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INTRODUCTION: Protonpumpinhibitors (PPIs) are associated with the onset of Clostridioides difficile infection (CDI). Although a new potassium-competitive acid blocker, vonoprazan, consistently shows a more potent acid inhibitory effect in comparison toPPIs, the risk of CDI in vonoprazan-treated patients relative to those treated with PPIs is unknown. In this retrospective case-control study, using a nationwide hospital-based administrative database in Japan, we investigated the association of the onset of CDI in patients treated with vonoprazan. METHODS: A CDI case was defined as a case in which a patient was diagnosed and treated for CDI. For each CDI case, 3 non-CDI patients were extracted as controls. Information on the usage of acid suppressants in the 2 months before the onset of CDI and other confounding factors was collected. Relative associations of gastric acid suppressants with the onset of CDI were estimated. RESULTS: A total of 4,466 CDI cases and 13,220 of non-CDI controls were extracted. A multivariate conditional regression analysis revealed that PPI or vonoprazan use was modestly, but significantly associated with CDI (odds ratio [95% confidence interval]: PPI, 1.3 [1.2-1.4]; vonoprazan, 1.4 [1.2-1.7]). With PPI users as a reference, vonoprazan did not show a stronger association with CDI (odds ratio [95% confidence interval]: 1.07 [0.91-1.26]). DISCUSSION: We found a significant positive association between vonoprazan use and CDI; however, the magnitude of the association was not beyond that in PPI users. This is the first report on any potential adverse effects of vonoprazan. [ABSTRACT FROM AUTHOR]

Background: There are only limited data in the literature on the thrombotic risk of patients with Clostridium difficile (CD) colitis, although this disease is widespread throughout the world. Objective: The aim of this study was to explore thrombin generation in these patients – the best way to evaluate their coagulation. Methods: A prospective observational study was conducted during 15 months on hospitalized patients with CD colitis. Thrombin generation was performed in platelet-poor plasma using a Ceveron® alpha analyzer and was compared with a group of volunteer control subjects. Results: Thirty-three patients and 51 control subjects were enrolled in the study. Two biomarkers – mean velocity index and peak thrombin – were significantly higher in patient group, compared to the control subjects (p = 0.010, respectively, p = 0.0395). This pattern of thrombin generation suggests that patients with CD colitis without septic shock have a potential thrombotic risk. The mean velocity index significantly correlated with the estimated related risk of death according to the Charlson age-comorbidity index. Conclusions: The higher values of thrombin generation suggest that CD colitis increases the thromboembolic risk. The pattern of thrombin generation could identify patients with particularly higher thromboembolic risk. They are potential candidates for thromboprophylaxis strategies and monitorization. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Clostridium strains were assessed for butanol production from agave hydrolysates. • 6.04 g/L of butanol was produced using Clostridium acetobutylicum ATCC 824. • Clostridium acetobutylicum ATCC 824 showed higher tolerance to inhibitors. • Co-culture was carried out using Bacillus subtilis and Clostridium acetobutylicum. • 8.28 g/L of butanol was produced in co-culture from agave hydrolysates. Abstract In this work, three Clostridium strains were tested for butanol production from Agave lechuguilla hydrolysates to select one for co-culturing. The agave hydrolysates medium was supplemented with nutrients and reducing agents to promote anaerobiosis. Clostridium acetobutylicum ATCC 824 had the highest butanol production (6.04 g/L) and was selected for further analyses. In the co-culture process, Bacillus subtilis CDBB 555 was used to deplete oxygen and achieve anaerobic conditions required for butanol production. The co-culture was prepared with C. acetobutylicum and B. subtilis without anaerobic pretreatment. Butanol production in co-culture from agave hydrolysates was compared with experiments using synthetic medium with glucose and a pure culture of C. acetobutylicum. The maximum butanol concentration obtained was 8.28 g/L in the co-cultured hydrolysate medium. Results obtained in the present work demonstrated that agave hydrolysates have the potential for butanol production using a co-culture of B. subtilis and C. acetobutylicum without anaerobic pretreatment. [ABSTRACT FROM AUTHOR]

Background/aim: Clostridium difficile is a frequent cause of nosocomial infections and has become a major public health concern in developed nations. In the present study, the prevalence and antimicrobial susceptibility pattern of toxigenic C. difficile strains isolated in Iran were investigated. Materials and methods: Between June 2016 and May 2017, 2947 inpatient fecal samples were taken from symptomatic adult hospitalized patients in different units of 32 care facilities in Tehran, Iran. C. difficile strains were identified by microbiological/biochemical methods. Susceptibility to 20 antimicrobials was measured by E-test method. Toxin-specific immunoassays and cytotoxicity assays were used to determine in vitro toxin production. Results: Out of 2947 fecal samples, 538 (18.25%) C. difficile isolates were obtained among those with suspected CDI. In E-test method, all C. difficile isolates were susceptible to fidaxomicin, vancomycin, amoxicillin/clavulanate, and meropenem and were resistant to penicillin G. The prevalence of multidrug resistant C. difficile was 69.33% (373/538). Among 538 C. difficile, 147 (27.32%), 169 (31.41%), and 222 (41.26%) isolates were TcdA+/TcdB+, TcdA-/TcdB+, and TcdA-/TcdB-, respectively. Conclusion: The results evidently support the hypothesis of a probable role of toxigenic strains of C. difficile in developing gastrointestinal complaints in patients with diarrhea. [ABSTRACT FROM AUTHOR]

