Complete genomic sequence and analysis of β2 toxin gene mapping of Clostridium perfringens JXJA17 isolated from piglets in China

Clostridium perfringens (Cp) is a ubiquitous opportunistic pathogen of humans and animals in the natural environment and animal intestines. The pathogenicity of Cp depends on the production of toxins encoded by genes on the chromosomes or plasmids. In contemporary literature, there is no clear consensus about the pathogenicity of CpA β2 toxin. To analyze the homology of the genome of piglet source CpA and its β2 toxin, we sequenced the whole genome of strain JXJA17 isolated from diarrhea piglets using the Illumina Miseq and Pacbio Sequel platforms. The genome was composed of a circular chromosome with 3,324,072 bp (G + C content: 28.51%) and nine plasmids. Genome and 16S rDNA homology analysis revealed a close relation of the JXJA17 strain with the JGS1495, Cp-06, Cp-16, and FORC_003 strains. These strains were isolated from different samples and belonged to different toxin-types. JXJA17 strain was found to carry two toxin genes (plc and cpb2). In contrast to other Cp strains, the cpb2 of JXJA17 was located on a large plasmid (58 kb) with no co-localization of other toxin genes or antibiotic resistance genes. Analysis of JXJA17 genome homology and its cpb2 would facilitate our further study the relationship between β2 toxin and piglet diarrhea.

all 205 Cp strains included in the NCBI genome database was constructed (Fig. 2). The dendrogram showed that the JXJA17 strain was more closely related to the JGS1495 (NZ_ABDU01000042.2, 84 contigs) strain and clustered with Cp-06(NZ_JAALNH010000001.1, 100 contigs) and Cp-16 (NZ_JAALMX010000001.1, 77 contigs) strains. However, the full genome sequences of these three C. perfringens were not de novo sequenced, and just spliced by some scaffolds and contigs. Therefore, the phylogenetic tree analysis is based on 16SrDNA genes and house-keeping genes between JXJA17 and 19 Cp strains complete genome using the i-sang platform (https :// www.i-sange r.com/) 13 (Fig. S2 and S3). The dendrogram showed that JXJA17 strain was more closely related to the Cp FORC_003 (NZ_CP009557.1). Therefore, we compared and analyzed the general features of four C. perfringens genomes, and the results are shown in Table 1. Surprisingly, strain JXJA17 and its four genetically related strains were isolated from different hosts and environments, and their toxinotype were different as well. If a comparison were based on de novo sequencing, toxin genotyping of JXJA17 and Cp FORC_003 would be type A.
Collinearity analyses of plasmid carrying cpb2 of JXJA17. The sequencing results showed consensus cpb2 (798 bp) in JXJA17. It (from 41,169 to 41,961 bp) was individually located on a large plasmid p1 (58,000 bp) and there was no co-carriage with other toxin (such as enterotoxin or ε toxin) genes and antibiotic resistance genes. In contrast to other plasmids carrying cpb2, no Tcp conjugation locus or insertion sequence (IS) was found in plasmid p1 of JXJA17 (Table 2 and Fig. 3). Collinearity analysis of plasmid carrying cpb2 was conducted between JXJA17 and eight reference plasmids which were all cpb2 encoding plasmids among the 50 Cp plasmids included in the NCBI. The results (Fig. 4) showed sharing of eight distinct locally collinear blocks (LCBs) by these plasmids, and cpb2 was located in the blue LCB.

Antibiotic resistance genes. Screening of the genome sequences against the Comprehensive Antibiotic
Resistance Database (CARD) identified 21 antibiotic resistance genes and 18 antibiotic targets in the JXJA17 strain. This strain was most likely to develop resistance to fluoroquinolone, daptomycin, tetracycline, streptomycin, and erythromycin. The 21 antibiotic resistance genes (Table S4) included one aminocoumarin gene, three fluoroquinolone genes, four daptomycin genes, two tetracycline genes, one transmembrane protein gene, one dihydropteroate synthase gene, two elfamycin genes, one streptomycin aminoglycoside adenylyl transferase gene, one UDP-glucose-6-dehydrogenase gene, one Van response regulator gene, one two-component response regulator (vanRF) gene, and three unknown product genes. In addition, the gene encoding erythromycin resistance was found in the JXJA17_p4 plasmid. However, no antibiotic resistance gene was carried with cpb2 in plasmid JXJA17_p1.

