Evaluation of Correlation between Pretest Probability for Clostridium difficile Infection and Clostridium difficile Enzyme Immunoassay Results.

PubMed

Kwon, Jennie H; Reske, Kimberly A; Hink, Tiffany; Burnham, C A; Dubberke, Erik R

2017-02-01

The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI. Copyright © 2017 American Society for Microbiology.

Clostridium difficile shows no trade-off between toxin and spore production within the human host.

PubMed

Blanco, Natalia; Walk, Seth; Malani, Anurag N; Rickard, Alexander; Benn, Michele; Eisenberg, Marisa; Zhang, Min; Foxman, Betsy

2018-05-01

This study aimed to describe the correlation between Clostridium difficile spore and toxin levels within the human host. In addition, we assessed whether overgrowth of Candida albicans modified this association. We measured toxin, spore and Candida albicans levels among 200 successively collected stool samples that tested positive for C. difficile, and PCR ribotyped these C. difficile isolates. Analysis of variance and linear regression were used to test the association between spore and toxin levels. Kruskal-Wallis tests and t-tests were used to compare the association between spore or toxin levels and host, specimen, or pathogen characteristics. C. difficile toxin and spore levels were positively associated (P<0.001); this association did not vary significantly with C. albicans overgrowth [≥5 logs of C. albicans colony-forming units (c.f.u.) g -1 ]. However, ribotypes 027 and 078-126 were significantly associated with higher levels of toxin and spores, and C. albicans overgrowth. The strong positive association observed between in vivo levels of C. difficile toxin and spores suggests that patients with more severe C. difficile infections may have increased spore production, enhancing C. difficile transmission. Although, on average, spore levels were higher in toxin-positive samples than in toxin-negative/PCR-positive samples, spores were found in almost all toxin-negative samples. The ubiquity of spore production among toxin-negative and formed stool samples emphasizes the importance of following infection prevention and control measures for all C. difficile-positive patients during their entire hospital stay.

The role of toxins in Clostridium difficile infection.

PubMed

Chandrasekaran, Ramyavardhanee; Lacy, D Borden

2017-11-01

Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease. Published by Oxford University Press on behalf of FEMS 2017.

Laboratory and Clinical features of EIA Toxin-positive and EIA Toxin-negative Community-acquired Clostridium difficile Infection.

PubMed

Patel, Hiren; Randhawa, Jeewanjot; Nanavati, Sushant; Marton, L Randy; Baddoura, Walid J; DeBari, Vincent A

2015-01-01

Studies have described the clinical course of patients with Clostridium difficile infection (CDI) with positive enzyme immunoassay (EIA) for toxins A and B. Limited information is available for the patients with negative EIA but positive for the toxin B gene (TcdB) by the PCR. The aim of our study is to determine if there are any differences that exist among the clinical and laboratory parameters in the patients tested to be positive by EIA for toxin and those who were negative. This is a retrospective cohort study conducted in a 700-bed teaching hospital. We reviewed charts of the patients with presumptive CDI between January 2006 and July 2013. We divided these patients into two groups, EIA-positive and EIA-negative, based on result of EIA for toxins A and B and the requirement for a positive PCR analysis of the TcdB gene. The EIA-positive group had significantly higher white blood cell counts (p<0.001), with a significantly greater percentage of bands (p<0.0001). Albumin and total protein both exhibit significantly (p<0.0001, both comparisons) lower values in the EIA-positive group. Among clinical findings, the EIA-positive group had significantly longer length of hospital stay (p=0.010). These data suggest that an infection with an EIA-negative strain of C. difficile presents laboratory markers closer to those of healthy subjects and clinical features suggesting considerably less severe than infection with EIA-positive C. difficile. © 2015 by the Association of Clinical Scientists, Inc.

Comparison of Diagnostic Algorithms for Detecting Toxigenic Clostridium difficile in Routine Practice at a Tertiary Referral Hospital in Korea.

PubMed

Moon, Hee-Won; Kim, Hyeong Nyeon; Hur, Mina; Shim, Hee Sook; Kim, Heejung; Yun, Yeo-Min

2016-01-01

Since every single test has some limitations for detecting toxigenic Clostridium difficile, multistep algorithms are recommended. This study aimed to compare the current, representative diagnostic algorithms for detecting toxigenic C. difficile, using VIDAS C. difficile toxin A&B (toxin ELFA), VIDAS C. difficile GDH (GDH ELFA, bioMérieux, Marcy-l'Etoile, France), and Xpert C. difficile (Cepheid, Sunnyvale, California, USA). In 271 consecutive stool samples, toxigenic culture, toxin ELFA, GDH ELFA, and Xpert C. difficile were performed. We simulated two algorithms: screening by GDH ELFA and confirmation by Xpert C. difficile (GDH + Xpert) and combined algorithm of GDH ELFA, toxin ELFA, and Xpert C. difficile (GDH + Toxin + Xpert). The performance of each assay and algorithm was assessed. The agreement of Xpert C. difficile and two algorithms (GDH + Xpert and GDH+ Toxin + Xpert) with toxigenic culture were strong (Kappa, 0.848, 0.857, and 0.868, respectively). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of algorithms (GDH + Xpert and GDH + Toxin + Xpert) were 96.7%, 95.8%, 85.0%, 98.1%, and 94.5%, 95.8%, 82.3%, 98.5%, respectively. There were no significant differences between Xpert C. difficile and two algorithms in sensitivity, specificity, PPV and NPV. The performances of both algorithms for detecting toxigenic C. difficile were comparable to that of Xpert C. difficile. Either algorithm would be useful in clinical laboratories and can be optimized in the diagnostic workflow of C. difficile depending on costs, test volume, and clinical needs.

Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection?

PubMed

Garvey, Mark I; Bradley, Craig W; Wilkinson, Martyn A C; Holden, Elisabeth

2017-01-01

Diagnosis of C. difficile infection (CDI) is controversial because of the many laboratory methods available and their lack of ability to distinguish between carriage, mild or severe disease. Here we describe whether a low C. difficile toxin B nucleic acid amplification test (NAAT) cycle threshold (CT) can predict toxin EIA, CDI severity and mortality. A three-stage algorithm was employed for CDI testing, comprising a screening test for glutamate dehydrogenase (GDH), followed by a NAAT, then a toxin enzyme immunoassay (EIA). All diarrhoeal samples positive for GDH and NAAT between 2012 and 2016 were analysed. The performance of the NAAT CT value as a classifier of toxin EIA outcome was analysed using a ROC curve; patient mortality was compared to CTs and toxin EIA via linear regression models. A CT value ≤26 was associated with ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. A CT value of ≤26 was significantly associated ( p  = 0.0262) with increased one month mortality, severe cases of CDI or failure of first line treatment. The ROC curve probabilities demonstrated a CT cut off value of 26.6. Here we demonstrate that a CT ≤26 indicates more severe CDI and is associated with higher mortality. Samples with a low CT value are often toxin EIA positive, questioning the need for this additional EIA test. A CT ≤26 could be used to assess the potential for severity of CDI and guide patient treatment.

Clostridium difficile Toxins A and B: Insights into Pathogenic Properties and Extraintestinal Effects

PubMed Central

Di Bella, Stefano; Ascenzi, Paolo; Siarakas, Steven; Petrosillo, Nicola; di Masi, Alessandra

2016-01-01

Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight junctions of target cells causing cell rounding and ultimately cell death. Detectable C. difficile toxemia is strongly associated with fulminant disease. However, besides the well-known intestinal damage, recent animal and in vitro studies have suggested a more far-reaching role for these toxins activity including cardiac, renal, and neurologic impairment. The creation of C. difficile strains with mutations in the genes encoding toxin A and B indicate that toxin B plays a major role in overall CDI pathogenesis. Novel insights, such as the role of a regulator protein (TcdE) on toxin production and binding interactions between albumin and C. difficile toxins, have recently been discovered and will be described. Our review focuses on the toxin-mediated pathogenic processes of CDI with an emphasis on recent studies. PMID:27153087

Clinical Characteristics and Outcomes of Hematologic Malignancy Patients With Positive Clostridium difficile Toxin Immunoassay Versus Polymerase Chain Reaction Test Results.

PubMed

Ziegler, Matthew; Landsburg, Daniel; Pegues, David; Alby, Kevin; Gilmar, Cheryl; Bink, Kristen; Gorman, Theresa; Moore, Amy; Bonhomme, Brittaney; Omorogbe, Jacqueline; Tango, Dana; Tolomeo, Pam; Han, Jennifer H

2018-04-25

In a cohort of inpatients with hematologic malignancy and positive enzyme immunoassay (EIA) or polymerase chain reaction (PCR) Clostridium difficile tests, we found that clinical characteristics and outcomes were similar between these groups. The method of testing is unlikely to predict infection in this population, and PCR-positive results should be treated with concern.Infect Control Hosp Epidemiol 2018;1-4.

Clostridium difficile carriage in adult cystic fibrosis (CF); implications for patients with CF and the potential for transmission of nosocomial infection.

PubMed

Burke, D G; Harrison, M J; Fleming, C; McCarthy, M; Shortt, C; Sulaiman, I; Murphy, D M; Eustace, J A; Shanahan, F; Hill, C; Stanton, C; Rea, M C; Ross, R P; Plant, B J

2017-03-01

Clostridium difficile is an anaerobic Gram-positive, spore-forming, toxin-producing bacillus transmitted among humans through the faecal-oral route. Despite increasing carriage rates and the presence of C. difficile toxin in stool, patients with CF rarely appear to develop typical manifestations of C. difficile infection (CDI). In this study, we examined the carriage, toxin production, ribotype distribution and antibiotic susceptibility of C. difficile in a cohort of 60 adult patients with CF who were pre-lung transplant. C. difficile was detected in 50% (30/60) of patients with CF by culturing for the bacteria. C. difficile toxin was detected in 63% (19/30) of C. difficile-positive stool samples. All toxin-positive stool samples contained toxigenic C. difficile strains harbouring toxin genes, tcdA and tcdB. Despite the presence of C. difficile and its toxin in patient stool, no acute gastrointestinal symptoms were reported. Ribotyping of C. difficile strains revealed 16 distinct ribotypes (RT), 11 of which are known to be disease-causing including the hyper-virulent RT078. Additionally, strains RT002, RT014, and RT015, which are common in non-CF nosocomial infection were described. All strains were susceptible to vancomycin, metronidazole, fusidic acid and rifampicin. No correlation was observed between carriage of C. difficile or any characteristics of isolated strains and any recorded clinical parameters or treatment received. We demonstrate a high prevalence of hypervirulent, toxigenic strains of C. difficile in asymptomatic patients with CF. This highlights the potential role of asymptomatic patients with CF in nosocomial transmission of C. difficile. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Comparison of five assays for detection of Clostridium difficile toxin.

PubMed

Chapin, Kimberle C; Dickenson, Roberta A; Wu, Fongman; Andrea, Sarah B

2011-07-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Molecular, microbiological and clinical characterization of Clostridium difficile isolates from tertiary care hospitals in Colombia.

PubMed

Salazar, Clara Lina; Reyes, Catalina; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita María; Paredes-Sabja, Daniel; Best, Emma; Fawley, Warren N; Wilcox, Mark; González, Ángel

2017-01-01

In Colombia, the epidemiology and circulating genotypes of Clostridium difficile have not yet been described. Therefore, we molecularly characterized clinical isolates of C.difficile from patients with suspicion of C.difficile infection (CDI) in three tertiary care hospitals. C.difficile was isolated from stool samples by culture, the presence of A/B toxins were detected by enzyme immunoassay, cytotoxicity was tested by cell culture and the antimicrobial susceptibility determined. After DNA extraction, tcdA, tcdB and binary toxin (CDTa/CDTb) genes were detected by PCR, and PCR-ribotyping performed. From a total of 913 stool samples collected during 2013-2014, 775 were included in the study. The frequency of A/B toxins-positive samples was 9.7% (75/775). A total of 143 isolates of C.difficile were recovered from culture, 110 (76.9%) produced cytotoxic effect in cell culture, 100 (69.9%) were tcdA+/tcdB+, 11 (7.7%) tcdA-/tcdB+, 32 (22.4%) tcdA-/tcdB- and 25 (17.5%) CDTa+/CDTb+. From 37 ribotypes identified, ribotypes 591 (20%), 106 (9%) and 002 (7.9%) were the most prevalent; only one isolate corresponded to ribotype 027, four to ribotype 078 and four were new ribotypes (794,795, 804,805). All isolates were susceptible to vancomycin and metronidazole, while 85% and 7.7% were resistant to clindamycin and moxifloxacin, respectively. By multivariate analysis, significant risk factors associated to CDI were, staying in orthopedic service, exposure to third-generation cephalosporins and staying in an ICU before CDI symptoms; moreover, steroids showed to be a protector factor. These results revealed new C. difficile ribotypes and a high diversity profile circulating in Colombia different from those reported in America and European countries.

Molecular, microbiological and clinical characterization of Clostridium difficile isolates from tertiary care hospitals in Colombia

PubMed Central

Salazar, Clara Lina; Reyes, Catalina; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita María; Paredes-Sabja, Daniel; Best, Emma; Fawley, Warren N.; Wilcox, Mark

2017-01-01

In Colombia, the epidemiology and circulating genotypes of Clostridium difficile have not yet been described. Therefore, we molecularly characterized clinical isolates of C.difficile from patients with suspicion of C.difficile infection (CDI) in three tertiary care hospitals. C.difficile was isolated from stool samples by culture, the presence of A/B toxins were detected by enzyme immunoassay, cytotoxicity was tested by cell culture and the antimicrobial susceptibility determined. After DNA extraction, tcdA, tcdB and binary toxin (CDTa/CDTb) genes were detected by PCR, and PCR-ribotyping performed. From a total of 913 stool samples collected during 2013–2014, 775 were included in the study. The frequency of A/B toxins-positive samples was 9.7% (75/775). A total of 143 isolates of C.difficile were recovered from culture, 110 (76.9%) produced cytotoxic effect in cell culture, 100 (69.9%) were tcdA+/tcdB+, 11 (7.7%) tcdA-/tcdB+, 32 (22.4%) tcdA-/tcdB- and 25 (17.5%) CDTa+/CDTb+. From 37 ribotypes identified, ribotypes 591 (20%), 106 (9%) and 002 (7.9%) were the most prevalent; only one isolate corresponded to ribotype 027, four to ribotype 078 and four were new ribotypes (794,795, 804,805). All isolates were susceptible to vancomycin and metronidazole, while 85% and 7.7% were resistant to clindamycin and moxifloxacin, respectively. By multivariate analysis, significant risk factors associated to CDI were, staying in orthopedic service, exposure to third-generation cephalosporins and staying in an ICU before CDI symptoms; moreover, steroids showed to be a protector factor. These results revealed new C. difficile ribotypes and a high diversity profile circulating in Colombia different from those reported in America and European countries. PMID:28902923

Comparison of Five Assays for Detection of Clostridium difficile Toxin

PubMed Central

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

Fidaxomicin Inhibits Clostridium difficile Toxin A-Mediated Enteritis in the Mouse Ileum

PubMed Central

Koon, Hon Wai; Ho, Samantha; Hing, Tressia C.; Cheng, Michelle; Chen, Xinhua; Ichikawa, Yoshi; Kelly, Ciarán P.

2014-01-01

Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins. PMID:24890583

Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

PubMed

Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J

2014-09-01

Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

[New methodological advances: algorithm proposal for management of Clostridium difficile infection].

PubMed

González-Abad, María José; Alonso-Sanz, Mercedes

2015-06-01

Clostridium difficile infection (CDI) is considered the most common cause of health care-associated diarrhea and also is an etiologic agent of community diarrhea. The aim of this study was to assess the potential benefit of a test that detects glutamate dehydrogenase (GDH) antigen and C. difficile toxin A/B, simultaneously, followed by detection of C. difficile toxin B (tcdB) gene by PCR as confirmatory assay on discrepant samples, and to propose an algorithm more efficient. From June 2012 to January 2013 at Hospital Infantil Universitario Niño Jesús, Madrid, the stool samples were studied for the simultaneous detection of GDH and toxin A/B, and also for detection of toxin A/B alone. When results between GDH and toxin A/B were discordant, a single sample for patient was selected for detection of C. difficile toxin B (tcdB) gene. A total of 116 samples (52 patients) were tested. Four were positive and 75 negative for toxigenic C. difficile (Toxin A/B, alone or combined with GDH). C. difficile was detected in the remaining 37 samples but not toxin A/B, regardless of the method used, except one. Twenty of the 37 specimens were further tested for C. difficile toxin B (tcdB) gene and 7 were positive. The simultaneous detection of GDH and toxin A/B combined with PCR recovered undiagnosed cases of CDI. In accordance with our data, we propose a two-step algorithm: detection of GDH and PCR (in samples GDH positive). This algorithm could provide a superior cost-benefit ratio in our population.

Intravenous immunoglobulin therapy for severe Clostridium difficile colitis

PubMed Central

Salcedo, J; Keates, S; Pothoulakis, C; Warny, M; Castagliuolo, I; LaMont, J; Kelly, C

1997-01-01

Background—Many individuals have serum antibodies against Clostridium difficile toxins. Those with an impaired antitoxin response may be susceptible to recurrent, prolonged, or severe C difficile diarrhoea and colitis. 
Aims—To examine whether treatment with intravenous immunoglobulin might be effective in patients with severe pseudomembranous colitis unresponsive to standard antimicrobial therapy. 
Patients—Two patients with pseudomembranous colitis not responding to metronidazole and vancomycin were given normal pooled human immunoglobulin intravenously (200-300 mg/kg). 
Methods—Antibodies against C difficile toxins were measured in nine immunoglobulin preparations by ELISA and by cytotoxin neutralisation assay. 
Results—Both patients responded quickly as shown by resolution of diarrhoea, abdominal tenderness, and distension. All immunoglobulin preparations tested contained IgG against C difficile toxins A and B by ELISA and neutralised the cytotoxic activity of C difficile toxins in vitro at IgG concentrations of 0.4-1.6 mg/ml. 
Conclusion—Passive immunotherapy with intravenous immunoglobulin may be a useful addition to antibiotic therapy for severe, refractory C difficile colitis. IgG antitoxin is present in standard immunoglobulin preparations and C difficile toxin neutralising activity is evident at IgG concentrations which are readily achieved in the serum by intravenous immunoglobulin administration. 

 Keywords: Clostridium difficile; toxin; diarrhoea; IgG; immunotherapy; antibiotic PMID:9378393

A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a Complete tcdA Gene

PubMed Central

Marín, Mercedes; Martín, Adoración; Rupnik, Maja

2014-01-01

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A− B+ CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site. PMID:25428159

Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile

PubMed Central

Lemee, Ludovic; Dhalluin, Anne; Testelin, Sabrina; Mattrat, Marie-Andre; Maillard, Karine; Lemeland, Jean-François; Pons, Jean-Louis

2004-01-01

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections. PMID:15583303

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

PubMed

Turgeon, David K; Novicki, Thomas J; Quick, John; Carlson, LaDonna; Miller, Pat; Ulness, Bruce; Cent, Anne; Ashley, Rhoda; Larson, Ann; Coyle, Marie; Limaye, Ajit P; Cookson, Brad T; Fritsche, Thomas R

2003-02-01

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Clinical application of polymerase chain reaction to diagnose Clostridium difficile in hospitalized patients with diarrhea.

PubMed

Morelli, Michael S; Rouster, Susan D; Giannella, Ralph A; Sherman, Kenneth E

2004-08-01

Clostridium difficile is a common cause of diarrhea in hospitalized patients and is associated with significant morbidity and cost. The current diagnostic standard, enzyme immunoassay (EIA), has low sensitivity, leading to duplicate testing and empiric treatment. We sought to show the usefulness and potential cost effectiveness of polymerase chain reaction (PCR) amplification of toxin B gene for diagnosis of C. difficile-induced diarrhea. A total of 148 stool samples from academic and community-based hospitals were sent for EIA testing and were evaluated prospectively for the presence of toxin B gene by PCR. Results were compared with EIA regarding sensitivity, specificity, and predictive values. Medical charts were reviewed to determine the following: (1) number of EIAs sent per admission, (2) number sent within a 24-hour time period, and (3) how caregivers practiced based on EIA results. The mean age of 130 patients was 55 years. EIA and PCR were positive in 6.8% and 13.6% of patients, respectively. EIA sensitivity was 40%, specificity was 98%, and positive and negative predictive values were 80% and 91%, respectively. The cost of the PCR was $22/sample. Empiric treatment for C. difficile was given unnecessarily in 42% of EIA-negative results. Thirty percent of patients had 3 or more EIAs sent during their hospital admission. Of patients with multiple samples sent, 57% had more than 1 sample sent in a 24-hour period. Many physicians do not conform to practice guidelines regarding recommended diagnosis and empiric treatment of C. difficile. Toxin B gene PCR represents a more sensitive and potentially cost-effective method to diagnose C. difficile-induced diarrhea than EIA and should be considered for use as an alternative diagnostic standard.

Clostridium difficile infection: molecular pathogenesis and novel therapeutics

PubMed Central

Rineh, Ardeshir; Kelso, Michael J; Vatansever, Fatma; Tegos, George P; Hamblin, Michael R

2015-01-01

The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed. PMID:24410618

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

PubMed

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

PubMed Central

Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

2015-01-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

Clostridium difficile infection

PubMed Central

Smits, Wiep Klaas; Lyras, Dena; Lacy, D. Borden; Wilcox, Mark H.; Kuijper, Ed J.

2017-01-01

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis — the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota. PMID:27158839

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291.

PubMed

Girinathan, Brintha P; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N; Sorg, Joseph A; Dupuy, Bruno; Govind, Revathi

2017-01-01

Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile -associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB , are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR . A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 a re linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile . In this study, we showed that a mutation in tcdR , the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291.

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

PubMed Central

Girinathan, Brintha P.; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N.; Dupuy, Bruno

2017-01-01

ABSTRACT Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291. PMID:28217744

The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

PubMed Central

Chen, Shuyi; Sun, Chunli; Wang, Haiying; Wang, Jufang

2015-01-01

Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB. PMID:26633511

Is there a relationship between the presence of the binary toxin genes in Clostridium difficile strains and the severity of C. difficile infection (CDI)?

PubMed

Berry, C E; Davies, K A; Owens, D W; Wilcox, M H

2017-12-01

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.

Sensitive assays enable detection of serum IgG antibodies against Clostridium difficile toxin A and toxin B in healthy subjects and patients with Clostridium difficile infection.

PubMed

Zhao, Xuemei; Bender, Florent; Shukla, Rajiv; Kang, John J; Caro-Aguilar, Ivette; Laterza, Omar F

2016-04-01

Pathogenic Clostridium difficile produces two proinflammatory exotoxins, toxin A and toxin B. Low level of serum antitoxin IgG antibodies is a risk factor for the development of primary and recurrent C. difficile infection (CDI). We developed and validated two sensitive, titer-based electrochemiluminescence assays for the detection of serum antibody levels against C. difficile toxins A and B. These assays demonstrated excellent precision. The sensitivity of the assays allowed the detection of antitoxin A and antitoxin B IgG antibodies in all tested serum samples during assay validation. The validated titer-based assays enable assessment of antitoxin A and antitoxin B IgG antibodies as potential biomarkers to identify patients with CDI at increased risk for CDI recurrence.

Evaluation of an interdisciplinary re-isolation policy for patients with previous Clostridium difficile diarrhea.

PubMed

Boone, N; Eagan, J A; Gillern, P; Armstrong, D; Sepkowitz, K A

1998-12-01

Diarrhea caused by Clostridium difficile is increasingly recognized as a nosocomial problem. The effectiveness and cost of a new program to decrease nosocomial spread by identifying patients scheduled for readmission who were previously positive for toxin was evaluated. The Memorial Sloan-Kettering Cancer Center is a 410-bed comprehensive cancer center in New York City. Many patients are readmitted during their course of cancer therapy. In 1995 as a result of concern about the nosocomial spread of C difficile, we implemented a policy that all patients who were positive for C difficile toxin in the previous 6 months with no subsequent toxin-negative stool as an outpatient would be placed into contact isolation on readmission pending evaluation of stool specimens. Patients who were previously positive for C difficile toxin were identified to infection control and admitting office databases via computer. Admitting personnel contacted infection control with all readmissions to determine whether a private room was required. Between July 1, 1995, and June 30, 1996, 47 patients who were previously positive for C difficile toxin were readmitted. Before their first scheduled readmission, the specimens for 15 (32%) of these patients were negative for C difficile toxin. They were subsequently cleared as outpatients and were readmitted without isolation. Workup of the remaining 32 patients revealed that the specimens for 7 patients were positive for C difficile toxin and 86 isolation days were used. An additional 25 patients used 107 isolation days and were either cleared after a negative specimen was obtained in-house or discharged without having an appropriate specimen sent. Four patients (9%) had reoccurring C difficile after having toxin-negative stools. We estimate (because outpatient specimens were not collected) the cost incurred at $48,500 annually, including the incremental cost of hospital isolation and equipment. Our policy to control the spread of nosocomial C difficile required interdisciplinary cooperation between infection control and the admitting department. By identifying patients who were positive for toxin through admitting, we were able to place all potentially infected patients into isolation. Our positivity rate of 15% on readmission demonstrates the importance of this policy. The cost of controlling C difficile can be significantly lowered by clearing patients who were previously positive for toxin before hospital readmission.

Comparing the economic and health benefits of different approaches to diagnosing Clostridium difficile infection.

PubMed

Bartsch, Sarah M; Umscheid, Craig A; Nachamkin, Irving; Hamilton, Keith; Lee, Bruce Y

2015-01-01

Accurate diagnosis of Clostridium difficile infection (CDI) is essential to effectively managing patients and preventing transmission. Despite the availability of several diagnostic tests, the optimal strategy is debatable and their economic values are unknown. We modified our previously existing C. difficile simulation model to determine the economic value of different CDI diagnostic approaches from the hospital perspective. We evaluated four diagnostic methods for a patient suspected of having CDI: 1) toxin A/B enzyme immunoassay, 2) glutamate dehydrogenase (GDH) antigen/toxin AB combined in one test, 3) nucleic acid amplification test (NAAT), and 4) GDH antigen/toxin AB combination test with NAAT confirmation of indeterminate results. Sensitivity analysis varied the proportion of those tested with clinically significant diarrhoea, the probability of CDI, NAAT cost and CDI treatment delay resulting from a false-negative test, length of stay and diagnostic sensitivity and specificity. The GDH/toxin AB plus NAAT approach leads to the timeliest treatment with the fewest unnecessary treatments given, resulted in the best bed management and generated the lowest cost. The NAAT-alone approach also leads to timely treatment. The GDH/toxin AB diagnostic (without NAAT confirmation) approach resulted in a large number of delayed treatments, but results in the fewest secondary colonisations. Results were robust to the sensitivity analysis. Choosing the right diagnostic approach is a matter of cost and test accuracy. GDH/toxin AB plus NAAT diagnosis led to the timeliest treatment and was the least costly. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Clostridium difficile binary toxin CDT

PubMed Central

Gerding, Dale N; Johnson, Stuart; Rupnik, Maja; Aktories, Klaus

2014-01-01

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed. PMID:24253566

Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile.

PubMed

Qutub, M O; AlBaz, N; Hawken, P; Anoos, A

2011-01-01

To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer's instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.

Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children

NASA Astrophysics Data System (ADS)

Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie

2014-12-01

Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.

Clostridium difficile and Clostridium perfringens from wild carnivore species in Brazil.

PubMed

Silva, Rodrigo Otávio Silveira; D'Elia, Mirella Lauria; Tostes Teixeira, Erika Procópio; Pereira, Pedro Lúcio Lithg; de Magalhães Soares, Danielle Ferreira; Cavalcanti, Álvaro Roberto; Kocuvan, Aleksander; Rupnik, Maja; Santos, André Luiz Quagliatto; Junior, Carlos Augusto Oliveira; Lobato, Francisco Carlos Faria

2014-08-01

Despite some case reports, the importance of Clostridium perfringens and Clostridium difficile for wild carnivores remains unclear. Thus, the objective of this study was to identify C. perfringens and C. difficile strains in stool samples from wild carnivore species in Brazil. A total of 34 stool samples were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota) and the beta-2 toxin (cpb2), enterotoxin (cpe) and NetB (netb) genes. C. difficile strains were analyzed by multiplex-PCR for toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB) and also submitted to a PCR ribotyping. Unthawed aliquots of samples positive for C. difficile isolation were subjected to the detection of A/B toxins by a cytotoxicity assay (CTA). C. perfringens was isolated from 26 samples (76.5%), all of which were genotyped as type A. The netb gene was not detected, whereas the cpb2 and cpe genes were found in nine and three C. perfringens strains, respectively. C. difficile was isolated from two (5.9%) samples. A non-toxigenic strain was recovered from a non-diarrheic maned wolf (Chrysocyon brachyurus). Conversely, a toxigenic strain was found in the sample of a diarrheic ocelot (Leopardus pardallis); an unthawed stool sample was also positive for A/B toxins by CTA, indicating a diagnosis of C. difficile-associated diarrhea in this animal. The present work suggests that wild carnivore species could carry C. difficile strains and that they could be susceptible to C. difficile infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prevalence of Clostridium difficile infection and colonization in a tertiary hospital and elderly community of North-Eastern Peninsular Malaysia.

PubMed

Zainul, N H; Ma, Z F; Besari, A; Siti Asma, H; Rahman, R A; Collins, D A; Hamid, N; Riley, T V; Lee, Y Y

2017-10-01

Little is known about Clostridium difficile infection (CDI) in Asia. The aims of our study were to explore (i) the prevalence, risk factors and molecular epidemiology of CDI and colonization in a tertiary academic hospital in North-Eastern Peninsular Malaysia; (ii) the rate of carriage of C. difficile among the elderly in the region; (iii) the awareness level of this infection among the hospital staffs and students. For stool samples collected from hospital inpatients with diarrhea (n = 76) and healthy community members (n = 138), C. difficile antigen and toxins were tested by enzyme immunoassay. Stool samples were subsequently analyzed by culture and molecular detection of toxin genes, and PCR ribotyping of isolates. To examine awareness among hospital staff and students, participants were asked to complete a self-administered questionnaire. For the hospital and community studies, the prevalence of non-toxigenic C. difficile colonization was 16% and 2%, respectively. The prevalence of CDI among hospital inpatients with diarrhea was 13%. Out of 22 C. difficile strains from hospital inpatients, the toxigenic ribotypes 043 and 017 were most common (both 14%). In univariate analysis, C. difficile colonization in hospital inpatients was significantly associated with greater duration of hospitalization and use of penicillin (both P < 0·05). Absence of these factors was a possible reason for low colonization in the community. Only 3% of 154 respondents answered all questions correctly in the awareness survey. C. difficile colonization is prevalent in a Malaysian hospital setting but not in the elderly community with little or no contact with hospitals. Awareness of CDI is alarmingly poor.

The roles of host and pathogen factors and the innate immune response in the pathogenesis of Clostridium difficile infection

PubMed Central

Sun, Xingmin; Hirota, Simon A.

2014-01-01

Clostridium difficile (C. difficile) is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of pseudomembranous colitis. The clinical manifestation of Clostridium difficile infection (CDI) is highly variable, from asymptomatic carriage, to mild self-limiting diarrhea, to the more severe pseudomembranous colitis. Furthermore, in extreme cases, colonic inflammation and tissue damage can lead to toxic megacolon, a condition requiring surgical intervention. C. difficile expresses two key virulence factors; the exotoxins, toxin A (TcdA) and toxin B (TcdB), which are glucosyltransferases that target host-cell monomeric GTPases. In addition, some hypervirulent strains produce a third toxin, binary toxin or C. difficile transferase (CDT), which may contribute to the pathogenesis of CDI. More recently, other factors such as surface layer proteins (SLPs) and flagellin have also been linked to the inflammatory responses observed in CDI. Although the adaptive immune response can influence the severity of CDI, the innate immune responses to C. difficile and its toxins play crucial roles in CDI onset, progression, and overall prognosis. Despite this, the innate immune responses in CDI have drawn relatively little attention from clinical researchers. Targeting these responses may prove useful clinically as adjuvant therapies, especially in refractory and/or recurrent CDI. This review will focus on recent advances in our understanding of how C. difficile and its toxins modulate innate immune responses that contribute to CDI pathogenesis. PMID:25242213

Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

PubMed Central

Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M.; Zhang, Yuhua; Arnold, Beth A.; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E.; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M.

2014-01-01

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

Cannabidiol restores intestinal barrier dysfunction and inhibits the apoptotic process induced by Clostridium difficile toxin A in Caco-2 cells.

PubMed

Gigli, Stefano; Seguella, Luisa; Pesce, Marcella; Bruzzese, Eugenia; D'Alessandro, Alessandra; Cuomo, Rosario; Steardo, Luca; Sarnelli, Giovanni; Esposito, Giuseppe

2017-12-01

Clostridium difficile toxin A is responsible for colonic damage observed in infected patients. Drugs able to restore Clostridium difficile toxin A-induced toxicity have the potential to improve the recovery of infected patients. Cannabidiol is a non-psychotropic component of Cannabis sativa, which has been demonstrated to protect enterocytes against chemical and/or inflammatory damage and to restore intestinal mucosa integrity. The purpose of this study was to evaluate (a) the anti-apoptotic effect and (b) the mechanisms by which cannabidiol protects mucosal integrity in Caco-2 cells exposed to Clostridium difficile toxin A. Caco-2 cells were exposed to Clostridium difficile toxin A (30 ng/ml), with or without cannabidiol (10 -7 -10 -9  M), in the presence of the specific antagonist AM251 (10 -7  M). Cytotoxicity assay, transepithelial electrical resistence measurements, immunofluorescence analysis and immunoblot analysis were performed in the different experimental conditions. Clostridium difficile toxin A significantly decreased Caco-2 cells' viability and reduced transepithelial electrical resistence values and RhoA guanosine triphosphate (GTP), bax, zonula occludens-1 and occludin protein expression, respectively. All these effects were significantly and concentration-dependently inhibited by cannabidiol, whose effects were completely abolished in the presence of the cannabinoid receptor type 1 (CB1) antagonist, AM251. Cannabidiol improved Clostridium difficile toxin A-induced damage in Caco-2 cells, by inhibiting the apoptotic process and restoring the intestinal barrier integrity, through the involvement of the CB1 receptor.

Clostridium difficile chimeric toxin receptor binding domain vaccine induced protection against different strains in active and passive challenge models.

PubMed

Tian, Jing-Hui; Glenn, Gregory; Flyer, David; Zhou, Bin; Liu, Ye; Sullivan, Eddie; Wu, Hua; Cummings, James F; Elllingsworth, Larry; Smith, Gale

2017-07-24

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB (027) ). TcdB (027) is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB (003) ). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB (003) , TcdB (027) and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB (003) /TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB (003) /TcdA/TcdB (027) ) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI). Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

PubMed Central

Alfa, Michelle J; Sepehri, Shadi

2013-01-01

BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile. PMID:24421808

A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage.

PubMed

Shaban, Lamyaa; Chen, Ying; Fasciano, Alyssa C; Lin, Yinan; Kaplan, David L; Kumamoto, Carol A; Mecsas, Joan

2018-04-01

Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O 2 conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections. Copyright © 2018 Elsevier Ltd. All rights reserved.

Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

PubMed Central

Castagliuolo, Ignazio; Riegler, Martin F.; Valenick, Leyla; LaMont, J. Thomas; Pothoulakis, Charalabos

1999-01-01

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease. PMID:9864230

Comparing ImmunoCard with two EIA assays for Clostridium difficile toxins.

PubMed

Chan, Edward L; Seales, Diane; Drum, Hong

2009-01-01

To compare three Clostridium difficile EIA kits for the detection of C. difficile toxins from clinical specimens. A total of 287 fresh and stored stool specimens were tested using all three assays. Stools with discrepant results were sent to a reference laboratory for tissue cytotoxin assay. Trinity Medical Center, a community hospital with network hospitals. Patients with diarrhea submitted stools for detection of C. difficile toxins. Of the 287 stool specimens, 116 were positive and 171 negative for C. difficile toxins. The sensitivity, specificity, and positive and negative predictive values of Meridian EIA assay were 99.1, 97.7, 96.6, and 99.4%; ImmunoCard were 100, 98.2, 97.5, and 100%; BioStar OIA assay were 94, 98.8, 98.2, and 96% respectively. ImmunoCardprovides the best sensitivity (100%) for C. difficile toxins A and B detection. The BioStar OIA rapid test missed seven positive stool specimens possibly due to failure to detect toxin B. ImmunoCard has slightly higher predictive values, shorter turnaround time and greater convenience compared to the Meridian EIA Assay. ImmunoCard may be cost effective not only in smaller laboratories, but also in high volume laboratories, when used on a STAT basis or single request.

Diagnostic trends in Clostridium difficile detection in Finnish microbiology laboratories.

PubMed

Könönen, Eija; Rasinperä, Marja; Virolainen, Anni; Mentula, Silja; Lyytikäinen, Outi

2009-12-01

Due to increased interest directed to Clostridium difficile-associated infections, a questionnaire survey of laboratory diagnostics of toxin-producing C. difficile was conducted in Finland in June 2006. Different aspects pertaining to C. difficile diagnosis, such as requests and criteria used for testing, methods used for its detection, yearly changes in diagnostics since 1996, and the total number of investigations positive for C. difficile in 2005, were asked in the questionnaire, which was sent to 32 clinical microbiology laboratories, including all hospital-affiliated and the relevant private clinical microbiology laboratories in Finland. The situation was updated by phone and email correspondence in September 2008. In June 2006, 28 (88%) laboratories responded to the questionnaire survey; 24 of them reported routinely testing requested stool specimens for C. difficile. Main laboratory methods included toxin detection (21/24; 88%) and/or anaerobic culture (19/24; 79%). In June 2006, 18 (86%) of the 21 laboratories detecting toxins directly from feces, from the isolate, or both used methods for both toxin A (TcdA) and B (TcdB), whereas only one laboratory did so in 1996. By September 2008, all of the 23 laboratories performing diagnostics for C. difficile used methods for both TcdA and TcdB. In 2006, the number of specimens processed per 100,000 population varied remarkably between different hospital districts. In conclusion, culturing C. difficile is common and there has been a favorable shift in toxin detection practice in Finnish clinical microbiology laboratories. However, the variability in diagnostic activity reported in 2006 creates a challenge for national monitoring of the epidemiology of C. difficile and related diseases.

Clostridium difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms ▿

PubMed Central

Novak-Weekley, Susan M.; Marlowe, Elizabeth M.; Miller, John M.; Cumpio, Joven; Nomura, Jim H.; Vance, Paula H.; Weissfeld, Alice

2010-01-01

The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study. PMID:20071552

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

The Regulatory Networks That Control Clostridium difficile Toxin Synthesis

PubMed Central

Martin-Verstraete, Isabelle; Peltier, Johann; Dupuy, Bruno

2016-01-01

The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. PMID:27187475

Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients.

PubMed

Erb, S; Frei, R; Strandén, A M; Dangel, M; Tschudin-Sutter, S; Widmer, A F

2015-11-01

The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12,481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/μL (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

PubMed

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Impact of toxigenic Clostridium difficile polymerase chain reaction testing on the clinical microbiology laboratory and inpatient epidemiology.

PubMed

Napierala, Maureen; Munson, Erik; Skonieczny, Patrice; Rodriguez, Sonia; Riederer, Nancy; Land, Gayle; Luzinski, Mary; Block, Denise; Hryciuk, Jeanne E

2013-08-01

Conversion from Clostridium difficile toxin A/B EIA to tcdB polymerase chain reaction for diagnosis of C. difficile infection (CDI) resulted in significant decreases in laboratory testing volume and largely unchanged C. difficile toxin detection rates. Decreases in healthcare-associated CDI rates (P ≤ 0.05) reflected a clinical practice benefit of this conversion. Copyright © 2013 Elsevier Inc. All rights reserved.

Binary toxin and its clinical importance in Clostridium difficile infection, Belgium.

PubMed

Pilate, T; Verhaegen, J; Van Ranst, M; Saegeman, V

2016-11-01

Binary toxin-producing Clostridium difficile strains such as ribotypes 027 and 078 have been associated with increased Clostridium difficile infection (CDI) severity. Our objective was to investigate the association between presence of the binary toxin gene and CDI severity and recurrence. We performed a laboratory-based retrospective study including patients between January 2013 and March 2015 whose fecal samples were analyzed by polymerase chain reaction (PCR) for the presence of the genes for toxin B and binary toxin and a deletion in the tcdC gene, specific for ribotype 027. Clinical and epidemiological characteristics were compared between 33 binary toxin-positive CDI patients and 33 binary toxin-negative CDI patients. Subsequently, the characteristics of 66 CDI patients were compared to those of 66 diarrhea patients who were carriers of non-toxigenic C. difficile strains. Fifty-nine of 1034 (5.7 %) fecal samples analyzed by PCR were binary toxin-positive, belonging to 33 different patients. No samples were positive for ribotype 027. Binary toxin-positive CDI patients did not differ from binary toxin-negative CDI patients in terms of disease recurrence, morbidity, or mortality, except for a higher peripheral leukocytosis in the binary toxin-positive group (16.30 × 10 9 /L vs. 11.65 × 10 9 /L; p = 0.02). The second part of our study showed that CDI patients had more severe disease, but not a higher 30-day mortality rate than diarrhea patients with a non-toxicogenic C. difficile strain. In our setting with a low prevalence of ribotype 027, the presence of the binary toxin gene is not associated with poor outcome.

[Pathogenicity factors of bacteria with glycosylating activity].

PubMed

Tartakovskaia, D I; Araslanova, V A; Belyĭ, Iu F

2011-01-01

A and B toxins of Clostridium difficile, a-toxin of C. novyi, lehal toxin of C. sordellii, and TpeL toxin of C. perfringens belong to the group of the so-called large Clostridium toxins. These toxins modify low-molecular weight guanosine triphosphate-binding proteins of the Rho/Ras family by their glycosylation that results in inactivation of major signal pathways in eukaryotic cells. Lgt glycosyltransferases, a new group of pathogenicity factors also capable of inactivating eukaryotic substrates via glycosylation, have recently been identified in Legionella. They are transported into cytoplasm of eukaryotic target cells by type 4 secretory system of Legionella. After translocation, the enzyme inhibits protein synthesis by attaching glucose residue to Ser53 of 1A elongation factor. The available data suggest an important role of bacterial glycosylating factors in the action of pathogens causing infectious diseases.

Immunization with Bacillus Spores Expressing Toxin A Peptide Repeats Protects against Infection with Clostridium difficile Strains Producing Toxins A and B ▿ †

PubMed Central

Permpoonpattana, Patima; Hong, Huynh A.; Phetcharaburanin, Jutarop; Huang, Jen-Min; Cook, Jenny; Fairweather, Neil F.; Cutting, Simon M.

2011-01-01

Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two toxins, A and B, produced by most strains of C. difficile are implicated as virulence factors, yet only recently has the requirement of these for infection been investigated by genetic manipulation. Current vaccine strategies are focused mostly on parenteral delivery of toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to evaluate the carboxy-terminal repeat domains of toxins A and B as protective antigens. Our findings are important and show that oral immunization of the repeat domain of toxin A is sufficient to confer protection in a hamster model of infection designed to closely mimic the human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain were shown to be cross-reactive with the analogous domain of toxin B and, being of high avidity, provided protection against challenge with a C. difficile strain producing toxins A and B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can mediate protection. Animals vaccinated with recombinant spores were fully able to survive reinfection, a property that is particularly important for a disease with which patients are prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to generate secretory IgA and that production of these neutralizing polymeric antibodies correlates with protection. This work demonstrates that an effective vaccine against C. difficile can be designed around two attributes, mucosal delivery and the repeat domain of toxin A. PMID:21482682

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

PubMed Central

Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

1996-01-01

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

PubMed

Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

2014-02-01

The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

C. difficile Infection

MedlinePlus

... Patients Home / Digestive Health Topic / C. Difficile Infection C. Difficile Infection Basics Overview Diarrhea is a frequent ... that change the normal colon bacteria allowing the C. difficile bacteria to grow and produce its toxins. ...

Evaluation of the Cepheid® Xpert®C. difficile binary toxin (BT) diagnostic assay.

PubMed

McGovern, Alan M; Androga, Grace O; Moono, Peter; Collins, Deirdre A; Foster, Niki F; Chang, Barbara J; Riley, Thomas V

2018-06-01

Strains of Clostridium difficile producing only binary toxin (CDT) are found commonly in animals but not humans. However, human diagnostic tests rarely look for CDT. The Cepheid Xpert C. difficile BT assay detects CDT with equal sensitivity (≥92%) in human and animal faecal samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

PubMed

Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan

2014-05-19

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

PubMed Central

Torres, J F; Lyerly, D M; Hill, J E; Monath, T P

1995-01-01

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization. PMID:7591115

Prevalence of Clostridium perfringens toxin in patients suspected of having antibiotic-associated diarrhea.

PubMed

Kim, Young Jin; Kim, Si Hyun; Ahn, Junggu; Cho, Soongmoon; Kim, Dongchun; Kim, Kwanghyun; Lee, Heegun; Son, Hyunwoo; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kim, Hye Ran; Shin, Jeong Hwan

2017-12-01

Although Clostridium perfringens has been reported as a cause of antibiotic-associated diarrhea (AAD), it is uncommon to detect this pathogen in clinical microbiology laboratories in Korea. The aim of this study was to investigate the prevalence of C. perfringens toxin in patients suspected of having AAD. A total of 135 stool specimens submitted to a clinical microbiology laboratory for C. difficile toxin assay were tested. We tried to detect both C. difficile and C. perfringens toxins using the Seeplex Diarrhea ACE Detection kit (Seegene, Seoul, Korea). We evaluated the prevalence of 10 bacteria and 5 viruses. A total of 40 Clostridium spp. were detected in 34 specimens (29.6%). The C. perfringens toxin was detected in 14 of 135 specimens (10.4%), while C. difficile toxin was detected in 26 specimens (19.3%). Other bacteria and viruses, including 8 Aeromonas spp., were detected in 15 specimens. All tests were negative in 92 of the 135 specimens (68.1%). Clostridium perfringens toxin is relatively common, and we should consider the possibility of its presence in patients suspected of having AAD, especially if C. difficile tests are negative. Copyright © 2017 Elsevier Ltd. All rights reserved.

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-04-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.

Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196.

PubMed Central

Perelle, S; Gibert, M; Bourlioux, P; Corthier, G; Popoff, M R

1997-01-01

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor. PMID:9119480

Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth.

PubMed

Weese, J S; Cote, N M; deGannes, R V G

2003-11-01

Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

The impact of Clostridum difficile on surgical rate among ulcerative colitis patients: A systemic review and meta-analysis.

PubMed

Peng, Jiang-Chen; Shen, Jun; Zhu, Qi; Ran, Zhi-Hua

2015-01-01

There is growing recognition of the impact of Clostridum difficile infection (CDI) on patients with inflammatory bowel disease. Clostridium difficile infection causes greater morbidity and mortality. This study aimed to evaluate the impact of C. difficile on surgical risk among ulcerative colitis (UC) patients. We searched the following databases: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, ACP Journal Club, DARE, CMR, and HTA. Studies were included if fulfilled the following criteria: (1) Cohort or case-control studies, which involved a comparison group that lacked CDI, (2) Patients were given a primary diagnosis of UC, (3) Comorbidity of CDI was evaluated by enzyme immunoassay of stool for C. difficile toxin A and B or C. difficile stool culture, (4) Studies evaluated surgical rate, and (5) Studies reported an estimate of odds ratio, accompanied by a corresponding measure of uncertainty. Five studies with 2380 patients fulfilled the inclusion criteria. Overall, meta-analysis showed that UC with CDI patients had a significant higher surgical rate than patients with UC alone. (OR=1.76, 95% CI=1.36-2.28). C. difficile infection increased the surgical rate in UC patients. However, results should be interpreted with caution, given the limitations of this stud.

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

PubMed Central

Gohari, Iman Mehdizadeh; Arroyo, Luis; MacInnes, Janet I.; Timoney, John F.; Parreira, Valeria R.; Prescott, John F.

2014-01-01

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered. PMID:24396174

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis.

PubMed

Gohari, Iman Mehdizadeh; Arroyo, Luis; Macinnes, Janet I; Timoney, John F; Parreira, Valeria R; Prescott, John F

2014-01-01

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.

Bezlotoxumab: anti-toxin B monoclonal antibody to prevent recurrence of Clostridium difficile infection.