Summary: Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σTcdR, which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σTcdR production by engineering cells to express tcdR from a tetracycline‐inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σD and CodY, revealed neither is required for bimodal toxin gene expression. However, σD biased cells toward the Toxin‐ON state, while CodY biased cells toward the Toxin‐OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σTcdR, which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs. Clostridioides difficile, the leading cause of antibiotic‐associated diarrhea, produces toxins that inactivate host Rho GTPases. We used a fluorescent reporter to visualize toxin gene expression in C. difficile within individual cells. Our findings imply toxin production is an example of bistability governed by cell‐to‐cell variation in the levels of the sigma factor TcdR, which is directly required for transcription of the toxin genes. TcdR levels are in turn controlled by several metabolic and genetic inputs. [ABSTRACT FROM AUTHOR]

Various bacterial protein toxins, including Clostridium difficile toxins A (TcdA) and B (TcdB), attack intracellular target proteins of host cells by glucosylation. After receptor binding and endocytosis, the toxins are translocated into the cytosol, where they modify target proteins (e.g., Rho proteins). Here we report that the activity of translocated glucosylating toxins depends on the chaperonin TRiC/CCT. The chaperonin subunits CCT4/5 directly interact with the toxins and enhance the refolding and restoration of the glucosyltransferase activities of toxins after heat treatment. Knockdown of CCT5 by siRNA and HSF1A, an inhibitor of TRiC/CCT, blocks the cytotoxic effects of TcdA and TcdB. In contrast, HSP90, which is involved in the translocation and uptake of ADP ribosylating toxins, is not involved in uptake of the glucosylating toxins. We show that the actions of numerous glycosylating toxins from various toxin types and different species depend on TRiC/CCT. Our data indicate that the TRiC/CCT chaperonin system is specifically involved in toxin uptake and essential for the action of various glucosylating protein toxins acting intracellularly on target proteins. [ABSTRACT FROM AUTHOR]

Purpose: The prospective assessment of Clostridium difficile infection (CDI) impact in inflammatory bowel disease (IBD) flare in outpatient setting has been poorly investigated. We aimed to evaluate the prevalence and the associated factors with CDI in IBD outpatients presenting colitis flares as well as the outcomes following treatment.Methods: In this prospective cohort study, conducted from October, 2014, to July, 2016, 120 IBD patients (55% presenting colitis flare) and 40 non-IBD controls were assessed for CDI. Multivariate regression analysis was performed to identify predictors of CDI. Outcome analysis was estimated for recurrent CDI, hospitalization, colectomy, and CDI-associated mortality.Results: The number of patients with CDI was significantly higher in IBD patients experiencing flares than in both inactive IBD and non-IBD groups (28.8 vs. 5.6 vs. 0%, respectively; p = 0.001). Females (OR = 1.39, 95% CI, 1.13-17.18), younger age (OR = 0.77, 95% CI, 0.65-0.92), steroid treatment (OR = 7.42, 95% CI, 5.17-40.20), and infliximab therapy (OR = 2.97, 95% CI, 1.99-24.63) were found to be independently associated with CDI. There was a dose-related increase in the risks of CDI on patients which had taken prednisone. Those treated with vancomycin had a satisfactory response to therapy, but 21% presented recurrent CDI and 16% were hospitalized. Neither necessity of colectomy nor mortality was noticed in any patient during the investigation.Conclusions: In IBD outpatients presenting colitis flares, CDI is highly prevalent. Females, younger age, infliximab, and notably steroid therapy were independently associated with CDI. Most patients with CDI experienced mild-to-moderate disease, and prompt treatment with vancomycin was highly effective, which seems to reduce the serious complication risks. [ABSTRACT FROM AUTHOR]

Little is known about the toxin profiles, toxinotypes and variations of toxin Clostridioides difficile C ( tcdC ) in Iranian C. difficile isolates. A total of 818 stool specimens were obtained from outpatients ( n = 45) and hospitalized patients ( n = 773) in Tehran, Iran, from 2011 to 2017. The 44 C. difficile isolates were subjected to PCR of toxin C. difficile A ( tcdA ), toxin C. difficile B ( tcdB ), tcdA 3′-end deletion, toxinotyping and sequencing of the tcdC gene. Thirty-eight isolates (86.36%) were identified as tcdA and tcdB positive, and the remaining six isolates (13.63%) were nontoxigenic. All tcdA - and tcdB -positive isolates yielded an amplicon of 2535 bp by PCR for the tcdA 3′ end. Fourteen (36.84%), seventeen (44.73%) and seven (18.43%) isolates belonged to wild-type, toxin C. difficile C subclone3 ( tcdC-sc3 ) and tcdC-A genotype of tcdC , respectively. Thirty-one isolates (81.57%) belonged to toxinotype 0, and seven isolates (18.42%) were classified as toxinotype V. This study provides evidence for the circulation of historical and hypervirulent isolates in the healthcare and community settings. Furthermore, it was also demonstrated that the tcdC-A genotype and toxinotype V are not uncommon among Iranian C. difficile isolates. [ABSTRACT FROM AUTHOR]

We present the description of Sanguibacter massiliensis sp. nov., Actinomyces minihominis sp. nov., Clostridium minihomine sp. nov., Neobittarella massiliensis gen. nov. and Miniphocibacter massiliensis gen. nov., new bacterial species isolated by culturomics from human stool samples. [ABSTRACT FROM AUTHOR]

Background A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l -histidine & l -phenylalanine) on C. difficile spore outgrowth were investigated. Methods Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine, l -histidine, l -phenyalanine). Results CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ± 0.02 vs 0.12 ± 0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l -histidine and l -phenylalanine. Conclusions We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine, l -histidine, l -phenylalanine) to C. difficile vegetative cell proliferation is also highlighted. [ABSTRACT FROM AUTHOR]

