Zero prevalence of Clostridium difficile in wild passerine birds in Europe

Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species, where it can cause significant medical problems. The major public health concern is the possibility of inapparent animal reservoirs of C. difficile and shedding of bacteria to noninfected individuals or populations, as well as being a source of food contamination. Migrating birds can be a key epizootiological factor for transmission and distribution of pathogens over a wide geographic range. Therefore, the purpose of this study was to investigate whether migrating passerine birds can be a source of spread of C. difficile along their migration routes. Cloacal samples were taken from 465 passerine birds during their migration south over the Alps. Selective enrichment was used for detection of C. difficile. Clostridium difficile was not isolated from any of the samples, which indicates that migrating passerine birds are unlikely to serve as a reservoir and a carrier of C. difficile.     

Clostridium difficile in vegetables, Canada

Background: Clostridium difficile is an important gastrointestinal pathogen of humans and animals. It has been isolated from various foods, including meat and ready‐to‐eat salads, and concern has been expressed regarding food as a possible source of human C. difficile infection (CDI). Aims: We sought to isolate C. difficile from a variety of vegetables obtained from local grocery stores and to characterize these isolates. Materials and Methods: Vegetables were purchased from 11 different grocery stores in Guelph, Ontario, Canada between May and August 2009. Enrichment culture was performed and isolates were characterized by ribotyping, PFGE, toxinotyping and PCR detection of toxin genes. Results: Clostridium difficile was isolated from 4.5% (5/111) of retail vegetables. Two different ribotypes and two different toxinotypes were identified. Three isolates were ribotype 078/NAP 7/toxinotype V, possessing all three toxin genes. The other two isolates shared a ribotype with a toxigenic strain previously found in humans with CDI in this region. Discussion: Contamination of vegetables was found at relatively low levels, however, all isolates were toxigenic and belonging to ribotypes previously associated with CDI. Conclusions: Contamination of vegetables with CDI‐associated isolates can occur and although the implications for food safety practices remain elusive, the presence of toxigenic isolates suggests vegetables could be a source of C. difficile in humans.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.     

A new Galeopid (Anomodontia,Galeopidae) from the Permian of Eastern Europe

A new anomodont genus and species, Parasuminia ivakhnenkoi sp. nov. (Galeopidae), from the Sundyr-1 locality (Upper Severodvinian Substage, Upper Permian) is described. Based on morphological analysis of tooth wear, the type of work of the jaw apparatus similar to that of Suminia Ivachnenko, 1994 is revealed.     

Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function

Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.     

Moist-heat resistance, spore aging, and superdormancy in Clostridium difficile

Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log(10) within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log(10) within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log(10), 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.     

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo . In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.     

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.     

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 Å x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a β-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a π-cation interaction within the β-flap that appear to be unique among the large clostridial cytotoxins.Clostridium difficile is a Gram-positive, spore-forming anaerobe that infects the colon and causes a range of disorders, including diarrhea, pseudomembranous colitis, and toxic megacolon (1, 2). Two large toxins, TcdA² and TcdB (308 and 270 kDa, respectively) are recognized as the main virulence factors of C. difficile, although their relative importance is the subject of on-going study (3, 4). These proteins belong to a class of homologous toxins called large clostridial toxins (LCTs) and have been classified more broadly as AB toxins, wherein a B moiety is involved in the delivery of an enzymatic A moiety into the cytosol of a target cell. In LCTs, the A subunit is an N-terminal glucosyltransferase that inactivates small G-proteins, such as Rho, leading to cell rounding and apoptosis of the intoxicated cell (5, 6). The B subunit corresponds to the remainder of the toxin and is responsible for binding the target cell through a C-terminal receptor-binding domain (7–9) and forming the membrane pore needed for translocation of the A subunit (10, 11). Unlike other known AB toxins, the glucosyltransferase A domains of LCTs are released from the B subunits by an autoproteolytic cleavage event (12). Cleavage is triggered by host inositol phosphates and the reducing environment of the cytosol (12).In LCTs, autoproteolysis has been attributed to a cysteine protease activity located within the N-terminal region of the B subunit (13). This region was identified based on homology with the cysteine protease domain (CPD) found in the multifunctional autoprocessing repeats in toxins (MARTX) toxins from Gram-negative bacteria (14). Autoprocessing in the MARTX toxin from Vibrio cholera (VcRTx) is also stimulated by InsP6 (15). A recent crystal structure of VcRTx CPD bound to InsP6 suggests a novel mechanism of InsP6-induced allosteric activation (16). The CPDs of TcdA and VcRTx share only 19% sequence identity. To gain insight into the mechanistic commonalities between these entirely different toxins and to delineate the LCT-specific modes of InsP6-induced processing, we performed structural and functional analyses on the cysteine protease from TcdA.     

Interaction of Clostridium difficile toxin A with L cells in culture

Toxin A of Clostridium difficile was purified by column chromatography and acetic acid precipitation. Cells exposed to toxin A showed polarization of nuclei towards one pole of the cells. Toxin A was conjugated to ferritin and applied to L cells to localize binding sites of this toxin to the cell surface. It was found that toxin A conjugate attached to the cell membrane in aggregated form. Antibody specific to toxin A was prepared and used for localization of intracellular toxins in intoxicated cells. Toxin A was found inside the cytoplasm 6 h after cell treatment, mainly in the form of aggregates inside the cytoplasmic vacuoles. At 24 h after exposure, toxin A could be detected within the cytoplasm. Tunicamycin treatment of cells reduced the cell-binding efficiency of toxin A to 50%, but neuraminidase did not effect toxin binding significantly.     