PubMed

Villafuerte Gálvez, Javier A; Kelly, Ciarán P

2017-07-01

Clostridium difficile infection (CDI) is the most common nosocomial infection in the U.S. 25% of CDI patients go on to develop recurrent CDI (rCDI) following current standard of care (SOC) therapy, leading to morbidity, mortality and economic loss. The first passive immunotherapy drug targeting C.difficile toxin B (bezlotoxumab) has been approved recently by the FDA and EMA for prevention of rCDI. Areas covered: A body of key studies was selected and reviewed by the authors. The unmet needs in CDI care were ascertained with emphasis in rCDI, including the epidemiology, pathophysiology and current management. The current knowledge about the immune response to C. difficile toxins and how this knowledge led to the development and the clinical use of bezlotoxumab is described. Current and potential future competitors to the drug were examined. Expert commentary: A single 10 mg/kg intravenous infusion of bezlotoxumab has been shown to decrease rCDI by ~40% (absolute reduction ~10%) in patients being treated for primary CDI or rCDI with SOC antibiotics. Targeting C.difficile toxins by passive immunotherapy is a novel mechanism for prevention of C.difficile infection. Bezlotoxumab will be a valuable adjunctive therapy to reduce the burden of CDI.

Clostridium difficile in retail baskets, trolleys, conveyor belts, and plastic bags in Saudi Arabia.

PubMed

Alqumber, Mohammed A

2014-10-01

To determine Clostridium difficile (C. difficile) prevalence on retail surfaces and shoppers plastic bags. From 20 June to 10 August 2011, in a cross-sectional epidemiological study, 17 supermarkets from 2 cities, Albaha and Altaif, Saudi Arabia were sampled. A total of 800 samples, which comprised 200 samples per surveyed surface, were studied. These included baskets, trolleys, conveyer belts, and outgoing shoppers' plastic bags. Clostridium difficile strains were isolated. The isolates were characterized using ribotyping and  polymerase chain reaction for the detection of toxin A (tcdA), toxin B (tcdB), binary toxin (cdtB), and toxin C (tcdC) genes. Susceptibility to antibiotics was determined on a Muller-Hinton agar with 5% sheep blood agar using E-tests. Overall, the C. difficile prevalence on sampled surfaces was 0.75%. The highest prevalence was found on retail baskets and trolleys, followed by plastic bags. A total of 5 different ribotypes were identified. Alterations in tcdC were detected in ribotype 027 and BT1. All the identified isolates were susceptible to vancomycin, but resistant to levofloxacin. In this study, C. difficile was present at a rate of 0.75% on supermarket surfaces. Spore disinfection of implicated surfaces may be necessary to control any community-acquired infections caused by this pathogen. 

Clinical and Microbiologic Characteristics of Clostridium difficile Infection Caused by Binary Toxin Producing Strain in Korea.

PubMed

Kim, Jieun; Seo, Mi-Ran; Kang, Jung Oak; Choi, Tae Yeal; Pai, Hyunjoo

2013-06-01

Binary toxin-producing Clostridium difficile infections (CDI) are known to be more severe and to cause higher case fatality rates than those by binary toxin-negative isolates. There has been few data of binary toxin-producing CDI in Korea. Objective of the study is to characterize clinical and microbiological trait of CDI cause by binary-toxin producing isolates in Korea. From September 2008 through January 2010, clinical characteristics, medication history and treatment outcome of all the CDI patients were collected prospectively. Toxin characterization, PCR ribotyping and antibiotic susceptibility were performed with the stool isolates of C. difficile. During the period, CDI caused by 11binary toxin-producing isolates and 105 toxin A & toxin B-positive binary toxin-negative isolates were identified. Comparing the disease severity and clinical findings between two groups, leukocytosis and mucoid stool were more frequently observed in patients with binary toxin-positive isolates (OR: 5.2, 95% CI: 1.1 to 25.4, P = 0.043; OR: 7.6, 95% CI: 1.6 to 35.6, P = 0.010, respectively), but clinical outcome of 2 groups did not show any difference. For the risk factors for acquisition of binary toxin-positive isolates, previous use of glycopeptides was the significant risk factor (OR: 6.2, 95% CI: 1.4 to 28.6, P = 0.019), but use of probiotics worked as an inhibitory factor (OR: 0.1, 95% CI: 0.0 to 0.8; P = 0.026). PCR ribotypes of binary toxinproducing C. difficile showed variable patterns: ribotype 130, 4 isolates; 027, 3 isolates; 267 and 122, 1 each isolate and unidentified C1, 2 isolates. All 11 binary toxin-positive isolates were highly susceptible to clindamycin, moxifloxacin, metronidazole, vancomycin and piperacillin-tazobactam, however, 1 of 11 of the isolates was resistant to rifaximin. Binary toxin-producing C. difficile infection was not common in Korea and those isolates showed diverse PCR ribotypes with high susceptibility to antimicrobial agents. Glycopeptide use was a risk factor for CDI by those isolates.

Clinical and Microbiologic Characteristics of Clostridium difficile Infection Caused by Binary Toxin Producing Strain in Korea

PubMed Central

Kim, Jieun; Seo, Mi-ran; Kang, Jung Oak; Choi, Tae Yeal

2013-01-01

Background Binary toxin-producing Clostridium difficile infections (CDI) are known to be more severe and to cause higher case fatality rates than those by binary toxin-negative isolates. There has been few data of binary toxin-producing CDI in Korea. Objective of the study is to characterize clinical and microbiological trait of CDI cause by binary-toxin producing isolates in Korea. Materials and Methods From September 2008 through January 2010, clinical characteristics, medication history and treatment outcome of all the CDI patients were collected prospectively. Toxin characterization, PCR ribotyping and antibiotic susceptibility were performed with the stool isolates of C. difficile. Results During the period, CDI caused by 11binary toxin-producing isolates and 105 toxin A & toxin B-positive binary toxin-negative isolates were identified. Comparing the disease severity and clinical findings between two groups, leukocytosis and mucoid stool were more frequently observed in patients with binary toxin-positive isolates (OR: 5.2, 95% CI: 1.1 to 25.4, P = 0.043; OR: 7.6, 95% CI: 1.6 to 35.6, P = 0.010, respectively), but clinical outcome of 2 groups did not show any difference. For the risk factors for acquisition of binary toxin-positive isolates, previous use of glycopeptides was the significant risk factor (OR: 6.2, 95% CI: 1.4 to 28.6, P = 0.019), but use of probiotics worked as an inhibitory factor (OR: 0.1, 95% CI: 0.0 to 0.8; P = 0.026). PCR ribotypes of binary toxinproducing C. difficile showed variable patterns: ribotype 130, 4 isolates; 027, 3 isolates; 267 and 122, 1 each isolate and unidentified C1, 2 isolates. All 11 binary toxin-positive isolates were highly susceptible to clindamycin, moxifloxacin, metronidazole, vancomycin and piperacillin-tazobactam, however, 1 of 11 of the isolates was resistant to rifaximin. Conclusions Binary toxin-producing C. difficile infection was not common in Korea and those isolates showed diverse PCR ribotypes with high susceptibility to antimicrobial agents. Glycopeptide use was a risk factor for CDI by those isolates. PMID:24265965

Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen.

PubMed

Awad, Milena M; Johanesen, Priscilla A; Carter, Glen P; Rose, Edward; Lyras, Dena

2014-01-01

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.

Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen

PubMed Central

Awad, Milena M; Johanesen, Priscilla A; Carter, Glen P; Rose, Edward; Lyras, Dena

2014-01-01

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions. PMID:25483328

Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

PubMed Central

Bouillaut, Laurent; Self, William T.

2013-01-01

Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the d-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions. PMID:23222730

Incorrect diagnosis of Clostridium difficile infection in a university hospital in Japan.

PubMed

Mori, Nobuaki; Yoshizawa, Sadako; Saga, Tomoo; Ishii, Yoshikazu; Murakami, Hinako; Iwata, Morihiro; Collins, Deirdre A; Riley, Thomas V; Tateda, Kazuhiro

2015-10-01

Physicians often fail to suspect Clostridium difficile infection (CDI) and many microbiology laboratories use suboptimal diagnostic techniques. To estimate the extent of and reasons for incorrect diagnosis of CDI in Japan, we investigated toxigenic C. difficile isolated from all stool culture samples and clinical course. Over a 12-month period in 2010, all stool culture samples (n = 975) submitted from inpatients in a university hospital in Japan were cultured for C. difficile and routine microbiological testing was conducted. In total, 177 C. difficile isolates were recovered, and 127 isolates were toxigenic. Among the toxin-A-positive/toxin-B-positive isolates, 12 were also positive for the binary toxin gene. However, clinically important ribotypes, such as 027 and 078, were not identified. A total of 58 (45.7%) cases with toxigenic C. difficile had unformed stool, and the incidence CDI was 1.6 cases per 10,000 patient-days. Of these 58 cases, 40 were not diagnosed in routine testing due to a lack of clinical suspicion (24.1%, 14/58) or a negative C. difficile toxin assay result (44.8%, 26/58). A stool toxin assay was performed in 54 patients (78.2%, 54/69) who did not have unformed stool. The present study demonstrated that a significant number of CDI cases in Japan might be overlooked or misdiagnosed in clinical practice due to a lack of clinical suspicion and limitations of microbiological testing for CDI in Japan. Providing education to promote awareness of CDI among physicians is important to improve the accuracy of diagnosis in Japan. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era.

PubMed

Polage, Christopher R; Gyorke, Clare E; Kennedy, Michael A; Leslie, Jhansi L; Chin, David L; Wang, Susan; Nguyen, Hien H; Huang, Bin; Tang, Yi-Wei; Lee, Lenora W; Kim, Kyoungmi; Taylor, Sandra; Romano, Patrick S; Panacek, Edward A; Goodell, Parker B; Solnick, Jay V; Cohen, Stuart H

2015-11-01

Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision making and public health reporting. Molecular tests are increasingly used to diagnose C difficile infection (CDI), but many molecular test-positive patients lack toxins that historically defined disease, making it unclear if they need treatment. To determine the natural history and need for treatment of patients who are toxin immunoassay negative and polymerase chain reaction (PCR) positive (Tox-/PCR+) for CDI. Prospective observational cohort study at a single academic medical center among 1416 hospitalized adults tested for C difficile toxins 72 hours or longer after admission between December 1, 2010, and October 20, 2012. The analysis was conducted in stages with revisions from April 27, 2013, to January 13, 2015. Patients undergoing C difficile testing were grouped by US Food and Drug Administration-approved toxin and PCR tests as Tox+/PCR+, Tox-/PCR+, or Tox-/PCR-. Toxin results were reported clinically. Polymerase chain reaction results were not reported. The main study outcomes were duration of diarrhea during up to 14 days of treatment, rate of CDI-related complications (ie, colectomy, megacolon, or intensive care unit care) and CDI-related death within 30 days. Twenty-one percent (293 of 1416) of hospitalized adults tested for C difficile were positive by PCR, but 44.7% (131 of 293) had toxins detected by the clinical toxin test. At baseline, Tox-/PCR+ patients had lower C difficile bacterial load and less antibiotic exposure, fecal inflammation, and diarrhea than Tox+/PCR+ patients (P < .001 for all). The median duration of diarrhea was shorter in Tox-/PCR+ patients (2 days; interquartile range, 1-4 days) than in Tox+/PCR+ patients (3 days; interquartile range, 1-6 days) (P = .003) and was similar to that in Tox-/PCR- patients (2 days; interquartile range, 1-3 days), despite minimal empirical treatment of Tox-/PCR+ patients. No CDI-related complications occurred in Tox-/PCR+ patients vs 10 complications in Tox+/PCR+ patients (0% vs 7.6%, P < .001). One Tox-/PCR+ patient had recurrent CDI as a contributing factor to death within 30 days vs 11 CDI-related deaths in Tox+/PCR+ patients (0.6% vs 8.4%, P = .001). Among hospitalized adults with suspected CDI, virtually all CDI-related complications and deaths occurred in patients with positive toxin immunoassay test results. Patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without C difficile by either method. Exclusive reliance on molecular tests for CDI diagnosis without tests for toxins or host response is likely to result in overdiagnosis, overtreatment, and increased health care costs.

1H, 13C, and 15N resonance assignments of an enzymatically active domain from the catalytic component (CDTa, residues 216-420) of a binary toxin from Clostridium difficile.

PubMed

Roth, Braden M; Godoy-Ruiz, Raquel; Varney, Kristen M; Rustandi, Richard R; Weber, David J

2016-04-01

Clostridium difficile is a bacterial pathogen and is the most commonly reported source of nosocomial infection in industrialized nations. Symptoms of C. difficile infection (CDI) include antibiotic-associated diarrhea, pseudomembranous colitis, sepsis and death. Over the last decade, rates and severity of hospital infections in North America and Europe have increased dramatically and correlate with the emergence of a hypervirulent strain of C. difficile characterized by the presence of a binary toxin, CDT (C. difficile toxin). The binary toxin consists of an enzymatic component (CDTa) and a cellular binding component (CDTb) that together form the active binary toxin complex. CDTa harbors a pair of structurally similar but functionally distinct domains, an N-terminal domain (residues 1-215; (1-215)CDTa) that interacts with CDTb and a C-terminal domain (residues 216-420; (216-420)CDTa) that harbors the intact ADP-ribosyltransferase (ART) active site. Reported here are the (1)H, (13)C, and (15)N backbone resonance assignments of the 23 kDa, 205 amino acid C-terminal enzymatic domain of CDTa, termed (216-420)CDTa. These NMR resonance assignments for (216-420)CDTa represent the first for a family of ART binary toxins and provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity.

PubMed

Considine, R V; Simpson, L L

1991-01-01

Clostridial organisms produce a number of binary toxins. Thus far, three complete toxins (botulinum, perfringens and spiroforme) and one incomplete toxin (difficile) have been identified. In the case of complete toxins, there is a heavy chain component (Mr approximately 100,000) that binds to target cells and helps create a docking site for the light chain component (Mr approximately 50,000). The latter is an enzyme that possesses mono(ADP-ribosyl)transferase activity. The toxins appear to proceed through a three step sequence to exert their effects, including a binding step, an internalization step and an intracellular poisoning step. The substrate for the toxins is G-actin. By virtue of ADP-ribosylating monomeric actin, the toxins prevent polymerization as well as promoting depolymerization. The most characteristic cellular effect of the toxins is alteration of the cytoskeleton, which leads directly to changes in cellular morphology and indirectly to changes in cell function (e.g. release of chemical mediators). Binary toxins capable of modifying actin are likely to be useful tools in the study of cell biology.

Toxin-positive Clostridium difficile latently infect mouse colonies and protect against highly pathogenic C. difficile.

PubMed

Etienne-Mesmin, Lucie; Chassaing, Benoit; Adekunle, Oluwaseyi; Mattei, Lisa M; Bushman, Frederic D; Gewirtz, Andrew T

2018-05-01

Clostridium difficile is a toxin-producing bacterium and a leading cause of antibiotic-associated disease. The ability of C. difficile to form spores and infect antibiotic-treated persons at low multiplicity of infection (MOI) underlies its large disease burden. However, C. difficile -induced disease might also result from long-harboured C. difficile that blooms in individuals administered antibiotics. Mice purchased from multiple vendors and repeatedly testing negative for this pathogen by quantitative PCR bloomed C. difficile following antibiotic treatment. This endogenous C. difficile strain, herein termed LEM1, which formed spores and produced toxin, was compared with highly pathogenic C. difficile strain VPI10463. Whole-genome sequencing revealed that LEM1 and VPI10463 shared 95% of their genes, including all known virulence genes. In contrast to VPI10463, LEM1 did not induce overt disease when administered to antibiotic-treated or germ-free mice, even at high doses. Rather, blooms of LEM1 correlated with survival following VPI10463 inoculation, and exogenous administration of LEM1 before or shortly following VPI10463 inoculation prevented C. difficile -induced death. Accordingly, despite similar growth properties in vitro, LEM1 strongly outcompeted VPI10463 in mice even at 100-fold lower inocula. These results highlight the difficulty of determining whether individual cases of C. difficile infection resulted from a bloom of endogenous C. difficile or a new exposure to this pathogen. In addition to impacting the design of studies using mouse models of C. difficile -induced disease, this study identified, isolated and characterised an endogenous murine spore-forming C. difficile strain able to decrease colonisation, associated disease and death induced by a pathogenic C. difficile strain. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

PubMed

Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

2018-06-01

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum , the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile . All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N -terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

Inhibition of Clostridium difficile toxin A and B by 1,2-cyclohexanedione modification of an arginine residue.

PubMed

Balfanz, J; Rautenberg, P

1989-12-29

Toxin A (enterotoxin) and toxin B (cytotoxin) of Clostridium difficile were both inactivated by the arginine specific reagent 1,2-cyclohexanedione. Molecular stability during the inactivation process was demonstrated by SDS-PAGE analysis showing the same migration rates for modified and unmodified forms of the 230 kDa toxin A and of the 250 kDa toxin B. Cytotoxicity of both toxins as well as mouse lethality of the enterotoxin were drastically decreased as a result of the arginine modification. The reaction followed pseudo-first-order kinetics. Analysis of the data suggested that modification of a single arginine residue was sufficient to abolish the activity of both toxins.

Transient Fecal Shedding and Limited Animal-to-Animal Transmission of Clostridium difficile by Naturally Infected Finishing Feedlot Cattle ▿

PubMed Central

Rodriguez-Palacios, Alexander; Pickworth, Carrie; Loerch, Steve; LeJeune, Jeffrey T.

2011-01-01

To longitudinally assess fecal shedding and animal-to-animal transmission of Clostridium difficile among finishing feedlot cattle as a risk for beef carcass contamination, we tested 186 ± 12 steers (mean ± standard deviation; 1,369 samples) in an experimental feedlot facility during the finishing period and at harvest. Clostridium difficile was isolated from 12.9% of steers on arrival (24/186; 0 to 33% among five suppliers). Shedding decreased to undetectable levels a week later (0%; P < 0.001), and remained low (<3.6%) until immediately prior to shipment for harvest (1.2%). Antimicrobial use did not increase fecal shedding, despite treatment of 53% of animals for signs of respiratory disease. Animals shedding C. difficile on arrival, however, had 4.6 times higher odds of receiving antimicrobials for respiratory signs than nonshedders (95% confidence interval for the odds ratio, 1.4 to 14.8; P = 0.01). Neither the toxin genes nor toxin A or B was detected in most (39/42) isolates based on two complementary multiplex PCRs and enzyme-linked immunosorbent assay testing, respectively. Two linezolid- and clindamycin-resistant PCR ribotype 078 (tcdA+/tcdB+/cdtB+/39-bp-type deletion in tcdC) isolates were identified from two steers (at arrival and week 20), but these ribotypes did not become endemic. The other toxigenic isolate (tcdA+/tcdB+/cdtB+/classic tcdC; PCR ribotype 078-like) was identified in the cecum of one steer at harvest. Spatio-temporal analysis indicated transient shedding with no evidence of animal-to-animal transmission. The association between C. difficile shedding upon arrival and the subsequent need for antimicrobials for respiratory disease might indicate common predisposing factors. The isolation of toxigenic C. difficile from bovine intestines at harvest highlights the potential for food contamination in meat processing plants. PMID:21441320

CLINICAL AND EPIDEMIOLOGIC CONSIDERATIONS OF CLOSTRIDIUM DIFFICILE IN HARBOR SEALS (PHOCA VITULINA) AT A MARINE MAMMAL REHABILITATION CENTER.

PubMed

Anderson, Chelsea E; Haulena, Martin; Zabek, Erin; Habing, Gregory; Raverty, Stephen

2015-06-01

Between 1998 and 2008, 15 cases of segmental to diffuse hemorrhagic to necrohemorrhagic enterocolitis were diagnosed in neonatal and weaned juvenile harbor seals (Phoca vitulina) presented from the Vancouver Aquarium Marine Mammal Rescue Centre for rehabilitation. Based on a combination of gross pathology, histopathology, bacterial isolation, and toxin testing, Clostridium difficile enterocolitis was diagnosed. Most pups were anorexic or inappetant and died acutely with few other premonitory signs. Due to ongoing clinical concerns and possible emergence of this pathogen at the facility, efforts to better characterize the disease and understand the epidemiology of C. difficile was initiated in 95 harbor seal pups presented for rehabilitation in a single stranding season. Fecal samples were collected on admission, following completion of antibiotic treatment, and also prerelease or postmortem. All samples were collected fresh and submitted either directly or stored frozen. Fecal samples were inoculated into selective media for culture and screened by enzyme-linked immunosorbant assay (ELISA) for C. difficile toxins A, B, or both. Results of the 95 seals in the study were as follows: on hospital admit 72 seals were sampled, 10 were culture positive, 12 were ELISA positive; following antibiotic therapy 46 seals were sampled noting three culture positive and nine ELISA positive; prior to release 58 seals were sampled noting zero culture positive and one ELISA positive; and on postmortem exam seven seals were sampled noting zero culture positive and two ELISA positive. Clostridium difficile was not deemed to be the cause of death in any of the animals. Although the exact mechanism of disease is unknown, this study suggests that C. difficile infection is not a significant cause of mortality and may be part of the normal flora in harbor seals undergoing rehabilitation. Morbidity and mortality from this bacterium can likely be minimized by judicious use of antibiotics, effective biosecurity-biocontainment protocols, and clean husbandry practices.

Bacteriophage treatment significantly reduces viable Clostridium difficile and prevents toxin production in an in vitro model system.

PubMed

Meader, Emma; Mayer, Melinda J; Gasson, Michael J; Steverding, Dietmar; Carding, Simon R; Narbad, Arjan

2010-12-01

Clostridium difficile is primarily a nosocomial pathogen, causing thousands of cases of antibiotic-associated diarrhoea in the UK each year. In this study, we used a batch fermentation model of a C. difficile colonised system to evaluate the potential of a prophylactic and a remedial bacteriophage treatment regime to control the pathogen. It is shown that the prophylaxis regime was effective at preventing the growth of C. difficile (p = <0.001) and precluded the production of detectable levels of toxins A and B. The remedial treatment regime caused a less profound and somewhat transient decrease in the number of viable C. difficile cells (p = <0.0001), but still resulted in a lower level of toxin production relative to the control. The numbers of commensal bacteria including total aerobes and anaerobes, Bifidobacterium sp., Bacteroides sp., Lactobacillus sp., total Clostridium sp., and Enterobacteriaceae were not significantly decreased by this therapy, whereas significant detrimental effects were observed with metronidazole treatment. Our study indicates that phage therapy has potential to be used for the control of C. difficile; it highlights the main benefits of this approach, and some future challenges. Copyright © 2010 Elsevier Ltd. All rights reserved.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile

PubMed Central

Hong, Huynh A.; Hitri, Krisztina; Hosseini, Siamand; Kotowicz, Natalia; Bryan, Donna; Mawas, Fatme; Wilkinson, Anthony J.; van Broekhoven, Annie; Kearsey, Jonathan

2017-01-01

ABSTRACT Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26–39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26–39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26–39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile. These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26–39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI. PMID:28167669

Clostridium difficile Infection in Production Animals and Avian Species: A Review.

PubMed

Moono, Peter; Foster, Niki F; Hampson, David J; Knight, Daniel R; Bloomfield, Lauren E; Riley, Thomas V

2016-12-01

Clostridium difficile is the leading cause of antibiotic-associated diarrhea and colitis in hospitalized humans. Recently, C. difficile infection (CDI) has been increasingly recognized as a cause of neonatal enteritis in food animals such as pigs, resulting in stunted growth, delays in weaning, and mortality, as well as colitis in large birds such as ostriches. C. difficile is a strictly anaerobic spore-forming bacterium, which produces two toxins A (TcdA) and B (TcdB) as its main virulence factors. The majority of strains isolated from animals produce an additional binary toxin (C. difficile transferase) that is associated with increased virulence. C. difficile is ubiquitous in the environment and has a wide host range. This review summarizes the epidemiology, clinical presentations, risk factors, and laboratory diagnosis of CDI in animals. Increased awareness by veterinarians and animal owners of the significance of clinical disease caused by C. difficile in livestock and avians is needed. Finally, this review provides an overview on methods for controlling environmental contamination and potential therapeutics available.

A Novel Regulator Controls Clostridium difficile Sporulation, Motility and Toxin Production

PubMed Central

Edwards, Adrianne N.; Tamayo, Rita; McBride, Shonna M.

2016-01-01

SUMMARY Clostridium difficile, is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. PMID:26915493

A novel regulator controls Clostridium difficile sporulation, motility and toxin production.

PubMed

Edwards, Adrianne N; Tamayo, Rita; McBride, Shonna M

2016-06-01

Clostridium difficile is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. © 2016 John Wiley & Sons Ltd.

Clostridial toxins active locally in the gastrointestinal tract.

PubMed

Wilkins, T; Krivan, H; Stiles, B; Carman, R; Lyerly, D

1985-01-01

Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens. They both produce toxins that are also produced by other clostridia. C. difficile toxins A and B are produced by C. sordellii and a few strains of C. perfringens whereas C. spiroforme produces the same toxins as C. perfringens Type E (iota toxin). Iota toxin activity may be the product of two proteins. Toxigenic strains of C. spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone. C. difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A). This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon. Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes. This receptor appears to be a glycoprotein. The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A. The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay. Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin. An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads. Monoclonal antibodies to toxin A also have been developed and are used in these and other assays.

Genome-Based Comparison of Clostridioides difficile: Average Amino Acid Identity Analysis of Core Genomes.

PubMed

Cabal, Adriana; Jun, Se-Ran; Jenjaroenpun, Piroon; Wanchai, Visanu; Nookaew, Intawat; Wongsurawat, Thidathip; Burgess, Mary J; Kothari, Atul; Wassenaar, Trudy M; Ussery, David W

2018-02-14

Infections due to Clostridioides difficile (previously known as Clostridium difficile) are a major problem in hospitals, where cases can be caused by community-acquired strains as well as by nosocomial spread. Whole genome sequences from clinical samples contain a lot of information but that needs to be analyzed and compared in such a way that the outcome is useful for clinicians or epidemiologists. Here, we compare 663 public available complete genome sequences of C. difficile using average amino acid identity (AAI) scores. This analysis revealed that most of these genomes (640, 96.5%) clearly belong to the same species, while the remaining 23 genomes produce four distinct clusters within the Clostridioides genus. The main C. difficile cluster can be further divided into sub-clusters, depending on the chosen cutoff. We demonstrate that MLST, either based on partial or full gene-length, results in biased estimates of genetic differences and does not capture the true degree of similarity or differences of complete genomes. Presence of genes coding for C. difficile toxins A and B (ToxA/B), as well as the binary C. difficile toxin (CDT), was deduced from their unique PfamA domain architectures. Out of the 663 C. difficile genomes, 535 (80.7%) contained at least one copy of ToxA or ToxB, while these genes were missing from 128 genomes. Although some clusters were enriched for toxin presence, these genes are variably present in a given genetic background. The CDT genes were found in 191 genomes, which were restricted to a few clusters only, and only one cluster lacked the toxin A/B genes consistently. A total of 310 genomes contained ToxA/B without CDT (47%). Further, published metagenomic data from stools were used to assess the presence of C. difficile sequences in blinded cases of C. difficile infection (CDI) and controls, to test if metagenomic analysis is sensitive enough to detect the pathogen, and to establish strain relationships between cases from the same hospital. We conclude that metagenomics can contribute to the identification of CDI and can assist in characterization of the most probable causative strain in CDI patients.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-08-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.

Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells.

PubMed Central

Popoff, M R; Milward, F W; Bancillon, B; Boquet, P

1989-01-01

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes. Images PMID:2545625

Saccharomyces boulardii Stimulates Intestinal Immunoglobulin A Immune Response to Clostridium difficile Toxin A in Mice

PubMed Central

Qamar, Amir; Aboudola, Samer; Warny, Michel; Michetti, Pierre; Pothoulakis, Charalabos; LaMont, J. Thomas; Kelly, Ciarán P.

2001-01-01

Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrent Clostridium difficile colitis. The administration of C. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses. PMID:11254650

Genome characterization of a novel binary toxin-positive strain of Clostridium difficile and comparison with the epidemic 027 and 078 strains.

PubMed

Peng, Zhong; Liu, Sidi; Meng, Xiujuan; Liang, Wan; Xu, Zhuofei; Tang, Biao; Wang, Yuanguo; Duan, Juping; Fu, Chenchao; Wu, Bin; Wu, Anhua; Li, Chunhui

2017-01-01

Clostridium difficile is an anaerobic Gram-positive spore-forming gut pathogen that causes antibiotic-associated diarrhea worldwide. A small number of C. difficile strains express the binary toxin (CDT), which is generally found in C. difficile 027 (ST1) and/or 078 (ST11) in clinic. However, we isolated a binary toxin-positive non-027, non-078 C. difficile LC693 that is associated with severe diarrhea in China. The genotype of this strain was determined as ST201. To understand the pathogenesis-basis of C. difficile ST201, the strain LC693 was chosen for whole genome sequencing, and its genome sequence was analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. The project finally generated an estimated genome size of approximately 4.07 Mbp for strain LC693. Genome size of the three ST201 strains ranged from 4.07 to 4.16 Mb, with an average GC content between 28.5 and 28.9%. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance. The ST201 strains contained a larger PaLoc with a Tn6218 element inserted than the 027/ST1 and 078/ST11 strains, and encoded a truncated TcdC. In addition, the ST201 strains contained intact binary toxin coding and regulation genes which are highly homologous to the 027/ST1 strain. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The presence of those genes also contributes to the pathogenesis of the ST201 strains. In this study, the genomic characterization of three binary toxin-positive C. difficile ST201 strains in clade 3 was discussed and compared to the genomes of the epidemic 027 and the 078 strains. Our analysis identified a number fitness and virulence associated genes/loci in the ST201 genomes that contribute to the pathogenesis of C. difficile ST201.

Prevalence and Characterization of a Binary Toxin (Actin-Specific ADP-Ribosyltransferase) from Clostridium difficile

PubMed Central

Gonçalves, Carina; Decré, Dominique; Barbut, Frédéric; Burghoffer, Béatrice; Petit, Jean-Claude

2004-01-01

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated. PMID:15131151

[Clinical and demographic profile and risk factors for Clostridium difficile infection].

PubMed

Carvajal, Carlos; Pacheco, Carlos; Jaimes, Fabián

2017-01-24

Clostridium difficile infection is the leading cause of nosocomial infectious diarrhea. The increasing incidence added to a lower rate of response to the initial treatment and higher rates of relapse has generated a higher burden of the disease. To determine the clinical characteristics of hospitalized patients with C. difficile infection. We made a nested case-cohort study. We reviewed medical records of the patients with nosocomial diarrhea for whom an assay for toxin A-B of C. difficile had been requested from February, 2010, to February, 2012. We defined case as a patient with diarrhea and a positive assay for the toxin, and control as those patients with a negative assay for the toxin. We collected data on demographic and clinical characteristics, risk factors, hospital length of stay, treatment, and complications. We collected data from 123 patients during the follow-up period, 30 of whom were positive for the toxin. Mean age in the study population was 49 years and 60% were men. The main symptoms were abdominal pain (35%) and fever (34%). The principal complications were electrolytic alteration and severe sepsis with secondary acute kidney injury. Mortality was 13% and independent factors associated to the appearance of the infection were the use of proton pump inhibitors and previous gastrointestinal tract surgery. The use of proton pump inhibitors and previous gastrointestinal tract surgery were factors associated to C. difficile infection.

Two-step glutamate dehydrogenase antigen real-time polymerase chain reaction assay for detection of toxigenic Clostridium difficile.

PubMed

Goldenberg, S D; Cliff, P R; Smith, S; Milner, M; French, G L

2010-01-01

Current diagnosis of Clostridium difficile infection (CDI) relies upon detection of toxins A/B in stool by enzyme immunoassay [EIA(A/B)]. This strategy is unsatisfactory because it has a low sensitivity resulting in significant false negatives. We investigated the performance of a two-step algorithm for diagnosis of CDI using detection of glutamate dehydrogenase (GDH). GDH-positive samples were tested for C. difficile toxin B gene (tcdB) by polymerase chain reaction (PCR). The performance of the two-step protocol was compared with toxin detection by the Meridian Premier EIA kit in 500 consecutive stool samples from patients with suspected CDI. The reference standard among samples that were positive by either EIA(A/B) or GDH testing was culture cytotoxin neutralisation (culture/CTN). Thirty-six (7%) of 500 samples were identified as true positives by culture/CTN. EIA(A/B) identified 14 of the positive specimens with 22 false negatives and two false positives. The two-step protocol identified 34 of the positive samples with two false positives and two false negatives. EIA(A/B) had a sensitivity of 39%, specificity of 99%, positive predictive value of 88% and negative predictive value of 95%. The two-step algorithm performed better, with corresponding values of 94%, 99%, 94% and 99% respectively. Screening for GDH before confirmation of positives by PCR is cheaper than screening all specimens by PCR and is an effective method for routine use. Current EIA(A/B) tests for CDI are of inadequate sensitivity and should be replaced; however, this may result in apparent changes in CDI rates that would need to be explained in national surveillance statistics. Copyright 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Effects of Surotomycin on Clostridium difficile Viability and Toxin Production In Vitro

PubMed Central

Bouillaut, Laurent; McBride, Shonna; Schmidt, Diane J.; Suarez, José M.; Tzipori, Saul; Mascio, Carmela; Chesnel, Laurent

2015-01-01

The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis. PMID:25941230

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

PubMed Central

Dubois, Thomas; Dancer-Thibonnier, Marie; Monot, Marc; Hamiot, Audrey; Bouillaut, Laurent; Soutourina, Olga; Martin-Verstraete, Isabelle

2016-01-01

The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. PMID:27297391

Utility of Clostridium difficile toxin B for inducing anti-tumor immunity.

PubMed

Huang, Tuxiong; Li, Shan; Li, Guangchao; Tian, Yuan; Wang, Haiying; Shi, Lianfa; Perez-Cordon, Gregorio; Mao, Li; Wang, Xiaoning; Wang, Jufang; Feng, Hanping

2014-01-01

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.

Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism.

PubMed

Francis, Michael B; Sorg, Joseph A

2016-01-01

Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis , 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile , spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC , spoVAD , and spoVAE . Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis ), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.

Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

PubMed

Bamber, A I; Fitzsimmons, K; Cunniffe, J G; Beasor, C C; Mackintosh, C A; Hobbs, G

2012-01-01

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

Presence of the epidemic North American Pulsed Field type 1 Clostridium difficile strain in hospitalized children.

PubMed

Toltzis, Philip; Kim, Jason; Dul, Michael; Zoltanski, Joan; Smathers, Sarah; Zaoutis, Theoklis

2009-04-01

A hypervirulent strain of Clostridium difficile-labeled North American Pulsed Field type 1 causes severe disease in adults. To determine the prevalence of NAP1 C. difficile in children, organisms from consecutive C. difficile toxin-positive stool samples at 2 children's hospitals microbiology laboratories were characterized. We found that 19.4% of these samples were NAP1.

Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system.

PubMed

Meader, Emma; Mayer, Melinda J; Steverding, Dietmar; Carding, Simon R; Narbad, Arjan

2013-08-01

Clostridium difficile is a leading cause of hospital-acquired diarrhoea and represents a major challenge for healthcare providers. Due to the decreasing efficacy and associated problems of antibiotic therapy there is a need for synergistic and alternative treatments. In this study we investigated the use of a specific bacteriophage, ΦCD27, in a human colon model of C. difficile infection. Our findings demonstrate a significant reduction in the burden of C. difficile cells and toxin production with phage treatment relative to an untreated control, with no detrimental effect on commensal bacterial populations. The results demonstrate the potential of phage therapy, and highlight the limitations of using phages that have lysogenic capacity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

PubMed

Secore, Susan; Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S; Bodmer, Jean-Luc; Heinrichs, Jon H

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains

PubMed Central

Wang, Su; Doughtry, Julie; Xie, Jinfu; Miezeiewski, Matt; Rustandi, Richard R.; Horton, Melanie; Xoconostle, Rachel; Wang, Bei; Lancaster, Catherine; Kristopeit, Adam; Wang, Sheng-Ching; Christanti, Sianny; Vitelli, Salvatore; Gentile, Marie-Pierre; Goerke, Aaron; Skinner, Julie; Strable, Erica; Thiriot, David S.; Bodmer, Jean-Luc; Heinrichs, Jon H.

2017-01-01

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile. PMID:28125650

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea.

PubMed

Wetterwik, Karl-Johan; Trowald-Wigh, Gunilla; Fernström, Lise-Lotte; Krovacek, Karel

2013-03-12

This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden. Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole. This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs.

Clostridium difficile – From Colonization to Infection

PubMed Central

Schäffler, Holger; Breitrück, Anne

2018-01-01

Clostridium difficile is the most frequent cause of nosocomial antibiotic-associated diarrhea. The incidence of C. difficile infection (CDI) has been rising worldwide with subsequent increases in morbidity, mortality, and health care costs. Asymptomatic colonization with C. difficile is common and a high prevalence has been found in specific cohorts, e.g., hospitalized patients, adults in nursing homes and in infants. However, the risk of infection with C. difficile differs significantly between these cohorts. While CDI is a clear indication for therapy, colonization with C. difficile is not believed to be a direct precursor for CDI and therefore does not require treatment. Antibiotic therapy causes alterations of the intestinal microbial composition, enabling C. difficile colonization and consecutive toxin production leading to disruption of the colonic epithelial cells. Clinical symptoms of CDI range from mild diarrhea to potentially life-threatening conditions like pseudomembranous colitis or toxic megacolon. While antibiotics are still the treatment of choice for CDI, new therapies have emerged in recent years such as antibodies against C. difficile toxin B and fecal microbial transfer (FMT). This specific therapy for CDI underscores the role of the indigenous bacterial composition in the prevention of the disease in healthy individuals and its role in the pathogenesis after alteration by antibiotic treatment. In addition to the pathogenesis of CDI, this review focuses on the colonization of C. difficile in the human gut and factors promoting CDI. PMID:29692762

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages

PubMed Central

Cairns, M. D.; Preston, M. D.; Hall, C. L.; Gerding, D. N.; Hawkey, P. M.; Kato, H.; Kim, H.; Kuijper, E. J.; Lawley, T. D.; Pituch, H.; Reid, S.; Kullin, B.; Riley, T. V.; Solomon, K.; Tsai, P. J.; Weese, J. S.

2016-01-01

ABSTRACT The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents. PMID:28031436

Clostridium difficile in retail meat and processing plants in Texas.

PubMed

Harvey, Roger B; Norman, Keri N; Andrews, Kathleen; Norby, Bo; Hume, Michael E; Scanlan, Charles M; Hardin, Margaret D; Scott, Harvey M

2011-07-01

The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to human beings. The objective of the present study was to determine the prevalence of C. difficile in pork from sausage manufacturing plants and retail meat in Texas. Twenty-three C. difficile isolates were detected from 243 meat samples (9.5%) from 3 sausage-manufacturing plants and 5 retail meat outlets from 2004 to 2009. Twenty-two isolates were positive for toxins A, B, and binary toxin, and were characterized as toxinotype V, PFGE type-NAP7, or "NAP7-variant." Susceptibilities to 11 antimicrobial agents in the current study were similar to those reported previously for toxinotype V isolates, although the results suggested somewhat reduced resistance than reported for other meat, animal, or human clinical toxinotype V isolates.

Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

PubMed

Stiles, Bradley G; Pradhan, Kisha; Fleming, Jodie M; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R

2014-09-05

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin.

Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

PubMed Central

Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

2014-01-01

Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

Faecal lactoferrin and calprotectin in patients with Clostridium difficile infection: a case-control study.

PubMed

Barbut, F; Gouot, C; Lapidus, N; Suzon, L; Syed-Zaidi, R; Lalande, V; Eckert, C

2017-12-01

Calprotectin and lactoferrin are released by the gastrointestinal tract in response to infection and mucosal inflammation. Our objective was to assess the usefulness of quantifying faecal lactoferrin and calprotectin concentrations in Clostridium difficile infection (CDI) patients with or without free toxins in the stools. We conducted a single-centre 22-month case-control study. Patients with a positive CDI diagnosis were compared to two control groups: group 1 = diarrhoeic patients negative for C. difficile and matched (1:1) to CDI cases on the ward location and age, and group 2 = diarrhoeic patients colonised with a non-toxigenic strain of C. difficile. Faecal lactoferrin and calprotectin concentrations in faeces were determined for patients with CDI and controls. Of 135 patients with CDI, 87 (64.4%) had a positive stool cytotoxicity assay (free toxin) and 48 (35.6%) had a positive toxigenic culture without detectable toxins in the stools. The median lactoferrin values were 26.8 μg/g, 8.0 μg/g and 15.8 μg/g in CDI patients and groups 1 and 2, respectively. The median calprotectin values were 218.0 μg/g, 111.5 μg/g and 111.3 μg/g, respectively. Among patients with CDI, faecal lactoferrin and calprotectin levels were higher in those with free toxins in their stools (39.2 vs. 10.2 μg/g, p = 0.003 and 274.0 vs. 166.0 μg/g, p = 0.051, respectively). Both faecal calprotectin and lactoferrin were higher in patients with CDI, especially in those with detectable toxin in faeces, suggesting a correlation between intestinal inflammation and toxins in stools.

Clostridium difficile in Ready-to-Eat Salads, Scotland

PubMed Central

Bakri, Marwah M.; Brown, Derek J.; Butcher, John P.

2009-01-01

Of 40 ready-to-eat salads, 3 (7.5%) were positive for Clostridium difficile by PCR. Two isolates were PCR ribotype 017 (toxin A–, B+), and 1 was PCR ribotype 001. Isolates were susceptible to vancomycin and metronidazole but variably resistant to other antimicrobial drugs. Ready-to-eat salads may be potential sources for virulent C. difficile. PMID:19402979

Suppression by Saccharomyces boulardii of toxigenic Clostridium difficile overgrowth after vancomycin treatment in hamsters.

PubMed Central

Elmer, G W; McFarland, L V

1987-01-01

Saccharomyces boulardii prevented the development of high counts of Clostridium difficile, high titers of toxin B, and positive latex agglutination tests after cessation of vancomycin treatment for hamsters. The protocol used was designed to stimulate relapse of human C. difficile-associated colitis. S. boulardii was protective in this model. PMID:3566236

Clostridium difficile and C. difficile Toxin Testing

MedlinePlus

... Blood Ketones Blood Smear Blood Typing Blood Urea Nitrogen (BUN) BNP and NT-proBNP Body Fluid Analysis ... other infections, typically for an extended period, the balance of the normal flora in the digestive tract ...

A genetic switch controls the production of flagella and toxins in Clostridium difficile.

PubMed

Anjuwon-Foster, Brandon R; Tamayo, Rita

2017-03-01

In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a "flagellar switch" that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins ("flg phase ON"). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion ("flg phase OFF"). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

PubMed

Mehta, Krunal K; Paskaleva, Elena E; Wu, Xia; Grover, Navdeep; Mundra, Ruchir V; Chen, Kevin; Zhang, Yongrong; Yang, Zhiyong; Feng, Hanping; Dordick, Jonathan S; Kane, Ravi S

2016-12-01

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Probiotic Saccharomyces boulardii CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal inflammation in hamsters

PubMed Central

Koon, Hon Wai; Su, Bowei; Xu, Chunlan; Mussatto, Caroline C.; Tran, Diana Hoang-Ngoc; Lee, Elaine C.; Ortiz, Christina; Wang, Jiani; Lee, Jung Eun; Ho, Samantha; Chen, Xinhua; Kelly, Ciaran P.

2016-01-01

C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-κB phosphorylation, and increased proinflammatory cytokine TNFα protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-κB phosphorylation, and TNFα protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins. PMID:27514478

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea

PubMed Central

2013-01-01

Background This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden. Findings Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole. Conclusions This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs. PMID:23497714

Case of antibiotic-associated diarrhea caused by Staphylococcus aureus enterocolitis.

PubMed

Avery, Lisa M; Zempel, Matt; Weiss, Erich

2015-06-01

A case of Staphylococcus aureus enterocolitis (SEC) misdiagnosed as toxin-negative Clostridium difficile is reported. An 82-year-old white man weighing 50 kg (body mass index, 16.8 kg/m(2)) was transported from an assisted living facility to the emergency department with the chief complaints of weakness, nausea, and diarrhea for one week and one bright-red stool on the morning of admission. Before hospital admission, he was treated for a urinary tract infection with ciprofloxacin 500 mg twice daily for 10 days. Stool cultures were negative for C. difficile but positive for S. aureus. The antimicrobial stewardship pharmacist recommended treatment with vancomycin 125 mg orally every 6 hours for staphylococcal colitis. Oral vancomycin was discontinued after three doses on the morning of hospital day 8 after a gastroenterology consultation. Within 48 hours of the discontinuation of oral vancomycin, the patient had eight stools per day. Vancomycin was reinitiated and the patient's symptoms began to again improve. On hospital day 19, the patient was discharged with a prescription for 7 more days of therapy with vancomycin (to complete a 15-day course) and a diagnosis of toxin-negative C. difficile, despite having symptoms consistent with SEC and an enteric culture positive for S. aureus. An 82-year-old man was transferred from an assisted living facility to the hospital with profuse diarrhea and dehydration. Enteric cultures were positive for methicillin-resistant S. aureus with multiple negative C. difficile toxin B assays. Appropriate therapy was delayed and the patient potentially misdiagnosed with toxin-negative C. difficile when the clinical symptoms and diagnostic testing were consistent with SEC. Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Comparison of Control of Clostridium difficile Infection in Six English Hospitals Using Whole-Genome Sequencing.

PubMed

Eyre, David W; Fawley, Warren N; Rajgopal, Anu; Settle, Christopher; Mortimer, Kalani; Goldenberg, Simon D; Dawson, Susan; Crook, Derrick W; Peto, Tim E A; Walker, A Sarah; Wilcox, Mark H

2017-08-01

Variation in Clostridium difficile infection (CDI) rates between healthcare institutions suggests overall incidence could be reduced if the lowest rates could be achieved more widely. We used whole-genome sequencing (WGS) of consecutive C. difficile isolates from 6 English hospitals over 1 year (2013-14) to compare infection control performance. Fecal samples with a positive initial screen for C. difficile were sequenced. Within each hospital, we estimated the proportion of cases plausibly acquired from previous cases. Overall, 851/971 (87.6%) sequenced samples contained toxin genes, and 451 (46.4%) were fecal-toxin-positive. Of 652 potentially toxigenic isolates >90-days after the study started, 128 (20%, 95% confidence interval [CI] 17-23%) were genetically linked (within ≤2 single nucleotide polymorphisms) to a prior patient's isolate from the previous 90 days. Hospital 2 had the fewest linked isolates, 7/105 (7%, 3-13%), hospital 1, 9/70 (13%, 6-23%), and hospitals 3-6 had similar proportions of linked isolates (22-26%) (P ≤ .002 comparing hospital-2 vs 3-6). Results were similar adjusting for locally circulating ribotypes. Adjusting for hospital, ribotype-027 had the highest proportion of linked isolates (57%, 95% CI 29-81%). Fecal-toxin-positive and toxin-negative patients were similarly likely to be a potential transmission donor, OR = 1.01 (0.68-1.49). There was no association between the estimated proportion of linked cases and testing rates. WGS can be used as a novel surveillance tool to identify varying rates of C. difficile transmission between institutions and therefore to allow targeted efforts to reduce CDI incidence. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Prevalence of Clostridium difficile in raw beef, cow, sheep, goat, camel and buffalo meat in Iran.

PubMed

Rahimi, Ebrahim; Jalali, Mohammad; Weese, J Scott

2014-02-05

Clostridium difficile has been shown to be a nosocomial pathogen associated with diarrhoea and pseudomembranous colitis in hospitalised patients and the infection is believed to be acquired nosocomially. Recent studies have shown the occurrence of C. difficile in food animals which may act as a source of infection to humans.The aim of this study was to determine the occurrence of C. difficile in retail raw beef, cow, sheep, goat, camel and buffalo meat in Iran. From April to October 2012, a total of 660 raw meat samples from beef, cow, sheep, goat, camel and buffalo were purchased from 49 butcheries in Isfahan and Khuzestan provinces, Iran, and were evaluated for the presence of C. difficile using a method including selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. In this study, 13 of 660 meat samples (2%) were contaminated with C. difficile. The highest prevalence of C. difficile was found in buffalo meat (9%), followed by goat meat (3.3%), beef meat (1.7%), cow (0.94%) and sheep meat (0.9%). Seven of the 13C. difficile strains (53.9%) were positive for tcdA, tcdB and cdtB toxin genes and were classified as ribotype 078. Four strains (30.8%) were positive tcdA, and tcdB, and one strain (7.7%) was possessed only tcdB. The remaining isolate was non-toxigenic. Susceptibilities of 13C. difficile isolates were determined for 11 antimicrobial drugs using the disk diffusion assay. Resistance to clindamycin, gentamycin, and nalidixic acid was the most common finding. To our knowledge, the present study is the first report of the isolation of C. difficile from raw buffalo meat. This study indicates the potential importance of food, including buffalo meat, as a source of transmission of C. difficile to humans.

