Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity

Background The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool. Findings The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation. Conclusions Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact.


Background
The human gut is an intricate environment which harbors a large variety of microorganisms. The interaction between the host and the gut microbiome has an effect on the host health. The human gut microbiome is dominated by two bacterial phyla, Firmicutes and Bacteroidetes [1,2]. The Firmicutes population in the gut is dominated by the genus Clostridium. Clostridia start to colonize the human gut from the first month of life. Clostridium species in the gut play vital roles in degradation of food products, production of vitamins and short chain fatty acids, maintaining gut homeostasis and shaping the mucosal immune system. Recent studies have shown that Clostridia are reduced in abundance in inflammatory conditions such as inflammatory bowel disease (IBD), which leads to the dysbiosis further leading to inflammation [2]. Thus, Clostridia are key members of the human gut microbiome.
Although this is true, some pathogenic Clostridium species like C. difficile, C. perfringens and C. tetani have been described. These organisms cause fatal infections like colitis, gangrene and tetanus respectively [3][4][5]. Hence, understanding the role of Clostridium species isolated from the human gut is of paramount importance. In this study, we carried out genome sequencing of a novel Clostridium species (isolate BL8), isolated from the stool sample of a healthy Indian individual, in order to decipher the potential role of this organism in the human gut ecosystem. Our study demonstrates that Clostridium sp BL8 has a potential to cause infections in humans and lays basis for further studies to characterize this potential novel pathogen.

Material and methods
Genomic DNA extraction and 16S rRNA gene PCR and antibiotic sensitivity Genomic DNA extraction and 16S rRNA gene sequencing was carried out as described earlier [6]. The antibiotic sensitivity of Clostridium sp. BL8 was carried out using antibiotic discs purchased from HiMedia laboratories, Mumbai following the CLSI guidelines [7].

Genome sequencing and bioinformatic analysis
The genome sequencing using an Ion Torrent PGM™ and bioinformatic analysis was carried out using the methodology described by Shetty et al. [8].

Cytotoxicity assay
The cell cytotoxicity assay was carried out by microculture tetrazolium (MTT) assay as described earlier [9].
Briefly: 96-well microplate was seeded with 100 μl medium containing 10,000 Vero cells in suspension. After 24 hr incubation and attachment, the cells were treated with 100 μl of 1 × 10 5 Clostridium sp. BL8 cell suspension or 100 μl cell free supernatant for 2 hrs and 4 hrs respectively, in triplicates. Cell suspension buffer and sterile culture medium were used as controls. Cell viability was determined by adding tetrazolium salt (5 mg/ml) (Sigma Aldrich, USA). After 3 hours of incubation at 37°C, media was removed and precipitated formazan was dissolve in 100 μl of DMSO. Absorbance was taken at 570 nm using an ELISA plate reader Spectra MAX250 (Molecular Devices, USA).

Findings
The isolate used in the study The Clostridium sp. BL8 (CCUG 64195) has been reported in our previous study [6]. Based on 16S rRNA sequence [GenBank: JN093128] the closest validly published species are Clostridium subterminale and Clostridium sulfidigenes Figure 1 Comparison of protein sequences of Clostridium sp. BL8 and C. tunisiense based on comparative genome analysis using SEED software. The lines on circle indicate the alignment of predicted protein sequences from both the genomes and the color indicates the percent similarity between the predicted protein sequences. Genome sequence of C. tunisiense [GenBank: AMQH00000000] is used as a reference for alignment of the genome sequence of Clostridium sp. BL8.
(97% sequence similarity in both the cases). This suggests that Clostridium sp. BL8 represents a novel species. Clostridium sp. BL8 shares 99% 16S rRNA sequence similarity with C. tunisiense, but C. tunisiense is not yet included in the list of standing nomenclature (http://www.bacterio.net). The comparison of genome sequences of BL8 and C. tunisiense [GenBank: AMQH00000000] suggested a very low sequence similarity between the genomes Figure 1. This further approves the fact that Clostridium sp. isolate BL8 represents a novel bacterial species belonging to the genus Clostridium.
Genomic features of Clostridium sp. BL8 The assembled genome of Clostridium sp. BL8 was 4,776,227 bp with an average coverage of 37×. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AUPA00000000. The list of all proteins annotated by RAST server is represented in Additional file 1: Table S1.
The genome of Clostridium sp. BL8 revealed the presence of adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. All these features are important for the survival of Clostridium sp. BL8 in the human gut [8].