Clostridium perfringens (C. perfringens) causes disease, generally, named as enterotoxemia in the animals. This bacterium is a normal inhabitant in gastro-intestinal tract (GIT) of animals and become harmful by increasing its colony counts as well as toxin liberation whenever gets an opportunity of favorable conditions. This study focused molecular typing of C. perfringens (α, β, ε, ι toxins) and type 'A' multidrug resistance profile in diarrheic goats in Pakistan. Diarrheic fecal samples (n=192) were collected from goats and 80.73% (155/192) of the samples were found positive for C. perfringens on the basis of culture growth and PCR. Elevated C. perfringens counts (>107 CFU/g) were recorded in 33.55% (52/155) of positive samples, while, 66.45% (103/155) of the positive sample appeared in normal range of bacterial counts (104-107 CFU/g). Molecular detection was carried out by targeting specific toxin genes i.e. cpa (α), cpb (β), etx (ε) and iap (ι) of C. perfringens using PCR. Among the positive samples, 78.06% of the isolates were found as type 'A', 5.16% isolates were type 'B', 3.23% isolates were type 'C' while, 13.55% of the positive samples were type 'D' C. perfringens. None of the isolates was found positive for iap toxin gene (type 'E'). C. perfringens type 'A' was subjected to in-vitro antibiotic sensitivity test. Penicillin, ciprofloxacin and ceftriaxone were found sensitive while bacitracin, amoxicillin and ampicillin were found least sensitive antibiotics. This study concludes that C. perfringens type 'A' is highly prevalent among goats in Pakistan and clinical cases of enterotoxemia can be effectively dealt with penicillin, ciprofloxacin and ceftriaxone antibiotics. [ABSTRACT FROM AUTHOR]

The pathogenesis of recurrent Clostridium difficile infection (CDI) is still poorly understood. The risk of recurrence is approximately 20% after an initial CDI episode and dramatically increases with subsequent CDI recurrences. Several factors may play a role in recurrent CDI (rCDI), including conditions influencing germination, metabolic pathways that influence toxin production of C. difficile , and the microbiota composition offering protection against colonization and disease caused by C. difficile . Paradoxically, the currently recommended treatment for acute symptomatic CDI, i.e. metronidazole or vancomycin, can cause modification of the intestinal flora. Indeed, administration of anti-CDI antibiotics leads to suppression of C. difficile , along with collateral damage of the protective intestinal microbiota and opening of a “window of vulnerability” for recurrence. Host factors also have a prominent role, including innate and acquired humoral immunity, i.e. passive antibodies administration or active vaccination as a prevention strategy. They play a crucial role in the protection against severe and recurrent CDI. The assessment of risk factors of recurrence and modeling prediction scores could help in preventing the troublesome experience of CDI recurrence. Six studies have methodologically assessed prediction scores for rCDI. However, the definition of recurrence was heterogeneous, external validation was often not performed, and immunological factors were often not considered. There is a need for further studies on the pathophysiology of recurrence to design models for prediction that are sound and applicable in clinical practice. [ABSTRACT FROM AUTHOR]

Introduction: Clostridium difficile is the major etiological agent of nosocomial antibiotics associated diarrhoea. C. difficile infection is associated with considerable morbidity and mortality among hospitalized patients worldwide. Despite its known importance, there is no study on this important pathogen in Algeria. Methodology: In this prospective study, undertaken between 2013 and 2015, faecal specimens were collected from 159 hospitalized patients with antibiotic-associated diarrhoea in two tertiary health care hospitals in Chlef, Algeria. Faecal samples were cultured on CLO plates Agar with cefoxitin, cycloserine antibiotics and sodium taurocholate. C. difficile suspected colonies were analysed by multiplex PCR for the detection of the toxin genes. C. difficile isolates were analysed by PCR ribotyping and multi-locus tandem repeat analysis. Antimicrobial susceptibility was determined by the E-test method, according to the Clinical and Laboratory Standards Institute protocol. Results: C. difficile was cultured from 11 of 159 stool specimen (6.9%). Seven strains were toxigenic, mainly represented by the 020 and 014 PCR ribotypes and four non toxigenic belong to PCR ribotype 084. All 11 isolates were susceptible to both vancomycin and metronidazole and resistant to ciprofloxacin. Conclusions: This study, which reported for the first time C. difficile ribotypes circulating in Algerian health care facilities, could paves the way for further more comprehensive studies on this important pathogen, and could be useful to the local health authorities to implement a surveillance program of C. difficile in Algeria. [ABSTRACT FROM AUTHOR]

Microbial Fe(III) reduction can make an excellent contribution to the bioremediation of contaminated environments and potentially reduce methanogenesis. Excessive input of phosphorus (P) by P fertilizer application and eutrophied irrigation water might have a substantial influence on the process of microbial Fe(III) reduction in flooded paddy soils. To evaluate the effect of P application on microbial Fe(III) reduction, the responses of Clostridium and Geobacteraceae communities to different concentrations of P addition (CK: 0 mmol P kg − 1 soil; P1: 3.3 mmol P kg − 1 soil; P2: 20 mmol P kg − 1 soil) were investigated in anaerobically incubated paddy slurries. P addition significantly inhibited Fe(III) reduction during the early stage of incubation (from days 0 to 20). Compared with the CK treatment, the maximum Fe(III) reduction rate ( V max ) in treatments P1 and P2 remarkably decreased by 0.281 and 0.439 mg·g − 1 ·d − 1 , respectively. However, the addition of P had no significant effect on Fe(III) reduction during the later stage of incubation (after 20 days). The abundances of Clostridium and Geobacteraceae were suppressed by P addition, and the suppression effect was more obvious with higher P concentration. P addition significantly changed the community structures of Clostridium and Geobacteraceae during the entire incubation. The communities of Clostridium and Geobacteraceae were closely correlated with the process of Fe(III) reduction. In conclusion, P addition could inhibit the microbial reduction of Fe(III) during the early stage of incubation by reducing the abundances and altering the community structures of Clostridium and Geobacteraceae , however, the inhibition could be eliminated with increased incubation time. This study demonstrates that soil microbial communities are sensitive to excessive P application, which can jointly impact relevant biogeochemical processes in flooded paddy soils. [ABSTRACT FROM AUTHOR]