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

PubMed Central

Banerjee, Pratik; Merkel, Glenn J; Bhunia, Arun K

2009-01-01

Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD). Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI), is potentially deadly. C. difficile associated diarrhea (CDAD) is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892) on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS) from LDB B-30892 in different dilutions (1:2 to 1:2048). In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16) on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin preparation (CFT) of separately cultured C. difficile, the LDB B-30892 CFS was inhibitory to C. difficile CFT-mediated cytotoxicity at a ratio of 1:8 (LDB B-30892 CFS:C. difficile CFT). We failed to find any similar inhibition of C. difficile-mediated cytotoxicity when other probiotic organisms were tested in parallel to LDB B-30892. Our data of cytotoxicity experiments suggest that LDB B-30892 releases one or more bioactive component(s) into the CFS, which neutralizes the cytotoxicity induced by C. difficile, probably by inactivating its toxin(s). Our data also indicate that CFS from LDB B-30892 reduced the adhesion of C. difficile by 81%, which is significantly (P <0.01) higher than all other probiotic organisms tested in this study. Conclusion This study reveals the very first findings that Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892) can eliminate C. difficile-mediated cytotoxicity, using Caco-2 cells as a model. The study also demonstrates that LDB B-30892 can reduce the colonization of C. difficile cells in colorectal cells. More study is warranted to elucidate the specific mechanism of action of such reduction of cytotoxicity and colonization. PMID:19397787

A genetic switch controls the production of flagella and toxins in Clostridium difficile

PubMed Central

2017-01-01

In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a “flagellar switch” that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins (“flg phase ON”). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion (“flg phase OFF”). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection. PMID:28346491

Rapid change of fecal microbiome and disappearance of Clostridium difficile in a colonized infant after transition from breast milk to cow milk.

PubMed

Davis, Manli Y; Zhang, Husen; Brannan, Lera E; Carman, Robert J; Boone, James H

2016-10-07

Clostridium difficile is the most common known cause of antibiotic-associated diarrhea. Upon the disturbance of gut microbiota by antibiotics, C. difficile establishes growth and releases toxins A and B, which cause tissue damage in the host. The symptoms of C. difficile infection disease range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Interestingly, 10-50 % of infants are asymptomatic carriers of C. difficile. This longitudinal study of the C. difficile colonization in an infant revealed the dynamics of C. difficile presence in gut microbiota. Fifty fecal samples, collected weekly between 5.5 and 17 months of age from a female infant who was an asymptomatic carrier of C. difficile, were analyzed by 16S rRNA gene sequencing. Colonization switching between toxigenic and non-toxigenic C. difficile strains as well as more than 100,000-fold fluctuations of C. difficile counts were observed. C. difficile toxins were detected during the testing period in some infant stool samples, but the infant never had diarrhea. Although fecal microbiota was stable during breast feeding, a dramatic and permanent change of microbiota composition was observed within 5 days of the transition from human milk to cow milk. A rapid decline and eventual disappearance of C. difficile coincided with weaning at 12.5 months. An increase in the relative abundance of Bacteroides spp., Blautia spp., Parabacteroides spp., Coprococcus spp., Ruminococcus spp., and Oscillospira spp. and a decrease of Bifidobacterium spp., Lactobacillus spp., Escherichia spp., and Clostridium spp. were observed during weaning. The change in microbiome composition was accompanied by a gradual increase of fecal pH from 5.5 to 7. The bacterial groups that are less abundant in early infancy, and that increase in relative abundance after weaning, likely are responsible for the expulsion of C. difficile.

Clostridium difficile infection: New insights into therapeutic options.

PubMed

Kachrimanidou, Melina; Sarmourli, Theopisti; Skoura, Lemonia; Metallidis, Symeon; Malisiovas, Nikolaos

2016-09-01

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings and represents a major social and economic burden. The major virulence determinants are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), encoded within the pathogenicity locus. Traditional therapies, such as metronidazole and vancomycin, frequently lead to a vicious circle of recurrences due to their action against normal human microbiome. New disease management strategies together with the development of novel therapeutic and containment approaches are needed in order to better control outbreaks and treat patients. This article provides an overview of currently available CDI treatment options and discusses the most promising therapies under development.

Evaluation of a focused virtual library of heterobifunctional ligands for Clostridium difficile toxins.

PubMed

Sanhueza, Carlos A; Cartmell, Jonathan; El-Hawiet, Amr; Szpacenko, Adam; Kitova, Elena N; Daneshfar, Rambod; Klassen, John S; Lang, Dean E; Eugenio, Luiz; Ng, Kenneth K-S; Kitov, Pavel I; Bundle, David R

2015-01-07

A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.

Probiotic Saccharomyces boulardii CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal inflammation in hamsters.

PubMed

Koon, Hon Wai; Su, Bowei; Xu, Chunlan; Mussatto, Caroline C; Tran, Diana Hoang-Ngoc; Lee, Elaine C; Ortiz, Christina; Wang, Jiani; Lee, Jung Eun; Ho, Samantha; Chen, Xinhua; Kelly, Ciaran P; Pothoulakis, Charalabos

2016-10-01

C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-κB phosphorylation, and increased proinflammatory cytokine TNFα protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-κB phosphorylation, and TNFα protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins. Copyright © 2016 the American Physiological Society.

Prevalence of Clostridium difficile toxinotypes in infected patients at a tertiary care center in Lebanon.

PubMed

Moukhaiber, Romy; Araj, George F; Kissoyan, Kohar Annie B; Cheaito, Katia A; Matar, Ghassan M

2015-07-30

Due to the increase in the incidence of Clostridium difficile associated diseases at a tertiary care center in Lebanon, this study was undertaken to determine the prevalent C. difficile toxinotypes. The immunocard method was used to test for toxins A and B in 88 collected stool samples, followed with API 20A to confirm for C. difficile. PCR amplification of the triose phosphate isomerase (tpi) gene, the toxin encoding genes tcdA, and tcdB, followed by toxinotyping, were performed on recovered isolates and stool specimens. Out of the 88 stool samples obtained, 30 (65.2%) were Immunocard positive, culture and or tpi positive for C. difficile. Of the 30 isolates, 4 were PCR negative for the tcdA and tcdB genes (A-B-), and 26 were PCR positive for the tcdA and / or tcdB genes with 4 being A+B+, 1 A+B-, and 21 A-B+. The results of toxinotyping showed that 2 isolates belonged to toxinotype 0, 4 to toxinotype XI, 2 to toxinotype XII, 1 to toxinotype XVI, 1(A+B-) and twenty (A-B+) designated as toxinotype 0-like. C. difficile was detected in 65.2% of patients' stools with prevalence of toxinotype 0-like. Identification of toxinotypes of C. difficile is important to determine the virulence potential of strains and control their spread.

Evaluation of the C.Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of clostridium difficile disease.

PubMed

Sharp, Susan E; Ruden, Lila O; Pohl, Julie C; Hatcher, Patricia A; Jayne, Linda M; Ivie, W Michael

2010-06-01

The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients.

Evaluation of the C.Diff Quik Chek Complete Assay, a New Glutamate Dehydrogenase and A/B Toxin Combination Lateral Flow Assay for Use in Rapid, Simple Diagnosis of Clostridium difficile Disease▿

PubMed Central

Sharp, Susan E.; Ruden, Lila O.; Pohl, Julie C.; Hatcher, Patricia A.; Jayne, Linda M.; Ivie, W. Michael

2010-01-01

The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients. PMID:20375230

Presence of Clostridium difficile in pig faecal samples and wild animal species associated with pig farms.

PubMed

Andrés-Lasheras, S; Bolea, R; Mainar-Jaime, R C; Kuijper, E; Sevilla, E; Martín-Burriel, I; Chirino-Trejo, M

2017-02-01

To determine the presence of Clostridium difficile on fattening pig farms in north-eastern Spain. Twenty-seven farms were sampled. Pools of pig faecal samples (n = 210), samples of intestinal content from common farm pest species (n = 95) and environment-related samples (n = 93) were collected. Isolates were tested for toxin genes of C. difficile, and typed by PCR-ribotyping and toxinotyping. The minimal inhibitory concentrations of six antimicrobial agents were determined using Etest. Thirty-four isolates were obtained from 12 farms, and 30 (88·2%) had toxin genes. Seven ribotypes were identified. Ribotype 078 and its variant 126 were predominant (52·9%). The same ribotypes were isolated from different animal species on the same farm. None of the isolates were resistant to metronidazole or vancomycin. Clostridium difficile was common within the pig farm environment. Most of the positive samples came from pest species or were pest-related environmental samples. Pest species were colonized with toxigenic and antimicrobial-resistant C. difficile strains of the same ribotypes that are found in humans and pigs. Rodents and pigeons may transmit toxigenic and antimicrobial-resistant C. difficile strains that are of the same ribotypes as those occuring in humans. © 2016 The Society for Applied Microbiology.

Efficacy of surotomycin in an in vitro gut model of Clostridium difficile infection.

PubMed

Chilton, C H; Crowther, G S; Todhunter, S L; Nicholson, S; Freeman, J; Chesnel, L; Wilcox, M H

2014-09-01

We investigated the efficacy of the cyclic lipopeptide surotomycin in treating clindamycin-induced Clostridium difficile infection (CDI) using an in vitro gut model. Two three-stage chemostat gut models were inoculated with human faeces, spiked with C. difficile spores (∼10(7) cfu/mL, PCR ribotype 027 or 001). Clindamycin (33.9 mg/L, four times daily for 7 days) was dosed to induce CDI. Following high-level toxin production, surotomycin (250 mg/L, twice daily for 7 days) was instilled. Microflora populations, C. difficile vegetative cells and spores, cytotoxin titres and antimicrobial levels (LC-MS/MS and bioassay) were determined. The emergence of C. difficile and enterococci with reduced susceptibility to surotomycin was monitored on breakpoint agar (4 × MIC). Counts of viable C. difficile were reduced to near the limit of detection on Days 1 and 3 of surotomycin instillation, and cytotoxin was undetectable on Days 3 and 4 of surotomycin instillation in the 027 and 001 models, respectively. Recurrence of vegetative growth and toxin production occurred 11 days (001 model) and 15 days (027 model) after surotomycin instillation had ceased, and remained for the duration of the experiment. Surotomycin instillation decreased populations of bifidobacteria, clostridia, enterococci and lactobacilli, but was sparing of Bacteroides fragilis group populations. All enumerated organisms had recovered to steady-state levels by 3 weeks post-surotomycin instillation. No evidence of the emergence of reduced susceptibility to surotomycin was observed. Surotomycin successfully reduced C. difficile vegetative cell counts and toxin levels in the gut model and was sparing of B. fragilis group populations. There was no evidence of decreased susceptibility to surotomycin during exposure or post-exposure. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Epidemiology, risk factors, and outcome of Clostridium difficile infection in heart and heart-lung transplant recipients.

PubMed

Bruminhent, Jackrapong; Cawcutt, Kelly A; Thongprayoon, Charat; Petterson, Tanya M; Kremers, Walter K; Razonable, Raymund R

2017-06-01

Clostridium difficile is a major cause of diarrhea in thoracic organ transplant recipients. We investigated the epidemiology, risk factors, and outcome of Clostridium difficile infection (CDI) in heart and heart-lung transplant (HT) recipients. This is a retrospective study from 2004 to 2013. CDI was defined by diarrhea and a positive toxigenic C. difficile in stool measured by toxin enzyme immunoassay (2004-2006) or polymerase chain reaction (2007-2013). Cox proportional hazards regression was used to model the association of risk factors with time to CDI and survival with CDI following transplantation. There were 254 HT recipients, with a median age of 53 years (IQR, 45-60); 34% were female. During the median follow-up of 3.1 years (IQR, 1.3-6.1), 22 (8.7%) patients developed CDI. In multivariable analysis, risk factors for CDI were combined heart-lung transplant (HR 4.70; 95% CI, 1.30-17.01 [P=.02]) and retransplantation (HR 7.19; 95% CI, 1.61-32.12 [P=.01]). Acute cellular rejection was associated with a lower risk of CDI (HR 0.34; 95% CI, 0.11-0.94 [P=.04]). CDI was found to be an independent risk factor for mortality (HR 7.66; 95% CI, 3.41-17.21 [P<.0001]). Clostridium difficile infection after HT is more common among patients with combined heart-lung and those undergoing retransplantation. CDI was associated with a higher risk of mortality in HT recipients. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection.

PubMed

Crobach, M J T; Planche, T; Eckert, C; Barbut, F; Terveer, E M; Dekkers, O M; Wilcox, M H; Kuijper, E J

2016-08-01

In 2009 the first European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guideline for diagnosing Clostridium difficile infection (CDI) was launched. Since then newer tests for diagnosing CDI have become available, especially nucleic acid amplification tests. The main objectives of this update of the guidance document are to summarize the currently available evidence concerning laboratory diagnosis of CDI and to formulate and revise recommendations to optimize CDI testing. This update is essential to improve the diagnosis of CDI and to improve uniformity in CDI diagnosis for surveillance purposes among Europe. An electronic search for literature concerning the laboratory diagnosis of CDI was performed. Studies evaluating a commercial laboratory test compared to a reference test were also included in a meta-analysis. The commercial tests that were evaluated included enzyme immunoassays (EIAs) detecting glutamate dehydrogenase, EIAs detecting toxins A and B and nucleic acid amplification tests. Recommendations were formulated by an executive committee, and the strength of recommendations and quality of evidence were graded using the Grades of Recommendation Assessment, Development and Evaluation (GRADE) system. No single commercial test can be used as a stand-alone test for diagnosing CDI as a result of inadequate positive predictive values at low CDI prevalence. Therefore, the use of a two-step algorithm is recommended. Samples without free toxin detected by toxins A and B EIA but with positive glutamate dehydrogenase EIA, nucleic acid amplification test or toxigenic culture results need clinical evaluation to discern CDI from asymptomatic carriage. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Intestinal Epithelial Cell Response to Clostridium difficile Flagella.

PubMed

Batah, Jameel; Kansau, Imad

2016-01-01

Clostridium difficile is the bacterium responsible for most antibiotic-associated diarrhea in North America and Europe. This bacterium, which colonizes the gut of humans and animals, produces toxins that are known to contribute directly to damage of the gut. It is known that bacterial flagella are involved in intestinal lesions through the inflammatory host response. The C. difficile flagellin recognizes TLR5 and consequently activates the NF-κB and the MAPK signaling pathways which elicit the synthesis of pro-inflammatory cytokines. Increasing interest on the role of C. difficile flagella in eliciting this cell response was recently developed and the development of tools to study cell response triggered by C. difficile flagella will improve our understanding of the pathogenesis of C. difficile.

Comparison of Simplexa universal direct PCR with cytotoxicity assay for diagnosis of Clostridium difficile infection: performance, cost, and correlation with disease.

PubMed

Landry, Marie L; Ferguson, David; Topal, Jeffrey

2014-01-01

Simplexa Clostridium difficile universal direct PCR, a real-time PCR assay for the detection of the C. difficile toxin B (tcdB) gene using the 3M integrated cycler, was compared with a two-step algorithm which includes the C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Three hundred forty-two liquid or semisolid stools submitted for diagnostic C. difficile testing, 171 GDH antigen positive and 171 GDH antigen negative, were selected for the study. All samples were tested by the C. Diff Chek-60 GDH antigen assay, cytotoxin neutralization, and Simplexa direct PCR. Of 171 GDH-positive samples, 4 were excluded (from patients on therapy or from whom duplicate samples were obtained) and 88 were determined to be true positives for toxigenic C. difficile. Of the 88, 67 (76.1%) were positive by the two-step method and 86 (97.7%) were positive by PCR. Seventy-nine were positive by the GDH antigen assay only. Of 171 GDH antigen-negative samples, none were positive by PCR. One antigen-negative sample positive by the cytotoxin assay only was deemed a false positive based on chart review. Simplexa C. difficile universal direct PCR was significantly more sensitive for detecting toxigenic C. difficile bacteria than cytotoxin neutralization (P = 0.0002). However, most PCR-positive/cytotoxin-negative patients did not have clear C. difficile disease. The estimated cost avoidance provided by a more rapid molecular diagnosis was outweighed by the cost of isolating and treating PCR-positive/cytotoxin-negative patients. The costs, clinical consequences, and impact on nosocomial transmission of treating and/or isolating patients positive for toxigenic C. difficile by PCR but negative for in vivo toxin production merit further study.

Disease Progression and Resolution in Rodent Models of Clostridium difficile Infection and Impact of Antitoxin Antibodies and Vancomycin.

PubMed

Warn, Peter; Thommes, Pia; Sattar, Abdul; Corbett, David; Flattery, Amy; Zhang, Zuo; Black, Todd; Hernandez, Lorraine D; Therien, Alex G

2016-11-01

Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Evaluation of NVB302 versus vancomycin activity in an in vitro human gut model of Clostridium difficile infection.

PubMed

Crowther, Grace S; Baines, Simon D; Todhunter, Sharie L; Freeman, Jane; Chilton, Caroline H; Wilcox, Mark H

2013-01-01

First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.

Proton pump inhibitor use and recurrent Clostridium difficile-associated disease: a case-control analysis matched by propensity score.

PubMed

Kim, Yong Gil; Graham, David Y; Jang, Byung Ik

2012-01-01

Clostridium difficile has been increasingly diagnosed in hospitalized patients. An association between proton pump inhibitors (PPIs) use and Clostridium difficile-associated disease (CDAD) and between recurrent CDAD has been suggested. The aim of this study is to investigate whether PPI use is associated with the development of recurrent CDAD. This was a retrospective case-control study of patients with CDAD at Yeungnam University Medical Center, seen from January 2004 to December 2008. C. difficile infection was diagnosed by the presence of C. difficile toxin in the stool. Those with recurrent disease were matched with nonrecurrent controls using multivariate matched sampling methods that incorporated the propensity score. Recurrent CDAD developed in 28 (14.1%) of the 198 patients with diarrhea and positive C. difficile stool toxin assays. Multivariate analysis of the total population of recurrent versus nonrecurrent CDAD revealed that additional use of non-C. difficile antimicrobial therapy (concomitant with the treatment or after or both), poor response to therapy with metronidazole or vancomycin, and recent gastrointestinal surgery were risk factors for recurrent CDAD. We were able to match 21 recurrent CDAD subjects with 21 without recurrent CDAD. Among the matched patients only PPI use was associated with recurrent CDAD (ie, 47.6% vs. 4.8%, P=0.004 for recurrent vs. nonrecurrent CDAD, respectively). Among the matched patient groups, only PPI therapy was associated with recurrent CDAD. Prospective studies are needed to clarify whether avoidance of PPIs or specific cotherapies will reduce the incidence of recurrent C. difficile-associated diarrhea.

Lactobacillus acidophilus modulates the virulence of Clostridium difficile.

PubMed

Yun, B; Oh, S; Griffiths, M W

2014-01-01

Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium that colonizes the human gastrointestinal tract. This pathogen causes antibiotic-associated diarrhea and colitis in animals and humans. Antibiotic-associated diseases may be treated with probiotics, and interest is increasing in such uses of probiotics. This study investigated the effect of Lactobacillus strains on the quorum-sensing signals and toxin production of C. difficile. In addition, an in vivo experiment was designed to assess whether Lactobacillus acidophilus GP1B is able to control C. difficile-associated disease. Autoinducer-2 activity was measured for C. difficile using the Vibrio harveyi coupled bioluminescent assay. Cell extract (10μg/mL) of L. acidophilus GP1B exhibited the highest inhibitory activity among 5 to 40μg/mL cell-extract concentrations. Real-time PCR data indicated decreased transcriptional levels in luxS, tcdA, tcdB, and txeR genes in the presence of 10μg/mL of cell extract of L. acidophilus GP1B. Survival rates at 5d for mice given the pathogen alone with L. acidophilus GP1B cell extract or L. acidophilus GP1B were 10, 70, and 80%, respectively. In addition, the lactic acid-produced L. acidophilus GP1B exhibits an inhibitory effect against the growth of C. difficile. Both the L. acidophilus GP1B and GP1B cell extract have significant antipathogenic effects on C. difficile. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genetic Relatedness of Clostridium difficile Isolates from Various Origins Determined by Triple-Locus Sequence Analysis Based on Toxin Regulatory Genes tcdC, tcdR, and cdtR▿

PubMed Central

Bouvet, Philippe J. M.; Popoff, Michel R.

2008-01-01

A triple-locus nucleotide sequence analysis based on toxin regulatory genes tcdC, tcdR and cdtR was initiated to assess the sequence variability of these genes among Clostridium difficile isolates and to study the genetic relatedness between isolates. A preliminary investigation of the variability of the tcdC gene was done with 57 clinical and veterinary isolates. Twenty-three isolates representing nine main clusters were selected for tcdC, tcdR, and cdtR analysis. The numbers of alleles found for tcdC, tcdR and cdtR were nine, six, and five, respectively. All strains possessed the cdtR gene except toxin A-negative toxin B-positive variants. All but one binary toxin CDT-positive isolate harbored a deletion (>1 bp) in the tcdC gene. The combined analyses of the three genes allowed us to distinguish five lineages correlated with the different types of deletion in tcdC, i.e., 18 bp (associated or not with a deletion at position 117), 36 bp, 39 bp, and 54 bp, and with the wild-type tcdC (no deletion). The tcdR and tcdC genes, though located within the same pathogenicity locus, were found to have evolved separately. Coevolution of the three genes was noted only with strains harboring a 39-bp or a 54-bp deletion in tcdC that formed two homogeneous, separate divergent clusters. Our study supported the existence of the known clones (PCR ribotype 027 isolates and toxin A-negative toxin B-positive C. difficile variants) and evidence for clonality of isolates with a 39-bp deletion (toxinotype V, PCR ribotype 078) that are frequently isolated worldwide from human infections and from food animals. PMID:18832125

Prevalence of Clostridium difficile infection among the patients attending a tertiary care teaching hospital.

PubMed

Segar, Lavanya; Easow, Joshy M; Srirangaraj, Sreenivasan; Hanifah, Mohammad; Joseph, Noyal M; Seetha, K S

2017-01-01

Clostridium difficile, a most important nosocomial enteric pathogen, is recognized globally as responsible for antibiotic-associated diarrhea and colitis. It is associated with considerable morbidity and mortality due to widespread use of antibiotics. The study was done to determine the prevalence of C. difficile infection (CDI) among the patients attending a tertiary care teaching hospital in Puducherry. We performed a prospective cohort study in Mahatma Gandhi Medical College and Research Institute. Around 150 patients were evaluated along with the patient details. C. difficile toxin detection was done as per the standard algorithm using the C. Diff Quik Chek Complete® assay (TECHLAB, Blacksburg, VA, USA). Analysis was done using statistics software (SPSS 16.0, SPSS Inc., Chicago, IL, USA). The prevalence of CDI was found to be 4%. More toxin-positive cases were between 50 and 60 years of age, and there was no difference in gender. Intensive Care Unit showed more toxin-positive cases; however, there was no significant association between the occurrence of CDI and the primary diagnosis of the patients. The prevalence of CDI in our hospital was found to be 4%, which was relatively lower compared to other Indian studies. However, awareness of the risk factors may assist in identifying patients at higher risk for CDI, guide implementation of appropriate preventive measures, and modulate potential intervention measure during management.

Pleiotropic roles of Clostridium difficile sin locus

PubMed Central

Ou, Junjun; Dupuy, Bruno

2018-01-01

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus’s role in C. difficile physiology. Transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR’ acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis. PMID:29529083

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

PubMed Central

Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

2015-01-01

Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

Optimizing the diagnostic testing of Clostridium difficile infection.

PubMed

Bouza, Emilio; Alcalá, Luis; Reigadas, Elena

2016-09-01

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and is associated with a considerable health and cost burden. However, there is still not a clear consensus on the best laboratory diagnosis approach and a wide variation of testing methods and strategies can be encountered. We aim to review the most practical aspects of CDI diagnosis providing our own view on how to optimize CDI diagnosis. Expert commentary: Laboratory diagnosis in search of C. difficile toxins should be applied to all fecal diarrheic samples reaching the microbiology laboratory in patients > 2 years old, with or without classic risk factors for CDI. Detection of toxins either directly in the fecal sample or in the bacteria isolated in culture confirm CDI in the proper clinical setting. Nuclear Acid Assay techniques (NAAT) allow to speed up the process with epidemiological and therapeutic consequences.

Clostridium difficile infection in solid organ transplant recipients.

PubMed

Nanayakkara, Deepa; Nanda, Neha

2017-08-01

Clostridium difficile infection (CDI) is a major healthcare-associated infection that causes significant morbidity and an economic impact in the United States. In this review, we provide an overview of Clostridium difficile infection in solid organ transplant recipients with an emphasis on recent literature. C. difficile in solid organ transplant population has unique risk factors. Fecal microbiota transplantation has shown favorable results in treatment of recurrent C. difficile in this population. Preliminary data from animal studies suggests excellent efficacy with immunization against C. difficile toxins. Over the last decade, number of individuals receiving solid organ transplants has increased exponentially making peri-transplant complications a common occurrence.C. difficile is a frequent cause of morbidity in solid organ transplant recipients. Early and accurate diagnosis of C. difficile requires a stepwise approach. Differentiating between asymptomatic carriage and infection is a diagnostic challenge. Microbial diversity is inversely proportional to risk of C. difficile infection. Antimicrobial stewardship programs help to retain microbial diversity in individuals susceptible to CDI. Recurrent or relapsing C. difficile infection require fecal microbiota transplantation for definitive cure.

Evaluation of the performance of C. DIFF QUIK CHEK COMPLETE and its usefulness in a hospital setting with a high prevalence of Clostridium difficile infection.

PubMed

Chung, Hae-Sun; Lee, Miae

2017-01-01

Rapid and accurate diagnosis of Clostridium difficile infection (CDI) is crucial for patient care, infection control, and efficient surveillance. We evaluated C. DIFF QUIK CHEK COMPLETE (QCC; TechLab), which detects glutamate dehydrogenase (GDH) antigen (QCC-Ag) and toxin A/B (QCC-Tox) simultaneously, and compared it to the laboratory diagnostics for CDI currently in use in a tertiary hospital setting with a high prevalence of CDI. QCC, RIDASCREEN C. difficile toxin A/B assay (Toxin EIA; R-Biopharm AG), chromID C. difficile agar (bioMérieux) culture (ChromID culture), and Xpert C. difficile PCR assay (Xpert PCR; Cepheid) were performed according to the manufacturers' instructions. Performances of the assays were compared against that of Xpert PCR as a reference. Of the 231 loose stool specimens, 83 (35.9%) were positive by Xpert PCR. The sensitivity, specificity, and positive and negative predictive values were 97.6%, 93.9%, 90.0%, and 98.6%, respectively, for QCC-Ag and 55.4%, 100%, 100%, and 80.0%, respectively, for QCC-Tox. The median threshold cycle values of the QCC-Tox(+) specimens were lower than those of the QCC-Tox(-) specimens. Results of QCC as an initial screening test were confirmed in 81.0% (187/231) of samples; these specimens did not require further testing. QCC is a rapid, easy, and cost-effective method that would be a useful first-line screening assay for laboratory diagnosis of CDI in a tertiary hospital with a high prevalence of CDI. A two-step algorithm using QCC as an initial screening tool, followed by Xpert PCR as a confirmatory test, is a practical and cost-effective approach. Copyright © 2016 American Federation for Medical Research.

Shedding of Clostridium difficile PCR ribotype 078 by zoo animals, and report of an unstable metronidazole-resistant isolate from a zebra foal (Equus quagga burchellii).

PubMed

Álvarez-Pérez, Sergio; Blanco, José L; Martínez-Nevado, Eva; Peláez, Teresa; Harmanus, Celine; Kuijper, Ed; García, Marta E

2014-03-14

Clostridium difficile is an emerging and potentially zoonotic pathogen, but its prevalence in most animal species, including exhibition animals, is currently unknown. In this study we assessed the prevalence of faecal shedding of C. difficile by zoo animals, and determined the ribotype, toxin profile and antimicrobial susceptibility of recovered isolates. A total of 200 samples from 40 animal species (36.5% of which came from plains zebra, Equus quagga burchellii) were analysed. C. difficile was isolated from 7 samples (3.5% of total), which came from the following animal species: chimpanzee (Pan troglodytes troglodytes), dwarf goat (Capra hircus), and Iberian ibex (Capra pyrenaica hispanica), with one positive sample each; and plains zebra, with 4 positive samples from 3 different individuals. Most recovered isolates (4/7, 57.1%) belonged to the epidemic PCR ribotype 078, produced toxins A and B, and had the genes encoding binary toxin (i.e. A(+)B(+)CDT(+) isolates). The remaining three isolates belonged to PCR ribotypes 039 (A(-)B(-)CDT(-)), 042 (A(+)B(+)CDT(-)) and 110 (A(-)B(+)CDT(-)). Regardless of their ribotype, all isolates displayed high-level resistance to the fluoroquinolones ciprofloxacin, enrofloxacin and levofloxacin. Some isolates were also resistant to meropenem and/or ertapenem. A ribotype 078 isolate recovered from a male zebra foal initially showed in vitro resistance to metronidazole (MIC ≥ 256 μg/ml), but lost that trait after subculturing on non-selective media. We conclude that zoo animals belonging to different species can carry ribotype 078 and other toxigenic strains of C. difficile showing resistance to antimicrobial compounds commonly used in veterinary and/or human medicine. Copyright © 2014 Elsevier B.V. All rights reserved.

Variability in testing policies and impact on reported Clostridium difficile infection rates: results from the pilot Longitudinal European Clostridium difficile Infection Diagnosis surveillance study (LuCID).

PubMed

Davies, K; Davis, G; Barbut, F; Eckert, C; Petrosillo, N; Wilcox, M H

2016-12-01

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.

Prevalence of diarrhea and enteropathogens in racing sled dogs.

PubMed

McKenzie, E; Riehl, J; Banse, H; Kass, P H; Nelson, S; Marks, S L

2010-01-01

Diarrhea is highly prevalent in racing sled dogs, although the underlying causes are poorly understood. Clostridium perfringens enterotoxin (CPE) and Clostridium difficile Toxin A and B are associated with diarrhea in racing sled dogs. One hundred and thirty-five sled dogs. Freshly voided feces were obtained from 55 dogs before racing and from 80 dogs after 400 miles of racing. Samples were visually scored for diarrhea, mucus, blood, and melena. CPE and C. difficile Toxin A and B were detected by ELISA. Samples were cultured for C. perfringens, C. difficile, Campylobacter, Salmonella, and Escherichia coli O157; Giardia and Cryptosporidium spp. were detected via immunofluorescence. Diarrhea occurred in 36% of dogs during racing, and hematochezia, fecal mucus or melena, or all 3 occurred in 57.5% of dogs. Salmonella was isolated from 78.2% of dogs before racing, and from 71.3% of dogs during racing. C. perfringens and C. difficile were isolated from 100 and 58.2% of dogs before racing, and from 95 and 36.3% of dogs during racing. Dogs were more likely to test positive for CPE during than before racing (18.8 versus 5.5%, P = .021); however, no enteropathogens or their respective toxins were significantly associated with hematochezia or diarrhea. Sled dogs participating in long distance racing have a high prevalence of diarrhea and hematochezia that is not associated with common enteropathogens. It is possible that diarrhea and hematochezia represent the effect of prolonged exercise on the gastrointestinal tract.

Prevention and treatment of Clostridium difficile associated diarrhea by reconstitution of the microbiota.

PubMed

Eliakim-Raz, Noa; Bishara, Jihad

2018-05-21

This review summarizes the latest advances in treating and preventing Clostridium difficile infection (CDI), the most common infectious disease cause of nosocomial diarrhea in adults in developed countries. As customary antibiotic therapies against C. difficile, metronidazole and vancomycin, are broad spectrum, they affect greatly the gut microbiota, which result in very high recurrence rates. Therefore, new strategies are researched intensively. New therapies focus on limiting further destruction of the gut microbiota or restoring the microbiota to its pre-destructed state. These include new antibiotics, such as fidaxomicin, which demonstrates reduced CDI recurrences, among other new drugs, biotherapeutic strategies, mainly fecal microbiota transplantation but also competitive inhibition with non-toxigenic strains of C. difficile, and finally, monoclonal antibodies against C. difficile toxins which offer protection against recurrences.

Clostridium perfringens enterotoxin and Clostridium difficile toxin A/B do not play a role in acute haemorrhagic diarrhoea syndrome in dogs.

PubMed

Busch, K; Suchodolski, J S; Kühner, K A; Minamoto, Y; Steiner, J M; Mueller, R S; Hartmann, K; Unterer, S

2015-03-07

Although an association between clostridial pathogens and canine idiopathic acute haemorrhagic diarrhoea syndrome (AHDS) has been described, the relevance of those bacteria and their toxins remains unclear. The aim of this study was to evaluate the association between severity of clinical signs and presence of Clostridium perfringens enterotoxin (CPE) and Clostridium difficile toxin A/B (CDT A/B) in faeces of dogs with AHDS. Faecal samples of 54 dogs with idiopathic AHDS were tested by qualitative CPE and CDT A/B ELISA, and PCR was performed to detect enterotoxin genes of C. perfringens (cpe) and toxin B genes of C. difficile (cdt b). Prevalence of cdt b and CDT A/B in dogs with AHDS was 10/54 and 2/54 versus 3/23 and 0/23 in control dogs. Prevalence of cpe was 35/54 in affected versus 9/23 in control dogs. Prevalence of CPE in dogs with AHDS (13/54) was higher compared with control dogs (0/23). No significant difference was detected between CPE-positive and -negative and between cpe-positive and -negative dogs in severity of clinical signs, duration of hospitalisation, mortality rate and selected laboratory parameters. This study suggests that CPE and CDT A/B do not play a role in idiopathic AHDS, are not associated with clinical parameters in affected dogs and cannot be used to predict disease outcome. British Veterinary Association.

Crystal structure of Clostridium difficile toxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chumbler, Nicole M.; Rutherford, Stacey A.; Zhang, Zhifen

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis eventmore » that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA 1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.« less

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

PubMed

Tsaloglou, M-N; Watson, R J; Rushworth, C M; Zhao, Y; Niu, X; Sutton, J M; Morgan, H

2015-01-07

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 μL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Comparison of the Vidas C. difficile GDH Automated Enzyme-Linked Fluorescence Immunoassay (ELFA) with Another Commercial Enzyme Immunoassay (EIA) (Quik Chek-60), Two Selective Media, and a PCR Assay for gluD for Detection of Clostridium difficile in Fecal Samples.

PubMed

Davies, K A; Berry, C E; Morris, K A; Smith, R; Young, S; Davis, T E; Fuller, D D; Buckner, R J; Wilcox, M H

2015-06-01

Prevention and management of Clostridium difficile infection (CDI) can be improved by rapid and reliable diagnostics. The Vidas C. difficile glutamate dehydrogenase assay had performance comparable to that of the Quik Chek-60 assay (overall agreement, 95%) and a sensitivity of >93%; thus, it is suitable as the first test in two-stage algorithms for a CDI diagnosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

PubMed

Silva, Rodrigo Otávio Silveira; Dorella, Fernanda Alves; Figueiredo, Henrique Cesar Pereira; Costa, Érica Azevedo; Pelicia, Vanessa; Ribeiro, Bruna Letícia Devidé; Ribeiro, Marcio Garcia; Paes, Antonio Carlos; Megid, Jane; Lobato, Francisco Carlos Faria

2017-12-01

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Clostridium difficile infection in the Lao People's Democratic Republic: first isolation and review of the literature.

PubMed

Cheong, Elaine; Roberts, Tamalee; Rattanavong, Sayaphet; Riley, Thomas V; Newton, Paul N; Dance, David A B

2017-09-21

Current knowledge of the epidemiology of Clostridium difficile infection in Asia, and in particular the Greater Mekong Subregion, is very limited. Only a few studies from Thailand and Vietnam have been reported from the region with variable testing methods and results, and no studies from Lao People's Democratic Republic (PDR). Therefore we investigated the presence of C. difficile in a single centre in the Lao PDR and determined the ribotypes present. Seventy unformed stool samples from hospital inpatients at Mahosot Hospital, Vientiane, were tested for the presence of C. difficile using selective differential agar and confirmed by latex agglutination. C. difficile isolates were further characterised by ribotyping and toxin gene detection. C. difficile was isolated from five of the 70 patients, and five different ribotypes were identified (014, 017, 020, QX 107 and QX 574). This is the first isolation of C. difficile from human stool samples in the Lao PDR. These results will add to the limited amount of data on C. difficile in the region. In addition, we hope this information will alert clinicians to the presence of C. difficile in the country and will help inform future investigations into the epidemiology and diagnosis of C. difficile in Lao PDR.

Clinical manifestations of Clostridium difficile infection in a medical center in Taiwan.

PubMed

Lai, Chih-Cheng; Lin, Sheng-Hsiang; Tan, Che-Kim; Liao, Chun-Hsing; Huang, Yu-Tsung; Hsueh, Po-Ren

2014-12-01

To investigate the clinical characteristics of Clostridium difficile infection (CDI) at a medical center in Taiwan. Patients with CDI were identified from medical records at the National Taiwan University Hospital (Taipei, Taiwan). The following information was gathered and analyzed to better understand the clinical manifestations of CDI: age; sex; underlying immunocompromised conditions; laboratory data; in-hospital mortality; and previous use of drugs such as antimicrobial agents, steroids, and antipeptic ulcer agents. During the years 2000-2010, 122 patients were identified as having CDI. This included 92 patients with nontoxigenic CDI (i.e., positive stool culture for C. difficile but negative results for toxins A and B) and 30 patients with toxigenic CDI (i.e., positive stool culture cultures for C. difficile and positive results for toxins A and B). Of the 122 patients, 48 (39%) patients were older than 65 years and most patients acquired the CDI while in the hospital. Active cancer was the most common reason for hospitalization, followed by diabetes mellitus, and end-stage renal disease. More than 90% of the patients had received antibiotics before acquiring CDI. The results of fecal leukocyte examinations were positive in 33 (27%) patients. The overall in-hospital mortality rate was 26.2%. There were no significant differences between patients with nontoxigenic CDI and patients with toxigenic CDI. Clostridium difficile infection can develop in healthcare facilities and in community settings, especially in immunocompromised patients. Copyright © 2013. Published by Elsevier B.V.

Discovery of LFF571: An Investigational Agent for Clostridium difficile Infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

LaMarche, Matthew J.; Leeds, Jennifer A.; Amaral, Adam

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3)more » and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.« less

Clostridium difficile infection

PubMed Central

Vedantam, Gayatri; Clark, Andrew; Chu, Michele; McQuade, Rebecca; Mallozzi, Michael; Viswanathan, V. K.

2012-01-01

Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets. PMID:22555464

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

PubMed

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M; Fawley, Warren N; Paredes-Sabja, Daniel; Wilcox, Mark; Gonzalez, Angel

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients' data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia

PubMed Central

Salazar, Clara Lina; Reyes, Catalina; Cienfuegos-Gallet, Astrid Vanessa; Best, Emma; Atehortua, Santiago; Sierra, Patricia; Correa, Margarita M.; Fawley, Warren N.; Paredes-Sabja, Daniel; Wilcox, Mark

2018-01-01

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia. PMID:29649308

Severity and frequency of community-onset Clostridium difficile infection on an Australian tertiary referral hospital campus.

PubMed

Clohessy, Penny; Merif, Juan; Post, Jeffrey John

2014-12-01

Clostridium difficile infection (CDI) is increasingly being found in populations without traditional risk factors. We compared the relative frequency, risk factors, severity, and outcomes of community-onset CDI with hospital-acquired infection. This was a retrospective, observational study of CDI at a tertiary hospital campus in Sydney, Australia. Patients aged 15 years and older with a first episode of CDI from January 1 to December 31, 2011 were included. CDI was defined as the presence of diarrhoea with a positive enzyme immunoassay in conjunction with a positive cell cytotoxicity assay, toxin culture, or organism culture. Main outcome measures were onset of infection (hospital or community), risk factors, markers of severity, and outcomes for the two groups. One hundred and twenty-nine cases of CDI infection were identified, of which 38 (29%) were community-onset. The community-onset infection group were less likely to have a recent history of antibiotic use (66% vs. 98%; p<0.001) or proton pump inhibitor use (38% vs. 69%; p=0.03) than the hospital-acquired infection group. Markers of severity and outcomes were similar in the two groups, with an overall mortality of 9%. Community-onset CDI accounts for a large proportion of C. difficile infections and has a similar potential for severe disease as hospital-acquired infection. Using a history of previous antibiotic use, proton pump inhibitor use, or recent hospitalization to predict cases is unreliable. We recommend that patients with diarrhoea being investigated in emergency departments and community practice are tested for Clostridium difficile infection. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Reactive Oxygen Species as Additional Determinants for Cytotoxicity of Clostridium difficile Toxins A and B

PubMed Central

Frädrich, Claudia; Beer, Lara-Antonia; Gerhard, Ralf

2016-01-01

Clostridium difficile infections can induce mild to severe diarrhoea and the often associated characteristic pseudomembranous colitis. Two protein toxins, the large glucosyltransferases TcdA and TcdB, are the main pathogenicity factors that can induce all clinical symptoms in animal models. The classical molecular mode of action of these homologous toxins is the inhibition of Rho GTPases by mono-glucosylation. Rho-inhibition leads to breakdown of the actin cytoskeleton, induces stress-activated and pro-inflammatory signaling and eventually results in apoptosis of the affected cells. An increasing number of reports, however, have documented further qualities of TcdA and TcdB, including the production of reactive oxygen species (ROS) by target cells. This review summarizes observations dealing with the production of ROS induced by TcdA and TcdB, dissects pathways that contribute to this phenomenon and speculates about ROS in mediating pathogenesis. In conclusion, ROS have to be considered as a discrete, glucosyltransferase-independent quality of at least TcdB, triggered by different mechanisms. PMID:26797634

Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile.

PubMed

Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N; Klassen, John S; Ng, Kenneth K S

2014-01-24

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile*

PubMed Central

Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N.; Klassen, John S.; Ng, Kenneth K. S.

2014-01-01

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents. PMID:24311789

Clostridium difficile carbohydrates: glucan in spores, PSII common antigen in cells, immunogenicity of PSII in swine and synthesis of a dual C. difficile-ETEC conjugate vaccine.

PubMed

Bertolo, Lisa; Boncheff, Alexander G; Ma, Zuchao; Chen, Yu-Han; Wakeford, Terra; Friendship, Robert M; Rosseau, Joyce; Weese, J Scott; Chu, Michele; Mallozzi, Michael; Vedantam, Gayatri; Monteiro, Mario A

2012-06-01

Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a D-glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester-phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Emerging monoclonal antibodies against Clostridium difficile infection.

PubMed

Péchiné, Séverine; Janoir, Claire; Collignon, Anne

2017-04-01

Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

Clostridium difficile

PubMed Central

Curry, Scott R.

2017-01-01

SYNOPSIS Clostridium difficile infections (CDI) have emerged as one of the principal threats to the health of hospitalized and immunocompromised patients. Nucleic acid testing for C. difficile toxin genes has eclipsed traditional clinical diagnostics for CDI in sensitivity and is now widespread in clinical use, but preliminary evidence suggests that this may have come at a cost of substantially reduced positive predictive value. The importance of C. difficile colonization is increasingly recognized not only as a source for false positive clinical testing but also as a source of new infections within hospitals and other healthcare environments. In the last five years, several new treatment strategies that capitalize on the increasing understanding of the altered microbiome and host defenses in CDI patients have completed clinical trials, including fecal microbiota transplantation (FMT). This article highlights the changing epidemiology, laboratory diagnostics, pathogenesis, and treatment of CDI. PMID:20513554

Clostridium difficile infection worsens the prognosis of ulcerative colitis

PubMed Central

Negrón, María E; Barkema, Herman W; Rioux, Kevin; De Buck, Jeroen; Checkley, Sylvia; Proulx, Marie-Claude; Frolkis, Alexandra; Beck, Paul L; Dieleman, Levinus A; Panaccione, Remo; Ghosh, Subrata; Kaplan, Gilaad G

2014-01-01

BACKGROUND: The impact of Clostridium difficile infections among ulcerative colitis (UC) patients is well characterized. However, there is little knowledge regarding the association between C difficile infections and postoperative complications among UC patients. OBJECTIVE: To determine whether C difficile infection is associated with undergoing an emergent colectomy and experiencing postoperative complications. METHODS: The present population-based case-control study identified UC patients admitted to Calgary Health Zone hospitals for a flare between 2000 and 2009. C difficile toxin tests ordered in hospital or 90 days before hospital admission were provided by Calgary Laboratory Services (Calgary, Alberta). Hospital records were reviewed to confirm diagnoses and to extract clinical data. Multivariate logistic regression analyses were performed among individuals tested for C difficile to examine the association between C difficile infection and emergent colectomy and diagnosis of any postoperative complications and, secondarily, an infectious postoperative complication. Estimates were presented as adjusted ORs with 95% CIs. RESULTS: C difficile was tested in 278 (58%) UC patients and 6.1% were positive. C difficile infection was associated with an increased risk for emergent colectomy (adjusted OR 3.39 [95% CI 1.02 to 11.23]). Additionally, a preoperative diagnosis of C difficile was significantly associated with the development of postoperative infectious complications (OR 4.76 [95% CI 1.10 to 20.63]). CONCLUSION: C difficile diagnosis worsened the prognosis of UC by increasing the risk of colectomy and postoperative infectious complications following colectomy. Future studies are needed to explore whether early detection and aggressive management of C difficile infection will improve UC outcomes. PMID:25157528

The Monoclonal Antitoxin Antibodies (Actoxumab-Bezlotoxumab) Treatment Facilitates Normalization of the Gut Microbiota of Mice with Clostridium difficile Infection.

PubMed

Džunková, Mária; D'Auria, Giuseppe; Xu, Hua; Huang, Jun; Duan, Yinghua; Moya, Andrés; Kelly, Ciarán P; Chen, Xinhua

2016-01-01

Antibiotics have significant and long-lasting impacts on the intestinal microbiota and consequently reduce colonization resistance against Clostridium difficile infection (CDI). Standard therapy using antibiotics is associated with a high rate of disease recurrence, highlighting the need for novel treatment strategies that target toxins, the major virulence factors, rather than the organism itself. Human monoclonal antibodies MK-3415A (actoxumab-bezlotoxumab) to C. difficile toxin A and toxin B, as an emerging non-antibiotic approach, significantly reduced the recurrence of CDI in animal models and human clinical trials. Although the main mechanism of protection is through direct neutralization of the toxins, the impact of MK-3415A on gut microbiota and its restoration has not been examined. Using a CDI murine model, we compared the bacterial diversity of the gut microbiome of mice under different treatments including MK-3415A, vancomycin, or vancomycin combined with MK-3415A, sampled longitudinally. Here, we showed that C. difficile infection resulted in the prevalence of Enterobacter species. Sixty percent of mice in the vehicle group died after 2 days and their microbiome was almost exclusively formed by Enterobacter . MK-3415A treatment resulted in lower Enterobacter levels and restoration of Blautia, Akkermansia , and Lactobacillus which were the core components of the original microbiota. Vancomycin treatment led to significantly lower survival rate than the combo treatment of MK-3415A and vancomycin. Vancomycin treatment decreased bacterial diversity with predominant Enterobacter and Akkermansia , while Staphylococcus expanded after vancomycin treatment was terminated. In contrast, mice treated by vancomycin combined with MK-3415A also experienced decreased bacterial diversity during vancomycin treatment. However, these animals were able to recover their initial Blautia and Lactobacillus proportions, even though episodes of Staphylococcus overgrowth were detected by the end of the experiments. In conclusion, MK-3415A (actoxumab-bezlotoxumab) treatment facilitates normalization of the gut microbiota in CDI mice. It remains to be examined whether or not the prevention of recurrent CDI by the antitoxin antibodies observed in clinical trials occurs through modulation of microbiota.

An investigation into the association between cpb2-encoding Clostridium perfringens type A and diarrhea in neonatal piglets

PubMed Central

Farzan, Abdolvahab; Kircanski, Jasmina; DeLay, Josepha; Soltes, Glenn; Songer, J. Glenn; Friendship, Robert; Prescott, John F.

