Complete genome sequence of Peptoclostridium difficile strain Z31

Peptoclostridium difficile, initially called Bacillus difficilis, was first isolated from the meconium of newborns by Hall and O’Toole in 1935 [1]. The name ‘Clostridium difficile’ was made official in 1980 in the Approved Lists of Bacterial Names [2] based on a phenotypic study by Prevót [3]. Recently, in a study based on 16S rRNA and ribosomal protein sequences, Yutin and Galperin [4] proposed the reallocation of some Clostridium species into six new genera, renaming C. difficile ‘Peptoclostridium difficile’.

The genus Peptoclostridium, in the phylum Firmicutes, class Clostridia, order Clostridiales, and family Peptostreptococcaceae [4], is characterized by strictly anaerobic, motile, pleomorphic Gram-positive bacteria, with dimensions of 0.5–1.9 × 3.0–16.9 µm, which form oval subterminal spores (Fig. 1) with a bacillus cell shape. The bacteria are spore-forming and mesophilic (20–37 °C), with an optimal pH range of neutral to alkaline. They ferment fructose, glucose, levulose, mannitol, mannose, salicin, and usually xylose, but not galactose, glycerol, inulin, lactose, raffinose, or sucrose. They are chemoorganotrophs and can use yeast extract as their sole carbon and energy source and peptone as their nitrogen source. Peptoclostridium difficile liquefies gelatin, but does not attack coagulated serum, milk, or meat proteins and is unable to reduce sulfate. It is negative for lecithinase, lipase, oxidase, and catalase. Acetate is produced as a major end product, but it also produces butyrate, formate, isobutyrate, isocaproate, isovalerate, lactate, and valerate [3‐5].

Until the late 1970s, P. difficile was not recognized as pathogenic bacteria. However, in this decade, P. difficile and its toxins were related in fecal contents of human patients with pseudomembranous colitis [6] and the disease was reproduced in hamsters [7], confirming the importance of this microorganism as an enteropathogen. Today, this bacterium is known to be the cause of P. difficile infection (PDI), the main cause of nosocomial diarrhea in humans worldwide and a possible cause of diarrhea in general community [8, 9].

In veterinary medicine, P. difficile is the most important uncontrolled cause of neonatal diarrhea in piglets in the USA and Europe, and also occurs in other domestic animals and some wild species [10, 11]. In piglets, CDI affects animals to 1–7 days of life, and it was demonstrated that until 1 day of life, 68–100 % of the animals are infected by the microorganism [12, 13]. The disease is subclinical, and just few animals show diarrhea, however, the infection can affect the development of the animals causing economic losses to the farmer [14].

The pathogeny of PDI involves the colonization of colon by some toxigenic strain of P. difficile and production of its toxins, the toxin A, an enterotoxin, and toxin B, a cytotoxin, that act synergistically causing cytoskeleton damages, cell rounding, disruption of tight junctions and cell death [15]. The genes responsible to produce toxins, the main difference between toxigenic and nontoxigenic strains, are localized in a pathogenicity locus of 19 kb, called PaLoc [16].

Despite the known importance of P. difficile in humans and animals, no vaccine is yet commercially available. Studies have shown that recombinant and classical immunogens expressing toxins A and B can prevent the occurrence of diarrhea or reduce the severity of P. difficile infection (PDI) in a rodent model [17]. These vaccines might limit, but cannot prevent, the fecal shedding of the microorganism, which is essential because P. difficile is a nosocomial pathogen. Because this bacterium is also a potential zoonotic agent, preventing its colonization of domestic animals should be a priority [10]. Among other alternative preventive strategies examined, the use of nontoxigenic P. difficile strains to prevent PDI has been shown to reduce the occurrence of the disease in humans and piglets by preventing their colonization by toxigenic strains [18‐21].

There has been no report of the complete genome sequence of a nontoxigenic P. difficile strain, a necessary step in understanding this candidate live vaccine. Therefore, in this study, we determined the complete genome sequence of P. difficile nontoxigenic strain Z31.

After the genome assembly, gap filling, and annotation process, an in silico PCR was performed through searching for genes related to virulence factor, antibiotic resistance, and other known toxins. Considering the perspective of using the nontoxigenic strain Z31 to prevent PDI by competitive exclusion, some non-toxin virulence factors are desirable, predominantly those factors responsible for spore production and stability and those that promote cell attachment and host colonization. Z31 is positive for Cwp84 and surface-layer protein A (SlpA). SlpA is considered the major factor responsible for bacterial intestinal adherence, and Cwp84 is essential for the formation of that protein [34, 35]. GroEL, Cwp66, and a fibronectin-binding protein (Fbp68), which are also important in host-cell adherence, were also found [34‐38]. Strain Z31 was also positive for genes encoding the flagellar proteins FliC and FliD, which play roles in the colonization and adherence of Z31 in vivo and are essential in later stages of biofilm formation [39‐41]. These factors found in Z31 related to cell attachment are extremely important, because non toxigenic strains have to be able to compete with toxigenic strains by the colonization sites to prevent the disease [23].

The gene encoding the major regulator of sporulation in P. difficile, Spo0A, was detected in this strain. An absence or deficiency of Spo0A can cripple or impair the sporulation process [35, 42]. Genes encoding five spore coat proteins (cotA, cotB, cotC, cotD, and sodA) were also detected. The cotA protein is the most important protein in stabilizing the spore coat and ensures the integrity of this structure [43]. The formation of stable spores is also important for a nontoxigenic strain candidate to prevent the disease, because the bacteria need to pass through the stomach and be able to colonize the colon [23]. Vegetative cells are sensible to low pH, on the other hand, the spores resist to this conditions, allowing a great number of viable particles reaches the colon [44]. Genes responsible for resistance to tetracycline (tetM) and erythromycin (ermG) were also detected with previously described PCR primers [45, 46]. In contrast, none of the genes encoding proteins directly linked to toxin production were detected (tcdA, tcdB, tcdC, cdtA, or cdtB) [47] confirming the absence of the pathogenicity locus (PaLoc), which is essential for P. difficile infection [48].

Furthermore, the complete genomes of this species available at GenBank were selected to perform a similarity analysis with Gegenees software [49] with sequence fragmentation length of 500 nucleotides and a threshold of 30 %. Also, two complete genomes of species of the Clostridium genus were included as an outgroup. The similarity matrix was used to generate a heatplot and a “.nexus” format for phylogenomic analysis (Additional file 3). Although the Z31 strain is a nontoxigenic strain, the Additional file 3 shows that clade of this strain is paraphyletic with the type strain ATCC9689, a known as toxigenic strain, suggesting an evolutionary derivation of a same organism. Thereby, the nontoxigenic behavior of the Z31 strain seems to be occasioned by the losses of the toxin genes.