Fatal clostridial infections and clostridial toxicoses are common in birds. Most fatalities are associated with toxin production and progress rapidly, often within 24 hours of infection. We describe an unusual and protracted course of disease in 6 captive brown pelicans ( Pelecanus occidentalis), which was believed to result from toxicosis by toxovar A produced by a mixed infection with Clostridium sordellii and Clostridium perfringens. Although the first death in the group occurred 3 days after signs of illness were documented, the remaining birds died over a 38-day period despite aggressive antibiotic and supportive therapy. Although the birds presented with classic signs of botulism, Clostridium botulinum was not identified in any tissues or environmental samples. Postmortem findings in all pelicans included extensive subacute myonecrosis, enteritis, and nonsuppurative hepatitis. Alpha-toxins and sordellilysin genes from C perfringens and C sordelli isolates, respectively, were detected via polymerase chain reaction. The source of the pathogenic bacteria was sediment within a water basin inside the affected birds' enclosure. [ABSTRACT FROM AUTHOR]

Some strains of Clostridium difficile produce a binary toxin, in addition to the main C. difficile virulence factors (toxins A and B). There have been conflicting reports regarding the role of binary toxin and its relationship to the severity of C. difficile infection (CDI). Samples, isolates and clinical data were collected as part of a prospective multicentre diagnostic study. Clostridium difficile isolates ( n = 1259) were tested by polymerase chain reaction (PCR) assay to detect binary toxin genes cdtA and cdtB. The PCR binary toxin gene results were compared with clinical severity and outcome data, including 30-day all-cause mortality. The 1259 isolates corresponded to 1083 different patients (October 2010 to September 2011). The prevalence of binary toxin positive strains was significantly higher in faecal samples with detectable toxin A/B than in those without toxin but that were positive by cytotoxigenic culture (26.3% vs. 10.3%, p < 0.001). The presence of binary toxin correlated moderately with markers of CDI severity (white cell count, serum albumin concentration and serum creatinine concentration). However, the risk ratio for all-cause mortality was 1.68 for binary toxin positive patients and patients were significantly less likely to survive if they had CDI caused by a binary toxin gene positive strain, even after adjusting for age ( p < 0.001). The presence of binary toxin genes does not predict the clinical severity of CDI, but it is significantly associated with the risk of all-cause mortality. [ABSTRACT FROM AUTHOR]

During faecal microbiota transplantation, stool from a healthy donor is transplanted to treat a variety of dysbiosis-associated gut diseases. Competent authorities are faced with the challenge to provide adequate regulation. Currently, regulatory harmonization is completely lacking and authorities apply non-existing to most stringent requirements. A regulatory approach for faecal microbiota transplantation could be inserting faecal microbiota transplantation in the gene-, cell- and tissue regulations, including the hospital exemption system in the European Advanced Therapy Medicinal Products regulation, providing a pragmatic and efficacy-risk balanced approach and granting all patients as a matter of principle access to this therapy. [ABSTRACT FROM AUTHOR]

The cellulolytic Clostridium cellulovorans has been engineered to produce n -butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans . First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n -butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues. [ABSTRACT FROM AUTHOR]

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Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the 'functional' region of Cwp2, consisting of residues 29-318 at 1.9Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. [ABSTRACT FROM AUTHOR]

Objectives: Clostridium difficile infection (CDI) is a significant cause of morbidity and mortality and is the most common nosocomial infection in the United States, with associated annual costs of approximately $3 billion. The epidemiology of CDI has changed with the identification of novel risk factors for incident and recurrent CDI. The aim of this study was to identify the predictors of 30-day mortality in hospitalized patients with CDI.Methods: We identified all of the patients diagnosed as having CDI from January 2011 to December 2014 at our university-setting hospital. Data were extracted using electronic medical records and chart review. The data of all of the patients who died within 30 days of incident CDI were compared with those who survived beyond 30 days of incident CDI. A multivariable logistic regression model was created for mortality after finding a subset of significant predictor variables by making bivariate comparisons also using logistic regression.Results: A total of 893 patients were diagnosed as having CDI during the study period. The mean age was 62 years and 49.5% were women. The mean length of hospital stay was 11.73 days. Of the 893 patients with CDI, 98 (10.97%) died within 30 days of incident CDI. CDI recurrence was noted in 76 patients (8.51%). On multivariate logistic regression analysis, peptic ulcer disease, advanced age, Charlson comorbidity index, and intensive care unit status were found to be significantly associated with 30-day mortality. There was no significant association between acid suppression and CDI mortality.Conclusions: Advanced age, Charlson comorbidity index, intensive care unit status, and peptic ulcer disease are predictors of all-cause 30-day mortality in hospitalized patients with CDI. [ABSTRACT FROM AUTHOR]

The Gram-positive pathogen Clostridium perfringens possesses a family of large conjugative plasmids that is typified by the tetracycline resistance plasmid pCW3. Since these plasmids may carry antibiotic resistance genes or genes encoding extracellular or sporulation-associated toxins, the conjugative transfer of these plasmids appears to be important for the epidemiology of C. perfringens -mediated diseases. Sequence analysis of members of this plasmid family identified a highly conserved 35 kb region that encodes proteins with various functions, including plasmid replication and partitioning. The tcp conjugation locus also was identified in this region, initially based on low-level amino acid sequence identity to conjugation proteins from the integrative conjugative element Tn 916 . Genetic studies confirmed that the tcp locus is required for conjugative transfer and combined with biochemical and structural analyses have led to the development of a functional model of the Tcp conjugation apparatus. This review summarises our current understanding of the Tcp conjugation system, which is now one of the best-characterized conjugation systems in Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