2013-01-01

To investigate the possible role of cpb2-positive type A Clostridium perfringens in neonatal diarrheal illness in pigs, the jejunum and colon of matched normal and diarrheic piglets from 10 farms with a history of neonatal diarrhea were examined grossly and by histopathology, and tested for C. perfringens, for C. perfringens beta2 (CPB2) toxin, as well as for Clostridium difficile toxins, Salmonella, enterotoxigenic Escherichia coli, rotavirus, transmissible gastroenteritis (TGE) virus, and coccidia. Clostridium perfringens isolates were tested using a multiplex real-time polymerase chain reaction (PCR) to determine the presence of cpa, consensus and atypical cpb2, and other virulence-associated genes. The numbers of C. perfringens in the intestinal contents were lower in diarrheic piglets (log10 5.4 CFU/g) compared with normal piglets (log10 6.5 CFU/g) (P < 0.05). The consensus cpb2 was present in 93% of isolates in each group, but atypical cpb2 was less common (56% healthy, 32% diarrheic piglets isolates, respectively, P < 0.05). The presence of CPB2 toxin in the intestinal contents of normal and diarrheic piglets did not differ significantly. Clostridium difficile toxins and rotavirus were each detected in 7 of the 21 (33%) diarrheic piglets. Rotavirus, C. difficile toxins, Salmonella, or enterotoxigenic E. coli were concurrently recovered in different combinations in 4 diarrheic piglets. The cause of diarrhea in 8 of the 21 (38%) piglets on 6 farms remained unknown. The etiological diagnosis of diarrhea could not be determined in any of the piglets on 2 of the farms. This study demonstrated that the number of cpb2-positive type A C. perfringens in the intestinal contents was not a useful approach for making a diagnosis of type A C. perfringens enteritis in piglets. Further work is required to confirm whether cpb2-carrying type A C. perfringens have a pathogenic role in enteric infection in neonatal swine. PMID:23814355

An investigation into the association between cpb2-encoding Clostridium perfringens type A and diarrhea in neonatal piglets.

PubMed

Farzan, Abdolvahab; Kircanski, Jasmina; DeLay, Josepha; Soltes, Glenn; Songer, J Glenn; Friendship, Robert; Prescott, John F

2013-01-01

To investigate the possible role of cpb2-positive type A Clostridium perfringens in neonatal diarrheal illness in pigs, the jejunum and colon of matched normal and diarrheic piglets from 10 farms with a history of neonatal diarrhea were examined grossly and by histopathology, and tested for C. perfringens, for C. perfringens beta2 (CPB2) toxin, as well as for Clostridium difficile toxins, Salmonella, enterotoxigenic Escherichia coli, rotavirus, transmissible gastroenteritis (TGE) virus, and coccidia. Clostridium perfringens isolates were tested using a multiplex real-time polymerase chain reaction (PCR) to determine the presence of cpa, consensus and atypical cpb2, and other virulence-associated genes. The numbers of C. perfringens in the intestinal contents were lower in diarrheic piglets (log₁₀ 5.4 CFU/g) compared with normal piglets (log₁₀ 6.5 CFU/g) (P < 0.05). The consensus cpb2 was present in 93% of isolates in each group, but atypical cpb2 was less common (56% healthy, 32% diarrheic piglets isolates, respectively, P < 0.05). The presence of CPB2 toxin in the intestinal contents of normal and diarrheic piglets did not differ significantly. Clostridium difficile toxins and rotavirus were each detected in 7 of the 21 (33%) diarrheic piglets. Rotavirus, C. difficile toxins, Salmonella, or enterotoxigenic E. coli were concurrently recovered in different combinations in 4 diarrheic piglets. The cause of diarrhea in 8 of the 21 (38%) piglets on 6 farms remained unknown. The etiological diagnosis of diarrhea could not be determined in any of the piglets on 2 of the farms. This study demonstrated that the number of cpb2-positive type A C. perfringens in the intestinal contents was not a useful approach for making a diagnosis of type A C. perfringens enteritis in piglets. Further work is required to confirm whether cpb2-carrying type A C. perfringens have a pathogenic role in enteric infection in neonatal swine.

High prevalence of Clostridium difficile on retail root vegetables, Western Australia.

PubMed

Lim, S C; Foster, N F; Elliott, B; Riley, T V

2018-02-01

The incidence of community-associated Clostridium difficile infection (CA-CDI) in Australia has increased since mid-2011. With reports of clinically important C. difficile strains being isolated from retail foods in Europe and North America, a foodborne source of C. difficile in cases of CA-CDI is a possibility. This study represents the first to investigate the prevalence and genotypes of C. difficile in Australian retail vegetables. A total of 300 root vegetables grown in Western Australia (WA) were collected from retail stores and farmers' markets. Three vegetables of the same kind bought from the same store/market were treated as one sample. Selective enrichment culture, toxin profiling and PCR ribotyping were performed. Clostridium difficile was isolated from 30% (30/100) of pooled vegetable samples, 55·6% of organic potatoes, 50% of nonorganic potatoes, 22·2% of organic beetroots, 5·6% of organic onions and 5·3% of organic carrots. Over half (51·2%, 22/43) the isolates were toxigenic. Many of the ribotypes of C. difficile isolated were common among human and Australian animals. Clostridium difficile could be found commonly on retail root vegetables of WA. This may be potential sources for CA-CDI. This study enhances knowledge of possible sources of C. difficile in the Australian community, outside the hospital setting. © 2017 The Society for Applied Microbiology.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

PubMed Central

Köberle, Martin; Göppel, David; Grandl, Tanja; Gaentzsch, Peer; Manncke, Birgit; Berchtold, Susanne; Müller, Steffen; Lüscher, Bernhard; Asselin-Labat, Marie-Liesse; Pallardy, Marc; Sorg, Isabel; Langer, Simon; Barth, Holger; Zumbihl, Robert; Autenrieth, Ingo B.; Bohn, Erwin

2012-01-01

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2. PMID:22792400

Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

PubMed Central

Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

1995-01-01

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification. PMID:7558302

Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

PubMed

Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

1995-10-01

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.

Construction of a fusion protein carrying antigenic determinants of enteric clostridial toxins.

PubMed

Belyi, Iouri F; Varfolomeeva, Nina A

2003-08-29

Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases. The main virulence factors of these microorganisms include toxins A and B of C. difficile (ToxA and ToxB) and enterotoxin of C. perfringens (Cpe). In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein. Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule. Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components. These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.

Clostridium difficile suppresses colonic vasoactive intestinal peptide associated with altered motility.

PubMed

Nassif, A; Longo, W E; Sexe, R; Stratton, M; Standeven, J; Vernava, A M; Kaminski, D L

1995-01-01

We investigated whether Clostridium difficile toxin alters colonic tissue levels of vasoactive intestinal peptide (VIP) at the expense of changes in colonic motility in the isolated perfused rabbit left colon. Colonic inflammation was induced by the intracolonic administration of 10(-8) M C. difflcile toxin. Strain gauge transducers were sewn onto the serosal surface of the colon to evaluate colonic motility. C. difflcile administration produced histologic changes consistent with epithelial damage. This was associated with an increased production of prostaglandin E(2) and thromboxane B(2). Tissue levels of VIP but not substance P were significantly reduced. This was associated with an increased number of contractions per minute and an average force of each colonic contraction. These results suggest that tissue levels of VIP are suppressed by C. difflcile and may participate in colonic dysmotility during active inflammation.

Community Environmental Contamination of Toxigenic Clostridium difficile

PubMed Central

Alam, M Jahangir; Walk, Seth T.; Endres, Bradley T.; Basseres, Eugenie; Khaleduzzaman, Mohammed; Amadio, Jonathan; Musick, William L.; Christensen, Jennifer L.; Kuo, Julie; Atmar, Robert L.

2017-01-01

Abstract Background. Clostridium difficile infection is often considered to result from recent acquisition of a C difficile isolate in a healthcare setting. However, C difficile spores can persist for long periods of time, suggesting a potentially large community environmental reservoir. The objectives of this study were to assess community environmental contamination of toxigenic C difficile and to assess strain distribution in environmental versus clinical isolates. Methods. From 2013 to 2015, we collected community environmental swabs from homes and public areas in Houston, Texas to assess C difficile contamination. All positive isolates were tested for C difficile toxins A and B, ribotyped, and compared with clinical C difficile isolates obtained from hospitalized patients in Houston healthcare settings. Results. A total of 2538 environmental samples were collected over the study period. These included samples obtained from homes (n = 1079), parks (n = 491), chain stores (n = 225), fast food restaurants (n = 123), other commercial stores (n = 172), and hospitals (n = 448). Overall, 418 environmental isolates grew toxigenic C difficile (16.5%; P < .001) most commonly from parks (24.6%), followed by homes (17.1%), hospitals (16.5%), commercial stores (8.1%), chain stores (7.6%), and fast food restaurants (6.5%). A similar distribution of ribotypes was observed between clinical and environmental isolates with the exception that ribotype 027 was more common in clinical isolates compared with environmental isolates (P < .001). Conclusions. We identified a high prevalence of toxigenic C difficile from community environs that were similar ribotypes to clinical isolates. These findings suggest that interventions beyond isolation of symptomatic patients should be targeted for prevention of C difficile infection. PMID:28480289

Carriage of Clostridium difficile by Wild Urban Norway Rats (Rattus norvegicus) and Black Rats (Rattus rattus)

PubMed Central

Patrick, David M.; Mak, Sunny; Jardine, Claire M.; Tang, Patrick; Weese, J. Scott

2014-01-01

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration. PMID:24317079

Carriage of Clostridium difficile by wild urban Norway rats (Rattus norvegicus) and black rats (Rattus rattus).

PubMed

Himsworth, Chelsea G; Patrick, David M; Mak, Sunny; Jardine, Claire M; Tang, Patrick; Weese, J Scott

2014-02-01

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 "novel" ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.

[Characteristics of Clostridium difficile infection in a high complexity hospital and report of the circulation of the NAP1/027 hypervirulent strain in Colombia].

PubMed

Gualtero, Sandra Milena; Abril, Lina Alejandra; Camelo, Nathalia; Sanchez, Susi Daniela; Davila, Fabián Antonio; Arias, Gerson; Silva, Edwin; Bustos, Ingrid Gissel; Josa, Diego Fernando; Torres, Isabel Cristina; Zambrano, Luis Carlos; Pareja, María José

2017-12-01

Clostridium difficile is the main pathogen related to healthcare-associated diarrhea and it is the cause of 20 to 30% of diarrhea cases caused by antibiotics. In Colombia and Latin America, the knowledge about the epidemiological behavior of this infection is limited. To describe the characteristics of a series of patients with C. difficile infection. We performed a descriptive case series study of patients with C. difficile infection hospitalized in the Fundación Clínica Shaio from January, 2012, to November, 2015. We analyzed 36 patients. The average age was 65 years. The risk factors associated with the infection were: previous use of antibiotics (94.4%), prior hospitalization in the last three months (66.7%) and use of proton pump inhibitors (50%). The most common comorbidities were chronic kidney disease (41.7%) and diabetes mellitus (30.6%). The most frequent symptoms were more than three loose stools per day (97.1%) and abdominal pain (42.9%). According to the severity of the disease, 44.4% of cases were classified as mild to moderate, 38.9% as severe, and 11.1% as complicated or severe. The detection of the toxin by PCR (GeneXpert) was the most common diagnostic procedure (63.8%). Global mortality during hospitalization was 8%. We identified four strains with serotype NAP1/027 and nine samples positive for binary toxin. Clostridium difficile infection should be suspected in patients with diarrhea and traditional risk factors associated with this disease. We report the circulation of the hypervirulent strain serotype NAP1/027 in Colombia, which should be countered with epidemiological surveillance and a prompt diagnosis.

[Prevalence of Clostridium difficile infection in hospitalized patients with diarrhea: results of a French prospective multicenter bi-annual point prevalence study].

PubMed

Barbut, Frédéric; Ramé, Laetitia; Petit, Amandine; Suzon, Laina; de Chevigny, Alix; Eckert, Catherine

2015-04-01

Clostridium difficile infections represent the major cause of healthcare-associated diarrhea. The objective of the study was to determine the incidence of C. difficile infection (CDI) in 2012 and to assess the under-estimation of the disease in France. Seventy healthcare facilities participated in a prospective point prevalence study. Each laboratory was requested to send all the diarrheal stool samples from hospitalized patients during 2 days (one in December 2012 and one in July 2013) to the National Reference Laboratory (NRL) for C. difficile, irrespective of a medical request for C. difficile. At the NRL, stool samples were analyzed using the Quik Chek Complete assay (Alere). Positive samples for glutamate deshydrogenase or toxins were confirmed by the toxigenic culture. Results obtained by the NRL were then compared to those given by each healthcare facility. Incidence of CDI in 2012 was provided by each healthcare facility through a specific questionnaire. Mean incidence of CDI reported in 2012 by the HCF was 3.6 ± 2.9 per 10,000 patient-days; the incidence was positively correlated to the density testing (defined by the number of tests per 10,000 patient-days), which varied across the HCF (median 29.0 per 10,000 patient-days, IQR 19-50). During the bi-annual point prevalence survey, 651 stool samples were included and 90 were positive for C. difficile in culture. The overall prevalence of patients infected by a toxigenic C. difficile strain was 9.7% (63/651) and the prevalence of patients colonized by a non-toxigenic strain was 4.2% (27/651). Among the 65 cases of CDI detected by the NRL, 35 (55.6%) were missed by the participating HCF because of a lack of sensitivity of the methods used for the diagnosis (16/63, 25.4%) or because of a lack of clinical suspicion (19/63, 30.2%). The incidence of CDI in 2012 has increased in France compared to that of 2009 but is still underestimated because of a lack of clinical suspicion or a lack of sensitivity of methods used for toxin detection. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

PubMed Central

COTTRELL, GRAEME S.; AMADESI, SILVIA; PIKIOS, STELLA; CAMERER, ERIC; WILLARDSEN, J. ADAM; MURPHY, BRETT R.; CAUGHEY, GEORGE H.; WOLTERS, PAUL J.; COUGHLIN, SHAUN R.; PETERSON, ANDERS; KNECHT, WOLFGANG; POTHOULAKIS, CHARALABOS; BUNNETT, NIGEL W.; GRADY, EILEEN F.

2008-01-01

Background & Aims We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. Results TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms. PMID:17570216

[Epidemiology, risk factors and prevention of Clostridium difficile nosocomial infections].

PubMed

Barbut, F; Petit, J C

2000-10-01

Clostridium difficile is responsible for 10-25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudo-membranous colitis (PMC). This anaerobic spore-forming bacterium has been identified as the leading cause of nosocomial infectious diarrhea in adults. Pathogenesis relies on a disruption of the normal bacterial flora of the colon, a colonization by C. difficile and the release of toxins A and B that cause mucosal damage and inflammation. Incidence of C. difficile intestinal disorders usually varies from one to 40 per thousand patient admissions. Risk factors for C. difficile-associated diarrhea include antimicrobial therapy, older age (> 65 years), antineoplastic chemotherapy, and length of hospital stay. Nosocomial transmission of C. difficile via oro-fecal route occurs in 3-30% of total patient admissions but it remains asymptomatic in more than 66% of cases. Persistent environmental contamination and carrying of the organism on the hands of hospital staff are common. Measures that are effective in reducing cross-infection consist of an accurate and rapid diagnosis, an appropriate treatment, an implementation of enteric precautions for symptomatic patients, a reinforcement of hand-washing and a daily environmental disinfection. C. difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.

Bezlotoxumab for the prevention of Clostridium difficile recurrence.

PubMed

Couture-Cossette, Antoine; Carignan, Alex; Ilangumaran, Subburaj; Valiquette, Louis

2017-11-01

Clostridium difficile infection is a major economic and clinical burden, due to its high frequency of recurrence. Currently recommended treatments are not efficient for prevention and may contribute to the risk of recurrent infection. In recent years, research has focused on strategies to lessen this risk. Bezlotoxumab is a monoclonal antibody that prevents recurrences of C. difficile infection through the antagonism of toxin B. Areas covered: In this review, the authors discuss the burden of C. difficile infection and its recurrences, the mechanisms underlying the recurrences, and current C. difficile treatments. They subsequently analyze the strategic therapeutic rationale for bezlotoxumab use, as well as the supporting clinical evidence. Expert opinion: Bezlotoxumab is an attractive solution for reducing the unacceptable level of recurrence that occurs with the currently recommended C. difficile treatments and other alternative therapies under consideration. Even though bezlotoxumab has not been tested in large-scale trials exclusively in cases of already established recurrent C.difficile infection (rCDI), it has an advantage over current treatments in that it does not interfere with the patient's gut flora while directly neutralizing the key virulence factor. Although cost remains an important factor against its widespread use, simpler administration, fewer side-effects, and better social acceptability justify its consideration for treating rCDI.

Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.

PubMed

Bakonyi, Daniel; Hummel, Werner

2017-04-01

A gene encoding a novel 7α-specific NADP + -dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP + using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type. Copyright © 2016. Published by Elsevier Inc.

Rifaximin therapy for metronidazole-unresponsive Clostridium difficile infection: a prospective pilot trial

PubMed Central

Patrick Basu, P.; Dinani, Amreen; Rayapudi, Krishna; Pacana, Tommy; Shah, Niraj James; Hampole, Hemant; Krishnaswamy, N. V.; Mohan, Vinod

2010-01-01

Background: Clostridium difficile infection (CDI) is a recent epidemic in the United States, particularly in the hospital setting. Oral metronidazole is standard therapy for C. difficile infection, but resistance to metronidazole is becoming a clinical challenge. Methods: We evaluated the efficacy of the nonsystemic oral antibiotic rifaximin for the treatment of metronidazole-resistant C. difficile infection. Twenty-five patients with C. difficile infection were enrolled in the study. All had mild-to-moderate C. difficile infection (5–10 bowel movements a day without sepsis) unresponsive to metronidazole (i.e. stools positive for toxins A and B after oral metronidazole 500 mg three times daily [t.i.d.] for 5 days). After discontinuation of metronidazole, rifaximin 400 mg t.i.d. for 14 days was prescribed. Patients were followed for 56 days and stool was tested for C. difficile using polymerase chain reaction (PCR) to assess the effect of treatment. A negative PCR test result was interpreted as a favorable response to rifaximin. Results: Sixteen of 22 patients (73%) were eligible for study inclusion and completed rifaximin therapy experienced eradication of infection (stool negative for C. difficile) immediately after rifaximin therapy and 56 days post-treatment. Three patients (12%) discontinued therapy because of abdominal distention. Rifaximin was generally well tolerated. Conclusions: In conclusion, rifaximin may be considered for treatment of mild-to-moderate C. difficile infection that is resistant to metronidazole. Larger randomized trials are needed to confirm these positive findings. PMID:21180604

Rifaximin therapy for metronidazole-unresponsive Clostridium difficile infection: a prospective pilot trial.

PubMed

Patrick Basu, P; Dinani, Amreen; Rayapudi, Krishna; Pacana, Tommy; Shah, Niraj James; Hampole, Hemant; Krishnaswamy, N V; Mohan, Vinod

2010-07-01

Clostridium difficile infection (CDI) is a recent epidemic in the United States, particularly in the hospital setting. Oral metronidazole is standard therapy for C. difficile infection, but resistance to metronidazole is becoming a clinical challenge. We evaluated the efficacy of the nonsystemic oral antibiotic rifaximin for the treatment of metronidazole-resistant C. difficile infection. Twenty-five patients with C. difficile infection were enrolled in the study. All had mild-to-moderate C. difficile infection (5-10 bowel movements a day without sepsis) unresponsive to metronidazole (i.e. stools positive for toxins A and B after oral metronidazole 500 mg three times daily [t.i.d.] for 5 days). After discontinuation of metronidazole, rifaximin 400 mg t.i.d. for 14 days was prescribed. Patients were followed for 56 days and stool was tested for C. difficile using polymerase chain reaction (PCR) to assess the effect of treatment. A negative PCR test result was interpreted as a favorable response to rifaximin. Sixteen of 22 patients (73%) were eligible for study inclusion and completed rifaximin therapy experienced eradication of infection (stool negative for C. difficile) immediately after rifaximin therapy and 56 days post-treatment. Three patients (12%) discontinued therapy because of abdominal distention. Rifaximin was generally well tolerated. In conclusion, rifaximin may be considered for treatment of mild-to-moderate C. difficile infection that is resistant to metronidazole. Larger randomized trials are needed to confirm these positive findings.

Laboratory diagnosis of Clostridium difficile infection: Comparison of Techlab C. diff Quik Chek Complete, Xpert C. difficile, and multistep algorithmic approach.

PubMed

Seo, Ja Young; Jeong, Ji Hun; Kim, Kyung Hee; Ahn, Jeong-Yeal; Park, Pil-Whan; Seo, Yiel-Hea

2017-11-01

Clostridium difficile is a major pathogen responsible for nosocomial infectious diarrhea. We explored optimal laboratory strategies for diagnosis of C. difficile infection (CDI) in our clinical settings, a 1400-bed tertiary care hospital. Using 191 fresh stool samples from adult patients, we evaluated the performance of Xpert C. difficile (Xpert CD), C. diff Quik Chek Complete (which simultaneously detects glutamate dehydrogenase [GDH] and C. difficile toxins [CDT]), toxigenic culture, and a two-step algorithm composed of GDH/CDT as a screening test and Xpert CD as a confirmatory test. Clostridium difficile was detected in 35 samples (18.3%), and all isolates were toxigenic strains. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of each assay for detecting CDI were as follows: Quik Chek Complete CDT (45.7%, 100%, 100%, 89.1%), Quik Chek Complete GDH (97.1%, 99.4%, 97.1%, 99.4%), Xpert CD (94.3%, 100%, 100%, 98.7%), and toxigenic culture (91.4%, 100%, 100%, 98.1%). A two-step algorithm performed identically with Xpert CD assay. Our data showed that most C. difficile isolates from adult patients were toxigenic. We demonstrated that a two-step algorithm based on GDH/CDT assay followed by Xpert CD assay as a confirmatory test was rapid, reliable, and cost effective for diagnosis of CDI in an adult patient setting with high prevalence of toxigenic C. difficile. © 2017 Wiley Periodicals, Inc.

Toxigenic Clostridium difficile colonization among hospitalised adults; risk factors and impact on survival.

PubMed

Behar, Laura; Chadwick, David; Dunne, Angela; Jones, Christopher I; Proctor, Claire; Rajkumar, Chakravarthi; Sharratt, Paula; Stanley, Philip; Whiley, Angela; Wilks, Mark; Llewelyn, Martin J

2017-07-01

To establish risk factors for Clostridium difficile colonization among hospitalized patients in England. Patients admitted to elderly medicine wards at three acute hospitals in England were recruited to a prospective observational study. Participants were asked to provide a stool sample as soon as possible after enrolment and then weekly during their hospital stay. Samples were cultured for C. difficile before ribotyping and toxin detection by PCR. A multivariable logistic regression model of risk factors for C. difficile colonization was fitted from univariable risk factors significant at the p < 0.05 level. 410/727 participants submitted ≥1 stool sample and 40 (9.8%) carried toxigenic C. difficile in the first sample taken. Ribotype 106 was identified three times and seven other ribotypes twice. No ribotype 027 strains were identified. Independent predictors of colonization were previous C. difficile infection (OR 4.53 (95% C.I. 1.33-15.48) and malnutrition (MUST score ≥2) (OR 3.29 (95% C.I. 1.47-7.35)). Although C. difficile colonised patients experienced higher 90-day mortality, colonization was not an independent risk for death. In a non-epidemic setting patients who have previously had CDI and have a MUST score of ≥2 are at increased risk of C. difficile colonization and could be targeted for active surveillance to prevent C. difficile transmission. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evaluation of Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile Assays for Direct Detection of Toxigenic Clostridium difficile in Stool Specimens.

PubMed

Shin, Bo-Moon; Yoo, Sun Mee; Shin, Won Chang

2016-03-01

We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.

Challenges and opportunities in the management of Clostridium difficile infection.

PubMed

DuPont, Herbert L

2014-11-01

Clostridium difficile infection (CDI) is increasing in all regions of the world where sought. There is no gold standard for diagnosis of CDI, with available tests having limitations. Prevention of CDI will be seen with antibiotic stewardship, improved disinfection of hospitals and nursing homes, chemo- and immuno-prophylaxis and next generation probiotics. The important therapeutic agents are oral vancomycin and fidaxomicin with metronidazole being used only in mild cases or when oral therapy cannot be given. Current therapy of CDI for 10 days is associated with high rate of recurrence that may be prevented by prolonging initial therapy. Future treatment strategies will focus on drugs that inhibit C. difficile, reduce toxin activity and inflammation in the gut, and improve colonic flora diversity.

Clostridium difficile suppresses colonic vasoactive intestinal peptide associated with altered motility

PubMed Central

Nassif, A.; Sexe, R.; Stratton, M.; Standeven, J.; Vernava, A. M.; Kaminski, D. L.

1995-01-01

We investigated whether Clostridium difficile toxin alters colonic tissue levels of vasoactive intestinal peptide (VIP) at the expense of changes in colonic motility in the isolated perfused rabbit left colon. Colonic inflammation was induced by the intracolonic administration of 10−8 M C. difflcile toxin. Strain gauge transducers were sewn onto the serosal surface of the colon to evaluate colonic motility. C. difflcile administration produced histologic changes consistent with epithelial damage. This was associated with an increased production of prostaglandin E2 and thromboxane B2. Tissue levels of VIP but not substance P were significantly reduced. This was associated with an increased number of contractions per minute and an average force of each colonic contraction. These results suggest that tissue levels of VIP are suppressed by C. difflcile and may participate in colonic dysmotility during active inflammation. PMID:18475679

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin.

PubMed

Popoff, M R; Boquet, P

1988-05-16

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

PubMed

Orth, Peter; Xiao, Li; Hernandez, Lorraine D; Reichert, Paul; Sheth, Payal R; Beaumont, Maribel; Yang, Xiaoyu; Murgolo, Nicholas; Ermakov, Grigori; DiNunzio, Edward; Racine, Fred; Karczewski, Jerzy; Secore, Susan; Ingram, Richard N; Mayhood, Todd; Strickland, Corey; Therien, Alex G

2014-06-27

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Epidemiology of Clostridium difficile in infants in Oxfordshire, UK: Risk factors for colonization and carriage, and genetic overlap with regional C. difficile infection strains.

PubMed

Stoesser, Nicole; Eyre, David W; Quan, T Phuong; Godwin, Heather; Pill, Gemma; Mbuvi, Emily; Vaughan, Alison; Griffiths, David; Martin, Jessica; Fawley, Warren; Dingle, Kate E; Oakley, Sarah; Wanelik, Kazimierz; Finney, John M; Kachrimanidou, Melina; Moore, Catrin E; Gorbach, Sherwood; Riley, Thomas V; Crook, Derrick W; Peto, Tim E A; Wilcox, Mark H; Walker, A Sarah

2017-01-01

Approximately 30-40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized. Predominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases. 338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0-10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%). In predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic overlap between some infant strains and those isolated from CDI cases suggest common community reservoirs of these C. difficile lineages, contrasting with those lineages found only in CDI cases, and therefore more consistent with healthcare-associated spread.

Impact and Time Course of Clostridium difficile Colonization in Very Low Birth Weight Infants.

PubMed

Pichler, Karin; Bausenhardt, Benjamin; Huhulescu, Steliana; Lindtner, Claudia; Indra, Alexander; Allerberger, Franz; Berger, Angelika

2018-06-12

Clostridium difficile is a gram-positive, anaerobic spore-forming, toxin-producing bacillus, which is one of the most common causes for health care-associated infections. High colonization rates in clinically asymptomatic neonates and infants have been described, although most studies go back to the early 1980 and 1990s, and were carried out in term and late preterm infants. The aim of our study was to determine both the impact and time course of C. difficile colonization in a cohort of very low birth weight infants (VLBWI) in an era of PCR-based technologies for diagnosis. Stool samples of VLBWI were analyzed for the presence of C. difficile strains in regular intervals during the hospital stay by PCR ribotyping. Analysis was continued throughout the first 2 years of life. A 32% C. difficile colonization rate during the first 2 years of life and an in-hospital colonization rate of 8% was found in a cohort of 190 VLBWI. C. difficile colonization occurred mainly in the first 6 months of life, which was similar to term neonates. In-hospital colonization accounted for only a small percentage of cases with no detection of hypervirulent strains. Also, C. difficile colonization was not related to an adverse outcome in this VLBWI cohort. Oral lactoferrin of bovine origin and treatment with piperacillin/tazobactam were negatively correlated with C. difficile colonization in our study. C. difficile colonization in our cohort of VLBWI was significantly lower than has been described in the literature and was not related to an adverse outcome. © 2018 S. Karger AG, Basel.

Comparison of Clostridium difficile Ribotypes Circulating in Australian Hospitals and Communities.

PubMed

Furuya-Kanamori, Luis; Riley, Thomas V; Paterson, David L; Foster, Niki F; Huber, Charlotte A; Hong, Stacey; Harris-Brown, Tiffany; Robson, Jenny; Clements, Archie C A

2017-01-01

Clostridium difficile infection (CDI) is becoming less exclusively a health care-associated CDI (HA-CDI). The incidence of community-associated CDI (CA-CDI) has increased over the past few decades. It has been postulated that asymptomatic toxigenic C. difficile (TCD)-colonized patients may play a role in the transfer of C. difficile between the hospital setting and the community. Thus, to investigate the relatedness of C. difficile across the hospital and community settings, we compared the characteristics of symptomatic and asymptomatic host patients and the pathogens from these patients in these two settings over a 3-year period. Two studies were simultaneously conducted; the first study enrolled symptomatic CDI patients from two tertiary care hospitals and the community in two Australian states, while the second study enrolled asymptomatic TCD-colonized patients from the same tertiary care hospitals. A total of 324 patients (96 with HA-CDI, 152 with CA-CDI, and 76 colonized with TCD) were enrolled. The predominant C. difficile ribotypes isolated in the hospital setting corresponded with those isolated in the community, as it was found that for 79% of the C. difficile isolates from hospitals, an isolate with a matching ribotype was isolated in the community, suggesting that transmission between these two settings is occurring. The toxigenic C. difficile strains causing symptomatic infection were similar to those causing asymptomatic infection, and patients exposed to antimicrobials prior to admission were more likely to develop a symptomatic infection (odds ratio, 2.94; 95% confidence interval, 1.20 to 7.14). Our findings suggest that the development of CDI symptoms in a setting without establishment of hospital epidemics with binary toxin-producing C. difficile strains may be driven mainly by host susceptibility and exposure to antimicrobials, rather than by C. difficile strain characteristics. Copyright © 2016 American Society for Microbiology.

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

PubMed Central

Wu, Xia; Paskaleva, Elena E.; Mehta, Krunal K.; Dordick, Jonathan S.; Kane, Ravi S.

2016-01-01

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract. PMID:27759081

Clostridium difficile infection among immunocompromised patients in Rio de Janeiro, Brazil and detection of moxifloxacin resistance in a ribotype 014 strain.

PubMed

Secco, Danielle Angst; Balassiano, Ilana Teruszkin; Boente, Renata Ferreira; Miranda, Karla Rodrigues; Brazier, Jon; Hall, Val; dos Santos-Filho, Joaquim; Lobo, Leandro Araujo; Nouér, Simone Aranha; Domingues, Regina Maria Cavalcanti Pilotto

2014-08-01

Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 → Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

Advances in the Microbiome: Applications to Clostridium difficile Infection

PubMed Central

Culligan, Eamonn P.; Sleator, Roy D.

2016-01-01

Clostridium difficile is a major cause of morbidity and mortality worldwide, causing over 400,000 infections and approximately 29,000 deaths in the United States alone each year. C. difficile is the most common cause of nosocomial diarrhoea in the developed world, and, in recent years, the emergence of hyper-virulent (mainly ribotypes 027 and 078, sometimes characterised by increased toxin production), epidemic strains and an increase in the number of community-acquired infections has caused further concern. Antibiotic therapy with metronidazole, vancomycin or fidaxomicin is the primary treatment for C. difficile infection (CDI). However, CDI is unique, in that, antibiotic use is also a major risk factor for acquiring CDI or recurrent CDI due to disruption of the normal gut microbiota. Therefore, there is an urgent need for alternative, non-antibiotic therapeutics to treat or prevent CDI. Here, we review a number of such potential treatments which have emerged from advances in the field of microbiome research. PMID:27657145

Fatal course of takotsubo cardiomyopathy in a female with recurrent Clostridium difficile infection.

PubMed

Elikowski, Waldemar; Małek-Elikowska, Małgorzata; Lisiecka, Monika; Mozer-Lisewska, Iwona

2017-06-23

Among diverse triggering factors of stress-induced takotsubo cardiomyopathy (TC), a viral or bacterial infection is rarely observed. Sepsis is an exception, regardless of the etiologic pathogen, in which case an excess of catecholamines may result in acute left ventricular dysfunction. TC precipitated by Clostridium difficile infection (CDI) has been reported only in two patients so far. The authors describe another case of TC triggered this time by recurrent C. difficile colitis which occurred in a 72-yearold female. Severe heart failure developed on the second day of a new episode of diarrhea. Echocardiography revealed apical ballooning, a typical form of TC, while the coronary arteries in coronary angiography were normal. Despite proper treatment of CDI, the course of the disease was fatal due to heart failure progression. In considerations of TC pathogenesis in the case presented, the impact of C. difficile toxins should be taken into account. One should remember about the potential extraintestinal complications of CDI, including sudden myocardial depression.

Efficacy and safety of fidaxomicin compared with oral vancomycin for the treatment of adults with Clostridium difficile-associated diarrhea: data from the OPT-80-003 and OPT-80-004 studies.

PubMed

Chen, Luke F; Anderson, Deverick J

2012-06-01

Clostridium difficile is emerging as one of the most important and devastating pathogens affecting hospitalized populations around the world. The incidence of C. difficile infection is increasing and disease severity is worsening. Thus, an effective alternative to metronidazole and oral vancomycin is urgently needed. Two Phase III trials, OPT-80-003 and OPT-80-004, showed that oral fidaxomicin for 10 days was noninferior compared with treatment with oral vancomycin among adult patients with toxin-positive C. difficile-associated diarrhea (CDAD). Furthermore, fidaxomicin was associated with a lower rate of recurrence of CDAD within 4 weeks of completion of therapy. The safety and tolerability of fidaxomicin was consistent with earlier studies and established that fidaxomicin is an efficacious and well-tolerated treatment option for CDAD. Despite these potential advantages, the cost-effectiveness of this expensive agent remains poorly understood.

Prevalence and characterization of enterotoxigenic Bacteroides fragilis and toxigenic Clostridium difficile in a Taipei emergency department.

PubMed

Ji, Dar-Der; Huang, I-Hsiu; Lai, Chao-Chih; Wu, Fang-Tzy; Jiang, Donald Dah-Shyong; Hsu, Bing-Mu; Lin, Wei-Chen

2017-02-01

Enterotoxigenic Bacteroides fragilis (ETBF) and toxin-encoding Clostridium difficile (TXCD) are associated with gastroenteritis. Routine anaerobic blood culture for recovery of these anaerobic pathogens is not used for the detection of their toxins, especially for toxin-variant TXCD. The aim of this study was to investigate the prevalence and risk factors of the genotypes of these anaerobes in patients with acute diarrheal illnesses. The data and samples of 513 patients with gastroenteritis were collected in a Taipei emergency department from March 1, 2006 to December 31, 2009. Nonenterotoxigenic B. fragilis (NTBF) and ETBF and the toxin genotypes of TXCD were detected by molecular methods. The prevalence rates of NTBF, ETBF, and TXCD infections were 33.14%, 1.56%, and 2.34%, respectively. ETBF infections often occurred in the elderly (average age = 67.13 years) and during the cold, dry winters. TXCD infections were widely distributed in age and often occurred in the warm, wet springs and summers. The symptoms of ETBF-infected patients were significantly more severe than those of NTBF-infected patients. This study identified and analyzed the prevalence, risk factors, and clinical presentations of these anaerobic infections. Future epidemiologic and clinical studies are needed to understand the role of ETBF and TXCD in human gastroenteritis. Copyright © 2015. Published by Elsevier B.V.

Clostridial binary toxins: iota and C2 family portraits.

PubMed

Stiles, Bradley G; Wigelsworth, Darran J; Popoff, Michel R; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host-cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.

The agr Locus Regulates Virulence and Colonization Genes in Clostridium difficile 027

PubMed Central

Martin, Melissa J.; Clare, Simon; Goulding, David; Faulds-Pain, Alexandra; Barquist, Lars; Browne, Hilary P.; Pettit, Laura; Dougan, Gordon; Lawley, Trevor D.

2013-01-01

The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage. PMID:23772065

Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.

PubMed

Martin, Jessica S H; Monaghan, Tanya M; Wilcox, Mark H

2016-04-01

Clostridium difficile infection (CDI) continues to affect patients in hospitals and communities worldwide. The spectrum of clinical disease ranges from mild diarrhoea to toxic megacolon, colonic perforation and death. However, this bacterium might also be carried asymptomatically in the gut, potentially leading to 'silent' onward transmission. Modern technologies, such as whole-genome sequencing and multi-locus variable-number tandem-repeat analysis, are helping to track C. difficile transmission across health-care facilities, countries and continents, offering the potential to illuminate previously under-recognized sources of infection. These typing strategies have also demonstrated heterogeneity in terms of CDI incidence and strain types reflecting different stages of epidemic spread. However, comparison of CDI epidemiology, particularly between countries, is challenging due to wide-ranging approaches to sampling and testing. Diagnostic strategies for C. difficile are complicated both by the wide range of bacterial targets and tests available and the need to differentiate between toxin-producing and non-toxigenic strains. Multistep diagnostic algorithms have been recommended to improve sensitivity and specificity. In this Review, we describe the latest advances in the understanding of C. difficile epidemiology, transmission and diagnosis, and discuss the effect of these developments on the clinical management of CDI.

Identification of an Essential Region for Translocation of Clostridium difficile Toxin B.

PubMed

Chen, Shuyi; Wang, Haiying; Gu, Huawei; Sun, Chunli; Li, Shan; Feng, Hanping; Wang, Jufang

2016-08-15

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756-1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756-1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756-1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756-1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells.

Premarket evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay.

PubMed

Stellrecht, K A; Espino, A A; Maceira, V P; Nattanmai, S M; Butt, S A; Wroblewski, D; Hannett, G E; Musser, K A

2014-05-01

Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratory's test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013.

PubMed

Davies, Kerrie A; Ashwin, Helen; Longshaw, Christopher M; Burns, David A; Davis, Georgina L; Wilcox, Mark H

2016-07-21

Clostridium difficile infection (CDI) is the major cause of infective diarrhoea in healthcare environments. As part of the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), the largest C. difficile epidemiological study of its type, PCR ribotype distribution of C. difficile isolates in Europe was investigated. PCR ribotyping was performed on 1,196 C. difficile isolates from diarrhoeal samples sent to the European coordinating laboratory in 2012-13 and 2013 (from two sampling days) by 482 participating hospitals from 19 European countries. A total of 125 ribotypes were identified, of which ribotypes 027 (19%, n =222), 001/072 (11%, n = 134) and 014/020 (10%, n = 119) were the most prevalent. Distinct regional patterns of ribotype distribution were noted. Of 596 isolates from patients with toxin-positive stools (CDI cases), ribotype 027 accounted for 22% (32/144) of infections in cases aged from 18 to less than 65 years, but the prevalence decreased in those aged ≥ 65 years (14% (59/412)) and further decreased in those aged ≥ 81 years (9% (18/195)). The prevalence of ribotype 027 and 176, but not other epidemic strains, was inversely proportional to overall ribotype diversity (R(2) = 0.717). This study highlights an increased diversity of C. difficile ribotypes across Europe compared with previous studies, with considerable intercountry variation in ribotype distribution. Continuous surveillance programmes are necessary to monitor the changing epidemiology of C. difficile. This article is copyright of The Authors, 2016.

The zoonotic potential of Clostridium difficile from small companion animals and their owners.

PubMed

Rabold, Denise; Espelage, Werner; Abu Sin, Muna; Eckmanns, Tim; Schneeberg, Alexander; Neubauer, Heinrich; Möbius, Nadine; Hille, Katja; Wieler, Lothar H; Seyboldt, Christian; Lübke-Becker, Antina

2018-01-01

Clostridium difficile infections (CDI) in humans range from asymptomatic carriage to life-threatening intestinal disease. Findings on C. difficile in various animal species and an overlap in ribotypes (RTs) suggest potential zoonotic transmission. However, the impact of animals for human CDI remains unclear. In a large-scale survey we collected 1,447 fecal samples to determine the occurrence of C. difficile in small companion animals (dogs and cats) and their owners and to assess potential epidemiological links within the community. The Germany-wide survey was conducted from July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR detection of toxin genes were used to characterize isolated C. difficile strains. A database was defined and logistic regression used to identify putative factors associated with fecal shedding of C. difficile. In total, 1,418 samples met the inclusion criteria. The isolation rates for small companion animals and their owners within the community were similarly low with 3.0% (25/840) and 2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in animals and humans, respectively, whereas three RTs were isolated in both, humans and animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078 were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals and humans sharing the same household. The results of the epidemiological analysis of factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic potential. Molecular characterization and epidemiological analysis revealed that the zoonotic risk for C. difficile associated with dogs and cats within the community is low but cannot be excluded.

[Clostridia: toxin masters. Botulism: from botox to sausages?].

PubMed

Buzzi, Marta; Rossel, Anne; Coen, Matteo; Kaiser, Laurent; Abbas, Mohamed

2016-04-13

Clostridia are ubiquitous Gram-positive bacteria whose toxins are responsible for serious diseases. In this article we report a case of foodborne botulism we have recently managed. Moreover, we briefly describe the major clinical syndromes caused by different species of Clostridium (except for C. difficile infections, as this subject has been previously extensively reviewed in this journal). Botulism causes a flaccid paralysis starting with cranial nerves. Administration of botulism anti-toxin should be rapidly considered as soon as botulism is suspected, as prognosis is largely dependent on timely treatment; alerting the public health authorities is equally important. In Switzerland botulinum antitoxin can be obtained from the pharmacy of the Swiss Army.

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

PubMed Central

Hunt, Jonathan J.

2013-01-01

SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade. PMID:24296572

CD44 Promotes intoxication by the clostridial iota-family toxins.

PubMed

Wigelsworth, Darran J; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D; Carman, Robert J; Wilkins, Tracy D; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G; Popoff, Michel R; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

PubMed Central

Wigelsworth, Darran J.; Ruthel, Gordon; Schnell, Leonie; Herrlich, Peter; Blonder, Josip; Veenstra, Timothy D.; Carman, Robert J.; Wilkins, Tracy D.; Van Nhieu, Guy Tran; Pauillac, Serge; Gibert, Maryse; Sauvonnet, Nathalie; Stiles, Bradley G.; Popoff, Michel R.; Barth, Holger

2012-01-01

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins. PMID:23236484

Clostridium difficile Infection Among US Emergency Department Patients With Diarrhea and No Vomiting.

PubMed

Abrahamian, Fredrick M; Talan, David A; Krishnadasan, Anusha; Citron, Diane M; Paulick, Ashley L; Anderson, Lydia J; Goldstein, Ellie J C; Moran, Gregory J

2017-07-01

The incidence of Clostridium difficile infection has increased and has been observed among persons from the community who have not been exposed to antibiotics or health care settings. Our aims are to determine prevalence of C difficile infection among emergency department (ED) patients with diarrhea and the prevalence among patients without traditional risk factors. We conducted a prospective observational study of patients aged 2 years or older with diarrhea (≥3 episodes/24 hours) and no vomiting in 10 US EDs (2010 to 2013). We confirmed C difficile infection by positive stool culture result and toxin assay. C difficile infection risk factors were antibiotic use or overnight health care stay in the previous 3 months or previous C difficile infection. We typed strains with pulsed-field gel electrophoresis. Of 422 participants, median age was 46 years (range 2 to 94 years), with median illness duration of 3.0 days and 43.4% having greater than or equal to 10 episodes of diarrhea during the previous 24 hours. At least one risk factor for C difficile infection was present in 40.8% of participants; 25.9% were receiving antibiotics, 26.9% had health care stay within the previous 3 months, and 3.3% had previous C difficile infection. Forty-three participants (10.2%) had C difficile infection; among these, 24 (55.8%) received antibiotics and 19 (44.2%) had health care exposure; 17 of 43 (39.5%) lacked any risk factor. Among participants without risk factors, C difficile infection prevalence was 6.9%. The most commonly identified North American pulsed-field gel electrophoresis (NAP) strains were NAP type 1 (23.3%) and NAP type 4 (16.3%). Among mostly adults presenting to US EDs with diarrhea and no vomiting, C difficile infection accounted for approximately 10%. More than one third of patients with C difficile infection lacked traditional risk factors for the disease. Among participants without traditional risk factors, prevalence of C difficile infection was approximately 7%. Copyright © 2016 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.

Epidemiology of Clostridium difficile in infants in Oxfordshire, UK: Risk factors for colonization and carriage, and genetic overlap with regional C. difficile infection strains

PubMed Central

Godwin, Heather; Pill, Gemma; Mbuvi, Emily; Vaughan, Alison; Griffiths, David; Martin, Jessica; Fawley, Warren; Dingle, Kate E.; Oakley, Sarah; Wanelik, Kazimierz; Finney, John M.; Kachrimanidou, Melina; Moore, Catrin E.; Gorbach, Sherwood; Riley, Thomas V.; Crook, Derrick W.; Peto, Tim E. A.; Wilcox, Mark H.; Walker, A. Sarah

2017-01-01

Background Approximately 30–40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized. Methods Predominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases. Results 338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0–10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%). Conclusions In predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic overlap between some infant strains and those isolated from CDI cases suggest common community reservoirs of these C. difficile lineages, contrasting with those lineages found only in CDI cases, and therefore more consistent with healthcare-associated spread. PMID:28813461

Pore-forming activity of clostridial binary toxins.

PubMed

Knapp, O; Benz, R; Popoff, M R

2016-03-01

Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of the BD Max Cdiff assay for the detection of toxigenic Clostridium difficile in human stool specimens.

PubMed

Putsathit, Papanin; Morgan, Justin; Bradford, Damien; Engelhardt, Nelly; Riley, Thomas V

2015-02-01

The Becton Dickinson (BD) PCR-based GeneOhm Cdiff assay has demonstrated a high sensitivity and specificity for detecting Clostridium difficile. Recently, the BD Max platform, using the same principles as BD GeneOhm, has become available in Australia. This study aimed to investigate the sensitivity and specificity of BD Max Cdiff assay for the detection of toxigenic C. difficile in an Australian setting. Between December 2013 and January 2014, 406 stool specimens from 349 patients were analysed with the BD Max Cdiff assay. Direct and enrichment toxigenic culture were performed on bioMérieux ChromID C. difficile agar as a reference method. isolates from specimens with discrepant results were further analysed with an in-house PCR to detect the presence of toxin genes. The overall prevalence of toxigenic C. difficile was 7.2%. Concordance between the BD Max assay and enrichment culture was 98.5%. The sensitivity, specificity, positive predictive value and negative predictive value for the BD Max Cdiff assay were 95.5%, 99.0%, 87.5% and 99.7%, respectively, when compared to direct culture, and 91.7%, 99.0%, 88.0% and 99.4%, respectively, when compared to enrichment culture. The new BD Max Cdiff assay appeared to be an excellent platform for rapid and accurate detection of toxigenic C. difficile.

Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates.

PubMed

Rodriguez, C; Taminiau, B; Brévers, B; Avesani, V; Van Broeck, J; Leroux, A A; Amory, H; Delmée, M; Daube, G

2014-08-06

Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual. Copyright © 2014 Elsevier B.V. All rights reserved.

[Recent advances in Saccharomyces boulardii research].

PubMed

Im, E; Pothoulakis, C

2010-09-01

This review summarizes the probiotic mechanisms of action of Saccharomyces boulardii (S. boulardii) against inflammatory and non-inflammatory diarrheal conditions. S. boulardii is distributed in lyophilized form in many countries and used for the prevention of diarrhea in children and adults, including Clostridium difficile (C. difficile) associated infection. The main mechanisms of action of S. boulardii include inhibition of activities of bacterial pathogenic products, trophic effects on the intestinal mucosa, as well as modification of host signaling pathways involved in inflammatory and non-inflammatory intestinal diseases. S. boulardii inhibits production of pro-inflammatory cytokines by inhibiting main regulators of inflammation, including nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAP kinases), ERK1/2 and p38, but stimulates production of anti-inflammatory molecules such as peroxisome proliferator-activated receptor-gamma (PPAR-γ). Moreover, S. boulardii suppresses bacterial infection by inhibiting adhesion and/or overgrowth of bacteria, produces a serine protease that cleaves C. difficile toxin A, and stimulates antibody production against this toxin. Furthermore, S. boulardii may interfere with pathogenesis of Inflammatory Bowel Disease (IBD) by acting on T cells and acts in diarrheal conditions by improving the fecal biostructure in patients with diarrhea. These diverse mechanisms exerted by S. boulardii provide molecular clues for its effectiveness in diarrheal diseases and intestinal inflammatory conditions with an inflammatory component. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Clinical and microbiological characterization of Clostridium difficile infection in a tertiary care hospital in Shanghai, China.

