Genome and Transcriptome Analyses of Salmonella enterica Virchow FORC_038 Isolated from Raw Chicken Meat

Abstract

Salmonella enterica is an ever-present worldwide public health threat and causes a range of diseases in humans such as gastroenteritis and invasive systemic infection. Especially, Salmonella enterica serovar Virchow shows antibiotic resistance and has been identified as an important cause of poultry meat outbreaks. The genome of S. Virchow FORC_038, which was obtained from raw chicken meat was completely sequenced using Illumina MiSeq and PacBio RS II platform. The genome consists of a circular chromosome of 4,938,076 bp with a GC content of 51.92%. The chromosome contains 4,632 open reading frames, 22 rRNAs, and 85 tRNA genes. BLAST analysis against VFDB (Virulence Factor Database) identified that the genome of FORC_038 contains the genes encoding SPI-1 (Salmonella pathogenic island 1) and SPI-2 effectors. Average nucleotide identity (ANI) analysis of FORC_038s genome with 40 other completely sequenced Salmonella genomes showed that the genome of FORC_038 is most closely related to those of S. Infantis 1326/28, isolated from chicken in 1973, UK. Comparative genome analysis of FORC_038 and S. Infantis 1326/28 revealed that the gene encoding to beta-lactamase, which contributes to resistance of beta-lactam antibiotics, and type III secretion system Salmonella outer protein (SopE), was also included on the genome of FORC_038 (FORC38_1065, FORC38_2974). To examine the role of SopE, the sopE mutant was constructed by integration of chloramphenicol resistance cassette into the chromosome of FORC_038 using red-mediated homologous recombination. Compared to the wild type, the sopE mutant displayed significantly reduced invasiveness toward the HeLa cells, indicating that SopE is essential for the invasion of S. Virchow to the host cells. Additional genomic analysis revealed that genes related to resistance to β-lactams (FORC38_0096, 1541, 3615), tetracyclines (FORC38_1059), aminoglycosides (FORC38_1321), macrolides (FORC38_3090, 3091), and multiple antibiotic resistance protein (FORC38_1327, 1328, 2505, 2506, 2507, 2508) were widely disseminated on the chromosome of FORC_038, implying that the strain might be resistant to various antibiotics. In silico prediction was phenotypically confirmed by the subsequent Kirby-Bauer Disk Susceptibility Test, which indicated that FORC_038 is resistant to the antibiotics bona fide. To additional analyze total mRNA profiles of the strain upon contact with chicken breast in raw chicken part, which is highly consumed in Korea, RNA-Sequencing was used. After that, transcriptome analysis hinted that S. Virchow mainly uses the raw chicken meat as a reservoir for growth and survival according to up regulation of genes related to adhesion, iron uptake, amino acid metabolisms and down regulation of amino acid synthesis, carbohydrates metabolism. In addition, all of the SPI1 effector encoding genes that are major factors of Salmonella virulence and host cell invasiveness were upregulated including sopE. The results suggested the FORC_038 strain may be pathogenic and there is high risk of infection when humans consume chicken meat contaminated with FORC_038. Providing new insight on S. Virchow, this report will support further research in prevention and epidemiological investigation of Salmonella outbreaks in South Korea.