Discussion
The genomic B LAST in NCBI and homology analysis of 16S rDNA revealed high homology of JXJA17 with CP-06, CP-16, JGS1495, and FORC_003 obtained from different hosts and geographical regions. The results indicated no significant correlation of the strains with the host sources, toxinotypes, or geographical distribution. Homology analysis based on 16S rDNA sequencing is a common method for C. perfringens taxonomy 14,15 . Among the 19 de novo sequenced C. perfringens included in the i-sang platform, the JXJA17 strain was most closely related to the Cp FORC_003 based on 16S rDNA homology analysis. The toxin genotyping of JXJA17 and  www.nature.com/scientificreports/ Cp FORC_003 were type A, notwithstanding the fact that Cp FORC_003 was isolated from aquarium water in South Korea. Based on the above aspects, there is no significant correlation of the strains with the host sources, toxinotypes, or geographical distribution. The location of the pathogenic toxin on plasmids may be a key characteristic that facilitates the spread of C. perfringens.
The complete genome sequences of strain JXJA17 was found to contain nine extrachromosomal elements or plasmids. One of the plasmids contains the cpb2, and another plasmid carries the erythromycin resistance gene. Many plasmid-containing strains have recently been reported. Profeta et al. reported two plasmids in the C. perfringens netB positive strain 2016TE7641_69 isolated from the intestine of a diseased turkey in Italy 16 . In C.    18 . The strain JP55 isolated from foal was found to contain five plasmids, and their G + C content was lower than that in the chromosome 19 .
The G + C content of chromosomes from the above reported strains was higher than that in their respective plasmids. The JXJA17 strain had nine plasmids, it was also consistent with the rule that the G + C content of each plasmid is lower than that of its chromosome. In this study, ten known virulence related genes were found in JXJA17. α toxin is the most toxic extracellular enzyme generated by C. perfringens type A 20 . This toxin is produced by all C. perfringens types and is essential for virulence. Furthermore, its core protein, phospholipase C, hydrolyzes phosphatidylcholine, and sphingomyelin, which are key constituents of eukaryotic cell membranes 21 . This toxin causes diseases such as myonecrosis 22 . The soluble toxins perfringolysin O and phospholipase C can cause host cell lysis and exhibit synergistic effects in C. perfringens-mediated gas gangrene 23 . β2 toxin is a very interesting compound. Some studies have indicated its lethal toxicity. Other reports indicate that β2 is an α auxiliary toxin 24,25 . Therefore, we are interested in the characteristics of β2 toxin from piglets CpA. In the JXJA17 strain, the size of plasmid carrying cpb2 was 58 kb. This is consistent with previous studies in which the size of the plasmids encoding β2 toxin was 45-97 kb. Among the fifty plasmids of C. perfringens included in the NCBI, only eight plasmids contain cpb2, including one recombinant plasmid (pCpb-CP1) 26 . Of the seven natural plasmids, four carried cpb2 along with another toxin (enterotoxin or ε toxin) and one plasmid was conjugated with antibiotic resistance gene. The plasmids pJFP55G and pJFP838D carrying the enterotoxin gene were from Cp JP55 and JP838 strains isolated from horses and dogs with necrotizing enteritis, respectively 19 . The plasmid pCPF5603 was from the Cp F5603 isolated from sporadic diarrhea (SPOR) 27 . The plasmid pCP8533etx carrying ε toxin was from CpB NCTC8533B4D. However, in JXJA17_p1, there were no other toxin genes, insertion sequence, antibiotic resistance gene, or Tcp conjugation locus conjugated with cpb2. By comparing these plasmids, JXJA17_p1 was most similar to plasmid pCP13, which was isolated from soil bacteria that cause gas gangrene. Plasmid pCP13 is a new family of conjugative toxin plasmids of C. perfringens strain 13 28 . CpCna, which encodes a putative collagen binding protein in the plasmid pCP13, is a potential virulence factor of porcine enteritis caused by C. perfringens 7 . The plasmid JXJA17_p1 contains the Pcp conjugation locus and CpCna. In addition, CRISPR is a prokaryotic immune system that recognizes foreign genes and silences their expression 29 . One CRISPR array predicted in the genome of JXJA 17 may lead to the sustained expression of toxin genes and their products. Further studies should investigate whether CRISPR in the genome would affect the expression of the toxin on JXJA17 plasmids.
In summary, we sequenced the complete genome and nine associated plasmids of the C. perfringens strain JXJA17. On 16SrDNA and whole-genome sequence analysis, the strain JXJA17 exhibited high homology with JGS1495, Cp-06, Cp-16, and Cp FORC_003. These strains were isolated from different hosts and environments, and their toxinotype were different as well. Public health authorities must be vigilant of the fact that C. perfringens carry multiple plasmids that harbor pathogenic toxin genes and are easily transmitted. Moreover, cpb2 in JXJA17 was located on plasmid p1, and was independent of other toxins, antibiotic resistance genes, or insertion sequences. Plasmid JXJA17_p1 contains no Tcp but Pcp conjugation locus, which is a new family of conjugative toxin plasmids in C. perfringens 28 . Analysis of JXJA17 genome homology and its cpb2 gene would facilitate our further study concerning the relationship between β2 toxin and piglet diarrhea.