PubMed

Dong, Danfeng; Peng, Yibing; Zhang, Lihua; Jiang, Cen; Wang, Xuefeng; Mao, Enqiang

2014-01-01

Over the last decade, Clostridium difficile infection (CDI) has emerged as a significant nosocomial infection, yet little has been reported from China. This study aimed to characterize the clinical and microbiological features of CDI from a hospital in Shanghai. Patients with CDI seen between December 2010 and March 2013 were included in this study, of which clinical data were retrospectively collected. The microbiological features of corresponding isolates were analyzed including genotype by multi-locus sequence typing (MLST), antimicrobial susceptibility, toxin production, sporulation capacity, biofilm formation, and motility. Ninety-four cases of CDI were included during this study period, 12 of whom were severe cases. By reviewing the clinical data, all patients were treated empirically with proton pump inhibitor or antibiotics or both, and they were distributed widely across various wards, most frequently to the digestive ward (28/94, 29.79%). Comparing the severe with mild cases, no significant differences were found in the basic epidemiological data or the microbiological features. Among the 94 isolates, 31 were toxin A-negative toxin B-positive all genotyped as ST37. They generated fewer toxins and spores, as well as similar amounts of biofilm and motility percentages, but exhibited highest drug resistance to cephalosporins, quinolones, macrolide-lincosamide and streptogramin (MLSB), and tetracycline. No specific clinical genotype or microbiological features were found in severe cases; antimicrobial resistance could be the primary reason for epidemic strains leading to the dissemination and persistence of CDI.

Clostridial Binary Toxins: Iota and C2 Family Portraits

PubMed Central

Stiles, Bradley G.; Wigelsworth, Darran J.; Popoff, Michel R.; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host–cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium. PMID:22919577

Burden of Clostridium difficile infection in the United States.

PubMed

Lessa, Fernanda C; Mu, Yi; Bamberg, Wendy M; Beldavs, Zintars G; Dumyati, Ghinwa K; Dunn, John R; Farley, Monica M; Holzbauer, Stacy M; Meek, James I; Phipps, Erin C; Wilson, Lucy E; Winston, Lisa G; Cohen, Jessica A; Limbago, Brandi M; Fridkin, Scott K; Gerding, Dale N; McDonald, L Clifford

2015-02-26

The magnitude and scope of Clostridium difficile infection in the United States continue to evolve. In 2011, we performed active population- and laboratory-based surveillance across 10 geographic areas in the United States to identify cases of C. difficile infection (stool specimens positive for C. difficile on either toxin or molecular assay in residents ≥ 1 year of age). Cases were classified as community-associated or health care-associated. In a sample of cases of C. difficile infection, specimens were cultured and isolates underwent molecular typing. We used regression models to calculate estimates of national incidence and total number of infections, first recurrences, and deaths within 30 days after the diagnosis of C. difficile infection. A total of 15,461 cases of C. difficile infection were identified in the 10 geographic areas; 65.8% were health care-associated, but only 24.2% had onset during hospitalization. After adjustment for predictors of disease incidence, the estimated number of incident C. difficile infections in the United States was 453,000 (95% confidence interval [CI], 397,100 to 508,500). The incidence was estimated to be higher among females (rate ratio, 1.26; 95% CI, 1.25 to 1.27), whites (rate ratio, 1.72; 95% CI, 1.56 to 2.0), and persons 65 years of age or older (rate ratio, 8.65; 95% CI, 8.16 to 9.31). The estimated number of first recurrences of C. difficile infection was 83,000 (95% CI, 57,000 to 108,900), and the estimated number of deaths was 29,300 (95% CI, 16,500 to 42,100). The North American pulsed-field gel electrophoresis type 1 (NAP1) strain was more prevalent among health care-associated infections than among community-associated infections (30.7% vs. 18.8%, P<0.001). C. difficile was responsible for almost half a million infections and was associated with approximately 29,000 deaths in 2011. (Funded by the Centers for Disease Control and Prevention.).

Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores

PubMed Central

Kochan, Travis J.; Kaiser, Alyssa M.; Hastie, Jessica L.; Giordano, Nicole P.; Smith, Ashley D.

2017-01-01

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI. PMID:28704538

Probiotics in Clostridium difficile infection: reviewing the need for a multistrain probiotic.

PubMed

Hell, M; Bernhofer, C; Stalzer, P; Kern, J M; Claassen, E

2013-03-01

In the past two years an enormous amount of molecular, genetic, metabolomic and mechanistic data on the host-bacterium interaction, a healthy gut microbiota and a possible role for probiotics in Clostridium difficile infection (CDI) has been accumulated. Also, new hypervirulent strains of C. difficile have emerged. Yet, clinical trials in CDI have been less promising than in antibiotic associated diarrhoea in general, with more meta-analysis than primary papers on CDI-clinical-trials. The fact that C. difficile is a spore former, producing at least three different toxins has not yet been incorporated in the rational design of probiotics for (recurrent) CDI. Here we postulate that the plethora of effects of C. difficile and the vast amount of data on the role of commensal gut residents and probiotics point towards a multistrain mixture of probiotics to reduce CDI, but also to limit (nosocomial) transmission and/or endogenous reinfection. On the basis of a retrospective chart review of a series of ten CDI patients where recurrence was expected, all patients on adjunctive probiotic therapy with multistrain cocktail (Ecologic®AAD/OMNiBiOTiC® 10) showed complete clinical resolution. This result, and recent success in faecal transplants in CDI treatment, are supportive for the rational design of multistrain probiotics for CDI.

Current knowledge on the laboratory diagnosis of Clostridium difficile infection.

PubMed

Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira

2017-03-07

Clostridium difficile ( C. difficile ) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.

Effects of Clostridium perfringens iota toxin in the small intestine of mice.

PubMed

Redondo, Leandro M; Redondo, Enzo A; Dailoff, Gabriela C; Leiva, Carlos L; Díaz-Carrasco, Juan M; Bruzzone, Octavio A; Cangelosi, Adriana; Geoghegan, Patricia; Fernandez-Miyakawa, Mariano E

2017-12-01

Iota toxin is a binary toxin solely produced by Clostridium perfringens type E strains, and is structurally related to CDT from C. difficile and CST from C. spiroforme. As type E causes hemorrhagic enteritis in cattle, it is usually assumed that associated diseases are mediated by iota toxin, although evidence in this regard has not been provided. In the present report, iota toxin intestinal effects were evaluated in vivo using a mouse model. Histological damage was observed in ileal loops treated with purified iota toxin after 4 h of incubation. Luminal iota toxin induced fluid accumulation in the small intestine in a dose dependent manner, as determined by the enteropooling and the intestinal loop assays. None of these changes were observed in the large intestine. These results suggest that C. perfringens iota toxin alters intestinal permeability, predominantly by inducing necrosis and degenerative changes in the mucosal epithelium of the small intestine, as well as changes in intestinal motility. The obtained results suggest a central role for iota toxin in the pathogenesis of C. perfringens type E hemorrhagic enteritis, and contribute to remark the importance of clostridial binary toxins in digestive diseases. Published by Elsevier Ltd.

Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.

PubMed

Koenigsknecht, Mark J; Theriot, Casey M; Bergin, Ingrid L; Schumacher, Cassie A; Schloss, Patrick D; Young, Vincent B

2015-03-01

Clostridium difficile infection (CDI) following antibiotic therapy is a major public health threat. While antibiotic disruption of the indigenous microbiota underlies the majority of cases of CDI, the early dynamics of infection in the disturbed intestinal ecosystem are poorly characterized. This study defines the dynamics of infection with C. difficile strain VPI 10463 throughout the gastrointestinal (GI) tract using a murine model of infection. After inducing susceptibility to C. difficile colonization via antibiotic administration, we followed the dynamics of spore germination, colonization, sporulation, toxin activity, and disease progression throughout the GI tract. C. difficile spores were able to germinate within 6 h postchallenge, resulting in the establishment of vegetative bacteria in the distal GI tract. Spores and cytotoxin activity were detected by 24 h postchallenge, and histopathologic colitis developed by 30 h. Within 36 h, all infected mice succumbed to infection. We correlated the establishment of infection with changes in the microbiota and bile acid profile of the small and large intestines. Antibiotic administration resulted in significant changes to the microbiota in the small and large intestines, as well as a significant shift in the abundance of primary and secondary bile acids. Ex vivo analysis suggested the small intestine as the site of spore germination. This study provides an integrated understanding of the timing and location of the events surrounding C. difficile colonization and identifies potential targets for the development of new therapeutic strategies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Prevalence of Clostridium Difficile Infection in Patients After Radical Cystectomy and Neoadjuvant Chemotherapy.

PubMed

Cotter, Katherine J; Fan, Yunhua; Sieger, Gretchen K; Weight, Christopher J; Konety, Badrinath R

2017-10-27

Clostridium Difficile is the most common cause of nosocomial infectious diarrhea. This study evaluates the prevalence and predictors of Clostridium Difficile infections in patients undergoing radical cystectomy with or without neoadjuvant chemotherapy. Retrospective chart review was performed of all patients undergoing cystectomy and urinary diversion at a single institution from 2011-2017. Infection was documented in all cases with testing for Clostridium Difficile polymerase chain reaction toxin B. Patient and disease related factors were compared for those who received neoadjuvant chemotherapy vs. those who did not in order to identify potential risk factors associated with C. Difficile infections. Chi squared test and logistic regression analysis were used to determine statistical significance. Of 350 patients who underwent cystectomy, 41 (11.7%) developed Clostridium Difficile in the 30 day post-operative period. The prevalence of C. Difficile infection was higher amongst the patients undergoing cystectomy compared to the non-cystectomy admissions at our hospital (11.7 vs. 2.9%). Incidence was not significantly different among those who underwent cystectomy for bladder cancer versus those who underwent the procedure for other reasons. Median time to diagnosis was 6 days (range 3-28 days). The prevalence of C. Diff infections was not significantly different among those who received neoadjuvant chemotherapy vs. those who did not (11% vs. 10.4% p  = 0.72). A significant association between C. Difficile infection was not seen with proton pump inhibitor use ( p  = 0.48), patient BMI ( p  = 0.67), chemotherapeutic regimen ( p  = 0.94), individual surgeon ( p  = 0.54), type of urinary diversion (0.41), or peri-operative antibiotic redosing ( p  = 0.26). Clostridium Difficile infection has a higher prevalence in patients undergoing cystectomy. No significant association between prevalence and exposure to neoadjuvant chemotherapy was seen.

Prevalence and risk factors for colonization by Clostridium difficile and extended-spectrum β-lactamase-producing Enterobacteriaceae in rehabilitation clinics in Germany.

PubMed

Arvand, M; Ruscher, C; Bettge-Weller, G; Goltz, M; Pfeifer, Y

2018-01-01

Rehabilitation clinics may vary widely in terms of type of care provided, duration of hospital stay, and case severity. Few data are available on prevalence of Clostridium difficile or extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) colonization in rehabilitation clinics in Germany. This study investigated the frequency of intestinal colonization by these pathogens among patients in rehabilitation clinics of different specialization. In the scope of a point prevalence study, faecal samples and demographic and clinical data were collected in five rehabilitation clinics. Samples were screened for C. difficile and ESBL-E by culture. Isolates were characterized by polymerase chain reaction for C. difficile toxins A and B, for β-lactamase genes, and by molecular typing including pulsed-field gel electrophoresis and PCR-based ribotyping. Of 305 patients screened, 11.1% were colonized by toxigenic C. difficile and 7.5% by ESBL-E. Colonization rates differed markedly between facilities, ranging from 1.6% to 26.3% for C. difficile and from zero to 23.7% for ESBL-E. Prevalence of colonization by C. difficile and ESBL-E was higher in neurological rehabilitation clinics than in clinics with other specialties (P<0.001). Molecular typing revealed six patients from one neurological rehabilitation clinic harbouring a unique C. difficile strain (ribotype 017). CTX-M-15 was the most prevalent ESBL type. We detected several indistinguishable pairs of ESBL-E isolates within some facilities. Significant differences were found in the prevalence of C. difficile and ESBL-E between rehabilitation clinics. Facilities providing specialized medical care for critically ill patients had higher prevalence rates. These results may help to delineate the requirements for infection prevention and control in rehabilitation clinics. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Stool Test: C. Difficile Toxin (For Parents)

MedlinePlus

... sealable container before taking it to the lab. Plastic wrap also can be used to line the diaper of an infant or toddler who isn't yet using the toilet. The wrap should be placed so that urine ... be collected into a clean, dry plastic jar with a screw-cap lid. For best ...

Nagging Presence of Clostridium difficile Associated Diarrhoea in North India

PubMed Central

Sharma, Nidhi; Gupta, Nitin; Kant, Kamla; Bahadur, Tej; Shende, Trupti M; Kumar, Lalit; Kabra, Sushil K

2017-01-01

Introduction Clostridium Difficile Associated Diarrhoea (CDAD) is a significant cause of morbidity in hospitalised patients worldwide. The data on clinical epidemiology of this disease in Indian subcontinent is scarce. Aim To evaluate the risk factors and clinical course of patients with CDAD. Materials and Methods A cross-sectional study was planned at our tertiary care centre, All India Institute of Medical Sciences, whereby, all patients who had nosocomial diarrhea between 2010 and 2014 were included in the study. Their clinical and laboratory profile were recorded using structured questionnaire and their stool samples were subjected to ELISA for detection of toxins A and B (Premier toxins A and B). Those patients who had toxins A and B in their stool samples were diagnosed as CDAD. The clinical and laboratory profile of CDAD patients were further analysed. Results A total of 791 patients with nosocomial diarrhea were included in this study. CDAD was diagnosed in a total of 48(6%) patients. The year wise breakdown of the positive patients is as follows: 7/135 (5.2%), 4/156 (2.6%), 5/141 (3.5%), 9/193 (4.7%) and 23/166 (13.8%), respectively. A total of 16/48 (33.3%) of CDAD cases belonged to the age group of 51-60 years. Malignancy (n=15, 31.25%) was the most common underlying pathological condition. All the patients had a history of antibiotic intake. Most common antibiotic used in the patients of CDAD was third generation cephalosporins (n=27, 56.25%). The use of clindamycin, carbapenems and colistin increased in the year 2014. Mean duration of hospital stay was 9.8 days. Diarrhoea was associated with fever in 50% of the patients while abdominal pain was seen in 39.6% of the patients. Conclusion The control of Clostridium difficile infection suffers from the rampant use of higher antibiotics. There is a need for proper implementation of antimicrobial stewardship programmes and better hospital infection control to stop the transmission of this nagging bug. PMID:29207702

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model.

PubMed

Guo, Shanguang; Yan, Weiwei; McDonough, Sean P; Lin, Nengfeng; Wu, Katherine J; He, Hongxuan; Xiang, Hua; Yang, Maosheng; Moreira, Maira Aparecida S; Chang, Yung-Fu

2015-03-24

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing.

PubMed

Killgore, George; Thompson, Angela; Johnson, Stuart; Brazier, Jon; Kuijper, Ed; Pepin, Jacques; Frost, Eric H; Savelkoul, Paul; Nicholson, Brad; van den Berg, Renate J; Kato, Haru; Sambol, Susan P; Zukowski, Walter; Woods, Christopher; Limbago, Brandi; Gerding, Dale N; McDonald, L Clifford

2008-02-01

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Comparison of planktonic and biofilm-associated communities of Clostridium difficile and indigenous gut microbiota in a triple-stage chemostat gut model.

PubMed

Crowther, Grace S; Chilton, Caroline H; Todhunter, Sharie L; Nicholson, Scott; Freeman, Jane; Baines, Simon D; Wilcox, Mark H

2014-08-01

Biofilms are characteristic of some chronic or recurrent infections and this mode of growth tends to reduce treatment efficacy. Clostridium difficile infection (CDI) is associated with a high rate of recurrent symptomatic disease. The presence and behaviour of C. difficile within intestinal biofilms remains largely unexplored, but may factor in recurrent infection. A triple-stage chemostat gut model designed to facilitate the formation of intestinal biofilm was inoculated with a pooled human faecal emulsion. Bacterial populations were allowed to equilibrate before simulated CDI was induced by clindamycin (33.9 mg/L, four times daily, 7 days) and subsequently treated with vancomycin (125 mg/L, four times daily, 7 days). Indigenous gut microbiota, C. difficile total viable counts, spores, cytotoxin and antimicrobial activity in planktonic and biofilm communities were monitored during the 10 week experimental period. Vancomycin successfully treated the initial episode of simulated CDI, but ∼18 days after therapy cessation, recurrent infection occurred. Germination, proliferation and toxin production were evident within planktonic communities in both initial and recurrent CDI. In contrast, sessile C. difficile remained in dormant spore form for the duration of the experiment. The effects of and recovery from clindamycin and vancomycin exposure for sessile populations was delayed compared with responses for planktonic bacteria. Intestinal biofilms provide a potential reservoir for C. difficile spore persistence, possibly facilitating their dispersal into the gut lumen after therapeutic intervention, leading to recurrent infection. Therapeutic options for CDI could have increased efficacy if they are more effective against sessile C. difficile. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile.

PubMed

Childress, Kevin O; Edwards, Adrianne N; Nawrocki, Kathryn L; Anderson, Sarah E; Woods, Emily C; McBride, Shonna M

2016-12-01

The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

PubMed Central

Childress, Kevin O.; Edwards, Adrianne N.; Nawrocki, Kathryn L.; Anderson, Sarah E.; Woods, Emily C.

2016-01-01

The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species. PMID:27647869

Community-acquired Clostridium difficile infection in children: A retrospective study.

PubMed

Borali, Elena; Ortisi, Giuseppe; Moretti, Chiara; Stacul, Elisabetta Francesca; Lipreri, Rita; Gesu, Giovanni Pietro; De Giacomo, Costantino

2015-10-01

Community acquired-Clostridium difficile infection (CDI) has increased also in children in the last years. To determine the incidence of community-acquired CDI and to understand whether Clostridium difficile could be considered a symptom-triggering pathogen in infants. A five-year retrospective analysis (January 2007-December 2011) of faecal specimens from 124 children hospitalized in the Niguarda Ca' Granda Hospital for prolonged or muco-haemorrhagic diarrhoea was carried out. Stool samples were evaluated for common infective causes of diarrhoea and for Clostridium difficile toxins. Patients with and without CDI were compared for clinical characteristics and known risk factors for infection. Twenty-two children with CDI were identified in 5 years. An increased incidence of community-acquired CDI was observed, ranging from 0.75 per 1000 hospitalizations in 2007 to 9.8 per 1000 hospitalizations in 2011. Antimicrobial treatment was successful in all 19 children in whom it was administered; 8/22 CDI-positive children were younger than 2 years. No statistically significant differences in clinical presentation were observed between patients with and without CDI, nor in patients with and without risk factors for CDI. Our study shows that Clostridium difficile infection is increasing and suggests a possible pathogenic role in the first 2 years of life. Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Current knowledge on the laboratory diagnosis of Clostridium difficile infection

PubMed Central

Martínez-Meléndez, Adrián; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Maldonado-Garza, Héctor Jesús; Villarreal-Treviño, Licet; Garza-González, Elvira

2017-01-01

Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from self-limited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI. PMID:28321156

[Infection frequency in patients with chronic idiopathic ulcerative colitis].

PubMed

Yamamoto-Furusho, J K; de León-Rendón, J L; Rodas, L

2012-01-01

Ulcerative Colitis (UC) is a chronic inflammatory bowel disease characterized by diffuse inflammation of the mucosa of the colon. Up to now, diverse observational studies have implicated a wide variety of pathogenic microorganisms as causal and exacerbating factors in UC. Clostridium difficile (C. difficile) infection has been associated with recurrence and treatment failure and its incidence in patients with UC has been on the rise in the last few years. To determine the frequency of infection by different microorganisms in Mexican UC patients. A total of 150 patients with definitive UC diagnosis were studied. All the stool tests for parasites and ova, stool cultures, tests for the C. difficile toxins A and B, and immunohistochemistry for Cytomegalovirus in colon segment biopsies were analyzed. Other demographic and clinical variables of the disease were recorded for their correlation with infection frequency. Infection frequency in UC patients was 28.00%. C. difficile infection was present in 0.013%. Other pathogens were found, such as Endolimax nana (9.00%), Entamoeba histolytica (3.00%), Cytomegalovirus (2.00%), Salmonella (2.00%), Shigella (0.70%), Toxoplasma gondii (0.70%) and Iodamoeba bütschlii (0.70%). Infection frequency was 28.00% in our study and C. difficile infection represented only 0.013%. Copyright © 2012 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.

PubMed

Vidunas, Eugene; Mathews, Antony; Weaver, Michele; Cai, Ping; Koh, Eun Hee; Patel-Brown, Sujata; Yuan, Hailey; Zheng, Zi-Rong; Carriere, Marjolaine; Johnson, J Erik; Lotvin, Jason; Moran, Justin

2016-07-01

A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and β-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

PubMed

Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

2017-01-01

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis , indicating that this protein would not be a good target for inhibiting spore formation.

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

PubMed Central

Ribis, John W.; Ravichandran, Priyanka; Putnam, Emily E.; Pishdadian, Keyan

2017-01-01

ABSTRACT The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation. PMID:28959733

Clostridium difficile Infection and Patient-Specific Antimicrobial Resistance Testing Reveals a High Metronidazole Resistance Rate.

PubMed

Barkin, Jodie A; Sussman, Daniel A; Fifadara, Nimita; Barkin, Jamie S

2017-04-01

Clostridium difficile (CD) infection (CDI) causes marked morbidity and mortality, accounting for large healthcare expenditures annually. Current CDI treatment guidelines focus on clinical markers of patient severity to determine the preferred antibiotic regimen of metronidazole versus vancomycin. The antimicrobial resistance patterns for patients with CD are currently unknown. The aim of this study was to define the antimicrobial resistance patterns for CD. This study included all patients with stools sent for CD testing to a private laboratory (DRG Laboratory, Alpharetta, Georgia) in a 6-month period from across the USA. Patient data was de-identified, with only age, gender, and zip-code available per laboratory protocol. All samples underwent PCR testing followed by hybridization for CD toxin regions A and B. Only patients with CD-positive PCR were analyzed. Antimicrobial resistance testing using stool genomic DNA evaluated presence of imidazole- and vancomycin-resistant genes using multiplex PCR gene detection. Of 2743, 288 (10.5%) stool samples were positive for CD. Six were excluded per protocol. Of 282, 193 (69.4%) were women, and average age was 49.4 ± 18.7 years. Of 282, 62 were PCR positive for toxins A and B, 160 for toxin A positive alone, and 60 for toxin B positive alone. Antimicrobial resistance testing revealed 134/282 (47.5%) patients resistant to imidazole, 17 (6.1%) resistant to vancomycin, and 9 (3.2%) resistant to imidazole and vancomycin. CD-positive patients with presence of imidazole-resistant genes from stool DNA extract was a common phenomenon, while vancomycin resistance was uncommon. Similar to treatment of other infections, antimicrobial resistance testing should play a role in CDI clinical decision-making algorithms to enable more expedited and cost-effective delivery of patient care.

Time-resolved cellular effects induced by TcdA from Clostridium difficile.

PubMed

Jochim, Nelli; Gerhard, Ralf; Just, Ingo; Pich, Andreas

2014-05-30

The anaerobe Clostridium difficile is a common pathogen that causes infection of the colon leading to diarrhea or pseudomembranous colitis. Its major virulence factors are toxin A (TcdA) and toxin B (TcdB), which specifically inactivate small GTPases by glucosylation leading to reorganization of the cytoskeleton and finally to cell death. In the present work a quantitative proteome analysis using the isotope-coded protein label (ICPL) approach was conducted to investigate proteome changes in the colon cell line Caco-2 after treatment with recombinant wild-type TcdA (rTcdA-wt) or a glucosyltransferase-deficient mutant TcdA (rTcdA-mut). Proteins from crude cell lysates or cellular subfractions were identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Two time points (5 h, 24 h) of toxin treatment were analyzed and about 4000 proteins were identified in each case. After 5 h treatment with rTcdA-wt, 150 proteins had a significantly altered abundance; rTcdA-mut caused regulation of 50 proteins at this time point. After 24 h treatment with rTcdA-wt changes in abundance of 61 proteins were observed, but no changes in protein abundance were detected after 24 h if cells were treated with rTcdA-mut. TcdA affected several proteins involved in signaling events, cytoskeleton and cell-cell contact organization, translation, and metabolic processes. The ICPL-dependent quantification was verified by label-free targeted MS techniques based on multiple reaction monitoring (MRM) and triple quadrupole mass spectrometry. LC/MS-based proteome analyses and the ICPL approach revealed comprehensive and reproducible proteome date and provided new insights into the cellular effects of clostridial glucosylating toxins (CGT). Copyright © 2014 John Wiley & Sons, Ltd.

Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells

PubMed Central

Edwards, Adrianne N.; Karim, Samiha T.; Pascual, Ricardo A.; Jowhar, Lina M.; Anderson, Sarah E.; McBride, Shonna M.

2016-01-01

Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands. PMID:27833595

Clostridial Binary Toxins: Basic Understandings that Include Cell Surface Binding and an Internal "Coup de Grâce".

PubMed

Stiles, Bradley G

2017-01-01

Clostridium species can make a remarkable number of different protein toxins, causing many diverse diseases in humans and animals. The binary toxins of Clostridium botulinum, C. difficile, C. perfringens, and C. spiroforme are one group of enteric-acting toxins that attack the actin cytoskeleton of various cell types. These enterotoxins consist of A (enzymatic) and B (cell binding/membrane translocation) components that assemble on the targeted cell surface or in solution, forming a multimeric complex. Once translocated into the cytosol via endosomal trafficking and acidification, the A component dismantles the filamentous actin-based cytoskeleton via mono-ADP-ribosylation of globular actin. Knowledge of cell surface receptors and how these usurped, host-derived molecules facilitate intoxication can lead to novel ways of defending against these clostridial binary toxins. A molecular-based understanding of the various steps involved in toxin internalization can also unveil therapeutic intervention points that stop the intoxication process. Furthermore, using these bacterial proteins as medicinal shuttle systems into cells provides intriguing possibilities in the future. The pertinent past and state-of-the-art present, regarding clostridial binary toxins, will be evident in this chapter.

Clostridium difficile infection in returning travellers

PubMed Central

Stevens, A. Michal; Esposito, Douglas H.; Stoney, Rhett J.; Hamer, Davidson H.; Flores-Figueroa, Jose; Bottieau, Emmanuel; Connor, Bradley A.; Gkrania-Klotsas, Effrossyni; Goorhuis, Abraham; Hynes, Noreen A.; Libman, Michael; Lopez-Velez, Rogelio; McCarthy, Anne E.; von Sonnenburg, Frank; Schwartz, Eli; van Genderen, Perry J.J.; Benson, L. Scott; Leung, Daniel T.

2017-01-01

Background There is increasing recognition of the contribution of community-acquired cases to the global burden of Clostridium difficile infection (CDI). The epidemiology of CDI among international travellers is poorly understood, and factors associated with international travel, such as antibiotic use and changes in gut microbiota, could potentially put travellers at higher risk. Methods We summarized demographic, travel-associated and geographic characteristics of travellers with CDI in the GeoSentinel database from 1997 to 2015. We also surveyed GeoSentinel sites to compare various testing indications, approaches, and diagnostic modalities. Results We identified 260 GeoSentinel records, including 187 that satisfied criteria for analysis (confirmed cases in non-immigrant travellers aged >2 years, seen <12 weeks post-travel). CDI was reported in all age groups and in travellers to all world regions; the largest proportions of cases having destinations in Asia (31%), Central/South America or the Caribbean (30%) and Africa (24%). Our site survey revealed substantial heterogeneity of testing approaches between sites; the most commonly used test was the C. difficile toxin gene PCR. Conclusions CDI is encountered in returning international travellers, although there is considerable variability in testing practices. These data underscore the importance of awareness of C. difficile as a potential cause of travel-associated diarrhoea. PMID:28355613

Correlation between fecal calprotectin levels, disease severity and the hypervirulent ribotype 027 strain in patients with Clostridium difficile infection.

PubMed

Peretz, Avi; Tkhawkho, Linda; Pastukh, Nina; Brodsky, Diana; Halevi, Chen Namimi; Nitzan, Orna

2016-06-22

Clostridium difficile is the most common infectious etiology of nosocomial diarrhea. Fecal calprotectin (fc) is a sensitive marker of intestinal inflammation, found to be associated with enteric bacterial infections and inflammatory bowel disease. We evaluated fc levels using a Chemiluminescent immunoassay method, in hospitalized patients with C. difficile infection (CDI) diagnosed by molecular stool examination and assessed correlation with virulent ribotype 027 strain infection, antibiotic susceptibility by gradient Etest strip performed on C. difficile colonies and clinical and laboratory measures of disease severity. Statistical analysis was performed for correlation of fc levels with clinical and laboratory parameters, disease severity and patient outcomes. Overall 29 patients with CDI were admitted at the Poria medical center in northern Israel, during June 2014-May 2015. Resistance to metronidazole was found in 3 (10.3 %) isolates and to vancomycin in 5 (17.2 %) isolates. Regarding patient outcomes, within 30 days of CDI diagnosis, recurrence of disease occurred in 10 (34.5 %) patients and 2 patients (6.9 %) died. Seven (24.1 %) isolates were C. difficile ribotype 027. Mean fc level was 331.4 μg/g (21-932). Higher fc levels were found in patients with C. difficile ribotype 027 (p < 0.0005). Fc levels were also correlated with elevated peripheral blood white cell count (p = 0.0007). A trend for higher fc levels was found in patients with a higher clostridium severity score index (p = 0.0633). No correlation was found between fecal calprotectin levels and age, sex, functional status, community versus hospital acquired CDI, antibiotic susceptibility, fever, and creatinine levels. Our study highlights the fact that fc has a potential role as a biomarker of disease severity and binary toxin producing ribotype associated disease.

Microbiome changes associated with sustained eradication of Clostridium difficile after single faecal microbiota transplantation in children with and without inflammatory bowel disease.

PubMed

Hourigan, S K; Chen, L A; Grigoryan, Z; Laroche, G; Weidner, M; Sears, C L; Oliva-Hemker, M

2015-09-01

Little data are available regarding the effectiveness and associated microbiome changes of faecal microbiota transplantation (FMT) for Clostridium difficile infection (CDI) in children, especially in those with inflammatory bowel disease (IBD) with presumed underlying dysbiosis. To investigate C. difficile eradication and microbiome changes with FMT in children with and without IBD. Children with a history of recurrent CDI (≥3 recurrences) underwent FMT via colonoscopy. Stool samples were collected pre-FMT and post-FMT at 2-10 weeks, 10-20 weeks and 6 months. The v4 hypervariable region of the 16S rRNA gene was sequenced. C. difficile toxin B gene polymerase chain reaction was performed. Eight children underwent FMT for CDI; five had IBD. All had resolution of CDI symptoms. All tested had eradication of C. difficile at 10-20 weeks and 6 months post-FMT. Pre-FMT patient samples had significantly decreased bacterial richness compared with donors (P = 0.01), in those with IBD (P = 0.02) and without IBD (P = 0.01). Post-FMT, bacterial diversity in patients increased. Six months post-FMT, there was no significant difference between bacterial diversity of donors and patients without IBD; however, bacterial diversity in those with IBD returned to pre-FMT baseline. Microbiome composition at 6 months in IBD-negative patients more closely approximated donor composition compared to IBD-positive patients. FMT gives sustained C. difficile eradication in children with and without IBD. FMT-restored diversity is sustained in children without IBD. In those with IBD, bacterial diversity returns to pre-FMT baseline by 6 months, suggesting IBD host-related mechanisms modify faecal microbiome diversity. © 2015 John Wiley & Sons Ltd.

Fidaxomicin for the treatment of Clostridium difficile infections.

PubMed

Whitman, Craig B; Czosnowski, Quinn A

2012-02-01

To evaluate the pharmacology, microbiology, safety, and efficacy of fidaxomicin for treatment of Clostridium difficile infections (CDI). Literature was identified through Ovid MEDLINE (1948-December 2011) and International Pharmaceutical Abstracts (1970-December 2011) using the search terms fidaxomicin, OPT-80, PAR-101, OP-118, difimicin, tiacumicin, lipiarmycin, Clostridium difficile, Clostridium difficile infection, Clostridium difficile-associated diarrhea, and cost. Drug monographs were retrieved from manufacturers' Web pages, and the Red Book component of Micromedex was used for cost information. All pertinent Phase 1, 2, and 3 studies published in English were included. Fidaxomicin is a macrocyclic compound bactericidal against C. difficile and inhibits toxin and spore production. It has poor oral absorption with high fecal concentrations. Available Phase 2 and 3 data with fidaxomicin 200 mg orally every 12 hours demonstrate similar effectiveness in treating CDI compared to oral vancomycin. Fidaxomicin was shown to have less frequency of recurrent infections. Adverse effects are uncommon and occur at similar rates as with oral vancomycin. The most frequently reported adverse effects are gastrointestinal, hematologic, and electrolyte disorders. Available data are lacking in several areas, including the efficacy and safety of fidaxomicin compared to established regimens for mild-to-moderate, life-threatening, and recurrent CDIs. The cost of a 10-day course of fidaxomicin is significantly more than that of metronidazole and vancomycin for treatment of mild-to-moderate CDI. Fidaxomicin appears to be an effective and safe alternative to oral vancomycin for treatment of mild-to-moderate and severe CDI. Data on its use compared to guideline-recommended therapies for mild-to-moderate and life-threatening CDI are needed. Further data assessing the cost-effectiveness of fidaxomicin are needed. Currently, it cannot be recommended over vancomycin for treatment of CDI. However, it may be considered for treatment of recurrent infections.

A combination of the probiotic and prebiotic product can prevent the germination of Clostridium difficile spores and infection.

PubMed

Rätsep, M; Kõljalg, S; Sepp, E; Smidt, I; Truusalu, K; Songisepp, E; Stsepetova, J; Naaber, P; Mikelsaar, R H; Mikelsaar, M

2017-10-01

Clostridium difficile infection (CDI) is one of the most prevalent healthcare associated infections in hospitals and nursing homes. Different approaches are used for prevention of CDI. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients. Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic Lactobacillus plantarum Inducia (DSM 21379) to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum Inducia, prebiotic xylitol (Xyl) and their combination as a synbiotic (Syn) product; c) the suppression of germination of C. difficile spores in vitro and in vivo in animal model of C. difficile infection with Inducia, Xyl and Syn treatment. The VPI strain 10463 (ATCC 43255), epidemic strain (M 13042) and clinical isolates (n = 12) of C. difficile from Norway and Estonia were susceptible and contrarily L. plantarum Inducia resistant to vancomycin, metronidazole and ciprofloxacin. The intact cells of Inducia, natural and neutralized cell free supernatant inhibited in vitro the growth of tested C. difficile reference strain VPI and Estonian and Norwegian clinical isolates of C. difficile after co-cultivation. This effect against C. difficile sustained in liquid media under ampicillin (0.75 μg/ml) and Xyl (5%) application. Further, incubation of Inducia in the media with 5% Xyl fully stopped germination of spores of C. difficile VPI strain after 48 h. In infection model the 48 hamsters were administered ampicillin (30 mg/kg) and 10-30 spores of C. difficile VPI strain. They also received five days before and after the challenge a pretreatment with a synbiotic (single daily dose of L. plantarum Inducia 1 ml of 10 10  CFU/ml and 20% xylitol in 1 ml by orogastric gavage). The survival rate of hamsters was increased to 78% compared to 13% (p = 0.003) survival rate of hamsters who received no treatment. When administered Xyl the survival rate of hamsters reached 56% vs.13% (p = 0.06). In both Syn (6/9, p = 0.003) and Xyl (3/9, p = 0.042) groups the number of animals not colonized with C. difficile significantly increased. In conclusion, the combination of xylitol with L. plantarum Inducia suppresses the germination of spores and outgrowth into vegetative toxin producing cells of C. difficile and reduces the colonization of gut with the pathogen. Putative therapeutical approach includes usage of the synbiotic during antimicrobial therapy for prevention of CDI and its potential to reduce recurrences of CDI. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ridinilazole: a novel therapy for Clostridium difficile infection.

PubMed

Vickers, Richard J; Tillotson, Glenn; Goldstein, Ellie J C; Citron, Diane M; Garey, Kevin W; Wilcox, Mark H

2016-08-01

Clostridium difficile infection (CDI) is the leading cause of infectious healthcare-associated diarrhoea. Recurrent CDI increases disease morbidity and mortality, posing a high burden to patients and a growing economic burden to the healthcare system. Thus, there exists a significant unmet and increasing medical need for new therapies for CDI. This review aims to provide a concise summary of CDI in general and a specific update on ridinilazole (formerly SMT19969), a novel antibacterial currently under development for the treatment of CDI. Owing to its highly targeted spectrum of activity and ability to spare the normal gut microbiota, ridinilazole provides significant advantages over metronidazole and vancomycin, the mainstay antibiotics for CDI. Ridinilazole is bactericidal against C. difficile and exhibits a prolonged post-antibiotic effect. Furthermore, treatment with ridinilazole results in decreased toxin production. A phase 1 trial demonstrated that oral ridinilazole is well tolerated and specifically targets clostridia whilst sparing other faecal bacteria. Phase 2 and 3 trials will hopefully further our understanding of the clinical utility of ridinilazole for the treatment of CDI. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Pseudomembranous Colitis

PubMed Central

Farooq, Priya D.; Urrunaga, Nathalie H.; Tang, Derek M.; von Rosenvinge, Erik C.

2015-01-01

Pseudomembranous colitis is an inflammatory condition of the colon characterized by elevated yellow-white plaques that coalesce to form pseudomembranes on the mucosa. Patients with the condition commonly present with abdominal pain, diarrhea, fever, and leukocytosis. Because pseudomembranous colitis is often associated with C. difficile infection, stool testing and empiric antibiotic treatment should be initiated when suspected. When results of C. difficile testing are negative and symptoms persist despite escalating empiric treatment, early gastroenterology consultation and lower endoscopy would be the next step in the appropriate clinical setting. If pseudomembranous colitis is confirmed endoscopically, colonic biopsies should be obtained, as histology can offer helpful clues to the underlying diagnosis. The less common non-C. difficile causes of pseudomembranous colitis should be entertained, as a number of etiologies can result in this condition. Examples include Behcet’s disease, collagenous colitis, inflammatory bowel disease, ischemic colitis, other infections organisms (e.g. bacteria, parasites, viruses), and a handful of drugs and toxins. Pinpointing the correct underlying etiology would better direct patient care and disease management. Surgical specialists would be most helpful in colonic perforation, gangrenous colon, or severe disease. PMID:25769243

Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 (Bio-K+): Characterization, Manufacture, Mechanisms of Action, and Quality Control of a Specific Probiotic Combination for Primary Prevention of Clostridium difficile Infection.

PubMed

Auclair, Julie; Frappier, Martin; Millette, Mathieu

2015-05-15

A specific probiotic formulation composed of Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2 (Bio-K+) has been marketed in North America since 1996. The strains and the commercial products have been evaluated for safety, identity, gastrointestinal survival, and stability throughout shelf life. The capacity of both the fermented beverages and the capsules to reduce incidences of antibiotic-associated diarrhea and Clostridium difficile infection (CDI) has been demonstrated in human clinical trials. Individual strains and the finished products have shown antimicrobial activity against C. difficile and toxin A/B neutralization capacity in vitro. The use of this specific probiotic formulation as part of a bundle of preventive measures to control CDI in healthcare settings is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Successful treatment of simulated Clostridium difficile infection in a human gut model by fidaxomicin first line and after vancomycin or metronidazole failure.

PubMed

Chilton, C H; Crowther, G S; Freeman, J; Todhunter, S L; Nicholson, S; Longshaw, C M; Wilcox, M H

2014-02-01

Fidaxomicin reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. We investigated fidaxomicin primary or secondary treatment efficacy using a gut model. Four triple-stage chemostat gut models were inoculated with faeces. After clindamycin induction of CDI, fidaxomicin (200 mg/L twice daily), vancomycin (125 mg/L four times daily) or metronidazole (9.3 mg/L three times daily) was administered for 7 days. Following failure/CDI recurrence, fidaxomicin (200 mg/L twice daily, 7 days) was instilled. C. difficile (CD) total viable counts (TVC), spore counts (SP), toxin titres (CYT), gut bacteria counts and antimicrobial concentrations were measured throughout. Fidaxomicin instillation reduced CD TVC/SP and CYT below the limit of detection (LOD) after 2 and 4 days, respectively, with no CDI recurrence. Metronidazole instillation failed to decrease CD TVC or CYT. Vancomycin instillation reduced CD TVC and CYT to LOD by day 4, but SP persisted. Recurrence occurred 13 days after vancomycin instillation; subsequent fidaxomicin instillation reduced CD TVC/SP/CYT below the LOD from day 2. CD was isolated sporadically, with no evidence of spore recrudescence or toxin production. Fidaxomicin had a minimal effect on the microflora, except for bifidobacteria. Fidaxomicin was detected for at least 21 days post-instillation, whereas other antimicrobials were undetectable beyond ∼4 days. Fidaxomicin successfully treated simulated primary and recurrent CDI. Fidaxomicin was superior to metronidazole in reducing CD TVC and SP, and superior to vancomycin in reducing SP without recurrence of vegetative cell growth. Fidaxomicin, but not vancomycin or metronidazole, persisted in the gut model for >20 days after instillation.

Diverticular disease of the colon does not increase risk of repeat C. difficile infection.

PubMed

Feuerstadt, Paul; Das, Rohit; Brandt, Lawrence J

2013-01-01

Studies have suggested that colonic diverticulosis might increase the likelihood of repeat Clostridium difficile infection (CDI). Our study was designed to compare rates of repeat infection in patients with and without colon diverticula. Patients who had a positive C. difficile toxin assay and colonoscopic evidence of diverticulosis were classified as CDI and diverticulosis (CDI-D), whereas those with a positive toxin assay but no such colonoscopic evidence were classified as CDI and no diverticulosis (CDI-ND). Various clinical and epidemiologic factors were recorded for each patient. Primary outcomes were "relapse" (repeat CDI within 3 mo of initial infection) and "recurrent" infection (repeat CDI≥3 mo after initial infection). Secondary outcomes 30 days after diagnosis were mortality, intensive care unit transfer, and continuous hospitalization. A total of 128 patients were classified as CDI-D, whereas 137 had CDI-ND. There were no significant differences between CDI-D and CDI-ND when comparing frequencies of repeat infection and its subclassifications, relapse or recurrence. There were, however, statistical associations seen between diverticulosis of the ascending colon and increased recurrence rates [hazard ratio (HR): 1.4±0.38, P<0.05] and decreased rates of relapse in diverticular disease of the descending (HR: 0.40±0.46, P<0.05), and sigmoid colon (HR: 0.39±0.49, P<0.05). The ascending colon association is limited by a small patient population. There were no significant differences in any of the 30-day outcomes including intensive care unit requirement, hospitalization stay, or mortality. Patients with diverticular disease of the colon are not at increased risk of repeat CDI.

[Individualized treatment strategies for Clostridium difficile infections].

PubMed

Solbach, P; Dersch, P; Bachmann, O

2017-07-01

Upon hospitalization, up to 15.5% of patients are already colonized with a toxigenic Clostridium difficile strain (TCD). The rate of asymptomatic colonization is 0-3% in healthy adults and up to 20-40% in hospitalized patients. The incidence and mortality of C. difficile infection (CDI) has significantly increased during recent years. Mortality lies between 3 and 14%. CDI is generally caused by intestinal dysbiosis, which can be triggered by various factors, including antibiotics or immune suppressants. If CDI occurs, ongoing antibiotic therapy should be discontinued. The choice of treatment is guided by the clinical situation: Mild courses of CDI should be treated with metronidazole. Oral vancomycin is suitable as a first-line therapy of mild CDI occurring during pregnancy and lactation, as well as in cases of intolerance or allergy to metronidazole. Severe courses should be treated with vancomycin. Recurrence should be treated with vancomycin or fidaxomicin. Multiple recurrences should be treated with vancomycin or fidaxomicin; if necessary, a vancomycin taper regimen may also be used. An alternative is fecal microbiota transplant (FMT), with healing rates of more than 80%. Bezlotoxumab is the first available monoclonal antibody which neutralizes the C. difficile toxin B, and in combination with an antibiotic significantly reduces the rate of a new C. difficile infection compared to placebo. A better definition of clinical and microbiota-associated risk factors and the ongoing implementation of molecular diagnostics are likely to lead to optimized identification of patients at risk, and an increasing individualization of prophylactic and therapeutic approaches.

The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin.

PubMed

Lin, Ni-Ni; Chen, Jia; Xu, Bin; Wei, Xia; Guo, Lei; Xie, Jian-Wei

2015-01-01

T-2 toxin poses a great threat to human health because it has the highest toxicity of the currently known trichothecene mycotoxins. To understand the in vivo toxicity and transformation mechanism of T-2 toxin, we investigated the role of one kind of principal phase I drug-metabolizing enzymes (cytochrome P450 [CYP450] enzymes) on the metabolism of T-2 toxin, which are crucial to the metabolism of endogenous substances and xenobiotics. We also investigated carboxylesterase, which also plays an important role in the metabolism of toxic substances. A chemical inhibition method and a recombinant method were employed to investigate the metabolism of the T-2 toxin by the CYP450 enzymes, and a chemical inhibition method was used to study carboxylesterase metabolism. Samples incubated with human liver microsomes were analyzed by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC- QqQ MS) after a simple pretreatment. In the presence of a carboxylesterase inhibitor, only 20 % T-2 toxin was metabolized. When CYP enzyme inhibitors and a carboxylesterase inhibitor were both present, only 3 % of the T-2 toxin was metabolized. The contributions of the CYP450 enzyme family to T-2 toxin metabolism followed the descending order CYP3A4, CYP2E1, CYP1A2, CYP2B6 or CYP2D6 or CYP2C19. Carboxylesterase and CYP450 enzymes are of great importance in T-2 toxin metabolism, in which carboxylesterase is predominant and CYP450 has a subordinate role. CYP3A4 is the principal member of the CYP450 enzyme family responsible for T-2 toxin metabolism. The primary metabolite produced by carboxylesterase is HT-2, and the main metabolite produced by CYP 3A4 is 3'-OH T-2. The different metabolites show different toxicities. Our results will provide useful data concerning the toxic mechanism, the safety evaluation, and the health risk assessment of T-2 toxin.

Clostridium difficile infection in returning travellers.