In this study it is intended to find out the prevalence of Clostridium difficile in modified atmosphere packaged (MAP) minced (n:50) and cubed beef meat samples (n :50); determine the toxin from the isolates and detect the antibiotic susceptibility against metronidazole, vancomycin and clindamycin. C. difficile isolates have been isolated from two (4%) of 50 minced beef meat samples; one (2%) of 50 cubed beef meat samples. All three isolates were confirmed by PCR as C. difficile by the detection of tpi gene. Three out of 5 C. difficile isolates showed toxigenic character, two of them were carried type B (tcdB) toxin genes, one of them harbored type A (tcdA) toxin gene. When antibiotic resistance profile was phenotypically analyzed, only C. difficile type A (tcdA) was found resistant against clindamycin. All isolates were found sensitive to vancomycin and metronidazole. The result of this study demonstrated that C. difficile strains detected in samples of modified atmosphere packaged (MAP) beef meat could be a potential problem to public health. [ABSTRACT FROM AUTHOR]

Several risk factors associated with the increased likelihood of healthcare-associated Clostridium difficile infection (CDI) have been identified in the literature. These risk factors are mainly related to age, previous CDI, antimicrobial exposure, and prior hospitalization. No model is available in the published literature that can be used to predict the CDI incidence using healthcare administration data. However, the administrative data can be imprecise and may challenge the building of classical statistical models. Fuzzy set theory can deal with the imprecision inherent in such data. This research aimed to develop a model based on deterministic and fuzzy mathematical techniques for the prediction of hospital-associated CDI by using the explanatory variables controllable by hospitals and health authority administration. Retrospective data on CDI incidence and other administrative data obtained from 22 hospitals within a regional health authority in British Columbia were used to develop a decision tree (deterministic technique based) and a fuzzy synthetic evaluation model (fuzzy technique based). The decision tree model had a higher prediction accuracy than that of the fuzzy based model. However, among the common results predicted by two models, 72 % were correct. Therefore, this relationship was used to combine their results to increase the precision and the strength of evidence of the prediction. These models were further used to develop an Excel-based tool called C. difficile Infection Incidence Prediction in Hospitals (CDIIPH). The tool can be utilized by health authorities and hospitals to predict the magnitude of CDI incidence in the following quarter. [ABSTRACT FROM AUTHOR]

Clostridium difficile infection ( CDI) is a challenging threat to human health. Infections occur after disruption of the normal microbiota, most commonly through the use of antibiotics. Current treatment for CDI largely relies on the broad-spectrum antibiotics vancomycin and metronidazole that further disrupt the microbiota resulting in frequent recurrence, highlighting the need for C. difficile-specific antimicrobials. The cell surface of C. difficile represents a promising target for the development of new drugs. C. difficile possesses a highly deacetylated peptidoglycan cell wall containing unique secondary cell wall polymers. Bound to the cell wall is an essential S-layer, formed of SlpA and decorated with an additional 28 related proteins. In addition to the S-layer, many other cell surface proteins have been identified, including several with roles in host colonization. This review aims to summarize our current understanding of these different C. difficile cell surface components and their viability as therapeutic targets. [ABSTRACT FROM AUTHOR]

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography-mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl-coenzyme A and butyryl-coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long-term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose-phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate-dependent. Kinetic labelling of hexose-phosphates and triose-phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed-sugar utilization. [ABSTRACT FROM AUTHOR]

Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38alpha -/- MSCV empty vector MEFs and the corresponding cell line with reconstituted p38alpha expression (p38alpha -/- MSCV p38alpha MEFs). Genetic deletion of p38alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38-/- MSCV empty vector MEFs and in p38alpha -/- MSCV p38alpha MEFs, suggesting that inhibition of p38beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38alpha deletion. In conclusion, the protective effects of SB203580 and of p38alpha deletion are likely not based on inhibition of the toxins' glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells. [ABSTRACT FROM AUTHOR]

Clostridium difficile-associated disease continues to be one of the leading health concerns worldwide. C. difficile is considered as a causative agent of nosocomial diarrhea that causes serious infection, which may result in death. The incidences of C. difficile infection (CDI) in developed countries have become increasingly high which may be attributed to the emergence of newer epidemic strains, extensive use of antibiotics, and limited alternative therapies. The available treatment options against CDI are expensive and promote resistance. Therefore, there is urgent need for new approaches to meet these challenges. This review discusses the current understanding of CDI, the existing clinical treatment strategies and future potential options as antidifficile agents based on the available published works. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a bacterial pathogen capable of causing life-threatening infections of the gastrointestinal tract characterized by severe diarrhea. Exposure to certain classes of antibiotics, advanced age, and prolonged hospitalizations are known risk factors for infection by this organism. Anecdotally, healthcare providers have reported that they can smell C. difficile infections in their patients, and several studies have suggested that there may indeed be an olfactory signal associated with C. difficile -associated diarrhea. In this study, we sought to characterize the volatile molecules produced by an epidemic strain of C. difficile (R20291) using headspace solid-phase microextraction (HS-SPME) followed by two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). We report on a set of 77 volatile compounds, of which 59 have not previously been associated with C. difficile growth in vitro . Amongst these reported compounds, we detect both straight-chain and branched-chain carboxylic acids, as well as p -cresol, which have been the primary foci of C. difficile volatile metabolomic studies to-date. We additionally report on novel sulfur-containing and carbonyl-containing molecules that have not previously been reported for C. difficile . With the identification of these novel C. difficile -associated volatile compounds, we demonstrate the superior resolution and sensitivity of GC × GC-TOFMS relative to traditional GC–MS. [ABSTRACT FROM AUTHOR]

