Salmonella is one of the food poisoning bacteria that can cause disease in people when they consume contaminated food (Ehuwa et al., 2021). This pathogen is found in various foods such as chicken, egg, and vegetable, and it is necessary to study Salmonella strain because it continuously causes foodborne outbreak (Kunwar et al., 2013). The serotypes of Salmonella have been classified by O and H antigens, and more than 2500 serotypes have been described (Zhang et al., 2015). Among the known serotypes, Salmonella enterica serotype Enteritidis (antigenic formula: 1,9,12:g,m:-) is the main cause of salmonellosis worldwide, but the traditional subtyping methods struggle to differentiate the serotype Enteritidis due to their high genetic homogeneity compared to many other serotypes (Olson et al., 2007; Banerji et al., 2020). In order to overcome these challenges new genomic typing tools such as whole-genome sequencing with next generation sequencing (NGS) have become popular.

In this study, we confirmed the complete genome sequence of Salmonella serotype Enteritidis MFDS1018147. According to Food Public Code provided by the Ministry of Food and Drug Safety in Korea, the strain was isolated and identified from a braised burdock linked to food poisoning accidents reported in kindergarten in Daejeon city, South Korea in 2020. Salmonella serotyping was performed according to the White-Kauffmann-Le Minor scheme using somatic (O) (Denka Seiken) and flagella (H) antisera (Becton & Dickinson).

For obtaining high-quality genomic DNA, the bacterium was incubated in tryptic soy agar at 37℃ and extracted using a Genomic DNA prep kit for bacteria (Bioneer). After measuring the quantity and quality of genomic DNA, it was used to prepare a sequencing library. To obtain the complete genome sequence of the bacterium, sequencing was performed using the Illumina MiSeq and Oxford Nanopore MinION platforms. The Illumina sequencing libraries were prepared by tagmentation and amplification according to the manufacturer’s protocols (Illumina). The libraries were quality assessed using the QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies), and then sequenced using the Illumina system. For nanopore sequencing, the Oxford Nanopore Rapid Barcoding Kit (Oxford Nanopore Technologies) was used for library construction.

Both Illumina sequencing data and nanopore sequencing data were processed and de novo assembled with Unicycler v0.4.8 by the Pathosystems Resource Integration Center (PATRIC) v3.6.12 web server. The completeness of genome assembly was evaluated as 100% by CheckM software (Parks et al., 2015). The genome was annotated by using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). The virulence-associated genes were predicted using the Virulence Factor Database (VFDB) (Liu et al., 2019), PATRIC_VF (Snyder et al., 2007), and the Comprehensive Antibiotic Resistance Database (McArthur et al., 2013). The complete genome sequence of the Salmonella strain MFDS 1018147 was 4,679,367 bp, with a GC content of 52.17% (Table 1). 4,497 genes were predicted by PGAP. In the similarity of the 16S rRNA gene sequence using NCBI BLASTn, the MFDS1018147 exhibited 100% identity to Salmonella enterica subsp. enterica. To calculate the average nucleotide identity (ANI) values, we selected strains from the same subspecies and used a web tool (Rodriguez-R and Konstantinidis, 2016). ANI values for the MFDS1018147 were calculated using Enteritidis strain R17.1476 (GenBank: CP100724.1), Enteritidis strain SE006 (GenBank: CP099973.1), Gallinarum strain SCPM-O-B-4548 (GenBank: CP088142.1), Worthington strain OLF-FSR1 (GenBank: CP051270.1), and Indiana strain SI85 (GenBank: CP050779.1). The ANI values were 99.98%, 99.98%, 99.91%, 98.66%, and 98.66% respectively. The result indicates that the MFDS1018147 is closely similar to Enteritidis. The serotype of the MFDS1018147 was confirmed as detecting Enteritidis specific marker, sdf gene by Seqsero2 v1.1.0 (Zhang et al., 2019). Salmonella pathogenicity islands (SPI) were known to confer virulence traits (Marcus et al., 2000). The genome of MFDS1018147 contained SPI genes such as invA (OF824_ 04750), hilA (OF824_04850), sipA (OF824_04820), sopB (OF824_14115), sopD (OF824_04500), ssrA (OF824_05630), and ssaR (OF824_10580) (Table 2). This genome information can be utilized as useful data to understand food poisoning pathogen and foodborne accidents.

Table 1

Genomic features	Value
Contig	2
Genome size (bp)	4,679,367 (Chromosome) 59,372 (Plasmid)
GC content (%)	52.17
Total number of CDSs	4,497
tRNA genes	85
rRNA genes	23

Table 2

Classification	Gene(s)	Function	Reference
Salmonella pathogenicity islands (SPI)	invA	Gene encoding an effector protein related to delivery of type III secretion system.	Marcus et al. (2000)
	hilA	Gene encoding a transcriptional activator of SPI genes	Marcus et al. (2000)
	sipA	Gene encoding Salmonella invasion protein. The protein causes destabilizing actin filaments of host cells by binding to actin in the host cell, directly.	Marcus et al. (2000)
	sopBD	Genes related to virulence factors. sopB encodes an inositol phosphate which enhances the secretion of the chloride.	Marcus et al. (2000)
	ssrA	Gene encoding a membrane-located sensor kinase which is involved in two-component system with SsrB. The system activates type III secretion system and effector proteins.	Marcus et al. (2000); Tomljenovic-Berube et al. (2010)
	ssaR	Gene related to translocation of effectors to the host cell via type III secretion system. These effectors involved in the processes of invasion, internalization, intracellular proliferation and survival.	Sever and Akan (2019)

Nucleotide sequence accession number(s)

Nucleotide sequence accession numbers. The complete genome sequence of Salmonella Enteriditis MFDS1018147 has been deposited at the NCBI GenBank database under the accession numbers CP110220.1 (chromosome, MFDS1018147) and CP110221.1 (plasmid, pMFDS1018147), and the strain has been deposited in the Korean Culture Collection for foodborne Pathogens under the strain number MFDS1018147.

살모넬라는 오염된 식품을 섭취하였을 때 식중독을 일으키는 병원균 중 하나이다. 본 연구에서는 2020년 대전 유치원에서 발생한 식중독 사고의 원인 식품으로 추정되는 우엉조림으로 분리된 Salmonella strain (MFDS1018147)의 유전체 분석을 진행하였다. Salmonella serotype Enteritidis MFDS1018147는 4,679,367 bp 길이의 chromosome과 59,372 bp 길이의 plasmid로 구성되어 있으며, 각각의 G + C contents는 52.2%와 52.0%로 확인되었다. 유전자 예측 결과, 4,497개의 CDS, 85개의 tRNA, 23개의 rRNA를 가지고 있는 것으로 확인되었다.

This research was financially supported by the Ministry of Food and Drug Safety, Republic of Korea (20161MFDS030, 21162MFDS027).

The authors have no conflict of interest to report.