PubMed

Michal Stevens, A; Esposito, Douglas H; Stoney, Rhett J; Hamer, Davidson H; Flores-Figueroa, Jose; Bottieau, Emmanuel; Connor, Bradley A; Gkrania-Klotsas, Effrossyni; Goorhuis, Abraham; Hynes, Noreen A; Libman, Michael; Lopez-Velez, Rogelio; McCarthy, Anne E; von Sonnenburg, Frank; Schwartz, Eli; van Genderen, Perry J J; Scott Benson, L; Leung, Daniel T

2017-05-01

There is increasing recognition of the contribution of community-acquired cases to the global burden of Clostridium difficile infection (CDI). The epidemiology of CDI among international travellers is poorly understood, and factors associated with international travel, such as antibiotic use and changes in gut microbiota, could potentially put travellers at higher risk. We summarized demographic, travel-associated and geographic characteristics of travellers with CDI in the GeoSentinel database from 1997 to 2015. We also surveyed GeoSentinel sites to compare various testing indications, approaches, and diagnostic modalities. We identified 260 GeoSentinel records, including 187 that satisfied criteria for analysis (confirmed cases in non-immigrant travellers aged >2 years, seen <12 weeks post-travel). CDI was reported in all age groups and in travellers to all world regions; the largest proportions of cases having destinations in Asia (31%), Central/South America or the Caribbean (30%) and Africa (24%). Our site survey revealed substantial heterogeneity of testing approaches between sites; the most commonly used test was the C. difficile toxin gene PCR. CDI is encountered in returning international travellers, although there is considerable variability in testing practices. These data underscore the importance of awareness of C. difficile as a potential cause of travel-associated diarrhoea. © International Society of Travel Medicine, 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Prevalence of Clostridium difficile colonization among healthcare workers

PubMed Central

2013-01-01

Background Clostridium difficile infection (CDI) has increased to epidemic proportions in recent years. The carriage of C. difficile among healthy adults and hospital inpatients has been established. We sought to determine whether C. difficile colonization exists among healthcare workers (HCWs) in our setting. Methods A point prevalence study of stool colonization with C. difficile among doctors, nurses and allied health staff at a large regional teaching hospital in Geelong, Victoria. All participants completed a short questionnaire and all stool specimens were tested by Techlab® C.diff Quik Check enzyme immunoassay followed by enrichment culture. Results Among 128 healthcare workers, 77% were female, of mean age 43 years, and the majority were nursing staff (73%). Nineteen HCWs (15%) reported diarrhoea, and 12 (9%) had taken antibiotics in the previous six weeks. Over 40% of participants reported having contact with a patient with known or suspected CDI in the 6 weeks before the stool was collected. C. difficile was not isolated from the stool of any participants. Conclusion Although HCWs are at risk of asymptomatic carriage and could act as a reservoir for transmission in the hospital environment, with the use of a screening test and culture we were unable to identify C. difficile in the stool of our participants in a non-outbreak setting. This may reflect potential colonization resistance of the gut microbiota, or the success of infection prevention strategies at our institution. PMID:24090343

Point-Counterpoint: What Is the Optimal Approach for Detection of Clostridium difficile Infection?

PubMed Central

Wilcox, Mark H.

2017-01-01

INTRODUCTION In 2010, we published an initial Point-Counterpoint on the laboratory diagnosis of Clostridium difficile infection (CDI). At that time, nucleic acid amplification tests (NAATs) were just becoming commercially available, and the idea of algorithmic approaches to CDI was being explored. Now, there are numerous NAATs in the marketplace, and based on recent proficiency test surveys, they have become the predominant method used for CDI diagnosis in the United States. At the same time, there is a body of literature that suggests that NAATs lack clinical specificity and thus inflate CDI rates. Hospital administrators are taking note of institutional CDI rates because they are publicly reported. They have become an important metric impacting hospital safety ratings and value-based purchasing; hospitals may have millions of dollars of reimbursement at risk. In this Point-Counterpoint using a frequently asked question approach, Ferric Fang of the University of Washington, who has been a consistent advocate for a NAAT-only approach for CDI diagnosis, will discuss the value of a NAAT-only approach, while Christopher Polage of the University of California Davis and Mark Wilcox of Leeds University, Leeds, United Kingdom, each of whom has recently written important articles on the value of toxin detection in the diagnosis, will discuss the impact of toxin detection in CDI diagnosis. PMID:28077697

Crystal structures of two nitroreductases from hypervirulent Clostridium difficile and functionally related interactions with the antibiotic metronidazole

PubMed Central

Wang, Bing; Powell, Samantha M.; Hessami, Neda; Najar, Fares Z.; Thomas, Leonard M.; Karr, Elizabeth A.; West, Ann H.; Richter-Addo, George B.

2016-01-01

Nitroreductases (NRs) are flavin mononucleotide (FMN)-dependent enzymes that catalyze the biotransformation of organic nitro compounds (RNO2; R = alkyl, aryl) to the nitroso RN=O, hydroxylamino RNHOH, or amine RNH2 derivatives. Metronidazole (Mtz) is a nitro-containing antibiotic that is commonly prescribed for lower-gut infections caused by the anaerobic bacterium Clostridium difficile. C. difficile infections rank number one among hospital acquired infections, and can result in diarrhea, severe colitis, or even death. Although NRs have been implicated in Mtz resistance of C. difficile, no NRs have been characterized from the hypervirulent R20291 strain of C. difficile. We report the first expression, purification, and three-dimensional X-ray crystal structures of two NRs from the C. difficile R20291 strain. The X-ray crystal structures of the two NRs were solved to 2.1 Å resolution. Their homodimeric structures exhibit the classic NR α+β fold, with each protomer binding one FMN cofactor near the dimer interface. Functional assays demonstrate that these two NRs metabolize Mtz with associated re-oxidation of the proteins. Importantly, these results represent the first isolation and characterization of NRs from the hypervirulent R20291 strain of relevance to organic RNO2 (e.g., Mtz) metabolism. PMID:27623089

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies*

PubMed Central

Herrera, Cristina; Tremblay, Jacqueline M.; Shoemaker, Charles B.; Mantis, Nicholas J.

2015-01-01

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. PMID:26396190

[Identifying gaps between guidelines and clinical practice in Clostridium difficile infection].

PubMed

Rodríguez-Martín, C; Serrano-Morte, A; Sánchez-Muñoz, L A; de Santos-Castro, P A; Bratos-Pérez, M A; Ortiz de Lejarazu-Leonardo, R

2016-01-01

The first aim was to determine whether patients are being treated in accordance with the Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America (IDSA/SHEA) Clostridium difficile guidelines and whether adherence impacts patient outcomes. The second aim was to identify specific action items in the guidelines that are not being translated into clinical practice, for their subsequent implementation. A retrospective, descriptive study was conducted over a 36 month period, on patients with compatible clinical symptoms and positive test for C. difficile toxins A and/or B in stool samples, in an internal medicine department of a tertiary medical centre. Patient demographic and clinical data (outcomes, comorbidity, risk factors) and compliance with guidelines, were examined A total of 77 patients with C. difficile infection were identified (87 episodes). Stratified by disease severity criteria, 49.3% of patients were mild-moderate, 35.1% severe, and 15.6% severe-complicated. Full adherence with the guidelines was observed in only 40.2% of patients, and was significantly better for mild-moderate (71.0%), than in severe (7.4%) or severe-complicated patients (16.6%) (P<.003). Adherence was significantly associated with clinical cure (57% vs 42%), fewer recurrences (22.2% vs 77.7%), and mortality (25% vs 75%) (P<.01). The stratification of severity of the episode, and the adequacy of antibiotic to clinical severity, need improvement. Overall adherence with the guidelines for management of Clostridium difficile infection was poor, especially in severe and severe-complicated patients, being associated with worse clinical outcomes. Educational interventions aimed at improving guideline adherence are warranted. Copyright © 2015 SECA. Published by Elsevier Espana. All rights reserved.

Consequences of Clostridium difficile infection: understanding the healthcare burden.

PubMed

Bouza, E

2012-12-01

Clostridium difficile is the leading cause of infectious nosocomial diarrhoea in developed countries, with a measured incidence of approximately five episodes per 10,000 days of hospital stay in Europe. Accurate diagnosis of C. difficile infection (CDI) is a prerequisite for obtaining reliable epidemiological data, but in many European countries diagnosis is probably suboptimal. A significant percentage of CDI cases are missed because clinicians often fail to request tests for C. difficile toxins in cases of unexplained diarrhoea. In addition, some laboratories continue to use tests of low sensitivity or apply them inappropriately. In one study in Spain, failure to request CDI testing in more than two-thirds of patients with unexplained diarrhoea led to significant underdiagnosis of cases. A recent pan-European survey revealed huge discrepancies in the rate of CDI testing across Europe, which suggests that epidemiological reports underestimate the true incidence of CDI in many parts of Europe. This is important because, as this review of the clinical and economic burden of CDI illustrates, infection with C. difficile imposes a significant burden not only on patients, owing to increased morbidity and mortality, but also on healthcare systems and society in general. On the basis of current incidence rates, annual costs for management of CDI amount to approximately $800 million in the USA and €3000 million in Europe. Moreover, estimates suggest that costs associated with recurrent CDI can exceed those of primary CDI. Measures to more effectively prevent CDI and reduce CDI recurrence rates may help to reduce this burden. © 2012 The Author Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

[Management of Clostridium difficile infections at German intensive care units - results from a survey among intensivists].

PubMed

Bruensing, Jan; Buendgens, Lukas; Jochum, Christoph; Herbers, Ulf; Canbay, Ali; Braun, Georg; Trautwein, Christian; Huber, Wolfgang; Koch, Alexander; Tacke, Frank

2018-06-01

 Clostridium difficile associated colitis is a frequent cause of nosocomial diarrhea at the intensive care unit (ICU) and is associated with poor prognosis in critically ill patients. Few studies have evaluated the efficacy of treatment options or adherence to guideline recommendations of Clostridium difficile infections at the ICU.  Therefore, on behalf of the Gastroenterology Intensive Care Medicine working group of the DGVS, we have conducted an online-based survey among leading intensivists in Germany.  Out of the 351 invited, 85 (24.2 %), primarily leading executive physicians at primary to tertiary care hospitals, completed the survey. They reported standardized diagnostic algorithms of 79.3 %, in line with current guideline recommendations (i. e., toxin testing in stool, possibly GDH screening, and endoscopy). First-line therapy of Clostridium difficile infections at the ICU was reported to be oral vancomycin in 48.3 % and oral metronidazole in 34.5 %. The success of first-line therapy was estimated at 67 % for clinical cure, 15 % persisting colitis, 5 % sepsis or megacolon, 10 % recurrence, and 3 % death. Hospitals of primary/secondary care more often used metronidazole compared to university hospitals. Standard treatments for recurrent infection were vancomycin orally (40 % alone, 29.1 % combined with metronidazole) or, more rarely, fidaxomicin (25.5 %). Fidaxomicin has been used at least once at the ICU in 79 % of the respondents. Eleven percent have used fecal microbiota transplant (FMT) in selected cases at the ICU.  Our survey indicated a high awareness of German intensivists for Clostridium difficile infections, but also marked differences in local therapeutic algorithms, especially in first-line treatment. © Georg Thieme Verlag KG Stuttgart · New York.

In silico analysis of antibiotic-induced Clostridium difficile infection: Remediation techniques and biological adaptations

PubMed Central

Carlson, Jean M.

2018-01-01

In this paper we study antibiotic-induced C. difficile infection (CDI), caused by the toxin-producing C. difficile (CD), and implement clinically-inspired simulated treatments in a computational framework that synthesizes a generalized Lotka-Volterra (gLV) model with SIR modeling techniques. The gLV model uses parameters derived from an experimental mouse model, in which the mice are administered antibiotics and subsequently dosed with CD. We numerically identify which of the experimentally measured initial conditions are vulnerable to CD colonization, then formalize the notion of CD susceptibility analytically. We simulate fecal transplantation, a clinically successful treatment for CDI, and discover that both the transplant timing and transplant donor are relevant to the the efficacy of the treatment, a result which has clinical implications. We incorporate two nongeneric yet dangerous attributes of CD into the gLV model, sporulation and antibiotic-resistant mutation, and for each identify relevant SIR techniques that describe the desired attribute. Finally, we rely on the results of our framework to analyze an experimental study of fecal transplants in mice, and are able to explain observed experimental results, validate our simulated results, and suggest model-motivated experiments. PMID:29451873

In silico analysis of antibiotic-induced Clostridium difficile infection: Remediation techniques and biological adaptations.

PubMed

Jones, Eric W; Carlson, Jean M

2018-02-01

In this paper we study antibiotic-induced C. difficile infection (CDI), caused by the toxin-producing C. difficile (CD), and implement clinically-inspired simulated treatments in a computational framework that synthesizes a generalized Lotka-Volterra (gLV) model with SIR modeling techniques. The gLV model uses parameters derived from an experimental mouse model, in which the mice are administered antibiotics and subsequently dosed with CD. We numerically identify which of the experimentally measured initial conditions are vulnerable to CD colonization, then formalize the notion of CD susceptibility analytically. We simulate fecal transplantation, a clinically successful treatment for CDI, and discover that both the transplant timing and transplant donor are relevant to the the efficacy of the treatment, a result which has clinical implications. We incorporate two nongeneric yet dangerous attributes of CD into the gLV model, sporulation and antibiotic-resistant mutation, and for each identify relevant SIR techniques that describe the desired attribute. Finally, we rely on the results of our framework to analyze an experimental study of fecal transplants in mice, and are able to explain observed experimental results, validate our simulated results, and suggest model-motivated experiments.

Slow intestinal transit contributes to elevate urinary p-cresol level in Italian autistic children.

PubMed

Gabriele, Stefano; Sacco, Roberto; Altieri, Laura; Neri, Cristina; Urbani, Andrea; Bravaccio, Carmela; Riccio, Maria Pia; Iovene, Maria Rosaria; Bombace, Francesca; De Magistris, Laura; Persico, Antonio M

2016-07-01

The uremic toxin p-cresol (4-methylphenol) is either of environmental origin or can be synthetized from tyrosine by cresol-producing bacteria present in the gut lumen. Elevated p-cresol amounts have been previously found in the urines of Italian and French autism spectrum disorder (ASD) children up until 8 years of age, and may be associated with autism severity or with the intensity of abnormal behaviors. This study aims to investigate the mechanism producing elevated urinary p-cresol in ASD. Urinary p-cresol levels were thus measured by High Performance Liquid Chromatography in a sample of 53 Italian ASD children assessed for (a) presence of Clostridium spp. strains in the gut by means of an in vitro fecal stool test and of Clostridium difficile-derived toxin A/B in the feces, (b) intestinal permeability using the lactulose/mannitol (LA/MA) test, (c) frequent use of antibiotics due to recurrent infections during the first 2 years of postnatal life, and (d) stool habits with the Bristol Stool Form Scale. Chronic constipation was the only variable significantly associated with total urinary p-cresol concentration (P < 0.05). No association was found with presence of Clostridium spp. in the gut flora (P = 0.92), augmented intestinal permeability (P = 0.18), or frequent use of antibiotics in early infancy (P = 0.47). No ASD child was found to carry C. difficile in the gut or to release toxin A/B in the feces. In conclusion, urinary p-cresol levels are elevated in young ASD children with increased intestinal transit time and chronic constipation. Autism Res 2016, 9: 752-759. © 2015 International Society for Autism Research, Wiley Periodicals, Inc. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

The host immune response to Clostridium difficile infection

PubMed Central

2013-01-01

Clostridium difficile infection (CDI) is the most common infectious cause of healthcare-acquired diarrhoea. Outcomes of C. difficile colonization are varied, from asymptomatic carriage to fulminant colitis and death, due in part to the interplay between the pathogenic virulence factors of the bacterium and the counteractive immune responses of the host. Secreted toxins A and B are the major virulence factors of C. difficile and induce a profound inflammatory response by intoxicating intestinal epithelial cells causing proinflammatory cytokine release. Host cell necrosis, vascular permeability and neutrophil infiltration lead to an elevated white cell count, profuse diarrhoea and in severe cases, dehydration, hypoalbuminaemia and toxic megacolon. Other bacterial virulence factors, including surface layer proteins and flagella proteins, are detected by host cell surface signal molecules that trigger downstream cell-mediated immune pathways. Human studies have identified a role for serum and faecal immunoglobulin levels in protection from disease, but the recent development of a mouse model of CDI has enabled studies into the precise molecular interactions that trigger the immune response during infection. Key effector molecules have been identified that can drive towards a protective anti-inflammatory response or a damaging proinflammatory response. The limitations of current antimicrobial therapies for CDI have led to the development of both active and passive immunotherapies, none of which have, as yet been formally approved for CDI. However, recent advances in our understanding of the molecular basis of host immune protection against CDI may provide an exciting opportunity for novel therapeutic developments in the future. PMID:25165542

Description and validation of a new automated surveillance system for Clostridium difficile in Denmark.

PubMed

Chaine, M; Gubbels, S; Voldstedlund, M; Kristensen, B; Nielsen, J; Andersen, L P; Ellermann-Eriksen, S; Engberg, J; Holm, A; Olesen, B; Schønheyder, H C; Østergaard, C; Ethelberg, S; Mølbak, K

2017-09-01

The surveillance of Clostridium difficile (CD) in Denmark consists of laboratory based data from Departments of Clinical Microbiology (DCMs) sent to the National Registry of Enteric Pathogens (NREP). We validated a new surveillance system for CD based on the Danish Microbiology Database (MiBa). MiBa automatically collects microbiological test results from all Danish DCMs. We built an algorithm to identify positive test results for CD recorded in MiBa. A CD case was defined as a person with a positive culture for CD or PCR detection of toxin A and/or B and/or binary toxin. We compared CD cases identified through the MiBa-based surveillance with those reported to NREP and locally in five DCMs representing different Danish regions. During 2010-2014, NREP reported 13 896 CD cases, and the MiBa-based surveillance 21 252 CD cases. There was a 99·9% concordance between the local datasets and the MiBa-based surveillance. Surveillance based on MiBa was superior to the current surveillance system, and the findings show that the number of CD cases in Denmark hitherto has been under-reported. There were only minor differences between local data and the MiBa-based surveillance, showing the completeness and validity of CD data in MiBa. This nationwide electronic system can greatly strengthen surveillance and research in various applications.

Hospital-based Clostridium difficile infection surveillance reveals high proportions of PCR ribotypes 027 and 176 in different areas of Poland, 2011 to 2013.

PubMed

Pituch, Hanna; Obuch-Woszczatyński, Piotr; Lachowicz, Dominika; Wultańska, Dorota; Karpiński, Paweł; Młynarczyk, Grażyna; van Dorp, Sofie M; Kuijper, Ed J

2015-01-01

As part of the European Clostridium difficile infections (CDI) surveillance Network (ECDIS-Net), which aims to build capacity for CDI surveillance in Europe, we constructed a new network of hospital-based laboratories in Poland. We performed a survey in 13 randomly selected hospital-laboratories in different sites of the country to determine their annual CDI incidence rates from 2011 to 2013. Information on C. difficile laboratory diagnostic testing and indications for testing was also collected. Moreover, for 2012 and 2013 respectively, participating hospital-laboratories sent all consecutive isolates from CDI patients between February and March to the Anaerobe Laboratory in Warsaw for further molecular characterisation, including the detection of toxin-encoding genes and polymerase chain reaction (PCR)-ribotyping. Within the network, the mean annual hospital CDI incidence rates were 6.1, 8.6 and 9.6 CDI per 10,000 patient-days in 2011, 2012, and 2013 respectively. Six of the 13 laboratories tested specimens only on the request of a physician, five tested samples of antibiotic-associated diarrhoea or samples from patients who developed diarrhoea more than two days after admission (nosocomial diarrhoea), while two tested all submitted diarrhoeal faecal samples. Most laboratories (9/13) used tests to detect glutamate dehydrogenase and toxin A/B either separately or in combination. In the two periods of molecular surveillance, a total of 166 strains were characterised. Of these, 159 were toxigenic and the majority belonged to two PCR-ribotypes: 027 (n=99; 62%) and the closely related ribotype 176 (n=22; 14%). The annual frequency of PCR-ribotype 027 was not significantly different during the surveillance periods (62.9% in 2012; 61.8% in 2013). Our results indicate that CDIs caused by PCR-ribotype 027 predominate in Polish hospitals participating in the surveillance, with the closely related 176 ribotype being the second most common agent of infection.

Spore formation and toxin production in Clostridium difficile biofilms.

PubMed

Semenyuk, Ekaterina G; Laning, Michelle L; Foley, Jennifer; Johnston, Pehga F; Knight, Katherine L; Gerding, Dale N; Driks, Adam

2014-01-01

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.

Spore Formation and Toxin Production in Clostridium difficile Biofilms

PubMed Central

Semenyuk, Ekaterina G.; Laning, Michelle L.; Foley, Jennifer; Johnston, Pehga F.; Knight, Katherine L.; Gerding, Dale N.; Driks, Adam

2014-01-01

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection. PMID:24498186

Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

PubMed Central

Donnelly, M. Lauren; Fimlaid, Kelly A.

2016-01-01

ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms. PMID:27044622

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

PubMed

Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

2015-11-13

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach.

PubMed

Neumann-Schaal, Meina; Metzendorf, Nicole G; Troitzsch, Daniel; Nuss, Aaron Mischa; Hofmann, Julia Danielle; Beckstette, Michael; Dersch, Petra; Otto, Andreas; Sievers, Susanne

2018-05-31

Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O 2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype. Copyright © 2018. Published by Elsevier Ltd.

Clostridium difficile infection in the elderly: an update on management.

PubMed

Asempa, Tomefa E; Nicolau, David P

2017-01-01

The burden of Clostridium difficile infection (CDI) is profound and growing. CDI now represents a common cause of health care-associated diarrhea, and is associated with significant morbidity, mortality, and health care costs. CDI disproportionally affects the elderly, possibly explained by the following risk factors: age-related impairment of the immune system, increasing antibiotic utilization, and frequent health care exposure. In the USA, recent epidemiological studies estimate that two out of every three health care-associated CDIs occur in patients 65 years or older. Additionally, the elderly are at higher risk for recurrent CDI. Existing therapeutic options include metronidazole, oral vancomycin, and fidaxomicin. Choice of agent depends on disease severity, history of recurrence, and, increasingly, the drug cost. Bezlotoxumab, a recently approved monoclonal antibody targeting C. difficile toxin B, offers an exciting advancement into immunologic therapies. Similarly, fecal microbiota transplantation is gaining popularity as an effective option mainly for recurrent CDI. The challenge of decreasing CDI burden in the elderly involves adopting preventative strategies, optimizing initial treatment, and decreasing the risk of recurrence. Expanded strategies are certainly needed to improve outcomes in this high-risk population. This review considers available data from prospective and retrospective studies as well as case reports to illustrate the merits and gaps in care related to the management of CDI in the elderly.

First clinical and microbiological characterization of Clostridium difficile infection in a Croatian University Hospital.

PubMed

Novak, Anita; Spigaglia, Patrizia; Barbanti, Fabrizio; Goic-Barisic, Ivana; Tonkic, Marija

2014-12-01

Clinical background and molecular epidemiology of Clostridium difficile infection (CDI) in the University Hospital Centre Split were investigated from January 2010 to December 2011. In total, 54 patients with first episode of CDI were consecutively included in the study based on the positive EIA test specific for A and B toxins. Demographic and clinical data were prospectively analyzed from medical records. CDI incidence rate was 0.6 per 10,000 patient-days. Thirty six cases (70.6%) were healthcare-associated, twelve cases (23.5%) were community-associated and three (5.9%) were indeterminate. Six patients (11.7%) had suffered one or more recurrences and 37 patients (72.5%) showed severe CDI. Prior therapy with third generation cephalosporin was significantly associated with severe CDI (P<0.021). Fifty four toxigenic C. difficile strains were isolated and 50 of them were available for PCR-ribotyping. Sixteen different PCR-ribotypes were identified. The most prevalent were PCR-ribotype 001 (27.8%) and 014/020 (24.1%). Twenty three strains were resistant to at least one of the antibiotics tested. Among resistant strains, three (13.0%)--all PCR-ribotype 001--were multi-resistant. Resistance to fluoroquinolones was significantly higher in strains that caused infection after previous use of fluoroquinolones (P=0.04). Copyright © 2014 Elsevier Ltd. All rights reserved.

Clostridium difficile infection in the elderly: an update on management

PubMed Central

Asempa, Tomefa E; Nicolau, David P

2017-01-01

The burden of Clostridium difficile infection (CDI) is profound and growing. CDI now represents a common cause of health care–associated diarrhea, and is associated with significant morbidity, mortality, and health care costs. CDI disproportionally affects the elderly, possibly explained by the following risk factors: age-related impairment of the immune system, increasing antibiotic utilization, and frequent health care exposure. In the USA, recent epidemiological studies estimate that two out of every three health care–associated CDIs occur in patients 65 years or older. Additionally, the elderly are at higher risk for recurrent CDI. Existing therapeutic options include metronidazole, oral vancomycin, and fidaxomicin. Choice of agent depends on disease severity, history of recurrence, and, increasingly, the drug cost. Bezlotoxumab, a recently approved monoclonal antibody targeting C. difficile toxin B, offers an exciting advancement into immunologic therapies. Similarly, fecal microbiota transplantation is gaining popularity as an effective option mainly for recurrent CDI. The challenge of decreasing CDI burden in the elderly involves adopting preventative strategies, optimizing initial treatment, and decreasing the risk of recurrence. Expanded strategies are certainly needed to improve outcomes in this high-risk population. This review considers available data from prospective and retrospective studies as well as case reports to illustrate the merits and gaps in care related to the management of CDI in the elderly. PMID:29123385

A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels of In Vitro Potency Shows In Vivo Protection in a Hamster Infection Model

PubMed Central

Davies, Nicola L.; Compson, Joanne E.; MacKenzie, Brendon; O'Dowd, Victoria L.; Oxbrow, Amanda K. F.; Heads, James T.; Turner, Alison; Sarkar, Kaushik; Dugdale, Sarah L.; Jairaj, Mark; Christodoulou, Louis; Knight, David E. O.; Cross, Amanda S.; Hervé, Karine J. M.; Tyson, Kerry L.; Hailu, Hanna; Doyle, Carl B.; Ellis, Mark; Kriek, Marco; Cox, Matthew; Page, Matthew J. T.; Moore, Adrian R.; Lightwood, Daniel J.

2013-01-01

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process. PMID:23324518

Molecular Epidemiology of Clostridium difficile Infection in Hospitalized Patients in Eastern China.

PubMed

Jin, Dazhi; Luo, Yun; Huang, Chen; Cai, Jian; Ye, Julian; Zheng, Yi; Wang, Liqian; Zhao, Peng; Liu, Anbing; Fang, Weijia; Wang, Xianjun; Xia, Shichang; Jiang, Jianmin; Tang, Yi-Wei

2017-03-01

Few studies on risk factors for and transmission of Clostridium difficile infection (CDI) in China have been reported. A cross-sectional study was conducted for 3 years in eastern China. Consecutive stool specimens from hospitalized patients with diarrhea were cultured for C. difficile. C. difficile isolates from these patients then were analyzed for toxin genes, genotypes, and antimicrobial resistance. A severity score for the CDI in each patient was determined by a blinded review of the medical record, and these scores ranged from 1 to 6. A total of 397 out of 3,953 patients (10.0%) with diarrhea were found to have CDI. Severity of CDI was mild to moderate, and the average (± standard deviation) severity score was 2.61 ± 1.01. C. difficile was isolated from stool specimens in 432 (10.9%) of all the patients who had diarrhea. C. difficile genotypes were determined by multilocus sequence analysis and PCR ribotyping; sequence type 37 (ST37)/ribotype 017 (RT017) ( n = 68, 16.5%) was the dominant genotype. Eleven patients (16.2%) with this genotype had a CDI severity score of 5. Overall, three RTs and four STs were predominant; these genotypes were associated with significantly different antimicrobial resistance patterns in comparison to all genotypes (χ 2 = 79.56 to 97.76; P < 0.001). Independent risk factors associated with CDI included age greater than 55 years (odds ratio [95% confidence interval], 26.80 [18.76 to 38.29]), previous hospitalization (12.42 [8.85 to 17.43]), previous antimicrobial treatment within 8 weeks (150.56 [73.11 to 310.06]), hospital stay more than 3 days before sampling (2.34 [1.71 to 3.22]), undergoing chemotherapy (3.31 [2.22 to 4.92]), and undergoing abdominal surgery (4.82 [3.54 to 6.55]). CDI is clearly a problem in eastern China and has a prevalence of 10.0% in hospitalized patients. Among risk factors for CDI, the advanced age threshold was younger for Chinese patients than that reported for patients in developed countries. Copyright © 2017 American Society for Microbiology.

The involvement of macrophage-derived tumour necrosis factor and lipoxygenase products on the neutrophil recruitment induced by Clostridium difficile toxin B.

PubMed Central

Souza, M H; Melo-Filho, A A; Rocha, M F; Lyerly, D M; Cunha, F Q; Lima, A A; Ribeiro, R A

1997-01-01

Clostridium difficile (Cd) toxins appear to mediate the inflammatory response in pseudomembranous colitis and/or colitis associated with the use of antibiotics. In contrast to Cd Toxin A (TxA), Cd Toxin B (TxB) has been reported not to promote fluid secretion or morphological damage in rabbits and hamsters and also does not induce neutrophil chemotaxis in vitro. However, TxB is about 1000 times more potent than TxA in stimulating the release of tumour necrosis factor-alpha (TNF-alpha) by cultured monocytes. In the present study, we investigated the ability of TxB to promote neutrophil migration into peritoneal cavities and subcutaneous air-pouches of rats. We also examined the role of resident peritoneal cells in this process as well as the inflammatory mediators involved. TxB caused a significant and dose-dependent neutrophil influx with a maximal response at 0.1 microgram/cavity after 4 hr. Depleting the peritoneal resident cell population by washing the peritoneal cavity or increasing this population by pretreating the animals with thioglycollate blocked and amplified the TxB-induced neutrophil migration, respectively. Pretreating the animals with MK886 (a lipoxygenase inhibitor), NDGA (a dual cyclo- and lipoxygenase inhibitor) or the glucocorticoid, dexamethasone, but not with indomethacin (a cyclo-oxygenase inhibitor), or BN52021 (a platelet-activating factor antagonist), inhibited the neutrophil migration evoked by TxB. Pretreatment with dexamethasone or the administration of anti-TNF-alpha serum into the air-pouches also significantly reduced the TxB-induced neutrophil migration. Supernatants from TxB-stimulated macrophages induced neutrophil migration when injected into the rat peritoneal cavity. This effect was attenuated by the addition of either MK886 or dexamethasone to the macrophage monolayer and by preincubating the supernatants with anti-TNF-alpha serum. TxB also stimulated the release of TNF-alpha by macrophages. Overall, these results suggest that TxB induces an intense neutrophil migration which is mediated by macrophage-derived TNF-alpha and lipoxygenase products. PMID:9227329

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

PubMed Central

Simpson, L L; Stiles, B G; Zepeda, H; Wilkins, T D

1989-01-01

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins. Images PMID:2521214

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

PubMed

Fawley, Warren N; Davies, Kerrie A; Morris, Trefor; Parnell, Peter; Howe, Robin; Wilcox, Mark H

2016-07-21

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant. This article is copyright of The Authors, 2016.

Clostridium difficile Diarrhea in the Elderly: Current Issues and Management Options.

PubMed

Mizusawa, Masako; Doron, Shira; Gorbach, Sherwood

2015-08-01

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in healthcare settings. Along with antimicrobial exposure, advanced age has been shown to be a significant risk factor for the development and recurrence of, and mortality from, CDI. The substantial burden of CDI in the elderly may be related to frequent healthcare exposure, the necessity for more medications, altered intestinal microbiota, and complicated comorbidities. A diagnosis of CDI is based on evidence of toxin, or the C. difficile organism itself, in a stool sample in the presence of clinical signs and symptoms. Only symptomatic patients should be tested for CDI, and routine surveillance or repeat testing on asymptomatic patients as a test of cure is discouraged. Antibiotic discontinuation alone can improve or resolve CDI in some patients, and concomitant use of antibiotics is associated with decreased response to CDI treatment. Metronidazole, vancomycin, and fidaxomicin are the therapeutic agents currently available for CDI, with the selection of these agents being based on disease severity, history of recurrence, and cost. The recurrence rate after initial treatment is 20-30%. The first recurrence can be treated with the same therapeutic agent and, for subsequent recurrences, vancomycin in a tapered and/or pulsed regimen is recommended. Fecal microbiota transplantation has shown remarkable effectiveness for recurrent anti-refractory CDI, although caution is advised in treating immunocompromised hosts and those with toxic megacolon. C. difficile can be transmitted directly and indirectly via contact with patients or their environment; therefore, isolation precautions should be initiated at the first suspicion of CDI. C. difficile spores can survive for a long time on environmental surfaces, and the patient's room and all equipment used in the room should be disinfected. In order to manage CDI in the elderly, timely diagnosis, appropriate treatment based on severity of illness, and effective infection control are essential.

Possible Application of Biotechnology to the Development of Biological Agents by Potential Enemies

DTIC Science & Technology

1987-06-01

of enzyme catalyzed reactions. Although cloning techniques are directly applicable to the manipulation of proteinaceous toxins, they would be less...useful for nonproteinaceous toxins because the corresponding gene for each enzyme must be cloned and expressed in a coordinated manner. Effective...to produce a synthetic DNA. The enzyme reverse transcriptase (RNA dependent DNA polymerase), which is obtained from retroviruses, is the only enzyme

Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax

NASA Astrophysics Data System (ADS)

Sellman, Bret R.; Mourez, Michael; John Collier, R.

2001-04-01

The protective antigen moiety of anthrax toxin translocates the toxin's enzymic moieties to the cytosol of mammalian cells by a mechanism that depends on its ability to heptamerize and insert into membranes. We identified dominant-negative mutants of protective antigen that co-assemble with the wild-type protein and block its ability to translocate the enzymic moieties across membranes. These mutants strongly inhibited toxin action in cell culture and in an animal intoxication model, suggesting that they could be useful in therapy of anthrax.

Clinical update for the diagnosis and treatment of Clostridium difficile infection

PubMed Central

IV, Edward C Oldfield; III, Edward C Oldfield; Johnson, David A

2014-01-01

Clostridium difficile infection (CDI) presents a rapidly evolving challenge in the battle against hospital-acquired infections. Recent advances in CDI diagnosis and management include rapid changes in diagnostic approach with the introduction of newer tests, such as detection of glutamate dehydrogenase in stool and polymerase chain reaction to detect the gene for toxin production, which will soon revolutionize the diagnostic approach to CDI. New medications and multiple medical society guidelines have introduced changing concepts in the definitions of severity of CDI and the choice of therapeutic agents, while rapid expansion of data on the efficacy of fecal microbiota transplantation heralds a revolutionary change in the management of patients suffering multiple relapses of CDI. Through a comprehensive review of current medical literature, this article aims to offer an intensive review of the current state of CDI diagnosis, discuss the strengths and limitations of available laboratory tests, compare both current and future treatments options and offer recommendations for best practice strategies. PMID:24729930

Structural Basis of Clostridium perfringens Toxin Complex Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams,J.; Gregg, K.; Bayer, E.

2008-01-01

The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the {mu}-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (Ka = 1.44 x 1011 M-1) between themore » X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The {mu}-toxin dockerin module in this complex is positioned {approx}180 relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.« less

New Class of Precision Antimicrobials Redefines Role of Clostridium difficile S-layer in Virulence and Viability

PubMed Central

Kirk, Joseph A.; Gebhart, Dana; Buckley, Anthony M.; Lok, Stephen; Scholl, Dean; Douce, Gillian R.; Govoni, Gregory R.; Fagan, Robert P.

2017-01-01

Avidocin-CDs are a new class of precision bactericidal agents that do not damage resident gut microbiota and are unlikely to promote the spread of antibiotic resistance. The precision killing properties result from the fusion of bacteriophage receptor binding proteins (RBPs) to a lethal contractile scaffold from an R-type bacteriocin. We recently described the prototypic Avidocin-CD, Av-CD291.2, that specifically kills C. difficile ribotype 027 strains and prevents colonization of mice. We have since selected two rare Av-CD291.2 resistant mutants of strain R20291 (RT027; S-layer cassette type-4, SCLT-4). These mutants have distinct point mutations in the slpA gene that result in an S-layer null phenotype. Reversion of the mutations to wild-type restored normal SLCT-4 S-layer formation and Av-CD291.2 sensitivity; however, complementation with other SCLT alleles did not restore Av-CD291.2 sensitivity despite restoring S-layer formation. Using newly identified phage RBPs, we constructed a panel of new Avidocin-CDs that kill C. difficile isolates in an SLCT-dependent manner, confirming the S-layer as the receptor in every case. In addition to bacteriophage adsorption, characterization of the S-layer null mutant also uncovered important roles for SlpA in sporulation, resistance to lysozyme and LL-37, and toxin production. Surprisingly, the S-layer-null mutant was found to persist in the hamster gut despite its completely attenuated virulence. Avidocin-CDs have significant therapeutic potential for the treatment and prevention of C. difficile Infection (CDI) given their exquisite specificity for the pathogen. Furthermore, the emergence of resistance forces mutants to trade virulence for continued viability and, therefore, greatly reduce their potential clinical impact. PMID:28878013

Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile▿†

PubMed Central

Forgetta, Vincenzo; Oughton, Matthew T.; Marquis, Pascale; Brukner, Ivan; Blanchette, Ruth; Haub, Kevin; Magrini, Vince; Mardis, Elaine R.; Gerding, Dale N.; Loo, Vivian G.; Miller, Mark A.; Mulvey, Michael R.; Rupnik, Maja; Dascal, Andre; Dewar, Ken

2011-01-01

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains. PMID:21508155

Bezlotoxumab: A Review in Preventing Clostridium difficile Infection Recurrence.

PubMed

Deeks, Emma D

2017-10-01

Bezlotoxumab (Zinplava™) is a fully human monoclonal antibody against Clostridium difficile toxin B indicated for the prevention of C. difficile infection (CDI) recurrence in patients with a high recurrence risk. It is the first agent approved for recurrence prevention and is administered as a single intravenous infusion in conjunction with standard-of-care (SoC) antibacterial treatment for CDI. In well-designed, placebo-controlled, phase 3 trials (MODIFY 1 and 2), a single infusion of bezlotoxumab, given in combination with SoC antibacterial therapy for CDI in adults, was effective in reducing CDI recurrence in the 12 weeks post-treatment, with this benefit being seen mainly in the patients at high recurrence risk. Bezlotoxumab did not impact the efficacy of the antibacterials being used to treat the CDI and, consistent with its benefits on CDI recurrence, appeared to reduce the need for subsequent antibacterials, thus minimizing further gut microbiota disruption. Longer term, there were no further CDI recurrences over 12 months' follow-up among patients who had received bezlotoxumab in MODIFY 2 and entered an extension substudy. Bezlotoxumab has low immunogenicity and is generally well tolerated, although the potential for heart failure in some patients requires consideration; cost-effectiveness data for bezlotoxumab are awaited with interest. Thus, a single intravenous infusion of bezlotoxumab during SoC antibacterial treatment for CDI is an emerging option for reducing CDI recurrence in adults at high risk of recurrence.

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE PAGES

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

2015-03-09

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

PubMed Central

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

2015-01-01

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Intake of Lactobacillus plantarum reduces certain gastrointestinal symptoms during treatment with antibiotics.

PubMed

Lönnermark, Elisabet; Friman, Vanda; Lappas, Georg; Sandberg, Torsten; Berggren, Anna; Adlerberth, Ingegerd

2010-02-01

To examine if intake of Lactobacillus plantarum can prevent gastrointestinal side effects in antibiotic-treated patients. Diarrhea is a common side effect of treatment with antibiotics. Some studies indicate that the risk of antibiotic-associated diarrhea can be reduced by administration of certain probiotic microorganisms. Patients treated for infections at a university hospital infectious diseases clinic were randomized to daily intake of either a fruit drink with L. plantarum 299v (10(10) colony forming units/d) or a placebo drink, until a week after termination of antibiotic treatment. Subjects recorded the number and consistency of stools as well as gastrointestinal symptoms until up to 3 weeks after last intake of test drink. Fecal samples were collected before the first intake of test drink and after termination of antibiotic therapy and analyzed for Clostridium difficile toxin. Clinical characteristics on admission were similar in the 2 groups. The overall risk of developing loose or watery stools was significantly lower among those receiving L. plantarum [odds ratio (OR), 0.69; 95% confidence interval (CI), 0.52-0.92; P=0.012], as was development of nausea (OR, 0.51; 95% CI, 0.30-0.85; P=0.0097). Diarrhea defined as > or =3 loose stools/24 h for > or =2 consecutive days was unaffected by the treatment (OR, 1.4; 95% CI, 0.33-6.0; P=0.86). No significant differences regarding carriage of toxin producing C. difficile were observed between the groups. Our results indicate that intake of L. plantarum could have a preventive effect on milder gastrointestinal symptoms during treatment with antibiotics.

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1).

PubMed Central

Simon-Plas, F.; Gomes, E.; Milat, M. L.; Pugin, A.; Blein, J. P.

1996-01-01

Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme. PMID:12226329

Toxin-Induced Autoimmune Hepatitis Caused by Raw Cashew Nuts.

PubMed

Crismale, James F; Stueck, Ashley; Bansal, Meena

2016-08-01

A 64-year-old man with no past medical history presented with abnormally elevated liver enzymes 1 year after developing a diffuse rash thought to be related to eating large quantities of raw cashew nuts. Liver biopsy was performed, which revealed features concerning for drug- or toxin-induced autoimmune hepatitis. The patient began treatment with azathioprine and prednisone, and liver enzymes normalized. We describe a unique case of a toxin-induced autoimmune hepatitis precipitated not by a drug or dietary supplement but by a food product.

Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Honke, K.; Taniguchi, N.; Makita, A.

1986-01-01

A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reactionmore » product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.« less

Localization of the action of cholera toxin on adenyl cyclase in mucosal epithelial cells of rabbit intestine

PubMed Central

Parkinson, David K.; Ebel, Hans; DiBona, Donald R.; Sharp, Geoffrey W. G.

1972-01-01

Brush borders and plasma membranes have been purified from mucosal epithelial cells of rabbit ileum under control conditions and after treatment for 3 hr with cholera toxin in vivo. The activity of several enzymes in these preparations was measured. It was concluded that adenyl cyclase, like NaK-ATPase, seems not to be a normal constituent of brush borders. Both these enzymes are present in plasma membrane preparations derived largely from the basal and lateral margins of the epithelial cells, both may be phospholipid dependent enzymes and both are affected by cholera toxin. Adenyl cyclase activity is increased while NaK-ATPase is decreased. The activities of alkaline phosphatase, leucineaminopeptidase, 5′-nucleotidase, glucose-6-phosphatase, and Mg-ATPase were not found to be affected by the toxin. Cholera toxin, which makes contact with the luminal side of the epithelial cells, in the natural disease and in the experimental model, would appear to exert its pathologic effect on adenyl cyclase at the opposite (basal and lateral) side of the cells. Images PMID:4344729

Emerging therapies for Clostridium difficile infections.

PubMed

McFarland, Lynne V

2011-09-01

Clostridium difficile infection (CDI) is the leading identifiable gastrointestinal disease in healthcare institutions, but the response rates to the two standard therapies for CDI are declining and so innovative therapies are being developed for CDI. The purpose of this paper is to review the data on the efficacy and safety of emerging therapies for CDI and assess their potential for effectiveness based on the clinical phase of development and marketing challenges. Emerging therapies for CDI are reviewed including new antibiotics, peptides, immune regulators, probiotics and toxin binders. PubMed, Medline and Google Scholar and online clinical trial registers are searched from 1976 to 2010 for articles unrestricted by language. Secondary searches by author, manufacturing companies and FDA websites are also performed. Of the emerging therapies for CDI, several may ultimately reduce the incidence of CDI and the economic burden of this disease on the healthcare system. Several emerging treatments (fidaxomicin, rifaximin and mAbs) show the most promise, although only one is currently being actively developed. Use of other clostridial strains, probiotic strains and immune enhancers have great potential as therapies, but require further development.

Assessment of treatment patterns and patient outcomes before vs after implementation of a severity-based Clostridium difficile infection treatment policy.

PubMed

Jardin, C G M; Palmer, H R; Shah, D N; Le, F; Beyda, N D; Jiang, Z; Garey, K W

2013-09-01

National guidelines recommend oral vancomycin for severe Clostridium difficile infection (CDI) based on results from recent clinical trials demonstrating improved clinical outcomes. However, real-world data to support these clinical trials are scant. To compare treatment patterns and patient outcomes of those treated for CDI before and after implementation of a severity-based CDI treatment policy at a tertiary teaching hospital. This study evaluated adult patients with a positive C. difficile toxin before and after implementation of a policy where patients with severe CDI given metronidazole were switched to oral vancomycin unless contra-indicated. Patients were stratified according to disease severity using a modified published severity score. Treatment patterns based on CDI severity and rates of refractory CDI were assessed. In total, 256 patients with CDI (mean age 66 years, standard deviation 17, 52% female) were evaluated (before implementation: N = 144; after implementation: N = 112). Use of oral vancomycin for severe CDI increased significantly from 14% (N = 8) to 91% (N = 48) following implementation of the policy (P < 0.0001). Refractory disease in patients with severe CDI decreased significantly from 37% to 15% following implementation of the policy (P = 0.035). No significant differences were noted among patients with mild to moderate CDI. A severity-based CDI treatment policy at a tertiary teaching hospital increased the use of oral vancomycin and was associated with decreased rates of refractory CDI. Copyright © 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Development of SYN-004, an oral beta-lactamase treatment to protect the gut microbiome from antibiotic-mediated damage and prevent Clostridium difficile infection.

PubMed

Kaleko, Michael; Bristol, J Andrew; Hubert, Steven; Parsley, Todd; Widmer, Giovanni; Tzipori, Saul; Subramanian, Poorani; Hasan, Nur; Koski, Perrti; Kokai-Kun, John; Sliman, Joseph; Jones, Annie; Connelly, Sheila

2016-10-01

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

C3larvin toxin, an ADP-ribosyltransferase from Paenibacillus larvae.

PubMed

Krska, Daniel; Ravulapalli, Ravikiran; Fieldhouse, Robert J; Lugo, Miguel R; Merrill, A Rod

2015-01-16

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD(+) (Km = 34 ± 12 μm) and RhoA (Km = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (Ki = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting Staphylococcus aureus Toxins: A Potential form of Anti-Virulence Therapy

PubMed Central

Kong, Cin; Neoh, Hui-min; Nathan, Sheila

2016-01-01

Staphylococcus aureus is an opportunistic pathogen and the leading cause of a wide range of severe clinical infections. The range of diseases reflects the diversity of virulence factors produced by this pathogen. To establish an infection in the host, S. aureus expresses an inclusive set of virulence factors such as toxins, enzymes, adhesins, and other surface proteins that allow the pathogen to survive under extreme conditions and are essential for the bacteria’s ability to spread through tissues. Expression and secretion of this array of toxins and enzymes are tightly controlled by a number of regulatory systems. S. aureus is also notorious for its ability to resist the arsenal of currently available antibiotics and dissemination of various multidrug-resistant S. aureus clones limits therapeutic options for a S. aureus infection. Recently, the development of anti-virulence therapeutics that neutralize S. aureus toxins or block the pathways that regulate toxin production has shown potential in thwarting the bacteria’s acquisition of antibiotic resistance. In this review, we provide insights into the regulation of S. aureus toxin production and potential anti-virulence strategies that target S. aureus toxins. PMID:26999200

Role of intravenous immune globulin in streptococcal toxic shock syndrome and Clostridium difficile infection.

PubMed

Shah, Punit J; Vakil, Niyati; Kabakov, Anna

2015-06-15

The use of intravenous immune globulin (IVIG) in the management of streptococcal toxic shock syndrome (STSS) and Clostridium difficile infection (CDI) is reviewed. IVIG has a wide range of uses in clinical practice, including STSS and CDI. It is an attractive option for these two infections because both infections are toxin mediated, and IVIG may contain antibodies that neutralize these toxins. For STSS and CDI, IVIG is often considered for use in critically ill patients who are not responding to traditional therapies. Several encouraging case reports and retrospective chart reviews have been published, highlighting the potential benefit of IVIG in such patients. However, its definitive role remains unclear, mainly due to the lack of high-level evidence. Data supporting its use have been extrapolated from retrospective chart reviews and case reports in which profound heterogeneity in patient populations and treatment modalities exist. The use of IVIG must be weighed carefully because it is not a benign product. As with the use of IVIG for STSS, the role of IVIG for CDI is unclear. Nonetheless, IVIG may serve as a useful adjunct therapy for patients suffering from severe complicated CDI (shock, ileus, or megacolon) who do not respond to conventional treatment. Adverse reactions to IVIG are mild and transitory and occur during or immediately after drug infusion. Although randomized, controlled trials supporting the use of IVIG for STSS and CDI are lacking, IVIG may be considered a last-line adjunct therapy in those patients for whom the clinical benefit outweighs the potential adverse effects of therapy. Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Comparison between oral antibiotics and probiotics as bowel preparation for elective colon cancer surgery to prevent infection: prospective randomized trial.