Lack of standardised Clostridium difficile testing is a potential confounder when comparing infection rates. We used an observational, systematic, prospective large-scale sampling approach to investigate variability in C. difficile sampling to understand C. difficile infection (CDI) incidence rates. In-patient and institutional data were gathered from 60 European hospitals (across three countries). Testing methodology, testing/CDI rates and case profiles were compared between countries and institution types. The mean annual CDI rate per hospital was lowest in the UK and highest in Italy (1.5 vs. 4.7 cases/10,000 patient bed days [pbds], p < 0.001). The testing rate was highest in the UK compared with Italy and France (50.7/10,000 pbds vs. 31.5 and 30.3, respectively, p < 0.001). Only 58.4 % of diarrhoeal samples were tested for CDI across all countries. Overall, only 64 % of hospitals used recommended testing algorithms for laboratory testing. Small hospitals were significantly more likely to use standalone toxin tests (SATTs). There was an inverse correlation between hospital size and CDI testing rate. Hospitals using SATT or assays not detecting toxin reported significantly higher CDI rates than those using recommended methods, despite testing similar testing frequencies. These data are consistent with higher false-positive rates in such (non-recommended) testing scenarios. Cases in Italy and those diagnosed by SATT or methods NOT detecting toxin were significantly older. Testing occurred significantly earlier in the UK. Assessment of testing practice is paramount to the accurate interpretation and comparison of CDI rates. [ABSTRACT FROM AUTHOR]

Background: Clostridium difficile infections (CDI) are the most frequent cause of diarrhoea in hospitals. Geriatric patients are more often affected by the condition, by a relapse and complications. Therefore, a crucial question is how often colonization with toxigenic Clostridium difficile strains occurs in elderly patients without diarrhoea and whether there is a "risk pattern" of colonized patients that can be defined by geriatric assessment. Furthermore, the probability for those asymptomatic carriers to develop a symptomatic infection over time has not been sufficiently explored. Methods: We performed a cohort study design to assess the association of clinical variables with Clostridium difficile colonization. The first stool sample of 262 consecutive asymptomatic patients admitted to a geriatric unit was tested for toxigenic Clostridium difficile using PCR (GeneXpert, Cepheid). A comprehensive geriatric assessment (CGA) including Barthel Index, Mini Mental State Examination (MMSE) and hand grip-strength was performed. In addition, Charlson Comorbidity Index, body mass index, number and length of previous hospital stays, previous treatment with antibiotics, institutionalization, primary diagnoses and medication were recorded and evaluated as possible risk factors of colonization by means of binary logistic regression. Secondly, we explored the association of C. difficile colonization with subsequent development of CDI during hospital stay. Results: At admission, 43 (16.4%) patients tested positive for toxin B by PCR. Seven (16.3%) of these colonized patients developed clinical CDI during hospital stay, compared to one out of 219 patients with negative or invalid PCR testing (Odds ratio 12,3; Fisher's exact test: p = 0.000). Overall, 7 out of 8 (87.5%) CDI patients had been colonized at admission. Risk factors of colonization with C. difficile were a history of CDI, previous antibiotic treatment and hospital stays. The parameters of the CGA were not significantly associated with colonization. Conclusion: Colonization with toxigenic Clostridium difficile strains occurs frequently in asymptomatic patients admitted to a geriatric unit. Previous CDI, antibiotic exposure and hospital stay, but not clinical variables such as CGA, are the main factors associated with asymptomatic Clostridium difficile carriage. Colonization is a crucial risk factor for subsequent development of symptomatic CDI. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene (clostridial myonecrosis), remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. [ABSTRACT FROM AUTHOR]

Background: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). It restores the disrupted intestinal microbiota and subsequently suppresses C. difficile. The long-term stability of the intestinal microbiota and the recovery of mucosal microbiota, both of which have not been previously studied, are assessed herein. Further, the specific bacteria behind the treatment efficacy are also investigated. Methods: We performed a high-throughput microbiota profiling using a phylogenetic microarray analysis of 131 faecal and mucosal samples from 14 rCDI patients pre- and post-FMT during a 1-year follow-up and 23 samples from the three universal donors over the same period. Results: The FMT treatment was successful in all patients. FMT reverted the patients' bacterial community to become dominated by Clostridium clusters IV and XIVa, the major anaerobic bacterial groups of the healthy gut. In the mucosa, the amount of facultative anaerobes decreased, whereas Bacteroidetes increased. Post-FMT, the patients' microbiota profiles were more similar to their own donors than what is generally observed for unrelated subjects and this striking similarity was retained throughout the 1-year follow-up. Furthermore, the universal donor approach allowed us to identify bacteria commonly established in all CDI patients and revealed a commonly acquired core microbiota consisting of 24 bacterial taxa. Conclusions: FMT induces profound microbiota changes, therefore explaining the high clinical efficacy for rCDI. The identification of commonly acquired bacteria could lead to effective bacteriotherapeutic formulations. FMT can affect microbiota in the long-term and offers a means to modify it relatively permanently for the treatment of microbiotaassociated diseases. [ABSTRACT FROM AUTHOR]

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii , Clostridium botulinum , Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum , Clostridium ljungdahlii , Clostridium autoethanogenum and even Geobacillus thermoglucosidasius . [ABSTRACT FROM AUTHOR]

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production. [ABSTRACT FROM AUTHOR]

Background: Accurate diagnosis of Clostridium difficile infection (CDI) is paramount for patient management. The wrong diagnosis places patients at risk, delays treatment, and/ or contributes to transmission of infection in the healthcare setting. Although amplification of the toxin B gene by polymerase chain reaction (PCR) is a sensitive method for detecting toxigenic C. difficile, false negative results still occur and could impact the diagnosis and treatment of this infection.Methods: This study investigated 48 patients that tested negative for toxigenic C. difficile via GeneXpert C. difficile epi test, while simultaneously testing positive for toxigenic C. difficile via stool culture. Fifty discrepant samples were collected over a 15-month period and all C. difficile isolates were characterized by ribotype. Patient charts were reviewed to assess whether discrepant results impacted the treatment course or clinical outcome of affected patients.Results: Fifty samples of a total of 2308 samples tested in an acute healthcare facility over a 15-month period had negative PCR and positive stool culture for toxigenic C. difficile. C. difficile isolated from the discrepant samples resulted in diverse ribotyping patterns suggesting they were derived from different strains. The samples belonged to patients who were distributed evenly between age groups and wards in the hospital. In the majority of cases, the false negative C. difficile test results did not seem to impact the clinical outcome in these patients.Conclusions: The PCR limit of detection may impact the results of molecular methods for C. difficile detection. Both clinical and analytical sensitivity of C. difficile tests should be considered when deciding which diagnostic assay to use, and clinical correlates should be examined carefully before excluding CDI as a cause of disease. [ABSTRACT FROM AUTHOR]

