Genomic Analysis of a Pathogenic Bacterium, Paeniclostridium sordellii CBA7122 Containing the Highest Number of rRNA Operons, Isolated from a Human Stool Sample

Citation: Kim JY, Kim YB, Song HS, Chung W-H, Lee C, Ahn SW, Lee SH, Jung MY, Kim T-W, Nam Y-D and Roh SW (2017) Genomic Analysis of a Pathogenic Bacterium, Paeniclostridium sordellii CBA7122 Containing the Highest Number of rRNA Operons, Isolated from a Human Stool Sample. Front. Pharmacol. 8:840. doi: 10.3389/fphar.2017.00840 Genomic Analysis of a Pathogenic Bacterium, Paeniclostridium sordellii CBA7122 Containing the Highest Number of rRNA Operons, Isolated from a Human Stool Sample


INTRODUCTION
Paeniclostridium sordellii was first isolated by Alfredo Sordelli in 1922 under the proposed name Bacillus oedematis, and was then renamed Bacillus sordellii in 1927 (Hall and Scott, 1927). Two years later, it was classified as Clostridium sordellii (Hall et al., 1929). Recently, this bacterium was reclassified as a species of the genus Paeniclostridium, named P. sordellii comb. nov. (Sasi Jyothsna et al., 2016). P. sordellii is an anaerobic, Gram-stain-positive, spore-forming rod bacterium with flagella. Most strains are non-pathogenic, but some strains have been associated with severe infections of humans and animals. In humans, P. sordellii is mainly associated with trauma, toxic shock, soft tissue skin infections, and gynecologic infections. Despite the serious consequences of infection with P. sordellii, treatment is difficult because of the rapid progression from recognition of the first symptoms to death (Aldape et al., 2006).
In this study, we performed whole-genome sequencing and genomic analysis of strain CBA7122 belonging to P. sordellii, which was isolated from the stool sample of an 85-year-old healthy female residing in the Republic of Korea. This genomic information of P. sordellii CBA7122 should motivate further research on related strains, which may provide new insight into the pathogenesis of P. sordellii toward development of new strategies for the control, prevention, and treatment of life-threatening infections.

MATERIALS AND METHODS
Strain Isolation, Culture, and DNA Extraction Strain CBA7122 was isolated from the stool sample of an 85-year-old healthy female by the standard dilution plating technique on modified Eggerth-Gagnon agar medium (containing per liter of distilled water: 10 g peptone, 4 g Na 2 HPO 4 , 2 g porcine gastric mucin, 50 ml sheep blood, 15 g agar) at 37 • C for 24 h in an anaerobic chamber (Coy Laboratory Products) with an atmosphere of N 2 /CO 2 /H 2 (90:5:5, by volume). Routine cultivation of strain CBA7122 was performed under the same conditions. Genomic DNA of strain CBA7122 was extracted using the QIAamp DNA extraction kit (Qiagen, USA) and QuickGene DNA tissue kit S (Kurabo, Japan), and quantified using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen, USA). The condition of extracted DNA was assessed by agarose gel electrophoresis on a 1% agarose gel.

Genome Sequencing, Assembly, and Annotation
The genome sequencing of strain CBA7122 was performed using a PacBio RS II sequencing platform as described previously . The library based on the genomic DNA of strain CBA7122 was constructed according to the manufacturer's instructions, and sequenced using a Pacific Biosciences RS II instrument. The 150,292 generated reads were filtered and assembled using the HGAP 2 protocol with default parameters in SMRT Analysis version 2.3. Gene prediction and the basic annotation for the assembled genome of strain CBA7122 were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). In brief, 16S rRNAs and 23S rRNAs were predicted using BLASTn, and 5S rRNAs and small ncRNAs were predicted using cmsearch 1.1.1. tRNAscan-SE was used to predict tRNA gene sequences. Coding sequences (CDSs) were detected using GenMarkS+. Functional gene annotations of the genome of strain CBA7122 were performed against various databases, including the catalytic families (CatFam) (Yu et al., 2009), Clusters of Orthologous Groups (COG) (Tatusov et al., 2000), NCBI reference sequence (RefSeq) (O'Leary et al., 2016), and SEED (Overbeek et al., 2014) databases. CRISPRs were confirmed using CRISPRFinder (Grissa et al., 2007). Prophage analysis was performed by PHAST (Arndt et al., 2016).

