Characterisation of Clostridium difficile Biofilm Formation,a Role for Spo0A

Clostridium difficile is a Gram-positive anaerobic, spore-forming bacillus that is the leading cause of nosocomial diarrhoea worldwide. We demonstrate that C. difficile aggregates and forms biofilms in vitro on abiotic surfaces. These polymicrobial aggregates are attached to each other and to an abiotic surface by an extracellular polymeric substance (EPS). The EPS matrix provides the scaffold bonding together vegetative cells and spores, as well as forming a protective barrier for vegetative cells against oxygen stress. The master regulator of sporulation, Spo0A, may play a key role in biofilm formation, as genetic inactivation of spo0A in strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 exhibits decreased biofilm formation. Our findings highlight an important attribute of C. difficile pathogenesis, which may have significant implications for infection, treatment and relapse.     

Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.     

Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile

Background

Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of ‘hypervirulent’ strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, two further CTns were described.

Results

CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.

Conclusions

The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.     

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.     

A mariner-Based Transposon System for In Vivo Random Mutagenesis of Clostridium difficile

Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10⁻⁴ per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.Clostridium difficile infection is widely recognized as the leading cause of health care-associated diarrhea in North America and Europe. Infection usually follows antibiotic treatment, which disrupts the native gastrointestinal microflora and thus allows C. difficile to proliferate. The emergence of so-called “epidemic” or “hypervirulent” strains of C. difficile over the last 5 to 10 years has compounded an already serious problem. Classed as BI/NAP1/027, these epidemic strains are believed to cause a more severe disease and lead to increased mortality and relapse rates (11, 20, 24).Understanding the genetic and molecular basis of C. difficile infection will be a crucial step in the development of effective countermeasures. Methods for directed gene inactivation in C. difficile have recently been described (7, 21). This has opened the way for reverse genetic studies, in which the exact role of a specific gene, hypothesized to be important in a given phenotype, can be elucidated experimentally. By way of contrast, forward genetic studies aim to identify the genetic basis of a particular phenotype without making any assumptions about the genes involved. In forward genetic studies, transposons are often used to generate libraries of random insertion mutants. Libraries are then screened to identify mutants that are defective in a particular phenotype. Identification of the gene or genes which have been inactivated by transposon insertion then implicates them as having a role in that particular phenotype. Recently, just such an approach was used to identify a novel toxin-regulatory locus in Clostridium perfringens (29). This study elegantly demonstrated the power of forward genetic studies in bacterial pathogens.A number of transposon mutagenesis systems have been described for Gram-positive bacteria (2, 3, 15, 16, 29, 32). Two different systems have recently been developed for use in C. perfringens (15, 29). Both are in vitro mutagenesis systems which rely on being able to transform the recipient organism. As such, they are not suitable for use in C. difficile because in the laboratory at present, recombinant DNA can be transferred into C. difficile only via conjugation. The conjugative transposons Tn916 and Tn5397 have been studied in C. difficile, but both have been found either to have a strong target site preference or to yield multiple insertions in individual clones (9, 30). Therefore, neither is well suited to generating libraries of random C. difficile mutants.We reasoned that a mariner-based transposon mutagenesis system would be an effective tool for generating libraries of random C. difficile mutants. The mariner-transposable element Himar1 has been shown to insert randomly into the genomes of many bacterial species (3, 6, 16, 17, 32). The cognate Himar1 transposase is the only factor required for transposition, which occurs via a cut-and-paste mechanism (13, 14). The transposon itself is defined by inverted terminal repeats (ITRs) at either end and inserts into a TA target site. This is highly appropriate for an organism with a low-GC content such as C. difficile. In this study, we have developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile. Moreover, we have demonstrated the system in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. This new genetic tool opens the way for forward genetic studies of C. difficile.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.     

SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate

Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Δerm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 was found to sporulate more slowly and produce fewer spores than 630Δerm. Furthermore, fewer {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.The Gram-positive anaerobe Clostridium difficile causes diarrheal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis (8, 30). This organism is resistant to various broad-spectrum antibiotics and capitalizes on disruption of the normal intestinal flora to colonize and cause disease symptoms through the action of toxins A and B (16, 40). While these toxins are the principal virulence factors, the ability of the organism to produce endospores is necessary for disease transmission.Clostridial spores are extremely resistant to all kinds of chemical and physical agents and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants (35). Thus, the spores shed in fecal matter are very difficult to eradicate and can persist on contaminated surfaces in health care facilities for extended periods of time (35). This leads to infection or reinfection of cohabitating individuals through inadvertent ingestion of infected material (10, 32). Once in the anaerobic environment of the gut, spores presumably germinate to form toxin-producing vegetative cells and, in susceptible individuals, diarrheal disease.Spore germination is defined as the events that result in the irreversible loss of spore characteristics. However, current mechanistic knowledge of the germination process is based principally on data derived from studying Bacillus subtilis. Little is known about spore germination in clostridia and, in particular, in C. difficile. Germination is initiated when the bacterial spore senses specific effectors, termed germinants. These effectors can include nutrients, cationic surfactants, peptidoglycan, and a 1:1 chelate of pyridine-2,6-dicarboxylic acid (dipicolinic acid) and Ca²⁺ (CaDPA) (23, 34, 36). Spores of B. subtilis can germinate in response to nutrients through the participation of three sensory receptors located in the spore inner membrane, GerA, GerB, and GerK (23). After activation, the events include the release of monovalent cations (H⁺, K⁺, and Na⁺) and CaDPA (accounting for approximately 10% of the spore dry weight) (36). The third major step in germination involves hydrolysis of the spore peptidoglycan (PG) cortex. It is during this hydrolysis that the previously low water content of the spore is restored to the water content of a normal vegetative cell and the core is able to expand, which in turn allows enzyme activity, metabolism, and spore outgrowth (36).CwlJ and SleB are two specific spore cortex-lytic enzymes (SCLEs) involved in Bacillus cortex hydrolysis, which break down PG containing muramic-δ-lactam (28). SleB has been shown to localize in both the inner and outer layers of B. subtilis spores through interaction of the enzyme peptidoglycan-binding motif and the δ-lactam structure of the cortex (7, 19) and in association with YpeB, which is required for sleB expression during sporulation (4, 7). SleB is a lytic transglycosylase muramidase, but so far its mode of activation is unknown (21). CwlJ is localized to the spore coat during sporulation (3) and is required for CaDPA-induced germination in B. subtilis. Activation can be due to either CaDPA released from the spore core at the onset of germination or exogenous CaDPA (22). Neither enzyme is individually essential for complete cortex hydrolysis during nutrient germination, although inactivation of both cwlJ and sleB in B. subtilis results in a spore unable to complete this process (15). The role of SleL has recently been studied in Bacillus anthracis. Mutants unable to produce this enzyme are still able to germinate, but the process is retarded (18).The SCLEs of Clostridium are less well studied than those of Bacillus. The SCLEs SleC (20) and SleM (6) have been identified in Clostridium perfringens, and a recent study demonstrated that SleC is required during germination for complete cortex hydrolysis (26). Although SleM can degrade spore cortex peptidoglycan and inactivation of both sleC and sleM decreased the ability of spores to germinate more than inactivation of sleC alone did, SleM was not essential (26). It has also been shown that the germination-specific serine protease CspB is essential for cortex hydrolysis and converts the inactive pro-SleC found in dormant spores to an active enzyme (24). So far, there has been no detailed study of any gene responsible for spore germination in C. difficile, although genes showing homology to cwlJ and sleB of B. subtilis (CD3563) and sleC of C. perfringens (CD0551) have now been identified in the C. difficile 630 genome (33).With germinant receptors in C. difficile yet to be identified, the mechanism by which the spores sense a suitable environment for germination is unclear. Recent studies have suggested that this process may involve the interaction of C. difficile with bile. Taurocholate has been shown to enhance recovery of C. difficile spores in nutrient-rich medium (42), and it has been proposed that glycine and taurocholate act as cogerminants (38), while chenodeoxycholate inhibits C. difficile spore germination (39).The emergence of C. difficile B1/NAP1/027 strains has increased the burden on health care services worldwide. Such strains have been shown to produce higher levels of toxin in the laboratory than many other types of strains (41), although the mechanism behind this production is not fully understood. However, while the observed higher levels of toxin production is doubtless important, perhaps the recent attention given to B1/NAP1/027 strains has focused too much on toxins. As spores represent the infectious stage of C. difficile, processes such as spore germination may also contribute to the greater virulence of these strains. In this study we evaluated the sporulation and germination efficiencies of an “epidemic” B1/NAP1/027 C. difficile strain ({"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, isolated from the Stoke Mandeville outbreak in 2004 and 2005) and the “nonepidemic” strain 630Δerm (14). We then constructed strains with mutations in CD3563 (a cwlJ/sleB homologue) and a sleC homologue to analyze the role of these genes in the germination of C. difficile spores.     

Cwp22, a novel peptidoglycan cross-linking enzyme,plays pleiotropic roles in Clostridioides difficile

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.     

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.     

Comparative genomics of Riemerella anatipestifer reveals genetic diversity

Background

Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.