Background: A prospective study was performed to investigate the prevalence of colonization among ICU patients and to examine whether asymptomatic carriers were the source of subsequent C. difficile infection (CDI) and acquisition of toxigenic C. difficile.Methods: Rectal swabs were collected from adult patients on admission to and at discharge from a 50-bed medical ICU of a major referral hospital in western China, from August to November 2014. Stools were collected from patients who developed ICU-onset diarrhea. Both swabs and stools were screened for tcdB (toxin B gene) by PCR. Samples positive to tcdB were cultured for C. difficile and isolates recovered were screened for tcdB and the binary toxin genes by PCR. Strain typing was performed using multilocus sequence typing and isolates belonging to the same sequence type (ST) were further typed using multiple-locus variable number tandem repeat analysis (MLVA).Results: During the 4-month period, rectal swabs were collected from 360 (90.9 %) out of 396 patients who were admitted to the ICU. Among the 360 patients, 314 had stayed in the ICU more than 3 days, of which 213 (73.6 %) had a rectal swab collected within the 3 days prior to discharge from ICU. The prevalence of toxigenic C. difficile colonization was 1.7 % (6 cases) and 4.3 % (10 cases) on admission and discharge, respectively. Only four (1.1 %) out of 360 patients had CDI, corresponding to 10.7 cases per 10,000 ICU days. None of the four cases had toxigenic C. difficile either on admission or at discharge. Toxigenic C. difficile isolates were recovered from all swabs and stool samples positive for tcdB by PCR and belonged to 7 STs (ST2, 3, 6, 37, 54, 103 and 129). None of the isolates belonging to the same ST had identical MLVA patterns. Binary toxin genes were detected in one ST103 isolate that caused colonization.Conclusion: The prevalence of colonization with toxigenic C. difficile among patients on admission to ICU was low in our setting. ICU-acquired toxigenic C. difficile were not linked to those detected on admission. Active screening for toxigenic C. difficile may not be a resource-efficient measure in settings with a low prevalence of colonization. [ABSTRACT FROM AUTHOR]

Probiotics are live microorganisms ingested for the purpose of conferring a health benefit on the host. Development of new probiotics includes the need for safety evaluations that should consider factors such as pathogenicity, infectivity, virulence factors, toxicity, and metabolic activity. Clostridium butyricum MIYAIRI 588® (CBM 588®), an anaerobic spore-forming bacterium, has been developed as a probiotic for use by humans and food animals. Safety studies of this probiotic strain have been conducted and include assessment of antimicrobial sensitivity, documentation of the lack of Clostridium toxin genes, and evaluation of CBM 588® on reproductive and developmental toxicity in a rodent model. With the exception of aminoglycosides, to which anaerobes are intrinsically resistant, CBM 588® showed sensitivity to all antibiotic classes important in human and animal therapeutics. In addition, analysis of the CBM 588® genome established the absence of genes for encoding for α, β, or ε toxins and botulin neurotoxins types A, B, E, or F. There were no deleterious reproductive and developmental effects observed in mice associated with the administration of CBM 588®. These data provide further support for the safety of CBM 588® for use as a probiotic in animals and humans. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains. [ABSTRACT FROM AUTHOR]

The vertebrate blood–brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE 194 – 319 variant cCPE Y306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPE Y306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPE Y306W/S313H . In an in vivo experiment, this study estimates the capability of cCPE Y306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPE Y306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPE Y306W/S313H has the capability – both in vitro and in vivo – to modulate BBB permeability temporarily by specific binding to Cldn5. [ABSTRACT FROM AUTHOR]

Background: Clostridium difficile infection (CDI) unresponsive to the standard treatments of metronidazole and oral vancomycin requires aggressive medical management and possible surgical intervention including colectomy. Intracolonic vancomycin therapy has been reported to be particularly promising in the setting of severe CDI in the presence of ileus. This is a descriptive case series exploring the effect of adjunctive intracolonic vancomycin therapy on the morbidity and mortality in patients with moderate to severe CDI.Methods: A retrospective chart review was conducted on 696 patients with CDI seen at a single institution. Each patient was assigned a severity score and 127 patients with moderate to severe CDI were identified. We describe the clinical presentation, risk factors and hospital course comparing those that received adjunctive intracolonic vancomycin to those that only received standard therapy.Results: The group that received adjunctive intracolonic vancomycin had higher rates of toxic megacolon, intensive care unit (ICU) admission, and colectomy, and yet maintained a similar mortality rate as the group that received only standard treatment.Conclusion: The intracolonic vancomycin group experienced more complications but showed a similar mortality rate to the standard therapy group, suggesting that intracolonic vancomycin may impart a protective effect. This study adds further evidence for the need of a randomized controlled study using intracolonic vancomycin as adjunctive therapy in patients presenting with severe CDI. [ABSTRACT FROM AUTHOR]