PubMed

Sadahiro, Sotaro; Suzuki, Toshiyuki; Tanaka, Akira; Okada, Kazutake; Kamata, Hiroko; Ozaki, Toru; Koga, Yasuhiro

2014-03-01

We have already reported that, for patients undergoing elective colon cancer operations, perioperative infection can be prevented by a single intravenous dose of an antibiotic given immediately beforehand if mechanical bowel preparation and the administration of oral antibiotics are implemented. Synbiotics has been reported to reduce the rate of infection in patients after pancreatic cancer operations. The effectiveness of oral antibiotics and probiotics in preventing postoperative infection in elective colon cancer procedures was examined in a randomized controlled trial. Three hundred ten patients with colon cancer randomly were assigned to one of three groups. All patients underwent mechanical bowel preparation and received a single intravenous dose of flomoxef immediately before operation. Probiotics were administered in Group A; oral antibiotics were administered in Group B; and neither probiotics nor oral antibiotics were administered in Group C. Stool samples were collected 9 and 2 days before and 7 and 14 days after the procedure. Clostridium difficile toxin and the number of bacteria in the intestine were determined. The rates of incisional surgical-site infection were 18.0%, 6.1%, and 17.9% in Groups A, B, and C, and the rates of leakage were 12.0%, 1.0%, and 7.4% in Groups A, B, and C, respectively, indicating that both rates were lesser in Group B than in Groups A and C (P = .014 and P = .004, respectively). The detection rates of C. difficile toxin were not changed among the three groups. We recommend oral antibiotics, rather than probiotics, as bowel preparation for elective colon cancer procedures to prevent surgical-site infections. Copyright © 2014 Mosby, Inc. All rights reserved.

Epidemiology of Clostridium difficile infections in Australia: enhanced surveillance to evaluate time trends and severity of illness in Victoria, 2010-2014.

PubMed

Worth, L J; Spelman, T; Bull, A L; Brett, J A; Richards, M J

2016-07-01

With epidemic strains of Clostridium difficile posing a substantial healthcare burden internationally, there is a need for longitudinal evaluation of Clostridium difficile infection (CDI) events in Australia. To evaluate time trends and severity of illness for CDI events in Australian healthcare facilities. All CDI events in patients admitted to Victorian public hospitals between 1(st) October 2010 and 31(st) December 2014 were reported to the Victorian Healthcare Associated Infection Surveillance System. CDI was defined as the isolation of a toxin-producing C. difficile organism in a diarrhoeal specimen, and classified as community-associated (CA-CDI) or healthcare-associated (HA-CDI). Severe disease was defined as admission to an intensive care unit, requirement for surgery and/or death due to infection. Time trends were examined using a mixed-effects Poisson regression model, and the Walter and Edward test of seasonality was applied to evaluate potential cyclical patterns. In total, 6736 CDI events were reported across 89 healthcare facilities. Of these, 4826 (71.6%) were HA-CDI, corresponding to a rate of 2.49/10,000 occupied bed days (OBDs). The incidence of HA-CDI was highest in the fifth quarter of surveillance (3.6/10,000 OBDs), followed by a reduction. Severe disease was reported in 1.66% of events, with the proportion being significantly higher for CA-CDI compared with HA-CDI (2.21 vs 1.45%, P = 0.03). The highest and lowest incidence of HA-CDI occurred in March and October, respectively. A low incidence of HA-CDI was reported in Victoria compared with US/European surveillance reports. Seasonality was evident, together with diminishing HA-CDI rates in 2012-2014. Severe infections were more common in CA-CDI, supporting future enhanced surveillance in community settings. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

PubMed Central

2018-01-01

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration. PMID:29503973

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection.

PubMed

Wilcox, Mark H; Gerding, Dale N; Poxton, Ian R; Kelly, Ciaran; Nathan, Richard; Birch, Thomas; Cornely, Oliver A; Rahav, Galia; Bouza, Emilio; Lee, Christine; Jenkin, Grant; Jensen, Werner; Kim, You-Sun; Yoshida, Junichi; Gabryelski, Lori; Pedley, Alison; Eves, Karen; Tipping, Robert; Guris, Dalya; Kartsonis, Nicholas; Dorr, Mary-Beth

2017-01-26

Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, -10.1 percentage points; 95% confidence interval [CI], -15.9 to -4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, -9.9 percentage points; 95% CI, -15.5 to -4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, -11.6 percentage points; 95% CI, -17.4 to -5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, -10.7 percentage points; 95% CI, -16.4 to -5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239 .).

Freshwater Cyanobacteria (Blue-Green Algae) Toxins: Isolation and Characterization

DTIC Science & Technology

1985-10-01

Another study involves detailing the enzyme kinetics and membrane ion effects of a new anticholinesterase compound. 4) Collaborative studies to...poisoned with Microcystis and may have diagnostic significance in differentiating algal poisoning from other plant hepatotoxicities. These sheep... activation of the toxin by the liver enzyme systems, but to date ao one has investigated this possibility. Female mice were slightly more sensitive to

Perceptions of Clostridium difficile infections among infection control professionals in Taiwan.

PubMed

Hung, Yuan-Pin; Lee, Jen-Chieh; Lin, Hsiao-Ju; Chiu, Chun-Wei; Wu, Jia-Ling; Liu, Hsiao-Chieh; Huang, I-Hsiu; Tsai, Pei-Jane; Ko, Wen-Chien

2017-08-01

High Clostridium difficile colonization and infection rates among hospitalized patients had been noted in Taiwan. Nevertheless, the cognition about clinical diagnosis and management of CDI among infection control professionals in Taiwan is not clear. A 24-item survey questionnaire about the diagnosis, therapy, or infection control policies toward CDI was distributed in the annual meeting of the Infectious Disease Society of Taiwan (IDST) in October 2015 and Infectious Control Society of Taiwan (ICST) in April 2016. Totally 441 individuals responded to the survey, and 280 (63.5%) participants would routinely monitor the prevalence of CDI and 347 (78.7%) reported the formulation of infection control policies of CDI in their hospital, including contact precaution (75.7%), wearing gloves (88.9%) or dressing (80.0%) at patient care, single room isolation (49.7%), preference of soap or disinfectant-based sanitizer (83.2%) and avoidance of alcohol-based sanitizer (63.3%), and environmental disinfection with 1000 ppm bleach (87.1%). For the timing of contact precaution discontinuation isolation for CDI patients, most (39.9%) participants suggested the time point of the absence of C. difficile toxin in feces. To treat mild CDI, most (61.9%) participants preferred oral metronidazole, and for severe CDI 26.1% would prescribe oral vancomycin as the drug of choice. There were substantial gaps in infection control polices and therapeutic choices for CDI between international guidelines and the perceptions of medical professionals in Taiwan. Professional education program and the setup of guideline for CDI should be considered in Taiwan. Copyright © 2017. Published by Elsevier B.V.

Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples

USDA-ARS?s Scientific Manuscript database

Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their effectiveness in detecting and differentiating between Shiga toxin 1 and 2 (Stx1 and Stx2) produced by Shiga toxin-producing E. coli (STEC) inoculated into food and water samples. Each kit incorporated monoclonal antibodies ...

[Diarrhea from the infectologist's point of view].

PubMed

Nemes, Zsuzsanna

2009-02-22

Gastroenteritis is a nonspecific term for various pathologic states of the gastrointestinal tract. Gastroenteritis causing pathogens are the second leading cause of morbidity and mortality worldwide. In the developed countries diarrhea is the most common reason for missing work, while in the developing world, it is a leading cause of death. Internationally, the mortality rate is 5-10 million deaths each year. "Traveller's diarrhea" is a polyetiologic common health problem of international travellers which affects travellers generally for days, but it can result in chronic postinfectious irritable bowel syndrome as well. Infectious agents usually cause acute gastroenteritis either by adherence of the intestinal mucosa, or by mucosal invasion, enterotoxin production, and/or cytotoxin production. The incubation period can often suggest the cause of etiology. When symptoms occur within 6 hours of eating, ingestion of preformed toxin of S. aureus or Bacillus cereus should be suspected. The incidence of hypervirulent C. difficile associated colitis is an emerging problem as a healthcare system associated infection. While infectious agents do not commonly cause chronic diarrhea, those that do include C. difficile, Giardia lamblia, Entamoeba histolytica, Cryptosporidium, Aeromonas and Yersinia . Amoebiasis is the second to malaria as a protozoal cause of death. Infection with HIV is also a common cause of diarrhea.

[Current treatment and epidemiology of Clostridium difficile infections].

PubMed

Dinh, A; Bouchand, F; Le Monnier, A

2015-09-01

During the past 10years, Clostridium difficile infections (CDI) have become a major public health challenge. Their epidemiology has changed with a rise in the number of cases and an increase in severe episodes. Recurrence and failure of conventional treatments have become more common. Furthermore, a spread of CDI has been observed in the general population-involving subjects without the usual risk factors (unexposed to antibiotic treatment, young people, pregnant women, etc.). All these change are partially due to the emergence of the hypervirulent and hyperepidemic clone NAP1/B1/027. New therapeutic strategies (antimicrobial treatment, immunoglobulins, toxin chelation, fecal microbiota transplantation) are now available and conventional treatments (metronidazole and vancomycin) have been reevaluated with new recommendations. Recent studies show a better efficacy of vancomycin compared to metronidazole for severe episodes. Fidaxomicin is a novel antibiotic drug with interesting features, including an efficacy not inferior to vancomycin and a lower risk of recurrence. Finally, for multi-recurrent forms, fecal microbiota transplantation seems to be the best option. We present the available data in this review. Copyright © 2015 Société nationale française de médecine interne (SNFMI). Published by Elsevier SAS. All rights reserved.

An in silico evaluation of treatment regimens for recurrent Clostridium difficile infection

PubMed Central

Blanco, Natalia; Foxman, Betsy; Malani, Anurag N.; Zhang, Min; Walk, Seth; Rickard, Alexander H.

2017-01-01

Background Clostridium difficile infection (CDI) is a significant nosocomial infection worldwide, that recurs in as many as 35% of infections. Risk of CDI recurrence varies by ribotype, which also vary in sporulation and germination rates. Whether sporulation/germination mediate risk of recurrence and effectiveness of treatment of recurring CDI remains unclear. We aim to assess the role of sporulation/germination patterns on risk of recurrence, and the relative effectiveness of the recommended tapered/pulsing regimens using an in silico model. Methods We created a compartmental in-host mathematical model of CDI, composed of vegetative cells, toxins, and spores, to explore whether sporulation and germination have an impact on recurrence rates. We also simulated the effectiveness of three tapered/pulsed vancomycin regimens by ribotype. Results Simulations underscored the importance of sporulation/germination patterns in determining pathogenicity and transmission. All recommended regimens for recurring CDI tested were effective in reducing risk of an additional recurrence. Most modified regimens were still effective even after reducing the duration or dosage of vancomycin. However, the effectiveness of treatment varied by ribotype. Conclusion Current CDI vancomycin regimen for treating recurrent cases should be studied further to better balance associated risks and benefits. PMID:28800598

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

2016-01-01

The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

[Identification of two Fusarium isolates and their crude toxin allelopathic effect on Cucumis melo seedlings].

PubMed

Zhao, Juan; Xue, Quan-Hong; Du, Jun-Zhi; Chen, Jiao-Jiao

2013-01-01

Eight fungi isolates were obtained from Fusarium-infected Cucumis melo (melon) plants and their rhizosphere soils. Taking melon cultivar 'Xitian 1' as test material, the re-inoculation and seed germination experiments were conducted to investigate the pathogenicity and growth inhibition effect of these fungi isolates on melon. Through the determination of the induced enzyme activities, resistant substance contents, and cell membrane permeability of potted melon roots, the allelopathic effect of the crude toxins of two harmful fungi was studied, and according to the morphological characteristics and Internal Transcribed Spacer (ITS) sequencing, the two harmful fungi were identified. The crude toxins of the two harmful fungi TF and HF had strong inhibition effects on the germination and growth of the melon seeds. The MDA and soluble protein contents and the cell membrane permeability of the 'Xitian 1' seedlings roots all increased, among which, the MDA content and cell membrane permeability increased by 108.6% and 40.6%, respectively when treated with the stock solution of TF toxin, compared with the control. The crude toxins of the two harmful fungi improved the induced enzyme activities of the melon roots, with the increment of the PAL and POD activities under the treatment of 10-fold dilution of TF crude toxin increased by 25.6% and 23.2%, respectively. When treated with the stock solution of HF toxin, the PAL activity significantly increased by 30.0%. The two harmful fungi TF and HF were primarily identified as Fusarium equisti and F. proliferatum, respectively. This study showed that the two Fusarium isolates could not infect melon via re-inoculation, but could negatively affect the melon's normal growth and normal physiological and biochemical metabolism via toxins excretion, and in the meantime, improve the root protective enzyme activities, with the effects of both benefit and harmfulness on melon plants. The allelopathic hazard of the crude toxins of the isolates could be one of the main causes of continuous cropping obstacle of melon.

Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

PubMed Central

Gharamah, Abdullah A; Moharram, Ahmed M; Ismail, Mady A; AL-Hussaini, Ashraf K

2014-01-01

Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2), sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin). Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss. PMID:24008795

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

PubMed

Barth, Holger; Aktories, Klaus; Popoff, Michel R; Stiles, Bradley G

2004-09-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

PubMed Central

Barth, Holger; Aktories, Klaus; Popoff, Michel R.; Stiles, Bradley G.

2004-01-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria. PMID:15353562

Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain.

PubMed

Haug, Gerd; Wilde, Christian; Leemhuis, Jost; Meyer, Dieter K; Aktories, Klaus; Barth, Holger

2003-12-30

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

Effects of cyanobacterial toxins and cyanobacterial cell-free crude extract on germination of alfalfa (Medicago sativa) and induction of oxidative stress.

PubMed

Pflugmacher, Stephan; Jung, Katharina; Lundvall, Linn; Neumann, Stefanie; Peuthert, Anja

2006-09-01

Cyanobacterial toxins have adverse effects on both terrestrial and aquatic plants. Microcystins are cyclic heptapeptides and an important group of cyanotoxins. When lake water contaminated with cyanobacterial blooms is used for spray irrigation, these toxins can come in contact with agricultural plants. During the exposure to these toxins, reactive oxygen species can form. These reactive oxygen species have a strong reactivity and are able to interact with other cellular compounds (lipids, protein, and DNA). Plants have antioxidative systems that will limit the negative effects caused by reactive oxygen species. These systems consist of enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, and nonenzymatic substances, such as reduced glutathione or vitamins. The aim of the present study was to investigate the effects of cyanobacterial toxins (microcystins and anatoxin-a) and cyanobacterial cell-free crude extract on alfalfa (Medicago sativa) seedlings. Inhibition of germination and root growth was observed with toxin concentrations of 5.0 microg/L. Also, oxidative damage, such as lipid peroxidation, was detected after the exposure of alfalfa seedlings to the toxin. Reactive oxygen detoxifying enzymes were elevated, showing a marked response in alfalfa to oxidative stress caused by the exposure to cyanobacterial metabolites that might influence the growth and development of these plants negatively.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

PubMed

Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

2017-03-01

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D ) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10 -8  M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL -1 , respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL -1 ), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Ribosomal Biosynthesis of the Cyclic Peptide Toxins of Amanita Mushrooms

PubMed Central

Walton, Jonathan D.; Hallen-Adams, Heather E.; Luo, Hong

2014-01-01

Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as α- and β-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys cross-bridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34–35 amino acids in length and have no predicted signal peptides. The genes for α-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable “toxin” region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide. PMID:20564017

Predicting the risk for hospital-onset Clostridium difficile infection (HO-CDI) at the time of inpatient admission: HO-CDI risk score.

PubMed

Tabak, Ying P; Johannes, Richard S; Sun, Xiaowu; Nunez, Carlos M; McDonald, L Clifford

2015-06-01

To predict the likelihood of hospital-onset Clostridium difficile infection (HO-CDI) based on patient clinical presentations at admission Retrospective data analysis Six US acute care hospitals Adult inpatients We used clinical data collected at the time of admission in electronic health record (EHR) systems to develop and validate a HO-CDI predictive model. The outcome measure was HO-CDI cases identified by a nonduplicate positive C. difficile toxin assay result with stool specimens collected >48 hours after inpatient admission. We fit a logistic regression model to predict the risk of HO-CDI. We validated the model using 1,000 bootstrap simulations. Among 78,080 adult admissions, 323 HO-CDI cases were identified (ie, a rate of 4.1 per 1,000 admissions). The logistic regression model yielded 14 independent predictors, including hospital community onset CDI pressure, patient age ≥65, previous healthcare exposures, CDI in previous admission, admission to the intensive care unit, albumin ≤3 g/dL, creatinine >2.0 mg/dL, bands >32%, platelets ≤150 or >420 109/L, and white blood cell count >11,000 mm3. The model had a c-statistic of 0.78 (95% confidence interval [CI], 0.76-0.81) with good calibration. Among 79% of patients with risk scores of 0-7, 19 HO-CDIs occurred per 10,000 admissions; for patients with risk scores >20, 623 HO-CDIs occurred per 10,000 admissions (P<.0001). Using clinical parameters available at the time of admission, this HO-CDI model demonstrated good predictive ability, and it may have utility as an early risk identification tool for HO-CDI preventive interventions and outcome comparisons.

Predictors of first recurrence in Clostridium difficile-associated disease. A study of 306 patients hospitalized in a Romanian tertiary referral center.

PubMed

Lupse, Mihaela; Flonta, Mirela; Cioara, Andreea; Filipescu, Irina; Todor, Nicolae

2013-12-01

Clostridium difficile is recognized as the major cause of nosocomial gastroenteritis usually related to antibiotic treatment. Although treatable, C. difficile--associated disease (CDAD) tends to recur in many patients. The purpose of the study was to analyze the risk factors for recurrence in patients with CDAD after the first treatment with vancomycin, metronidazole or both. We conducted a retrospective study of all patients admitted to the Teaching Hospital of Infectious Diseases Cluj-Napoca, Romania, between January 2011 and October 2012 with the diagnosis of CDAD or who developed diarrhoea after admission. A clinical diagnosis was made and culture and toxin A and B detection were carried out. We performed a statistical analysis taking into consideration: age, gender, previous hospital exposure, previous antibiotic treatment, and treatment duration. The patients were followed-up for at least 60 days. We included 306 patients (177 women and 129 men) with a median age of 71 years; 208 patients (68%) had prior hospitalization and 195 (64%) had received prior antibiotic treatment. Actual treatment consisted of vancomycin in 76 (25%) patients, metronidazole in 132 (43%) and both combined in 98 (32%) patients. The average duration of treatment was 10 days. Sixty patients (20%) experienced 95 recurrences and 9 patients died (3%). Treatment with metronidazole, vancomycin or both for 10 or more days did not prevent recurrences. Age over 70 (RR 1.5, CI 95%: 1.055-2.71) and use of PPI (RR 1.3, CI 95%: 1.16-3.1) significantly increased the risk of first recurrence of CDAD. CDAD recurrence rates were similar to those reported in the literature. The risk of first recurrence was significantly higher in patients older than 70 who also received PPI treatment.

Epidemiology of Clostridium difficile-associated disease at University Hospital Basel including molecular characterisation of the isolates 2006-2007.

PubMed

Fenner, L; Frei, R; Gregory, M; Dangel, M; Stranden, A; Widmer, A F

2008-12-01

A prospective study was conducted during a one-year period between 2006 and 2007 to describe the epidemiology of Clostridium difficile-associated disease (CDAD) at University Hospital Basel, Switzerland (UHBS) and to determine phenotypic and genotypic features of C. difficile strains isolated at the Microbiology Laboratory UHBS including strains from regional non-university hospitals. We prospectively identified 78 CDAD cases at UHBS with an incidence of 2.65/1,000 hospitalised patients or 2.3/10,000 patient-days. Sixteen patients (20.5%) were infected with clindamycin-resistant strains of PCR-ribotype 027 during an outbreak at the geriatric hospital. Among 124 single-patient isolates, 28 (22.6%) were resistant to moxifloxacin and 34 (27.4%) were resistant to clindamycin, but all remained susceptible to metronidazole and vancomycin. Of 102 toxigenic isolates, 19 (18.7%) had an 18-bp deletion in the tcdC gene, eight (7.8%) a 39-bp deletion, and one (1.0%) a 54-bp deletion. Genes for binary toxin were present in 27 (21.8%). PCR-ribotype 027 was associated with older age (median age 83.5 vs. 65.5 years, p < 0.0001) and longer duration of hospitalisation before onset of disease (median 15.5 vs. 9 days, p = 0.014) with a trend towards higher crude mortality, more severe disease, and previous use of macrolides compared to ribotype non-027. Overall, severe disease correlated with use of a nasogastric tube and surprisingly shorter duration of hospitalisation before onset of disease. Today, laboratory-based and epidemiological surveillance systems are required to monitor CDAD cases and emergence of new epidemic strains.

Clostridium difficile the hospital plague.

PubMed

Czepiel, J; Kozicki, M; Panasiuk, P; Birczyńska, M; Garlicki, A; Wesełucha-Birczyńska, A

2015-04-07

Clostridium difficile infection (CDI) has become one of the major public health threats in the last two decades. An increase has been observed not only in the rate of CDI, but also in its severity and mortality. Symptoms caused by this pathogen are accompanied by intense local and systemic inflammation. We confirmed that Raman microspectroscopy can help us in understanding CDI pathogenesis. A single erythrocyte of patients with CDI shows a difference, approximately 10 times, in the intensity of the Raman spectra at the beginning of hospitalization and after one week of treatment. The intensity level is an indicator of the spread of the inflammation within the cell, confirmed by standard laboratory tests. Many of the observed bands with enormously enhanced intensity, e.g. 1587, 1344, 1253, 1118 and 664 cm(-1), come from the symmetric vibration of the pyrrole ring. Heme variation of recovered cells in the acute CDI state between the first and the seventh day of treatment seems to show increased levels of oxygenated hemoglobin. Intense inflammation alters the conformation of the protein which is reflected in the significant changes in the amide I, II and III bands. There is an observed shift and a significant intensity increase of 1253 and 970 cm(-1) amide III and skeletal protein backbone CC stretching vibration bands, respectively. Principal Component Analysis (PCA) was used to find the variance in the data collected on the first and seventh day. PC2 loading in the 1645-1500 cm(-1) range shows an increase of heme, Tyr, Trp, or Phe vibrations because of changes in the protein microenvironment due to their exposure. Positive maxima at 1621, 1563 and 1550 in the PC2 loading originated from the ring vibrations. These observations indicate that Clostridium difficile toxins induce cytopathogenicity by altering cellular proteins.

Detection of Clostridium difficile infection clusters, using the temporal scan statistic, in a community hospital in southern Ontario, Canada, 2006-2011.

PubMed

Faires, Meredith C; Pearl, David L; Ciccotelli, William A; Berke, Olaf; Reid-Smith, Richard J; Weese, J Scott

2014-05-12

In hospitals, Clostridium difficile infection (CDI) surveillance relies on unvalidated guidelines or threshold criteria to identify outbreaks. This can result in false-positive and -negative cluster alarms. The application of statistical methods to identify and understand CDI clusters may be a useful alternative or complement to standard surveillance techniques. The objectives of this study were to investigate the utility of the temporal scan statistic for detecting CDI clusters and determine if there are significant differences in the rate of CDI cases by month, season, and year in a community hospital. Bacteriology reports of patients identified with a CDI from August 2006 to February 2011 were collected. For patients detected with CDI from March 2010 to February 2011, stool specimens were obtained. Clostridium difficile isolates were characterized by ribotyping and investigated for the presence of toxin genes by PCR. CDI clusters were investigated using a retrospective temporal scan test statistic. Statistically significant clusters were compared to known CDI outbreaks within the hospital. A negative binomial regression model was used to identify associations between year, season, month and the rate of CDI cases. Overall, 86 CDI cases were identified. Eighteen specimens were analyzed and nine ribotypes were classified with ribotype 027 (n = 6) the most prevalent. The temporal scan statistic identified significant CDI clusters at the hospital (n = 5), service (n = 6), and ward (n = 4) levels (P ≤ 0.05). Three clusters were concordant with the one C. difficile outbreak identified by hospital personnel. Two clusters were identified as potential outbreaks. The negative binomial model indicated years 2007-2010 (P ≤ 0.05) had decreased CDI rates compared to 2006 and spring had an increased CDI rate compared to the fall (P = 0.023). Application of the temporal scan statistic identified several clusters, including potential outbreaks not detected by hospital personnel. The identification of time periods with decreased or increased CDI rates may have been a result of specific hospital events. Understanding the clustering of CDIs can aid in the interpretation of surveillance data and lead to the development of better early detection systems.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

PubMed

Féraudet-Tarisse, Cécile; Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R; Dorner, Brigitte G; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

PubMed Central

Mazuet, Christelle; Pauillac, Serge; Krüger, Maren; Lacroux, Caroline; Popoff, Michel R.; Dorner, Brigitte G.; Andréoletti, Olivier; Plaisance, Marc; Volland, Hervé; Simon, Stéphanie

2017-01-01

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test. PMID:28700661

Structural Insight into the Clostridium difficile Ethanolamine Utilisation Microcompartment

PubMed Central

Faulds-Pain, Alexandra; Lewis, Richard J.; Marles-Wright, Jon

2012-01-01

Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen. PMID:23144756

Healthcare resource utilization for recurrent Clostridium difficile infection in a large university hospital in Houston, Texas.

PubMed

Aitken, Samuel L; Joseph, Tiby B; Shah, Dhara N; Lasco, Todd M; Palmer, Hannah R; DuPont, Herbert L; Xie, Yang; Garey, Kevin W

2014-01-01

There are limited data examining healthcare resource utilization in patients with recurrent Clostridium difficile infection (CDI). Patients with CDI at a tertiary-care hospital in Houston, TX, were prospectively enrolled into an observational cohort study. Recurrence was assessed via follow-up phone calls. Patients with one or more recurrence were included in this study. The location at which healthcare was obtained by patients with recurrent CDI was identified along with hospital length of stay. CDI-attributable readmissions, defined as a positive toxin test within 48 hours of admission and a primary CDI diagnosis, were also assessed. 372 primary cases of CDI were identified of whom 64 (17.2%) experienced at least one CDI recurrence. Twelve of 64 patients experienced 18 further episodes of CDI recurrence. Of these 64 patients, 33 (50.8%) patients with recurrent CDI were readmitted of which 6 (18.2%) required ICU care, 29 (45.3%) had outpatient care only, and 2 (3.1%) had an ED visit. Nineteen (55.9%) readmissions were defined as CDI-attributable. For patients with CDI-attributable readmission, the average length of stay was 6 ± 6 days. Recurrent CDI leads to significant healthcare resource utilization. Methods of reducing the burden of recurrent CDI should be further studied.

Verification of performance specifications for a US Food and Drug Administration-approved molecular microbiology test: Clostridium difficile cytotoxin B using the Becton, Dickinson and Company GeneOhm Cdiff assay.

PubMed

Schlaberg, Robert; Mitchell, Michael J; Taggart, Edward W; She, Rosemary C

2012-01-01

US Food and Drug Administration (FDA)-approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturer's performance standards. We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. Performance data from the verification studies conducted by the laboratory were consistent with the manufacturer's performance standards and the assay was implemented into the laboratory's test menu. Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.

Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria.

PubMed

Engler, K H; Efstratiou, A

2000-04-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.

Rapid Enzyme Immunoassay for Determination of Toxigenicity among Clinical Isolates of Corynebacteria

PubMed Central

Engler, Kathryn H.; Efstratiou, Androulla

2000-01-01

A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day. PMID:10747112

Models for the study of Clostridium difficile infection

PubMed Central

Best, Emma L.; Freeman, Jane; Wilcox, Mark H.

2012-01-01

Models of Clostridium difficile infection (C. difficile) have been used extensively for Clostridium difficile (C. difficile) research. The hamster model of C. difficile infection has been most extensively employed for the study of C. difficile and this has been used in many different areas of research, including the induction of C. difficile, the testing of new treatments, population dynamics and characterization of virulence. Investigations using in vitro models for C. difficile introduced the concept of colonization resistance, evaluated the role of antibiotics in C. difficile development, explored population dynamics and have been useful in the evaluation of C. difficile treatments. Experiments using models have major advantages over clinical studies and have been indispensible in furthering C. difficile research. It is important for future study programs to carefully consider the approach to use and therefore be better placed to inform the design and interpretation of clinical studies. PMID:22555466

Binding Assays for the Quantitative Detection of P. brevis Polyether Neurotoxins in Biological Samples and Antibodies as Therapeutic Aids for Polyether Marine Intoxication

DTIC Science & Technology

1990-05-15

was also linked to urease and toxin-enzyme conjugates were evaluated. 4. Toxin Enzyme Conjugates. Brevetoxins linked to either Jack Bean urease or...described in materials and methods. For urease conjugates, 1:2, 1:4 and 1:6 molar ratios were investigated. The following protocol yielded the most...fold excess urease in 1 volume equivalent of water, in three equal aliquots. Total volume after addition is 2-fold the volume in step [2], final

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

PubMed

Harms, Alexander; Brodersen, Ditlev Egeskov; Mitarai, Namiko; Gerdes, Kenn

2018-06-07

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules. Copyright © 2018 Elsevier Inc. All rights reserved.

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle.

PubMed

Gregory, A R; Ellis, T M; Jubb, T F; Nickels, R J; Cousins, D V

1996-02-01

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.

ADP-ribosylation of proteins: Enzymology and biological significance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Althaus, F.R.; Richter, C.

1987-01-01

This book presents an overview of the molecular and biological consequences of the posttranslational modification of proteins with ADP-ribose monomers and polymers. Part one focuses on chromatin-associated poly ADP-ribosylation reactions which have evolved in higher eukaryotes as modulators of chromatin functions. The significance of poly ADP-ribosylation in DNA repair, carcinogenesis, and gene expression during terminal differentiation is discussed. Part two reviews mono ADP-ribosylation reactions which are catalyzed by prokaryotic and eukaryotic enzymes. Consideration is given to the action of bacterial toxins, such as cholera toxin, pertussis toxin, and diphtheria toxin. These toxins have emerged as tools for the molecular probingmore » of proteins involved in signal transduction and protein biosynthesis.« less

Prevalence and Duration of Asymptomatic Clostridium difficile Carriage among Healthy Subjects in Pittsburgh, Pennsylvania

PubMed Central

Galdys, Alison L.; Nelson, Jemma S.; Shutt, Kathleen A.; Schlackman, Jessica L.; Pakstis, Diana L.; Pasculle, A. William; Marsh, Jane W.; Harrison, Lee H.

2014-01-01

Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens. PMID:24759727

Validation of the 3-day rule for stool bacterial tests in Japan.

PubMed

Kobayashi, Masanori; Sako, Akahito; Ogami, Toshiko; Nishimura, So; Asayama, Naoki; Yada, Tomoyuki; Nagata, Naoyoshi; Sakurai, Toshiyuki; Yokoi, Chizu; Kobayakawa, Masao; Yanase, Mikio; Masaki, Naohiko; Takeshita, Nozomi; Uemura, Naomi

2014-01-01

Stool cultures are expensive and time consuming, and the positive rate of enteric pathogens in cases of nosocomial diarrhea is low. The 3-day rule, whereby clinicians order a Clostridium difficile (CD) toxin test rather than a stool culture for inpatients developing diarrhea >3 days after admission, has been well studied in Western countries. The present study sought to validate the 3-day rule in an acute care hospital setting in Japan. Stool bacterial and CD toxin test results for adult patients hospitalized in an acute care hospital in 2008 were retrospectively analyzed. Specimens collected after an initial positive test were excluded. The positive rate and cost-effectiveness of the tests were compared among three patient groups. The adult patients were divided into three groups for comparison: outpatients, patients hospitalized for ≤3 days and patients hospitalized for ≥4 days. Over the 12-month period, 1,597 stool cultures were obtained from 992 patients, and 880 CD toxin tests were performed in 529 patients. In the outpatient, inpatient ≤3 days and inpatient ≥4 days groups, the rate of positive stool cultures was 14.2%, 3.6% and 1.3% and that of positive CD toxin tests was 1.9%, 7.1% and 8.5%, respectively. The medical costs required to obtain one positive result were 9,181, 36,075 and 103,600 JPY and 43,200, 11,333 and 9,410 JPY, respectively. The 3-day rule was validated for the first time in a setting other than a Western country. Our results revealed that the "3-day rule" is also useful and cost-effective in Japan.

Structure-Function Relationship of Hydrophiidae Postsynaptic Neurotoxins

DTIC Science & Technology

1992-03-11

monster venom concluded that gila toxin is an arginine esterase with kallikrein-like activity causing lethality and gyration in mice. However, it is not a...Fractionation of Lapemis venom ............ 49 Fig 3-4 Fractionation of Gila Toxin ............... 50 Fig 3-5 Fibrinogenolytic Activity of Gila toxin...Sequence of 8 kD Fragment of Lapemis PLA ..... 8 7 Tab 3-9 Enzyme Activity of Native and Metal Pl . 88 Tab 3-10 Amino Acid Analysis of Lapemis 9 kD prorein

Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin.

PubMed

Aktories, Klaus; Barth, Holger

2004-04-01

Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

Review article: anti-inflammatory mechanisms of action of Saccharomyces boulardii.

PubMed

Pothoulakis, C

2009-10-15

Saccharomyces boulardii, a well-studied probiotic, can be effective in inflammatory gastrointestinal diseases with diverse pathophysiology, such as inflammatory bowel disease (IBD), and bacterially mediated or enterotoxin-mediated diarrhoea and inflammation. To discuss the mechanisms of action involved in the intestinal anti-inflammatory action of S. boulardii. Review of the literature related to the anti-inflammatory effects of this probiotic. Several mechanisms of action have been identified directed against the host and pathogenic microorganisms. S. boulardii and S. boulardii secreted-protein(s) inhibit production of proinflammatory cytokines by interfering with the global mediator of inflammation nuclear factor kappaB, and modulating the activity of the mitogen-activated protein kinases ERK1/2 and p38. S. boulardii activates expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) that protects from gut inflammation and IBD. S. boulardii also suppresses 'bacteria overgrowth' and host cell adherence, releases a protease that cleaves C. difficile toxin A and its intestinal receptor and stimulates antibody production against toxin A. Recent results indicate that S. boulardii may interfere with IBD pathogenesis by trapping T cells in mesenteric lymph nodes. The multiple anti-inflammatory mechanisms exerted by S. boulardii provide molecular explanations supporting its effectiveness in intestinal inflammatory states.

Low cost bioluminescence imaging as an alternative to in vivo bioassays for quantifying biologically active staphylococcal enterotoxin type E

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a major causative agent implicated in outbreaks of food poisoning, acting through the production of a range of toxins including staphylococcal enterotoxin type E (SEE). While tests such as enzyme-linked immunosorbent (ELISA) exist to detect the toxin molecules, existing meth...

A highly rapid and simple competitive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poisoning toxins in shellfish.

PubMed

Kawatsu, Kentaro; Kanki, Masashi; Harada, Tetsuya; Kumeda, Yuko

2014-11-01

Using a streptavidin-coated well plate, a biotin-labelled anti-gonyautoxin 2/3 monoclonal antibody GT-13A, and a decarbamoyl saxitoxin-peroxidase conjugate, a direct competitive enzyme-linked immunosorbent assay (PSP-ELISA) was developed for monitoring paralytic shellfish poisoning (PSP) toxins in shellfish. This assay is simple to perform and can be completed in approximately 20 min. The PSP-ELISA was compared to the mouse bioassay (MBA) for the detection of PSP toxins in shellfish samples (n=83) collected from the coast of Osaka Prefecture, Japan. When positive and negative results were indicated based on the regulatory limit for PSP toxins (4 mouse unit(MU)/g of shellfish meat), the PSP-ELISA results showed a sensitivity of 100% (25 of 25) and a specificity of 89.7% (52 of 58 samples) compared to the MBA results. These results suggest that the PSP-ELISA could be used as a rapid and simple screening method prior to the MBA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preliminary Fractionation of Tiger Rattlesnake (Crotalus tigris) Venom

DTIC Science & Technology

1990-01-31

J., ZEPEDA , 11. and SCtIWARTZMAN, R. J. (1988) Gyroxin, a toxin from the venom of Crot( _ d !1rissus j!2.rificius, is a thrombin-like enzyme. Toxicon...had low protease activity, lacked hemolytic activity and had an i.p. D 5 0 , of 0.070 mg/kg for mice. Lethal fractions obtained by anion and cation...L.. d . Lerrfic and C ihi IU±L WEINSTEIN et al. (1985) reported the presence of a toxin antigenically related to mojave toxin in £, ligris venom. The

Gut Microbiota Patterns Associated with Colonization of Different Clostridium difficile Ribotypes

PubMed Central

Skraban, Jure; Dzeroski, Saso; Zenko, Bernard; Mongus, Domen; Gangl, Simon; Rupnik, Maja

2013-01-01

C. difficile infection is associated with disturbed gut microbiota and changes in relative frequencies and abundance of individual bacterial taxons have been described. In this study we have analysed bacterial, fungal and archaeal microbiota by denaturing high pressure liquid chromatography (DHPLC) and with machine learning methods in 208 faecal samples from healthy volunteers and in routine samples with requested C. difficile testing. The latter were further divided according to stool consistency, C. difficile presence or absence and C. difficile ribotype (027 or non-027). Lower microbiota diversity was a common trait of all routine samples and not necessarily connected only to C. difficile colonisation. Differences between the healthy donors and C. difficile positive routine samples were detected in bacterial, fungal and archaeal components. Bifidobacterium longum was the single most important species associated with C. difficile negative samples. However, by machine learning approaches we have identified patterns of microbiota composition predictive for C. difficile colonization. Those patterns also differed between samples with C. difficile ribotype 027 and other C. difficile ribotypes. The results indicate that not only the presence of a single species/group is important but that certain combinations of gut microbes are associated with C. difficile carriage and that some ribotypes (027) might be associated with more disturbed microbiota than the others. PMID:23469128

Fecal Microbiota Transplantation: A Review of Emerging Indications Beyond Relapsing Clostridium difficile Toxin Colitis

PubMed Central

Lee, Woo Jung; Lattimer, Lakshmi D. N.; Stephen, Sindu; Borum, Marie L.

2015-01-01

The symbiotic relationship between gut microbiota and humans has been forged over many millennia. This relationship has evolved to establish an intimate partnership that we are only beginning to understand. Gut microbiota were once considered pathogenic, but the concept of gut microbiota and their influence in human health is undergoing a major paradigm shift, as there is mounting evidence of their impact in the homeostasis of intestinal development, metabolic activities, and the immune system. The disruption of microbiota has been associated with many gastrointestinal and nongastrointestinal diseases, and the reconstitution of balanced microbiota has been postulated as a potential therapeutic strategy. Fecal microbiota transplantation (FMT), a unique method to reestablish a sustained balance in the disrupted microbiota of diseased intestine, has demonstrated great success in the treatment of recurrent Clostridium difficile infection and has gained increasing acceptance in clinical use. The possibility of dysfunctional micro-biota playing a causative role in other gastrointestinal and nongas-trointestinal diseases, therefore, has also been raised, and there are an increasing number of studies supporting this hypothesis. FMT is emerging as a feasible therapeutic option for several diseases; however, its efficacy remains in question, given the lack of clinical trial data. Altering microbiota with FMT holds great promise, but much research is needed to further define FMT’s therapeutic role and optimize the microbiota delivery system. PMID:27099570

Fecal Microbiota Transplantation: A Review of Emerging Indications Beyond Relapsing Clostridium difficile Toxin Colitis.

PubMed

Jung Lee, Woo; Lattimer, Lakshmi D N; Stephen, Sindu; Borum, Marie L; Doman, David B

2015-01-01

The symbiotic relationship between gut microbiota and humans has been forged over many millennia. This relationship has evolved to establish an intimate partnership that we are only beginning to understand. Gut microbiota were once considered pathogenic, but the concept of gut microbiota and their influence in human health is undergoing a major paradigm shift, as there is mounting evidence of their impact in the homeostasis of intestinal development, metabolic activities, and the immune system. The disruption of microbiota has been associated with many gastrointestinal and nongastrointestinal diseases, and the reconstitution of balanced microbiota has been postulated as a potential therapeutic strategy. Fecal microbiota transplantation (FMT), a unique method to reestablish a sustained balance in the disrupted microbiota of diseased intestine, has demonstrated great success in the treatment of recurrent Clostridium difficile infection and has gained increasing acceptance in clinical use. The possibility of dysfunctional micro-biota playing a causative role in other gastrointestinal and nongas-trointestinal diseases, therefore, has also been raised, and there are an increasing number of studies supporting this hypothesis. FMT is emerging as a feasible therapeutic option for several diseases; however, its efficacy remains in question, given the lack of clinical trial data. Altering microbiota with FMT holds great promise, but much research is needed to further define FMT's therapeutic role and optimize the microbiota delivery system.

Fidaxomicin in Clostridium difficile infection: latest evidence and clinical guidance

PubMed Central

2014-01-01

The incidence of Clostridium difficile infection (CDI) has risen 400% in the last decade. It currently ranks as the third most common nosocomial infection. CDI has now crossed over as a community-acquired infection. The major failing of current therapeutic options for the management of CDI is recurrence of disease after the completion of treatment. Fidaxomicin has been proven to be superior to vancomycin in successful sustained clinical response to therapy. Improved outcomes may be due to reduced collateral damage to the gut microflora by fidaxomicin, bactericidal activity, inhibition of Clostridial toxin formation and inhibition of new sporulation. This superiority is maintained in groups previously reported as being at high risk for CDI recurrence including those: with relapsed infection after a single treatment course; on concomitant antibiotic therapy; aged >65 years; with cancer; and with chronic renal insufficiency. Because the acquisition cost of fidaxomicin far exceeds that of metronidazole or vancomycin, in order to rationally utilize this agent, it should be targeted to those populations who are at high risk for relapse and in whom the drug has demonstrated superiority. In this manuscript is reviewed the changing epidemiology of CDI, current treatment options for this infection, proposed benefits of fidaxomicin over currently available antimicrobial options, available analysis of cost effectiveness of the drug, and is given recommendations for judicious use of the drug based upon the available published literature. PMID:24587892

Fidaxomicin in Clostridium difficile infection: latest evidence and clinical guidance.

PubMed

Mullane, Kathleen

2014-03-01

The incidence of Clostridium difficile infection (CDI) has risen 400% in the last decade. It currently ranks as the third most common nosocomial infection. CDI has now crossed over as a community-acquired infection. The major failing of current therapeutic options for the management of CDI is recurrence of disease after the completion of treatment. Fidaxomicin has been proven to be superior to vancomycin in successful sustained clinical response to therapy. Improved outcomes may be due to reduced collateral damage to the gut microflora by fidaxomicin, bactericidal activity, inhibition of Clostridial toxin formation and inhibition of new sporulation. This superiority is maintained in groups previously reported as being at high risk for CDI recurrence including those: with relapsed infection after a single treatment course; on concomitant antibiotic therapy; aged >65 years; with cancer; and with chronic renal insufficiency. Because the acquisition cost of fidaxomicin far exceeds that of metronidazole or vancomycin, in order to rationally utilize this agent, it should be targeted to those populations who are at high risk for relapse and in whom the drug has demonstrated superiority. In this manuscript is reviewed the changing epidemiology of CDI, current treatment options for this infection, proposed benefits of fidaxomicin over currently available antimicrobial options, available analysis of cost effectiveness of the drug, and is given recommendations for judicious use of the drug based upon the available published literature.

A Comprehensive Study of Costs Associated With Recurrent Clostridium difficile Infection.

PubMed

Rodrigues, Rodrigo; Barber, Grant E; Ananthakrishnan, Ashwin N

2017-02-01

BACKGROUND Clostridium difficile infection (CDI) is the most common healthcare-associated infection and is associated with considerable morbidity. Recurrent CDI is a key contributing factor to this morbidity. Despite an estimated 83,000 recurrences annually in the United States, there are few accurate estimates of costs associated with recurrent CDI. OBJECTIVE We performed this study (1) to identify the health consequences of recurrent CDI including need for repeat hospitalization, intensive care unit (ICU) stay, and surgery; (2) to determine costs associated with recurrent CDI and identify determinants of such costs; and (3) to compare the outcomes and costs of recurrent CDI to those who develop reinfection. METHODS We identified all patients with confirmed recurrent CDI between January to December 2013 at a single referral center. Healthcare burden associated with recurrence including diagnostic testing, pharmacologic treatment, and inpatient and outpatient healthcare visits were identified in the 12 months following the first recurrence. Total healthcare costs were calculated, and the predictors of high healthcare utilization were identified. RESULTS Our study population included 98 patients with recurrent CDI. The median interval between the initial infection and recurrence was 37 days. The mean age of the cohort was 67 years, two-thirds were women (62%), and the mean Charlson index was 8.6. During the year following the first recurrence of CDI, each patient underwent a mean of 4.4 stool C. difficile toxin tests and received a mean of 2.5 prescriptions for oral vancomycin (range, 0-6). Most patients (84%) with recurrence had a CDI-related hospitalization, and 6% underwent colectomy. The mean total CDI-associated cost was $34,104 per patient, with hospitalization costs accounting for 68%, surgery 20%, and drug treatment 8% of this cost, respectively. Extrapolating to the United States overall, we estimate an annual cost of $2.8 billion related to recurrent CDI. CONCLUSION Recurrent CDI is associated with considerable morbidity and cost. Infect Control Hosp Epidemiol 2017;38:196-202.

Length of stay and hospital costs among high-risk patients with hospital-origin Clostridium difficile-associated diarrhea.

PubMed

Campbell, Rebecca; Dean, Bonnie; Nathanson, Brian; Haidar, Tracy; Strauss, Marcie; Thomas, Sheila

2013-01-01

Hospital-onset Clostridium difficile-associated diarrhea (HO-CDAD) has been associated with longer length of stay (LOS) and higher hospital costs among patients in general. The burden of HO-CDAD is unknown among patients who may be at particular risk of poor outcomes: older patients, those with complex or chronic conditions (renal disease, cancer, inflammatory bowel disease [IBD]), and those with concomitant antibiotic (CAbx) use during treatment for CDAD. A retrospective analysis (2005-2011) of the Health Facts® database (Cerner Corp., Kansas City, MO) containing comprehensive clinical records from 186 US hospitals identified hospitalized adult patients with HO-CDAD based on a positive C. difficile toxin collected >48 h after admission. Control patients were required to have total hospital LOS ≥2 days. Separate logistic regression models to estimate propensities were developed for each study group, with HO-CDAD vs controls as the outcome. Differences in LOS and costs were calculated between cases and controls for each group. A total of 4521 patients with HO-CDAD were identified. Mean age was 70 years, 54% were female, and 13% died. After matching, LOS was significantly greater among HO-CDAD patients (vs controls) in each group except IBD. The significant difference in LOS ranged from 3.0 (95% CI = 1.4-4.6) additional days in older patients to 7.8 (95% CI = 5.7-9.9) days in patients with CAbx exposure. HO-CDAD was associated with significantly higher costs among older patients (p < 0.001) and among those with renal impairment (p = 0.012) or CAbx use (p < 0.001). Missing cost data and potential misclassification of colonized patients as infected. Renal impairment, advanced age, cancer, and CAbx use are associated with significantly longer LOS among HO-CDAD patients, with CAbx users being the most resource intensive. Early identification and aggressive treatment of HO-CDAD in these groups may be warranted.

Economic burden of Clostridium difficile associated diarrhoea: a cost-of-illness study from a German tertiary care hospital.

PubMed

Heimann, S M; Vehreschild, J J; Cornely, O A; Wisplinghoff, H; Hallek, M; Goldbrunner, R; Böttiger, B W; Goeser, T; Hölscher, A; Baldus, S; Müller, F; Jazmati, N; Wingen, S; Franke, B; Vehreschild, M J G T

2015-12-01

Clostridium difficile associated diarrhoea (CDAD) is the most common cause of health-care-associated infectious diarrhoea. In the context of the German health-care system, direct and indirect costs of an initial episode of CDAD and of CDAD recurrence are currently unknown. We defined CDAD as presence of diarrhoea (≥3 unformed stools/day) in association with detection of Clostridium difficile toxin in an unformed faecal sample. Patients treated with metronidazole (PO or IV) and/or vancomycin (PO) were included. Comprehensive data of patients were retrospectively documented into a database using the technology of the Cologne Cohort of Neutropenic Patients (CoCoNut). Patients with CDAD were matched to control patients in a 1:1 ratio. Analysis was split in three groups: incidence group (CDAD patients without recurrence), recurrence group (CDAD patients with ≥1 recurrence) and control group (matched non-CDAD patients). Between 02/2010 and 12/2011, 150 patients with CDAD (114 patients in the incidence and 36 (24 %) in the recurrence group) and 150 controls were analysed. Mean length of stay was: 32 (95 %CI: 30-37), 94 (95 %CI: 76-112) and 24 days (95 %CI: 22-27; P = <0.001), resulting in mean overall direct treatment costs per patient of €18,460 (95 %CI: €14,660-€22,270), €73,900 (95 %CI: €50,340-€97,460) and €14,530 (95 %CI: €11,730-€17,330; P = <0.001). In the incidence and recurrence group, the mean cumulative number of antibiotic CDAD treatment days was 11 (95 %CI: 10-12) and 36 (95 %CI: 27-45; P = <0.001). Especially CDAD recurrence was associated with excessive costs, which were mostly attributable to a significantly longer overall length of stay. Innovative treatment strategies are warranted to reduce treatment costs and prevent recurrence of CDAD.

