Clostridium perfringens CBA7123 (=KCCM 43242) was isolated from the feces of a 73-year-old man, and a pure culture was obtained using serial dilution. The colony was cultured anaerobically in ATCC medium no. 2840, modified Eggerth-Gagnon medium (10 g peptone, 4 g Na2HPO4 2H2O, 2 g porcine gastric mucin, 50 ml sheep blood, and 15 g agar per liter) at 37  C for 24 h. The total genomic DNA of strain CBA7123 was extracted using QuickGene DNA tissue kit S (Kurabo, Japan) and the G-spin total DNA extraction kit (iNtRON Biotechnology, Korea). The purity, quality, and quantity of genomic DNA were measured using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) as described the manufacturer's instruction.
Detailed genome sequencing was performed as described previously. Briefly, the genomic DNA of strain CBA7123 was sheared according to the PacBio 20-kb Template Preparation using BluePippin Size-Selection System protocol, and SMRTbell library was prepared using P6-C4 chemistry (Pacific Biosciences, USA). The sequences were obtained using PacBio RS II system (Pacific Biosciences) as following an instruction of the manufacturer. The 150,292 reads were generated with 7090 bp of average read length by the PacBio RS II system from one SMRT cell.
De novo assembly of the genome sequence was performed using Hierarchical Genome Assembly Process (HGAP) version 2 software, with default parameters supported by PacBio SMRT Analysis ver. 2.3.0. rRNA and tRNA of the assembled sequence were identified using RNAmmer 1.2 and tRNAscan-SE 1.21, respectively. Genes were predicted using Glimmer3 of Rapid Annotation using Subsystem Technology (RAST) server (http://rast.nmpdr.org), and functional gene annotations were performed using the SEED, Clusters of Orthologous Groups (COG, http://www.ncbi.nlm.nih.gov/COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) databases. PathogenFinder 1.1 and ResFinder 2.1 were used to estimate the pathogenicity and antimicrobial resistance genes, respectively. The virulence factors were searched using the Basic Local Alignment Search Tool (BLAST) in the virulence factors of pathogenic bacteria database with default parameters and predicted with zero e-value.
The C. perfringens strains for comparative genomic analysis were selected using Microbial Nucleotide BLAST in the NCBI complete genome database. The four strains with BLAST total scores over 5.5e+06 were selected: strains FORC 003, JP55, FORC 025, and JP838. To compare the genomic structures between strain CBA7123 and these four strains, the progressive alignment algorithm in MAUVE multiple genome alignment software 2.4.0 was used. The OrthoANI algorithm was used to analyze genomic relatedness between strain CBA7123 and other C. perfringens species. OrthoANI percentages were calculated and a phylogenetic tree was constructed, as described by Lee et al.. Orthologs between strain CBA7123 and the reference strains were predicted and mapped using the reciprocal best hit method in UBLAST. Pan-genome orthologous groups (POGs) were estimated using the EzBioCloud Comparative Genomics Database (http://cg.ezbiocloud.net/), and their presence was calculated using the Jaccard coefficient. UPGMA clustering was then used to show clustering between strain CBA7123 and the reference strains as a dendrogram, based on the presence or absence of gene content. The intersections and differences of POG sets of the different strains were visualized as a Venn diagram, using the jvenn program.
The single colony of strain CBA7123 was transferred more than three times to obtain a pure culture and confirmed using a variable pressure field emission scanning electron microscope (Chuncheon Center, Korea Basic Science Institute, Korea) (Additional file 1: Figure S1) and a PCR-sequencing approach targeting 16S rRNA gene as described by Min et al..