Results

In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP003787","term_id":"403311769","term_text":"CP003787"}}CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP004020","term_id":"441482619","term_text":"CP004020"}}CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.

Conclusion

The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.     

Nucleotide sequence analysis of the enterotoxigenic Escherichia coli Ent plasmid

We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (LT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The LT and STIa region was located 13.5 kb apart and was surrounded by three IS1s and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.     

LncRNA‐AK137033 inhibits the osteogenic potential of adipose‐derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation

ObjectivesBone tissue engineering based on adipose‐derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON‐ASCs), osteogenic potential of DOP‐ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP‐ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.Materials and methodsAn animal model of DOP was established in mice. CON‐ASCs and DOP‐ASCs were isolated from CON and DOP mice, respectively. {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 small interfering RNA (SiRNA) and an {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 overexpression plasmid were used to regulate the expression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in CON‐ASCs and DOP‐ASCs in vitro. Lentiviruses that carried shRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 or {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 cDNA were used to knockdown or overexpress {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033, respectively, in CON‐ASCs and DOP‐ASCs in vivo. Hematoxylin and eosin (H&E), Masson''s, alizarin red, and alkaline phosphatase (ALP) staining, micro‐computed tomography (Micro‐CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite‐specific PCR (BSP) were used to analyze the functional changes of ASCs.ResultsThe DOP mouse model was established successfully. Compared with CON‐ASCs, {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP‐ASCs. In vitro experiments showed that {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON‐ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of {"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 in DOP‐ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.ConclusionsLncRNA‐{"type":"entrez-nucleotide","attrs":{"text":"AK137033","term_id":"74205820","term_text":"AK137033"}}AK137033 inhibits the osteogenic potential of DOP‐ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients.     

Novel Variant Serotype of Streptococcus suis Isolated from Piglets with Meningitis

Streptococcus suis is an emerging zoonotic pathogen causing severe infections in pigs and humans. In previous studies, 33 serotypes of S. suis have been identified using serum agglutination. Here, we describe a novel S. suis strain, {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302, isolated from an outbreak of acute piglet meningitis in eastern China. Strong pathogenicity of meningitis caused by strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 was reproduced in the BALB/c mouse model. The strain showed a high fatality rate (8/10), higher than those for known virulent serotype 2 strains P1/7 (1/10) and 9801 (2/10). Cell adhesion assay results with bEnd.3 and HEp2 cells showed that {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 was significantly close to P1/7 and 9801. Both the agglutination test and its complementary test showed that strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 had no strong cross-reaction with the other 33 S. suis serotypes. The multiplex PCR assays revealed no specified bands for all four sets used to detect the other 33 serotypes. In addition, genetic analysis of the whole cps gene clusters of all serotypes was performed in this study. The results of comparative genomics showed that the cps gene cluster of {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302, which was not previously reported, showed no homology to the gene sequences of the other strains. Especially, the wzy, wzx, and acetyltransferase genes of strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 are phylogenetically distinct from strains of the other 33 serotypes. Therefore, this study suggested that strain {"type":"entrez-nucleotide","attrs":{"text":"CZ130302","term_id":"57728550","term_text":"CZ130302"}}CZ130302 represents a novel variant serotype of S. suis (designated serotype Chz) which has a high potential to be virulent and associated with meningitis in animals.     

Butlerius butleri Goodey, 1929 (Rhabditida) from Iran with the Phylogenetic Position of the Species

A population of Butlerius butleri Goodey, 1929 was isolated from vermicompost in Kerman in the Kerman Province of Iran during a nematode survey that was conducted during 2014. This population of B. butleri is characterized by the presence of a dorsal thorn-like tooth (4 to 5 μm long), long spicules (44 to 47 μm long), gubernaculum (33 to 37 μm or more than half of the spicule length), three pairs of precloacal papillae, five pairs of postcloacal papillae (papillae V3 and V5 comprising three small papillae), and a long filiform tail (304 to 409 μm in females, 312 to 380 μm in males). Molecular and phylogenetic analysis of B. butleri individuals from this Iranian population based on 18S ribosomal deoxyribonucleic acid (rDNA) sequence placed this species close to Pseudodiplogasteroides compositus ({"type":"entrez-nucleotide","attrs":{"text":"AB597237","term_id":"354681824","term_text":"AB597237"}}AB597237) and an unidentified Pseudodiplogasteroides species ({"type":"entrez-nucleotide","attrs":{"text":"AB597238","term_id":"354681825","term_text":"AB597238"}}AB597238). Measurements, illustrations, and the phylogenetic tree, including the position of B. butleri are provided.