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The complete genome sequence of Clostridium perfringens MFDS1012647, isolated from a barbeque
Korean J. Microbiol. 2022;58(4):327-329
Published online December 31, 2022
© 2022 The Microbiological Society of Korea.

Soohyun Sung1†, Suhyun Noh2†, Eunsu Ha2, Woojung Lee1, Eun Sook An1, Seung Hwan Kim1, Jinho Choi2*, and Soon Han Kim1*

1Food Microbiology Division, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea
2Sanigen Co., Ltd, Anyang 14059, Republic of Korea
Correspondence to: *(J. Choi) E-mail: cjh@sanigen.kr; Tel.: +82-10-3211-8406; Fax: +82-2-573-3134 / (S.H. Kim) E-mail: lambndog@korea.kr; Tel.: +82-43-719-4301; Fax: +82-43-719-4300
These authors contributed equally to this work.
Received October 14, 2022; Revised December 15, 2022; Accepted December 16, 2022.
Abstract
Clostridium perfringens is a bacterium that cause food poisoning outbreaks worldwide. This study presents the complete genome sequence of the C. perfringens strain MFDS1012647, isolated in 2018 from the barbeque of a cafeteria in Seoul, South Korea. The MFDS1012647 genome sequence contains 2,954,425 bp chromosomal DNA, a 61,476 bp plasmid, and a 28,917 bp plasmid, with 28%, 24.5%, and 26.5% GC contents, respectively. Gene prediction revealed that this strain possesses 2,668 coding sequences (CDSs), 94 tRNAs, and 30 rRNAs in the genome.
Keywords : Clostridium perfringens, barbeque, complete genome
Body

Clostridium perfringens is a spore-forming, anaerobic, Gram-positive, rod-shaped bacterium belonging to the phylum Firmicutes (Shimizu et al., 2002; Johansson et al., 2006; Wong et al., 2014) that inhabits diverse environments such as soil, sewage, and animal intestines (Minton et al., 2016). Although C. perfringens does not invade healthy cells, it acts as a pathogen by producing enterotoxin, alpha-toxin and various enzymes (nanH, nagH, nagJ); it is a common cause of food poisoning outbreaks. In this study, we determined the complete genome sequence of C. perfringens collected in Seoul, South Korea, in 2018.

Clostridium perfringens MFDS1012647 was identified during a food poisoning investigation by the South Korean Ministry of Food and Drug Safety (MFDS). The MFDS1012647 was isolated from the barbeque of a cafeteria and detected in the patients suffering from food poisoning. The MFDS1012647 isolated was incubated anaerobically in tryptic soy agar medium at 37℃ for 24 h. The total genomic DNA of MFDS1012647 was extracted using the Genomic DNA Prep Kit for Bacteria (Bioneer). The genomic DNA qualitatively and quantitatively was measured using a NanoDrop 2000 UV-visible spectrophotometer (Thermo Fisher Scientific) and a QubitTM 3.0 Fluorometer (Invitrogen) with dsDNA HS Assay Kit, respectively. DNA libraries were constructed using a Nextera DNA Flex Library Prep Kit (Illumina). The concentration and quality of the libraries were determined using a Qubit and 2100 Bioanalyzer instrument (Agilent Technologies). Sequencing was performed using a MiSeq sequencing system (Illumina) on a paired-end library using the MiSeq Reagent Kit v3 (600 cycles). After sequencing, individual sequence reads were analyzed using FastQC-v.0.11.8. The illumina sequencing yielded 2.5 M reads, including 637 Mb, with 207.9× coverage. The sequencing quality values Q20 and Q30 were 98.33% and 94.93% respectively. For nanopore sequencing, the Oxford Nanopore Rapid Barcoding Kit (Oxford Nanopore Technologies) was used to construct the libraries. Nanopore sequencing data were basecalled on guppy_barcoder v6.0.1. The Nanopore sequencing yielded 101 K reads, including 177 Mb, with 51.8 x coverage (N50, 3 kb). The sequencing mean quality was 11.7.

Both Illumina and nanopore sequencing data were processed and de novo assembled with Unicycler v0.4.8, using the Pathosystems Resource Integration Center (PATRIC) v3.6.12 web server. The complete genome sequence of C. perfringens MFDS1012647 was 3,044,818 bp (GC content 28%). The entire genome contains 2,668 coding sequences, 94 tRNA genes, and 30 rRNA genes (Table 1). The MFDS1012647 genome consists of a circular chromosome of 2,954,425 bp (28% G + C content) and two plasmids (61,476 bp, 24.5% G + C content; 28,917 bp, 26.5% G + C content) (Fig. 1).

Genome features of <italic>C. perfringens</italic> MFDS1012647
Genomic feature Value
Contig 3
Genome size (bp) 3,044,818
GC content (%) 28
CDSs 2,668
rRNAs (5S, 16S, 23S) 30 (10, 10, 10)
tRNAs 94


Fig. 1. The complete genome map of MFDS1012647. The circular genome visualization of the bacterium was performed using the Proksee (https://proksee.ca/) web server. The second inner circle with the green and purple histogram shows the GC skew +/− whereas the fourth inner circle with the black histogram indicates the GC content. (A) The chromosome genome map of MFDS1012647. (B) The first plasmid of MFDS1012647 (pMFDS1012647-1) genome map. (C) The second plasmid of MFDS1012647 ((pMFDS1012647-2) genome map.

The genome was annotated on the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016; Haft et al., 2018; Li et al., 2021), with a total of 2,850 genes were predicted. Prediction of the virulence-associated genes was conducted using the Virulence Factor Database (Liu et al., 2019), PATRIC_VF (Snyder et al., 2007), and the Comprehensive Antibiotic Resistance Database (McArthur et al., 2013) on the PATRIC web server. The virulence associated genes predicted are enterotoxin (cpe), alpha-toxin (plc), and exo-alpha-sialidase (nanH), hyaluronidase (mu-toxin) (nagH and nagJ). Additionally, the bacterium had putative antibiotic resistance genes (tetA(P) and tetB(P)). The complete C. perfringens genome reported here will be useful for future investigations regarding the understanding of foodborne pathogens, and provides the genetic basis for a more detailed analysis of virulence factors.

Nucleotide sequence accession number(s)

Nucleotide sequence accession numbers. The complete genome sequence of Clostridium perfringens MFDS1012647 has been deposited at the NCBI GenBank database under the accession numbers CP106926 (chromosome, MFDS1012647), CP106927 (the first plasmid, pMFDS1012647-1) and CP106928 (the second plasmid, pMFDS1012647-2). The strain has been deposited in the Korean Culture Collection for foodborne Pathogens under the strain number MFDS1012647.

적 요

클로스트리디움 퍼프린젠스는 전 세계적으로 식중독을 일으키는 세균 중 하나이다. 본 연구에서는 2018년 서울의 대학교 기숙사 식당에서 일어난 식중독 사고의 원인식품으로 추정되는 바비큐 구이로부터 분리된 Clostridium perfringens (MFDS 1012647)의 유전체 분석을 수행하였다. Clostridium perfringens MFDS1012647는 한 개의 chromosome (2,954,425 bp)과 두 개의 plasmid (61,476 bp; 28,917 bp)로 구성되어 있었고, 각각의 G + C contents는 28%, 24.5%, 26.5%로 확인되었다. 또한, 유전체에서 2,668개의 단백질 코딩 유전자(CDSs), 94개의 tRNA, 30개의 rRNA를 확인하였다.

Acknowledgments

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

Conflict of Interest

There are no conflicts of interest to declare.

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