Pseudomembranous colitis is a frequent nosocomial infection associated with significant morbidity and mortality. Clostridium difficile infection incidence most frequently increases due to unreasonable antibiotic use and the appearance of new hypervirulent bacterial strains, which leads to prolonged hospitalization and an increase in the total cost of hospital treatment. This is a retrospective design study conducted at Clinical Centre Kragujevac from January to December 2014. The patient data were obtained from the protocol of the Virological Laboratory and from medical documentation. All statistical analyses were performed using the computer program SPSS. The descriptive statistical data are expressed as percentage values. Continuous variables are expressed as the arithmetic mean with the standard deviation. Clostridium difficile infection occurred more frequently with elderly patients (123 patients were over 65 years old). Out of 154 patients on antibiotic treatment, 110 patients were treated with a combination of two or more antibiotics from different pharmacological groups. The most represented antibiotics were from the cephalosporin (71.4%) and quinolone (46.3%) groups. A total of 85.8% of the patients used proton pump inhibitors and H2 blockers. Our results describe the clinical and demographic characteristics of patients with diagnosed Clostridium difficile infection. The most prevalent characteristics (age, antibiotic therapy, PPI and H2 blocker use), which other researchers have also mentioned as risk factors, were present in our study as well. [ABSTRACT FROM AUTHOR]

Introduction: In the last few years, the use of collagenase clostridium histolyticum for management of Dupuytren's contracture has increased. The procedure of enzymatic fasciectomy has become popular because it is noninvasive, safe and fast to perform. Sources of data: A systematic search was performed on Medline (PubMed), Web of Science and Scopus databases using the combined keywords 'Dupuytren collagenase' and 'Dupuytren clostridium histolyticum'. Forty-three studies were identified. The quality of the studies was assessed using the Coleman Methodological Score. Areas of agreement: The use of collagenase clostridium histolyticum provides better outcomes in patients with mild-moderate joint contracture, with lower complications and side effects than open fasciectomy. Manipulation can be performed 2-7 days after the injection. The use of collagenase is cost-effective. Areas of controversy: Most of the studies did not report patient-related outcomes. The role of dynamic splint has to be investigated with randomized clinical trials. Growing points: The shorter recovery time and the low incidence of serious or major adverse effects are the main advantages of this new technology. Areas timely for developing research: There is a need to perform studies with longer follow-up because the recurrence rate seems to increase with time. Further investigations are necessary to assess whether it is safe and effective to inject two or more cords at the same time. [ABSTRACT FROM AUTHOR]

Background: We report herein a case of fecal microbiota transplantation (FMT) used for severe Clostridium difficile infection for a 65-year-old Lebanese man who underwent left ventricular assist device implantation. To the best of our knowledge this is the first case report from Lebanon and the region presenting such technique.Case Presentation: The patient experienced diarrhea and rectal bleeding and was diagnosed of pseudomembranous colitis (PMC). His condition failed to improve on maximal pharmacological therapy. Protocolectomy, an invasive operation consisting in resection of the entire colon and rectum seemed to be the last resort before the patient responded to FMT given through gastroscopy.Conclusion: Despite the increasing experience with FMT for C. difficile infection, published evidence in severe related cases from this region is very limited. Hence, we promote adjunctive FMT, an effective noninvasive method, to be considered as a promising early treatment option in severe C. difficile infection. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate Clostridium botulinum growth and toxin production in the industrially manufactured Italian Parma ham. The study focuses on the Parma ham production phase identified as maximum risk to C. botulinum proliferation, i.e. the transition from cold phase (salting and resting) to a phase carried out at temperature between 15 and 23°C (drying). A preliminary in vitro test was carried out in order to verify the capability of 6 C. botulinum strains (1 type A, 4 type B, and 1 type E strains) to grow in conditions of temperature, pH and NaCl concentration comparable to those of the beginning stage of ham drying. Five C. botulinum strains grew at 20°C and pH 6, four strains produced toxin when inoculated at a concentration equal to 103 cfu/mL at NaCl concentration of 4%, while when the inoculum concentration was 10 cfu/mL, NaCl concentration of 3% resulted the toxin-genesis limiting factor. An experimental contamination with a mixture of the 5 C. botulinum strains selected by the preliminary in vitro test was performed on 9 thighs inoculated at the end of the resting phase. The study was designed to evaluate the potential growth and toxin production in extremely favourable conditions for the bacterium. Type B proteolytic C. botulinum toxin was produced after 14 days of incubation at 20°C in 2 thighs characterised by high weight, low number of days of resting and anomalous physiochemical characteristics [one for very low NaCl concentration (1.59%), the other for elevated pH (6.27) and both for high water activity values (>0.970)]. The results of this research confirm that the cold resting step is a critical phase in the production process of Parma ham for the investigated hazard. Based on the present study, the long resting phase adopted in the manufacturing of Parma ham is proven effective to prevent the growth of C. botulinum, an event which could not otherwise be excluded if the hams were processed under less stringent technological conditions. [ABSTRACT FROM AUTHOR]

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1-93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. [ABSTRACT FROM AUTHOR]

The present study investigated the influence of the influent concentration of substrate, feeding time and temperature on the production of biohydrogen from cheese whey in an AnSBBR with liquid phase recirculation. The highest hydrogen yield (0.80 molH 2. molLactose −1 ) and productivity (660 mLH 2 L −1 d −1 ) were achieved for influent concentrations of 5400 mgDQO L −1 . No significant difference was noted in the biological hydrogen production for the feeding time conditions analyzed. The lowest temperature tested (15 °C) promoted the highest hydrogen yield and productivity (1.12 molH 2 molLactose −1 and 1080 mLH 2 L −1 d −1 ), and for the highest temperature (45 °C), hydrogen production did not occur. The indicator values for the hydrogen production obtained with this configuration were higher than those obtained in other studies using traditional configurations such as UASBr and CSTR. A phylogenetic analysis showed that the majority of the analyzed clones were similar to Clostridium . In addition, clones phylogenetically similar to the Lactobacilaceae family, notably Lactobacillus rhamnosus , and clones with similar sequences to Acetobacter indonesiensi s were observed in small proportion in the reactor. [ABSTRACT FROM AUTHOR]