faculty of Veterinary Medicine, Department of Microbiology. CRISPR sequences were amplified by PCR using appropriate primers, and the strains resulting from the band were sent for sequence analysis. CRISPR region analysis was performed using NCBI-Blast and CRISPRCasFinder databases. From the sequences uploaded to the CRISPRCasFinder program for analysis of CRISPR1 and CRISPR2 gene regions, 17 CRISPR1 and 12 CRISPR2 regions were identified. There are 118 spacers in the CRISPR1 gene region of broiler isolates, 44 spacers in the CRISPR1 gene region of laying hens isolates and in total, 162 spacers in CRISPR1 were detected in this thesis study. In the CRISPR2 gene region, a total of 70 spacers were detected, 43 in broiler isolates and 27 in laying hen isolates. When CRISPR sequences are compared proportionally, the rate of CRISPR1 in broiler isolates is 21.6% and CRISPR1 is 13.3% in laying hen isolates while the rate of CRISPR2 in broilers is 11.6% and 16.6% in laying hen isolates. CRISPR sequence was not detected in turkey strains. The conserved repeat sequence in most CRISPR1 gene regions is determined as 5'- CGGTTTATCCCCGCTGGCGCGGGGAACAC-3' and is 29 bp long. There is a single nucleotide difference in 6 samples containing CRISPR1. In CRISPR2, it is the only isolate containing the conserved repeat sequence 5'-CGGTTTATCCCCGCTGGCGCGGGGAACAC-3', while the others vary between 23 and 31 bp. In this thesis study, many spacer sequences compatible with phage and plasmid were identified. In the light of the data obtained as a result of this study, it has been observed that the polymorphisms seen in S. Infantis CRISPR sequences can be evaluated and used in typing studies, and according to the spacer analysis results, information can be obtained about the phages, plasmids and other genetic elements to which the bacteria are exposed. In order to better understand the CRISPR regions of S. Infantis isolates, it would be useful to continue diversifying and monitoring them with more samples from different geographical regions and based on different breeding types. Additionally, evaluating Salmonella CRISPR sequences together with Cas genes will provide a better understanding. Keywords: CRISPR, Molecular Typing, PCR, Poultry, Salmonella Infantis