Resistance to antibiotics
The in vitro antibiotic sensitivity test demonstrated multidrug resistance pattern of Clostridium sp. BL8, with resistance to 11 antibiotics belonging to 6 different classes. The genome sequencing revealed the presence of antibiotic resistance genes conferring resistance against these tested as well as other non tested antibiotics. The list of antibiotic resistance genes and tested antibiotics is represented in Table 1. MDR-type ABC transporters, drug/metabolite transporters (DMT), multidrug and toxin extrusion (MATE) family efflux pumps were also detected in the genome of BL8.
Horizontal gene transfer between pathogens and human commensals is an important factor for the dissemination of antibiotic resistance [10]. BL8 genome has mobile genetic elements like integrase and transposes such as IS1380-Spn, ISSod4, IS1294, IS3/IS911, IS110 and IS4. Type IV secretion system are important for transfer of DNA and secretion of proteins in bacteria [11]. BL8 genome encodes for type IV secretion system with presence of PilABC and PilMNO proteins and pilus regulator PilR. These systems would enable exchange of DNA between BL8 and other gut commensals.

Genes coding for virulence factors
The virulence genes were categorized into two categories 1) toxins, 2) exoenzyme, adhesins and others. 1) Toxins and hemolysins: C. perfringens is known to produce alpha toxin (Phospholipase C) which is responsible for  myonecrosis eventually causing gangrene [4]. Phospholipase C (EC 3.1.4.3) was detected in the genome of BL8 suggesting that it has a potential to cause muscle necrosis. The genes coding for hemolysin and hemolysin III were also detected in BL8 genome. All these genes are important virulence factors for C. perfringens [4]. Proteins similar to aureolysin and exfoliative toxin A of Stahpyhlococcus were also detected in BL8 genome. Exfoliative toxin A is known as 'epidermolytic toxins' which cleaves upper epidermal layer of skin, while aureolysin is a known complement inhibitor [12,13]. Both these genes are important for S. aureus pathogenesis.
2) Exoenzymes, adhensins and others: Secreted enzymes like proteases and serine proteases degrade host extracellular matrix allowing the entry of pathogens in the tissue, thus are important factors for virulence in bacterial pathogens. Secreted proteins like prepilin peptidase, HtrA family serine proteases, subtilisin-type proteinase, peptidase M23B were detected in BL8 genome. All these proteins play an important role in pathogenesis of various pathogens like Legionella pneumophila, Streptococcus pyogenes, Streptococcus suis, Pseudomonas aeruginosa, Salmonella typhimurium, Campylobacter jejuni and Helicobacter pylori [14][15][16][17][18].
Along with secreted proteins the genome of BL8 had myosin reactive antigen, which is reported to be important for virulence in Shigella flexneri [19]. BL8 genome also contains platelet-activating factor acetylhydrolase and iron ABC uptake transporter similar to anthrachelin. The platelet-activating factor acetylhydrolase is a proinflammatory enzyme required for virulence, while anthrachelin is essential for virulence of Bacillus anthracis [20,21]. Fibronectin/fibrinogen-binding protein, adhensins are important for adhesion and colonization of the pathogen on wound tissue and blood clots [5]. Genes coding for both these factors were present in the genome of BL8. To aid the regulations of these genes, several transcriptional regulators belonging to AraC, DeoR, GntR, LysR, MarR and TetR families were detected in the genome of BL8. All these regulators have been reported to be important in the regulation of several virulence genes in pathogens [22,23]. The presence of all these virulence genes and regulatory factors suggests that Clostridium sp. BL8 has a high potential for pathogenicity.

Cytotoxicity assay
Pathogens such as C. perfringens and C. difficile are known to have cytotoxic activity [24,25]. To investigate whether Clostridium sp. BL8 possesses any cytotoxic activity, we tested the in vitro cytotoxic effect of Clostridium sp. BL8 on Vero cells by MTT assay. Vero cells were used for the assay as this cell line has been recommended for the assay of C. perfringens enterotoxin [24]. Both BL8 cells and cell free supernatant showed cytotoxic activity against Vero cell line (Figure 2). BL8 cells had significantly higher cytotoxicity compared to cell free supernatant, both after