Comparative Genomic Analysis
To find unique features of the genome of strain CBA7122, the genomes of the following P. sordellii and Paraclostridium bifermentans strains were selected to perform comparative genomic analysis using the NCBI genome database (http:// www.ncbi.nlm.nih.gov/genome/): P. sordellii strains ATCC 9714 T (GCA_000444075.1), VPI 9048 (GCA_000444095.1), and JGS6382 (GCA_000953555.1), and P. bifermentans ATCC 638 T (GCA_000452245.1). Genome similarities between strain CBA7122 and the reference strains were determined using Orthologous Average Nucleotide Identity (OrthoANI) values, and used to reconstruct the phylogenetic tree using the Orthologous Average Nucleotide Identity Tool (Lee et al., 2016). For comparisons at the whole-genome level, the genomes of strain CBA7122 and related strains were aligned using the progressive MAUVE algorithm in the MAUVE multiple genome alignment software 2.4.0 (Darling et al., 2004). Pangenome analysis was performed by BIOiPLUG (Chunlab, Korea).

Virulence Factor Identification
To determine the virulence factors of strain CBA7122, Basic Local Alignment Search Tool (BLAST) was used with the core dataset containing information on genes associated with experimentally verified virulence factors in the virulence factor database (VFDB) (Chen et al., 2016), with the expected e-value 0.0001.

Ethics Statement
The study protocol was approved by the institutional review board of the Theragen ETEX Bio Institute (700062-20160804-JR-005-02). Before the current study, the purpose, experimental procedure, and benefits were fully explained to the participants. Oral consents were obtained from each volunteer and consent procedure was witnessed and documented on the research record.

The Number of rRNA Gene Copies of P. sordellii CBA7122
Interestingly, the genome of strain CBA7122 had 17 rRNA operons, which represented the highest number of rRNA operons known so far in the domain Bacteria. Of all P. sordellii genomes FIGURE 1 | Circular map of the Paeniclostridium sordellii CBA7122 genome. RNA genes (red, rRNA; blue, tRNA), forward and reverse strands (colored according to COG categories) are indicated from the outer fringe to the center. Inner circles show the GC content in yellow and blue and the GC skew is shown with red and green indicating positive and negative values, respectively. This genome map was generated by CLgenomics 1.52 (Chun Lab Inc.). listed in the NCBI genome database, strain CBA7122 had the highest 16S and 23S rRNA genes copies, whereas other genomes had lower 16S (3.7 in average) and 23S (5.5 in average) rRNA genes copies (Supplementary Figure S1). In the case of the number of 5S rRNA genes, all genomes had an average of 13.7 gene copies, and strain CBA7122 had the second most number of gene copies. According to Roller et al. (2016), the number of rRNA operons is known to be associated with these two factors of reproduction, growth rate, and growth efficiency.

Virulence Factors
We identified several known virulence factors in the genome of strain CBA7122, including perfringolysin O (pfoA), sialidase (nanH), thiol-activated cytolysin (ALO), polysialic acid capsule biosynthesis protein SiaC (siaC), Hsp60, 60K heat shock protein HtpB (htpB), capsular polysaccharide synthesis enzyme Cap8D (cap8D), UDP-galactopyranose mutase (cpsI), UDP-glucose 6dehydrogenase (hasB), ATPase EscN (escN), glycosyl transferase CpsE (cpsE), Listeria adhesion protein Lap (lap), ATP-dependent protease (clpE), sigma 54-dependent response regulator (fleR/flrC), collagenase (colA), and UDP-galactopyranose mutase (glf ). However, there were no large clostridial cytotoxin (LCC) genes identified in the genome of strain CBA7122, whereas these genes were identified in strains ATCC 9714 T and VPI 9048, which are known as the key factors of human infection leading to death. Our data based on genomic analyses provides basic information of P. sordellii, which should serve as a useful reference for detailed studies focused on gaining a better understanding of the virulence factors in the genomes of these strains and their effects on human health. In addition, since strain CBA7122 contains a prophage genome, it is necessary to check whether the human infection status will be changed through phage therapy in further studies.

DATA ACCESS
The genome sequence of Paeniclostridium sordellii CBA7122 were deposited in the DDBJ/ENA/GenBank under accession numbers BDJI01000001-BDJI01000003.

AUTHOR CONTRIBUTIONS
SWR and Y-DN designed and coordinated all the experiments. HSS performed cultivation, DNA extraction, and purification. JYK, YBK, SHL, and MYJ performed the sequence assembly, gene prediction, gene annotation, comparative genomic analysis, and wrote manuscript. W-HJ, CL, SWA, and T-WK checked and edited the manuscript. All authors have read and approved the manuscript.