Methods
Isolation and DNA extraction of Clostridium perfringens JXJA17. The study was approved by the Institution Animal Care and Use Committee of Jiangxi Agricultural University and performed according to its guidelines. All the piglets involved in the study were obtained after informed consent of the pig farm owner. We obtained the rectal contents by dipping rectal contents directly with sterilized cotton swabs through the anus of piglets. JXJA17 is a CpA strain isolated from the rectal contents of diarrhea piglets in the Jiangxi region of China and identified by biochemical tests, 16S rDNA sequencing, and toxin subtyping. A pure culture of the C. perfringens JXJA17 strain was obtained utilizing tryptose-sulfite-cycloserine agar (Haibo Biological Engineering Co. Ltd., Qingdao, China). Genomic DNA was extracted and sequenced by the Shanghai Personal Biotechnology Co. Ltd (Shanghai, China). The whole-genome sequence data was deposited in GenBank (Accession number CP028149).

Homology analysis.
The complete genome sequence and annotation of 205 Cp strains were obtained from the GenBank database based on the whole-genome sequence of the JXJA17 strain. These strains were isolated from different hosts and the surroundings. To construct a dendrogram, we performed a homology analysis of 206 Cp in the NCBI database based on approximately 2967 genes of the JXJA17 using the genomic BLAST; the phylogenetic tree of 16S rRNA genes or house-keeping gene of 19 Cp which de novo sequencing for complete genome using the i-sang platform (https ://www.i-sange r.com/). A list of the general features of the C. perfringens genomes which exhibit homology with JXJA17 was built.
Prediction of virulence and antibiotic resistance genes. Potential virulence and antibiotic resistance genes in the complete genome were predicted using the virulence factors database (VFDB, http://www.mgc. ac.cn/VFs/main.htm) and the comprehensive antibiotic resistance database (CARD, http://arpca rd.mcmas ter. card) 32 , respectively. Using BLAST (ftp://ftp.ncbi.nlm.nih.gov/blast /) to predict genes in the genome that are associated with virulence factors and antibiotic resistance, E-value threshold were set to 1e-6, more than 45% amino acid sequence consistency, and the ratio of the length of the sequence alignment to the length of the sequence not less than 70%.