Clostridium difficile toxins A and B: Receptors,pores, and translocation into cells

The most potent toxins secreted by pathogenic bacteria contain enzymatic moieties that must reach the cytosol of target cells to exert their full toxicity. Toxins such as anthrax, diphtheria, and botulinum toxin all use three well-defined functional domains to intoxicate cells: a receptor-binding moiety that triggers endocytosis into acidified vesicles by binding to a specific host-cell receptor, a translocation domain that forms pores across the endosomal membrane in response to acidic pH, and an enzyme that translocates through these pores to catalytically inactivate an essential host cytosolic substrate. The homologous toxins A (TcdA) and Toxin B (TcdB) secreted by Clostridium difficile are large enzyme-containing toxins that for many years have eluded characterization. The cell-surface receptors for these toxins, the non-classical nature of the pores that they form in membranes, and mechanism of translocation have remained undefined, exacerbated, in part, by the lack of any structural information for the central ～1000 amino acid translocation domain. Recent advances in the identification of receptors for TcdB, high-resolution structural information for the translocation domain, and a model for the pore have begun to shed light on the mode-of-action of these toxins. Here, we will review TcdA/TcdB uptake and entry into mammalian cells, with focus on receptor binding, endocytosis, pore formation, and translocation. We will highlight how these toxins diverge from classical models of translocating toxins, and offer our perspective on key unanswered questions for TcdA/TcdB binding and entry into mammalian cells.     

A group II intron in a conjugative transposon from the gram-positive bacterium,Clostridium difficile

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.     

Clostridium difficile toxin B causes apoptosis in epithelial cells by thrilling mitochondria. Involvement of ATP-sensitive mitochondrial potassium channels

Targeting to mitochondria is emerging as a common strategy that bacteria utilize to interact with these central executioners of apoptosis. Several lines of evidence have in fact indicated mitochondria as specific targets for bacterial protein toxins, regarded as the principal virulence factors of pathogenic bacteria. This work shows, for the first time, the ability of the Clostridium difficile toxin B (TcdB), a glucosyltransferase that inhibits the Rho GTPases, to impact mitochondria. In living cells, TcdB provokes an early hyperpolarization of mitochondria that follows a calcium-associated signaling pathway and precedes the final execution step of apoptosis (i.e. mitochondria depolarization). Importantly, in isolated mitochondria, the toxin can induce a calcium-dependent mitochondrial swelling, accompanied by the release of the proapoptogenic factor cytochrome c. This is consistent with a mitochondrial targeting that does not require the Rho-inhibiting activity of the toxin. Of interest, the mitochondrial ATP-sensitive potassium channels are also involved in the apoptotic response to TcdB and appear to be crucial for the cell death execution phase, as demonstrated by using specific modulators of these channels. To our knowledge, the involvement of these mitochondrial channels in the ability of a bacterial toxin to control cell fate is a hitherto unreported finding.     

A survey of metronidazole and vancomycin resistance in strains of Clostridium difficile isolated in Warsaw, Poland

The drug of choice used to treat Clostridium difficile-associated diarroea (CDAD) are metronidazole and vancomycin. Information about emergence of antimicrobial resistance among C. difficile strains to metronidazole and intermediate resistance to vancomycin in some countries are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 193 C. difficile strains isolated in our diagnostic laboratory between year 1998 and 2003 from patients adults and children suffering from CDAD. Among these strains, 142 produced toxin A and B (TcdA(+)TcdB(+)), 43 only B (TcdA(-)TcdB(+)) and 8 were nontoxigenic. We have not observed any differences in susceptibility to metronidazole and vancomycin between all C. difficile strains under investigation (toxinogenic and non-toxinogenic). Resistance to metronidazole and vancomycin was not observed.     

Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.     

A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB ⁺, n =  111; tcdB ⁻, n  = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours.In a further study (tcdB ⁺, n =  47; tcdB ⁻, n  = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB ⁺, n =  25; tcdB ⁻, n  = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.     

Prevalence of Clostridium difficile in uncooked ground meat products from Pittsburgh, Pennsylvania

The prevalence of Clostridium difficile in retail meat samples has varied widely. The food supply may be a source for C. difficile infections. A total of 102 ground meat and sausage samples from 3 grocers in Pittsburgh, PA, were cultured for C. difficile. Brand A pork sausages were resampled between May 2011 and January 2012. Two out of 102 (2.0%) meat products initially sampled were positive for C. difficile; both were pork sausage from brand A from the same processing facility (facility A). On subsequent sampling of brand A products, 10/19 samples from processing facility A and 1/10 samples from 3 other facilities were positive for C. difficile. The isolates recovered were inferred ribotype 078, comprising 6 genotypes. The prevalence of C. difficile in retail meat may not be as high as previously reported in North America. When contamination occurs, it may be related to events at processing facilities.     

Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12

Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits. A gene bank of C. difficile DNA in Escherichia coli was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified. One of these clones lambda tA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A. The size of the lambda tA5 insert DNA was 14.3 kb.