Association Between Outpatient Antibiotic Prescribing Practices and Community-Associated Clostridium difficile Infection

PubMed Central

Dantes, Raymund; Mu, Yi; Hicks, Lauri A.; Cohen, Jessica; Bamberg, Wendy; Beldavs, Zintars G.; Dumyati, Ghinwa; Farley, Monica M.; Holzbauer, Stacy; Meek, James; Phipps, Erin; Wilson, Lucy; Winston, Lisa G.; McDonald, L. Clifford; Lessa, Fernanda C.

2015-01-01

Background. Antibiotic use predisposes patients to Clostridium difficile infections (CDI), and approximately 32% of these infections are community-associated (CA) CDI. The population-level impact of antibiotic use on adult CA-CDI rates is not well described. Methods. We used 2011 active population- and laboratory-based surveillance data from 9 US geographic locations to identify adult CA-CDI cases, defined as C difficile-positive stool specimens (by toxin or molecular assay) collected from outpatients or from patients ≤3 days after hospital admission. All patients were surveillance area residents and aged ≥20 years with no positive test ≤8 weeks prior and no overnight stay in a healthcare facility ≤12 weeks prior. Outpatient oral antibiotic prescriptions dispensed in 2010 were obtained from the IMS Health Xponent database. Regression models examined the association between outpatient antibiotic prescribing and adult CA-CDI rates. Methods. Healthcare providers prescribed 5.2 million courses of antibiotics among adults in the surveillance population in 2010, for an average of 0.73 per person. Across surveillance sites, antibiotic prescription rates (0.50–0.88 prescriptions per capita) and unadjusted CA-CDI rates (40.7–139.3 cases per 100 000 persons) varied. In regression modeling, reducing antibiotic prescribing rates by 10% among persons ≥20 years old was associated with a 17% (95% confidence interval, 6.0%–26.3%; P = .032) decrease in CA-CDI rates after adjusting for age, gender, race, and type of diagnostic assay. Reductions in prescribing penicillins and amoxicillin/clavulanic acid were associated with the greatest decreases in CA-CDI rates. Conclusions and Relevance. Community-associated CDI prevention should include reducing unnecessary outpatient antibiotic use. A modest reduction of 10% in outpatient antibiotic prescribing can have a disproportionate impact on reducing CA-CDI rates. PMID:26509182

Surveillance of Clostridium difficile Infections: Results from a Six-Year Retrospective Study in Nine Hospitals of a North Italian Local Health Authority

PubMed Central

Roncarati, Greta; Dallolio, Laura; Leoni, Erica; Panico, Manuela; Zanni, Angela; Farruggia, Patrizia

2017-01-01

Clostridium difficile is an emerging cause of healthcare associated infections. In nine hospitals of an Italian Local Health Authority the episodes of C. difficile infection (CDI) were identified using the data registered by the centralized Laboratory Information System, from 2010 to 2015. CDI incidence (positive patients for A and/or B toxins per patients-days) was analysed per year, hospital, and ward. A number of cases approximately equivalent to the mean of identified cases per year were studied retrospectively to highlight the risk factors associated to CDI and their severity. Nine hundred and forty-two patients affected by CDI were identified. The overall incidence was 3.7/10,000 patients-days, with a stable trend across the six years and the highest rates observed in smaller and outlying hospitals (up to 17.8/10,000), where the admitted patients were older and the wards with the highest incidences (long-term-care: 7.6/10,000, general medicine: 5.7/10,000) were more represented. The mean age of patients in each hospital was correlated with CDI rates. Of the 101 cases selected for the retrospective study, 86.1% were healthcare associated, 10.9% community acquired; 9.1% met the criteria for recurrent case and 23.8% for severe case of CDI. The overall mortality rate was 28.7%. Comorbidity conditions occurred in 91.1%, previous exposure to antibiotics in 76.2%, and proton pump inhibitors in 77.2%. Recurrent and severe cases were significantly associated with renal insufficiency and creatinine levels ≥2 mg/dL. The survey based on the centralized laboratory data was useful to study CDI epidemiology in the different centres in order to identify possible weaknesses and plan control strategies, in particular the reinforcement of staff training, mainly targeted at compliance with contact precautions and hand hygiene. PMID:28075419

Prolonged ileus as a sole manifestation of pseudomembranous enterocolitis.

PubMed

Elinav, Eran; Planer, David; Gatt, Moshe E

2004-05-01

Pseudomembranous colitis usually manifests as fever and diarrhea in hospitalized patients treated with systemic antibiotics. We present a case that represents a unique variant. The 44-year-old man suffered of several weeks of abdominal pain, low-grade fever, nausea, vomiting, and lack of bowel movements. Upper gastrointestinal barium swallow and passage series revealed evidence of severe intestinal hypomotility. A thorough evaluation for the cause of the patient's ileus and abdominal pain was unrevealing, and symptomatic treatment was ineffective. Following the administration of opiates and dietary fiber supplementation the patient's abdominal pain and distention rapidly worsened, requiring an urgent subtotal colectomy. The macroscopic and microscopic appearance of the excised colon as well as results of the colonic cytotoxin essay and fecal enzyme-linked immunosorbent assay essay confirmed the diagnosis of severe Clostridium difficile induced pseudomembranous colitis as the cause of the patient's illness. To our knowledge, this is the first reported case of Clostridium-difficile induced disease consisting of prolonged ileus in the absence of diarrhea in a patient not previously taking antibiotics.

T-2 Toxin Alters the Levels of Collagen II and Its Regulatory Enzymes MMPs/TIMP-1 in a Low-Selenium Rat Model of Kashin-Beck Disease.

PubMed

Zhou, Xiaorong; Yang, Haojie; Guan, Fang; Xue, Senhai; Song, Daiqin; Chen, Jinghong; Wang, Zhilun

2016-02-01

The objectives of this study are to assess T-2 toxin's involvement in low selenium (Se)-induced Kashin-Beck disease (KBD) in rats and unveil the mechanisms underlying this disease. Two hundred thirty rats were randomly divided into two groups after weaning and fed normal or low-Se diets (n = 115), respectively, for a month. After low-Se model confirmation, rats in each group were subdivided into five: two subgroups (n = 20) were fed their current diets (normal or low-Se diets, respectively) for 30 and 90 days, respectively; two other subgroups (n = 25) received their current diets + low T-2 toxin (100 ng/g BW/day) for 30 and 90 days, respectively; and 25 rats were fed their current diets + high T-2 toxin (200 ng/g BW/day) for 30 days. Articular cartilage samples were extracted for hematoxylin and eosin (H&E) staining and immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to assess protein and mRNA levels, respectively, of collagen II, matrix metalloproteinase (MMP-1), MMP -3, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). Low Se and T-2 toxin synergistically affected animal fitness. Interestingly, low Se + T-2 toxin groups showed KBD characteristics. MMP-1, -3, and -13 mRNA and protein levels generally increased in low-Se groups, while collagen II and TIMP-1 levels showed a downward trend, compared with normal diet fed animals for the same treatment (P < 0.05). T-2 toxin's effect was dose but not time dependent. Low Se and T-2 toxin synergistically alter the expression levels of collagen II as well as its regulatory enzymes MMP-1, MMP-3, MMP-13, and TIMP-1, inducing cartilage damage. Therefore, T-2 toxin may cause KBD in low-Se conditions.

Validation of two new immunoassays for sensative detection of a broad range of shiga toxins

USDA-ARS?s Scientific Manuscript database

The objective of this study was to evaluate two newly developed commercial assays, Abraxis Stx1 and Stx2 enzyme-linked immunosorbent assays (ELISAs), for their ability to detect Shiga toxin (Stx) produced by Escherichia coli (E. coli). The performance of these two assays were compared to a widely us...

In Vitro and In Vivo Activities of Nitazoxanide against Clostridium difficile

PubMed Central

McVay, Catherine S.; Rolfe, Rial D.

2000-01-01

We have used the hamster model of antibiotic-induced Clostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against C. difficile, (ii) toxicity, (iii) ability to prevent C. difficile-associated ileocecitis, and (iv) propensity to induce C. difficile-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficile were comparable to those of vancomycin or metronidazole. C. difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C. difficile in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-C. difficile challenge. During the treatment period, NTZ (≥7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C. difficile intestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of C. difficile disease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C. difficile. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C. difficile intestinal disease, nor was C. difficile detected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal C. difficile intestinal disease in 20 and 70% of recipients, respectively. PMID:10952564

Ancient Venom Systems: A Review on Cnidaria Toxins

PubMed Central

Jouiaei, Mahdokht; Yanagihara, Angel A.; Madio, Bruno; Nevalainen, Timo J.; Alewood, Paul F.; Fry, Bryan G.

2015-01-01

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or “venom” that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design. PMID:26094698

Ancient Venom Systems: A Review on Cnidaria Toxins.

PubMed

Jouiaei, Mahdokht; Yanagihara, Angel A; Madio, Bruno; Nevalainen, Timo J; Alewood, Paul F; Fry, Bryan G

2015-06-18

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or "venom" that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

The integration of nutrients, cyanobacterial biomass and ...

EPA Pesticide Factsheets

This presentation is an integrated evaluation of cyanobacterial growth and toxin production, from a reservoir through drinking water treatment - where biomass and toxin removal are achieved. Data is generated by a variety of methods: online instrumentation for chlorophyll, dissolved oxygen, temperature and pH; enzyme linked immune substrate (ELISA) and liquid chromatography/mass spectrometric (LC/MS) methods for toxin analysis; microscopic methods for species identification; quantitative PCR methods for species identification; and bench-scale engineering studies for removal of toxins and biomass through drinking water treatment. This presentation is an integrated evaluation of cyanobacterial growth and toxin production, from a reservoir through drinking water treatment. The content will be useful for EPA regional office staff, state primacy personnel, state and local health personnel, drinking water treatment managers and consulting engineers.

Discovery of a widely distributed toxin biosynthetic gene cluster

PubMed Central

Lee, Shaun W.; Mitchell, Douglas A.; Markley, Andrew L.; Hensler, Mary E.; Gonzalez, David; Wohlrab, Aaron; Dorrestein, Pieter C.; Nizet, Victor; Dixon, Jack E.

2008-01-01

Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes. PMID:18375757

Clostridium Difficile Infections

MedlinePlus

Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Loss of appetite Nausea Abdominal pain or tenderness C. difficile is more common in people who need ...

Genomic study of the Type IVC secretion system in Clostridium difficile: understanding C. difficile evolution via horizontal gene transfer.

PubMed

Zhang, Wen; Cheng, Ying; Du, Pengcheng; Zhang, Yuanyuan; Jia, Hongbing; Li, Xianping; Wang, Jing; Han, Na; Qiang, Yujun; Chen, Chen; Lu, Jinxing

2017-01-01

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

A case of toxic megacolon caused by clostridium difficile infection and treated with fecal microbiota transplantation.

PubMed

Gweon, Tae Geun; Lee, Kyung Jin; Kang, Dong Hoon; Park, Sung Soo; Kim, Kyung Hoon; Seong, Hyeon Jin; Ban, Tae Hyun; Moon, Sung Jin; Kim, Jin Su; Kim, Sang Woo

2015-03-01

Clostridium difficile infection. The mortality rate of fulminant C. difficile infection is reported to be as high as 50%. Fecal microbiota transplantation is a highly effective treatment in patients with recurrent or refractory C. difficile infection. However, there are few published articles on the use of such transplantation for fulminant C. difficile infection. Here, we report on a patient with toxic megacolon complicated by C. difficile infection who was treated successfully with fecal mi-crobiota transplantation. (Gut Liver, 2015;9:247-250).

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A/sub 1/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gill, D.M.; Coburn, J.

1987-10-06

The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTP..gamma..S), forms over a period of 10-15 min at 37/sup 0/C. Both guanosine 5'-O-(2-thiodiphosphate)more » and GTP block this quasi-permanent activation. Some S x GTP..gamma..S forms in membranes that are exposed to CF alone and then to GTP..gamma..S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTP..gamma..S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45/sup 0/C is decreased by GTP..gamma..S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A/sub 1/ fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified.« less

High Variability in Nosocomial Clostridium difficile Infection Rates Across Hospitals After Colorectal Resection.

PubMed

Aquina, Christopher T; Probst, Christian P; Becerra, Adan Z; Hensley, Bradley J; Iannuzzi, James C; Noyes, Katia; Monson, John R T; Fleming, Fergal J

2016-04-01

Hospital-acquired Clostridium difficile infection is associated with adverse patient outcomes and high medical costs. The incidence and severity of C. difficile has been rising in both medical and surgical patients. Our aim was to assess risk factors and variation associated with the development of nosocomial C. difficile colitis among patients undergoing colorectal resection. This was a retrospective cohort study. The study included segmental colectomy and proctectomy cases in New York State from 2005 to 2013. The study cohort included 150,878 colorectal resections. Patients with a documented previous history of C. difficile infection or residence outside of New York State were excluded. A diagnosis of C. difficile colitis either during the index hospital stay or on readmission within 30 days was the main measure. C. difficile colitis occurred in 3323 patients (2.2%). Unadjusted C. difficile colitis rates ranged from 0% to 11.3% among surgeons and 0% to 6.8% among hospitals. After controlling for patient, surgeon, and hospital characteristics using mixed-effects multivariable analysis, significant unexplained variation in C. difficile rates remained present across hospitals but not surgeons. Patient factors explained only 24% of the total hospital-level variation, and known surgeon and hospital-level characteristics explained an additional 8% of the total hospital-level variation. Therefore, ≈70% of the hospital variation in C. difficile infection rates remained unexplained by captured patient, surgeon, and hospital factors. Furthermore, there was an ≈5-fold difference in adjusted C. difficile rates across hospitals. A limited set of hospital and surgeon characteristics was available. Colorectal surgery patients appear to be at high risk for C. difficile infection, and alarming variation in nosocomial C. difficile infection rates currently exists among hospitals after colorectal resection. Given the high morbidity and cost associated with C. difficile colitis, adopting institutional quality improvement programs and maintaining strict prevention strategies are of the utmost importance.

Structure of the gangrene alpha-toxin: the beauty in the beast.

PubMed

Derewenda, Z S; Martin, T W

1998-08-01

The crystal and molecular structure of the Clostridium perfringens alpha-toxin crowns over a century-long research into the mechanisms of pathogenesis of gas gangrene. The structure reveals a two-domain enzyme, with a catalytic all-helical N-terminal domain, and a C-terminal domain similar in its jelly-roll topology to those found in pancreatic lipase and lipoxygenases.

Structure of a bacterial toxin-activating acyltransferase.

PubMed

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

Do piperacillin/tazobactam and other antibiotics with inhibitory activity against Clostridium difficile reduce the risk for acquisition of C. difficile colonization?

PubMed

Kundrapu, Sirisha; Sunkesula, Venkata C K; Jury, Lucy A; Cadnum, Jennifer L; Nerandzic, Michelle M; Musuuza, Jackson S; Sethi, Ajay K; Donskey, Curtis J

2016-04-18

Systemic antibiotics vary widely in in vitro activity against Clostridium difficile. Some agents with activity against C. difficile (e.g., piperacillin/tazobactam) inhibit establishment of colonization in mice. We tested the hypothesis that piperacillin/tazobactam and other agents with activity against C. difficile achieve sufficient concentrations in the intestinal tract to inhibit colonization in patients. Point-prevalence culture surveys were conducted to compare the frequency of asymptomatic rectal carriage of toxigenic C. difficile among patients receiving piperacillin/tazobactam or other inhibitory antibiotics (e.g. ampicillin, linezolid, carbapenems) versus antibiotics lacking activity against C. difficile (e.g., cephalosporins, ciprofloxacin). For a subset of patients, in vitro inhibition of C. difficile (defined as a reduction in concentration after inoculation of vegetative C. difficile into fresh stool suspensions) was compared among antibiotic treatment groups. Of 250 patients, 32 (13 %) were asymptomatic carriers of C. difficile. In comparison to patients receiving non-inhibitory antibiotics or prior antibiotics within 90 days, patients currently receiving piperacillin/tazobactam were less likely to be asymptomatic carriers (1/36, 3 versus 7/36, 19 and 15/69, 22 %, respectively; P = 0.024) and more likely to have fecal suspensions with in vitro inhibitory activity against C. difficile (20/28, 71 versus 3/11, 27 and 4/26, 15 %; P = 0.03). Patients receiving other inhibitory antibiotics were not less likely to be asymptomatic carriers than those receiving non-inhibitory antibiotics. Our findings suggest that piperacillin/tazobactam achieves sufficient concentrations in the intestinal tract to inhibit C. difficile colonization during therapy.

[Clostridium difficile infection: epidemiology, disease burden and therapy].

PubMed

Gulácsi, László; Kertész, Adrienne; Kopcsóné Németh, Irén; Banai, János; Ludwig, Endre; Prinz, Gyula; Reményi, Péter; Strbák, Bálint; Zsoldiné Urbán, Edit; Baji, Petra; Péntek, Márta; Brodszky, Valentin

2013-07-28

C. difficile causes 25 percent of the antibiotic associated infectious nosocomial diarrhoeas. C. difficile infection is a high-priority problem of public health in each country. The available literature of C. difficile infection's epidemiology and disease burden is limited. Review of the epidemiology, including seasonality and the risk of recurrences, of the disease burden and of the therapy of C. difficile infection. Review of the international and Hungarian literature in MEDLINE database using PubMed up to and including 20th of March, 2012. The incidence of nosocomial C. difficile associated diarrhoea is 4.1/10 000 patient day. The seasonality of C. difficile infection is unproved. 20 percent of the patients have recurrence after metronidazole or vancomycin treatment, and each recurrence increases the chance of a further one. The cost of C. difficile infection is between 130 and 500 thousand HUF (430 € and 1665 €) in Hungary. The importance of C. difficile infection in public health and the associated disease burden are significant. The available data in Hungary are limited, further studies in epidemiology and health economics are required.

Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

PubMed

McClain, Mark S; Cover, Timothy L

2007-04-01

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.

Clostridium difficile colonization in preoperative colorectal cancer patients

PubMed Central

Lv, Yinxiang; Huang, Chen; Sheng, Qinsong; Zhao, Peng; Ye, Julian; Jiang, Weiqin; Liu, Lulu; Song, Xiaojun; Tong, Zhou; Chen, Wenbin; Lin, Jianjiang; Tang, Yi-Wei; Jin, Dazhi; Fang, Weijia

2017-01-01

The entire process of Clostridium difficile colonization to infection develops in large intestine. However, the real colonization pattern of C. difficile in preoperative colorectal cancer patients has not been studied. In this study, 33 C. difficile strains (16.1%) were isolated from stool samples of 205 preoperative colorectal cancer patients. C. difficile colonization rates in lymph node metastasis patients (22.3%) were significantly higher than lymph node negative patients (10.8%) (OR=2.314, 95%CI=1.023-5.235, P =0.025). Meanwhile, patients positive for stool occult blood had lower C. difficile colonization rates than negative patients (11.5% vs. 24.0%, OR=0.300, 95%CI=0.131-0.685, P =0.019). A total of 16 sequence types were revealed by multilocus sequence typing. Minimum spanning tree and time-space cluster analysis indicated that all C. difficile isolates were epidemiologically unrelated. Antibiotic susceptibility testing showed all isolates were susceptible to vancomycin and metronidazole. The results suggested that the prevalence of C. difficile colonization is high in preoperative colorectal cancer patients, and the colonization is not acquired in the hospital. Since lymph node metastasis colorectal cancer patients inevitably require adjuvant chemotherapy and C. difficile infection may halt the ongoing treatment, the call for sustained monitoring of C. difficile in those patients is apparently urgent. PMID:28060753

Clostridium difficile colonization in preoperative colorectal cancer patients.

PubMed

Zheng, Yi; Luo, Yun; Lv, Yinxiang; Huang, Chen; Sheng, Qinsong; Zhao, Peng; Ye, Julian; Jiang, Weiqin; Liu, Lulu; Song, Xiaojun; Tong, Zhou; Chen, Wenbin; Lin, Jianjiang; Tang, Yi-Wei; Jin, Dazhi; Fang, Weijia

2017-02-14

The entire process of Clostridium difficile colonization to infection develops in large intestine. However, the real colonization pattern of C. difficile in preoperative colorectal cancer patients has not been studied. In this study, 33 C. difficile strains (16.1%) were isolated from stool samples of 205 preoperative colorectal cancer patients. C. difficile colonization rates in lymph node metastasis patients (22.3%) were significantly higher than lymph node negative patients (10.8%) (OR=2.314, 95%CI=1.023-5.235, P =0.025). Meanwhile, patients positive for stool occult blood had lower C. difficile colonization rates than negative patients (11.5% vs. 24.0%, OR=0.300, 95%CI=0.131-0.685, P =0.019). A total of 16 sequence types were revealed by multilocus sequence typing. Minimum spanning tree and time-space cluster analysis indicated that all C. difficile isolates were epidemiologically unrelated. Antibiotic susceptibility testing showed all isolates were susceptible to vancomycin and metronidazole. The results suggested that the prevalence of C. difficile colonization is high in preoperative colorectal cancer patients, and the colonization is not acquired in the hospital. Since lymph node metastasis colorectal cancer patients inevitably require adjuvant chemotherapy and C. difficile infection may halt the ongoing treatment, the call for sustained monitoring of C. difficile in those patients is apparently urgent.

Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview

PubMed Central

Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

2012-01-01

The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na+ and K+ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins. PMID:23015776

Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview.

PubMed

Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

2012-08-01

The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na⁺ and K⁺ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins.

Costs of Clostridium difficile infection in pediatric operations: A propensity score-matching analysis.

PubMed

Kulaylat, Afif N; Rocourt, Dorothy V; Podany, Abigail B; Engbrecht, Brett W; Twilley, Marianne; Santos, Mary C; Cilley, Robert E; Hollenbeak, Christopher S; Dillon, Peter W

2017-05-01

The purpose of this analysis was to assess the burden of Clostridium difficile infection in the hospitalized pediatric surgical population and to characterize its influence on the costs of care. There were 313,664 patients age 1-18 years who underwent a general thoracic or abdominal procedure in the Kids' Inpatient Database during 2003, 2006, 2009, and 2012. Logistic regression was used to model factors associated with the development of C difficile infection. A propensity score-matching analysis was performed to evaluate the influence of C difficile infection on mortality, duration of stay, and costs in similar patient cohorts. Population weights were used to estimate the national excess burden of C difficile infection on these outcomes. The overall prevalence of C difficile infection in the sampled cohort was 0.30%, with an increasing trend of C difficile infection over time in non-children's hospitals (P < .001). C difficile infection was associated with younger age, nonelective procedures, increasing comorbidities, and urban teaching hospital status (P < .001). An estimated 1,438 children developed C difficile infection after operation. After propensity score matching, the mean excess duration of stay and costs attributable to C difficile infection were 5.8 days and $12,801 (P < .001), accounting for 8,295 days spent in the hospital and $18.4 million (2012 USD) in spending annually. C difficile infection is a relatively uncommon but costly complication after pediatric operative procedures. Given the increasing trend of C difficile infection among hospitalized surgical patients, there is substantial opportunity for reduction of inpatient burden and associated costs in this potentially preventable nosocomial infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

PubMed

Pyo, Dongjin; Hahn, Jong Hoon

2009-01-01

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

RNA repair: an antidote to cytotoxic eukaryal RNA damage.

PubMed

Nandakumar, Jayakrishnan; Schwer, Beate; Schaffrath, Raffael; Shuman, Stewart

2008-07-25

RNA healing and sealing enzymes drive informational and stress response pathways entailing repair of programmed 2',3' cyclic PO(4)/5'-OH breaks. Fungal, plant, and phage tRNA ligases use different strategies to discriminate the purposefully broken ends of the anticodon loop. Whereas phage ligase recognizes the tRNA fold, yeast and plant ligases do not and are instead hardwired to seal only the tRNA 3'-OH, 2'-PO(4) ends formed by healing of a cyclic phosphate. tRNA anticodon damage inflicted by secreted ribotoxins such as fungal gamma-toxin underlies a rudimentary innate immune system. Yeast cells are susceptible to gamma-toxin because the sealing domain of yeast tRNA ligase is unable to rectify a break at the modified wobble base of tRNA(Glu(UUC)). Plant andphage tRNA repair enzymes protect yeast from gamma-toxin because they are able to reverse the damage. Our studies underscore how a ribotoxin exploits an Achilles' heel in the target cell's tRNA repair system.

Evaluation of eight cephalosporins in hamster colitis model.

PubMed Central

Ebright, J R; Fekety, R; Silva, J; Wilson, K H

1981-01-01

Eight commonly used cephalosporins were evaluated in the hamster colitis mode. They were all found to cause hemorrhagic cecitis and death within 10 days of being given as subcutaneous or oral challenges. Necropsy findings were indistinguishable from clindamycin-induced cecitis. Bacteria-free cecal filtrate obtained from hamsters dying of cephalosporin-induced cecitis contained toxin similar or identical to hat produced by Clostridium difficile isolated from the cecum of a hamster. Daily oral administration of poorly absorbed cephalosporins protected hamsters from clindamycin-induced cecitis and death as long as the cephalosporins were continued. The absorbable cephalosporins were ineffective in protecting hamsters from clindamycin-induced cecitis. This difference probably relates to the lower concentrations of absorbable cephalosporins maintained in the ceca of the hamsters. The possible correlation of these findings to human cases of cephalosporin-induced pseudomembranous colitis is discussed. PMID:6973951

Gonzalez Regimen (PDQ®)—Health Professional Version

Cancer.gov

The Gonzalez regimen is a specialized diet that uses enzymes, supplements, and other factors in cancer management. It is based on a theory that involves the use of pancreatic enzymes to help the body get rid of toxins that lead to cancer. Read about existing clinical data in this expert-reviewed summary.

The effect of hospital-acquired infection with Clostridium difficile on length of stay in hospital.

PubMed

Forster, Alan J; Taljaard, Monica; Oake, Natalie; Wilson, Kumanan; Roth, Virginia; van Walraven, Carl

2012-01-10

The effect of hospital-acquired infection with Clostridium difficile on length of stay in hospital is not yet fully understood. We determined the independent impact of hospital-acquired infection with C. difficile on length of stay in hospital. We conducted a retrospective observational cohort study of admissions to hospital between July 1, 2002, and Mar. 31, 2009, at a single academic hospital. We measured the association between infection with hospital-acquired C. difficile and time to discharge from hospital using Kaplan-Meier methods and a Cox multivariable proportional hazards regression model. We controlled for baseline risk of death and accounted for C. difficile as a time-varying effect. Hospital-acquired infection with C. difficile was identified in 1393 of 136,877 admissions to hospital (overall risk 1.02%, 95% confidence interval [CI] 0.97%-1.06%). The crude median length of stay in hospital was greater for patients with hospital-acquired C. difficile (34 d) than for those without C. difficile (8 d). Survival analysis showed that hospital-acquired infection with C. difficile increased the median length of stay in hospital by six days. In adjusted analyses, hospital-acquired C. difficile was significantly associated with time to discharge, modified by baseline risk of death and time to acquisition of C. difficile. The hazard ratio for discharge by day 7 among patients with hospital-acquired C. difficile was 0.55 (95% CI 0.39-0.70) for patients in the lowest decile of baseline risk of death and 0.45 (95% CI 0.32-0.58) for those in the highest decile; for discharge by day 28, the corresponding hazard ratios were 0.74 (95% CI 0.60-0.87) and 0.61 (95% CI 0.53-0.68). Hospital-acquired infection with C. difficile significantly prolonged length of stay in hospital independent of baseline risk of death.

Interobserver variability and feasibility of polymerase chain reaction-based assay in distinguishing ischemic colitis from Clostridium difficile colitis in endoscopic mucosal biopsies.

PubMed

Wiland, Homer O; Procop, Gary W; Goldblum, John R; Tuohy, Marion; Rybicki, Lisa; Patil, Deepa T

2013-06-01

Polymerase chain reaction (PCR)-based assays using stool samples are currently the most effective method of detecting Clostridium difficile. This study examines the feasibility of this assay using mucosal biopsy samples and evaluates the interobserver reproducibility in diagnosing and distinguishing ischemic colitis from C difficile colitis. Thirty-eight biopsy specimens were reviewed and classified by 3 observers into C difficile and ischemic colitis. The findings were correlated with clinical data. PCR was performed on 34 cases using BD GeneOhm C difficile assay. The histologic interobserver agreement was excellent (κ= 0.86) and the agreement between histologic and clinical diagnosis was good (κ = 0.84). All 19 ischemic colitis cases tested negative (100% specificity) and 3 of 15 cases of C difficile colitis tested positive (20% sensitivity). C difficile colitis can be reliably distinguished from ischemic colitis using histologic criteria. The C difficile PCR test on endoscopic biopsy specimens has excellent specificity but limited sensitivity.

Review article: Anti-inflammatory mechanisms of action of Saccharomyces boulardii

PubMed Central

Pothoulakis, C.

2009-01-01

SUMMARY Background Saccharomyces boulardii (S. boulardii), a well-studied probiotic, can be effective in inflammatory gastrointestinal diseases with diverse pathophysiology, such as Inflammatory Bowel Disease (IBD), and bacterially – or enterotoxin-mediated diarrhea and inflammation. Aim Discuss the mechanisms of action involved in the intestinal anti-inflammatory action of S. boulardii Methods Review of the literature related to the anti-inflammatory effects of this probiotic. Results Several mechanisms of action have been identified directed against the host and pathogenic microorganisms. S. boulardii and S. boulardii secreted protein(s) inhibit production of proinflammatory cytokines by interfering with the global mediator of inflammation nuclear factor κB, and modulating the activity of the mitogen-activated protein kinases ERK1/2 and p38. S. boulardii activates expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) that protects from gut inflammation and IBD. S. boulardii also suppresses “bacteria overgrowth” and host cell adherence, releases a protease that cleaves C. difficile toxin A and its intestinal receptor, and stimulates antibody production against toxin A. Recent results indicate that S. boulardii may interfere with IBD pathogenesis by trapping T cells in mesenteric lymph nodes. Conclusions The multiple anti-inflammatory mechanisms exerted by S. boulardii provide molecular explanations supporting its effectiveness in intestinal inflammatory states. PMID:19706150

The potential value of Clostridium difficile vaccine: an economic computer simulation model.

PubMed

Lee, Bruce Y; Popovich, Michael J; Tian, Ye; Bailey, Rachel R; Ufberg, Paul J; Wiringa, Ann E; Muder, Robert R

2010-07-19

Efforts are currently underway to develop a vaccine against Clostridium difficile infection (CDI). We developed two decision analytic Monte Carlo computer simulation models: (1) an Initial Prevention Model depicting the decision whether to administer C. difficile vaccine to patients at-risk for CDI and (2) a Recurrence Prevention Model depicting the decision whether to administer C. difficile vaccine to prevent CDI recurrence. Our results suggest that a C. difficile vaccine could be cost-effective over a wide range of C. difficile risk, vaccine costs, and vaccine efficacies especially, when being used post-CDI treatment to prevent recurrent disease. (c) 2010 Elsevier Ltd. All rights reserved.

The Potential Value of Clostridium difficile Vaccine: An Economic Computer Simulation Model

PubMed Central

Lee, Bruce Y.; Popovich, Michael J.; Tian, Ye; Bailey, Rachel R.; Ufberg, Paul J.; Wiringa, Ann E.; Muder, Robert R.

2010-01-01

Efforts are currently underway to develop a vaccine against Clostridium difficile infection (CDI). We developed two decision analytic Monte Carlo computer simulation models: (1) an Initial Prevention Model depicting the decision whether to administer C. difficile vaccine to patients at-risk for CDI and (2) a Recurrence Prevention Model depicting the decision whether to administer C. difficile vaccine to prevent CDI recurrence. Our results suggest that a C. difficile vaccine could be cost-effective over a wide range of C. difficile risk, vaccine costs, and vaccine efficacies especially when being used post-CDI treatment to prevent recurrent disease. PMID:20541582

Neutrophil-mediated inflammation in the pathogenesis of Clostridium difficile infections

PubMed Central

Jose, Shinsmon; Madan, Rajat

2016-01-01

Clostridium difficile is the most important cause of nosocomial infectious diarrhea in the western world. C. difficile infections are a major healthcare burden with approximately 500,000 new cases every year and an estimated annual cost of nearly $1 billion in the U.S. Furthermore, the infections are no longer restricted to health care facilities, and recent studies indicate spread of C. difficile infection to the community as well. The clinical spectrum of C. difficile infection ranges from asymptomatic colonization to severe diarrhea, fulminant colitis and death. This spectrum results from a complex interplay between bacterial virulence factors, the colonic microbiome and the host inflammatory response. The overall vigor of host inflammatory response is believed to be an important determinant of C. difficile disease severity, and a more robust immune response is associated with worse outcomes. Neutrophils are the primary cells that respond to C. difficile invasion and neutrophilic inflammation is the hallmark of C. difficile-associated disease. In this review, we will focus on the role of neutrophils (infiltration to infected tissue, pathogen clearance and resolution of inflammation) in the immuno-pathogenesis of C. difficile-associated disease (CDAD). PMID:27063896

Neutrophil-mediated inflammation in the pathogenesis of Clostridium difficile infections.

PubMed

Jose, Shinsmon; Madan, Rajat

2016-10-01

Clostridium difficile is the most important cause of nosocomial infectious diarrhea in the western world. C. difficile infections are a major healthcare burden with approximately 500,000 new cases every year and an estimated annual cost of nearly $1 billion in the U.S. Furthermore, the infections are no longer restricted to health care facilities, and recent studies indicate spread of C. difficile infection to the community as well. The clinical spectrum of C. difficile infection ranges from asymptomatic colonization to severe diarrhea, fulminant colitis and death. This spectrum results from a complex interplay between bacterial virulence factors, the colonic microbiome and the host inflammatory response. The overall vigor of host inflammatory response is believed to be an important determinant of C. difficile disease severity, and a more robust immune response is associated with worse outcomes. Neutrophils are the primary cells that respond to C. difficile invasion and neutrophilic inflammation is the hallmark of C. difficile-associated disease. In this review, we will focus on the role of neutrophils (infiltration to infected tissue, pathogen clearance and resolution of inflammation) in the immuno-pathogenesis of C. difficile-associated disease (CDAD). Copyright © 2016 Elsevier Ltd. All rights reserved.

Membrane lipid physiology and toxin catabolism underlie ethanol and acetic acid tolerance in Drosophila melanogaster.

PubMed

Montooth, Kristi L; Siebenthall, Kyle T; Clark, Andrew G

2006-10-01

Drosophila melanogaster has evolved the ability to tolerate and utilize high levels of ethanol and acetic acid encountered in its rotting-fruit niche. Investigation of this phenomenon has focused on ethanol catabolism, particularly by the enzyme alcohol dehydrogenase. Here we report that survival under ethanol and acetic acid stress in D. melanogaster from high- and low-latitude populations is an integrated consequence of toxin catabolism and alteration of physical properties of cellular membranes by ethanol. Metabolic detoxification contributed to differences in ethanol tolerance between populations and acclimation temperatures via changes in both alcohol dehydrogenase and acetyl-CoA synthetase mRNA expression and enzyme activity. Independent of changes in ethanol catabolism, rapid thermal shifts that change membrane fluidity had dramatic effects on ethanol tolerance. Cold temperature treatments upregulated phospholipid metabolism genes and enhanced acetic acid tolerance, consistent with the predicted effects of restoring membrane fluidity. Phospholipase D was expressed at high levels in all treatments that conferred enhanced ethanol tolerance, suggesting that this lipid-mediated signaling enzyme may enhance tolerance by sequestering ethanol in membranes as phophatidylethanol. These results reveal new candidate genes underlying toxin tolerance and membrane adaptation to temperature in Drosophila and provide insight into how interactions between these phenotypes may underlie the maintenance of latitudinal clines in ethanol tolerance.

Presence of Clostridium difficile in poultry and poultry meat in Egypt.

PubMed

Abdel-Glil, Mostafa Y; Thomas, Prasad; Schmoock, Gernot; Abou-El-Azm, Kamel; Wieler, Lothar H; Neubauer, Heinrich; Seyboldt, Christian

2018-06-01

C. difficile has been recognized as a potential zoonotic agent encouraging investigations of C. difficile prevalence and ribotypes in animals. Here we report the prevalence and diversity of Egyptian C. difficile in I) samples from healthy poultry (n = 50), II) samples from diseased poultry (n = 54), and III) poultry meat (n = 150). Thirteen isolates were obtained from seven healthy and five diseased animals, but no C. difficile was cultured from poultry meat. The isolated C. difficile strains belonged to 3 different PCR-ribotypes (039/2, 205 and 001/FLI01). The detection of strains related to RT 001 known for its ability to cause disease in humans makes poultry a potential reservoir for pathogenic C. difficile. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Protein crystallography and infectious diseases.

PubMed Central

Verlinde, C. L.; Merritt, E. A.; Van den Akker, F.; Kim, H.; Feil, I.; Delboni, L. F.; Mande, S. C.; Sarfaty, S.; Petra, P. H.; Hol, W. G.

1994-01-01

The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man. We present here 2 illustrations of structure-based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity. Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure-based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen. PMID:7849584

An Evaluation of Food as a Potential Source for Clostridium difficile Acquisition in Hospitalized Patients.

PubMed

Kwon, Jennie H; Lanzas, Cristina; Reske, Kimberly A; Hink, Tiffany; Seiler, Sondra M; Bommarito, Kerry M; Burnham, Carey-Ann D; Dubberke, Erik R

2016-12-01

OBJECTIVE To determine whether Clostridium difficile is present in the food of hospitalized patients and to estimate the risk of subsequent colonization associated with C. difficile in food. METHODS This was a prospective cohort study of inpatients at a university-affiliated tertiary care center, May 9, 2011-July 12, 2012. Enrolled patients submitted a portion of food from each meal. Patient stool specimens and/or rectal swabs were collected at enrollment, every 3 days thereafter, and at discharge, and were cultured for C. difficile. Clinical data were reviewed for evidence of infection due to C. difficile. A stochastic, discrete event model was developed to predict exposure to C. difficile from food, and the estimated number of new colonization events from food exposures per 1,000 admissions was determined. RESULTS A total of 149 patients were enrolled and 910 food specimens were obtained. Two food specimens from 2 patients were positive for C. difficile (0.2% of food samples; 1.3% of patients). Neither of the 2 patients was colonized at baseline with C. difficile. Discharge colonization status was available for 1 of the 2 patients and was negative. Neither was diagnosed with C. difficile infection while hospitalized or during the year before or after study enrollment. Stochastic modeling indicated contaminated hospital food would be responsible for less than 1 newly colonized patient per 1,000 hospital admissions. CONCLUSIONS The recovery of C. difficile from the food of hospitalized patients was rare. Modeling suggests hospital food is unlikely to be a source of C. difficile acquisition. Infect Control Hosp Epidemiol 2016;1401-1407.

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

The action of the bacterial toxin, microcin B17, on DNA gyrase.

PubMed

Parks, William M; Bottrill, Andrew R; Pierrat, Olivier A; Durrant, Marcus C; Maxwell, Anthony

2007-04-01

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

PubMed Central

Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.

1998-01-01

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463

Determination of the median toxic dose of type C botulism in lactating dairy cows

USGS Publications Warehouse

Moeller, R.B.; Puschner, B.; Walker, R.L.; Rocke, Tonie E.; Galey, F.D.; Cullor, J.S.; Ardans, A.A.

2003-01-01

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

NASA Astrophysics Data System (ADS)

Lange, Adam; Giller, Karin; Hornig, Sönke; Martin-Eauclaire, Marie-France; Pongs, Olaf; Becker, Stefan; Baldus, Marc

2006-04-01

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane-similar to the catalytic function of the active site of an enzyme-and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa).

PubMed

Ponce, Dalia; Brinkman, Diane L; Potriquet, Jeremy; Mulvenna, Jason

2016-04-05

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.

Clostridium difficile infection in the twenty-first century

PubMed Central

Ghose, Chandrabali

2013-01-01

Clostridium difficile is a spore-forming gram-positive bacillus, and the leading cause of antibiotic-associated nosocomial diarrhea and colitis in the industrialized world. With the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027), the epidemiology of C. difficile infection has rapidly changed in the last decade. C. difficile infection, once thought to be an easy to treat bacterial infection, has evolved into an epidemic that is associated with a high rate of mortality, causing disease in patients thought to be low-risk. In this review, we discuss the changing face of C .difficile infection and the novel treatment and prevention strategies needed to halt this ever growing epidemic. PMID:26038491

Clostridium difficile infection: Evolution, phylogeny and molecular epidemiology.

PubMed

Elliott, Briony; Androga, Grace O; Knight, Daniel R; Riley, Thomas V

2017-04-01

Over the recent decades, Clostridium difficile infection (CDI) has emerged as a global public health threat. Despite growing attention, C. difficile remains a poorly understood pathogen, however, the exquisite sensitivity offered by next generation sequencing (NGS) technology has enabled analysis of the genome of C. difficile, giving us access to massive genomic data on factors such as virulence, evolution, and genetic relatedness within C. difficile groups. NGS has also demonstrated excellence in investigations of outbreaks and disease transmission, in both small and large-scale applications. This review summarizes the molecular epidemiology, evolution, and phylogeny of C. difficile, one of the most important pathogens worldwide in the current antibiotic resistance era. Copyright © 2016 Elsevier B.V. All rights reserved.

Clostridium difficile-associated reactive arthritis in two children.

PubMed

Löffler, Helga A; Pron, Benedicte; Mouy, Richard; Wulffraat, Nico M; Prieur, Anne-Marie

2004-01-01

In adults, reactive arthritis (ReA) following Clostridium difficile-enterocolitis has been documented. In children, only one case of C. difficile-associated ReA has been reported. We now describe two other cases of ReA associated with C. difficile in children. The characteristics of ReA due to C. difficile appear to be similar in adults and children. Both children show polyarthritis after an episode of diarrhoea with positive stool cultures for C. difficile. Arthritis is asymmetrical with a self-limiting course. Nonsteroidal antiinflammatory drug (NSAID) therapy is sufficient. One case is remarkable because of its prolonged course of ReA despite NSAID therapy, and its association with the presence of HLA-B27 antigen.

Clostridium difficile Drug Pipeline: Challenges in Discovery and Development of New Agents

PubMed Central

2015-01-01

In the past decade Clostridium difficile has become a bacterial pathogen of global significance. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals. In response to the increasing medical burden, a new C. difficile antibiotic, fidaxomicin, was approved in 2011 for the treatment of C. difficile-associated diarrhea. Rudimentary fecal transplants are also being trialed as effective treatments. Despite these advances, therapies that are more effective against C. difficile spores and less damaging to the resident gastrointestinal microbiome and that reduce recurrent disease are still desperately needed. However, bringing a new treatment for C. difficile infection to market involves particular challenges. This review covers the current drug discovery pipeline, including both small molecule and biologic therapies, and highlights the challenges associated with in vitro and in vivo models of C. difficile infection for drug screening and lead optimization. PMID:25760275

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

PubMed

Curry, Scott R; Marsh, Jane W; Schlackman, Jessica L; Harrison, Lee H

2012-06-01

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.

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

PubMed Central

Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

2015-01-01

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

The Ecology and Pathobiology of Clostridium difficile Infections: An Interdisciplinary Challenge

PubMed Central

Dubberke, Erik R.; Haslam, David B.; Lanzas, Cristina; Bobo, Linda D.; Burnham, Carey-Ann D.; Gröhn, Yrjö T.; Tarr, Phillip I.

2013-01-01

Summary Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, aging of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen. PMID:21223531

The epidemiology and economic burden of Clostridium difficile infection in Korea.

PubMed

Choi, Hyung-Yun; Park, So-Youn; Kim, Young-Ae; Yoon, Tai-Young; Choi, Joong-Myung; Choe, Bong-Keun; Ahn, So-Hee; Yoon, Seok-Jun; Lee, Ye-Rin; Oh, In-Hwan

2015-01-01

The prevalence of Clostridium difficile infection and the associated burden have recently increased in many countries. While the main risk factors for C. difficile infection include old age and antibiotic use, the prevalence of this infection is increasing in low-risk groups. These trends highlight the need for research on C. difficile infection. This study pointed out the prevalence and economic burden of C. difficile infection and uses the representative national data which is primarily from the database of the Korean Health Insurance Review and Assessment Service, for 2008-2011. The annual economic cost was measured using a prevalence approach, which sums the costs incurred to treat C. difficile infection. C. difficile infection prevalence was estimated to have increased from 1.43 per 100,000 in 2008 to 5.06 per 100,000 in 2011. Moreover, mortality increased from 69 cases in 2008 to 172 in 2011. The economic cost increased concurrently, from $2.4 million in 2008 to $7.6 million, $10.5 million, and $15.8 million in 2009, 2010, and 2011, respectively. The increasing economic burden of C. difficile infection over the course of the study period emphasizes the need for intervention to minimize the burden of a preventable illness like C. difficile infection.

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (5)--Six-month repeated oral dose toxicity study and supplement study in rats].

PubMed

Sameshima, H; Omori, M; Nishimura, Y; Chihaya, Y; Itoh, F; Mizushima, Y; Yabuuchi, K; Ohno, K; Furukawa, H; Yoshida, I; Ueno, M; Yahara, I; Kato, I

2001-05-01

Cefmatilen hydrochloride hydrate (S-1090) was orally administered to rats at dose levels of 100, 300 and 1000 mg potency/kg once daily for 6 months. All the S-1090 treated groups showed soft feces, reddish-brown feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet), abdominal distention, increased food and water consumption, lower urine pH, and a decrease of white blood cells counts (except for males of the 100 mg potency/kg group). One male in the 300 mg potency/kg group showed mucous feces and marked decrease in body weight, and diet in the middle stage of the administration period. In necropsy of the survivors of all treated groups, marked cecal enlargement was noted. No remarkable changes were observed in the other examination items. From the early stage of the withdrawal period, animals in the 1000 mg potency/kg group showed again soft or mucous feces and a marked decrease in body weight. Of these animals, one male died and another male was sacrificed in a moribund state at about 2 weeks of the withdrawal period. Enterocolitis was observed in these cases. Almost all animals recovered within 3 weeks of withdrawal. A supplemental study of the 6-month toxicity study was conducted to examine the mechanisms of enterocolitis and the changes observable in the 100 or 300 mg potency/kg groups after drug withdrawal. As a reference, cefdinir (CFDN), an oral cephem antibiotic the same as S-1090, was added in the 1000 mg potency/kg group. No deaths occurred in any groups. Decreased intestinal flora were noted in all the groups treated with S-1090 or CFDN at the end of the dosing period. At 2 weeks of the withdrawal period, C. difficile and its D-1 toxin in the cecal contents were highly detected in the S-1090 300 and 1000 mg potency/kg groups and CFDN group. Inflammatory changes in the cecum and colon were observed in these groups. At 4 weeks of the withdrawal period, intestinal flora in the S-1090 groups almost returned to the condition before dosing, but those in the CFDN group were retained highly. Cecal D-1 toxin in the CFDN group was positive and higher than in the S-1090 groups. It was thus considered that the critical condition with enterocolitis resulted from C. difficile, which proliferated more rapidly than the other bacteria and D-1 toxin produced by this bacteria in the withdrawal period. Above changes were commonly observed in the CFDN group. The NOAEL of S-1090 was assessed to be 100 mg potency/kg/day which induced no enteritis.

Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).

PubMed

Leira, F; Vieites, J M; Vieytes, M R; Botana, L M

2000-12-01

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.

[Shellfish monitoring system for paralytic shellfish toxins using enzyme-linked immunosorbent assay].

PubMed

Shinozaki, Takashi; Watanabe, Ryuichi; Kawatsu, Kentaro; Sakurada, Kiyonari; Takahi, Shinya; Ueno, Ken-ichi; Matsushima, Ryoji; Suzuki, Toshiyuki

2013-01-01

We investigated the applicability of enzyme-linked immunosorbent assay (PSP-ELISA) using a monoclonal antibody against paralytic shellfish toxins (PST) for screening oysters collected at several coastal areas in Kumamoto prefecture, Japan. Oysters collected between 2007 and 2010 were analyzed by PSP-ELISA. As an alternative calibrant, a naturally contaminated oyster extract was used to quantify toxins in the oyster samples. The toxicity of the calibrant oyster extract determined by the official testing method, mouse bioassay (MBA), was 4 MU/g. Oyster samples collected over 3 years showed a similar toxin profile to the alternative standard, resulting in good agreement between the PSP-ELISA and the MBA. The PSP-ELISA method was better than the MBA in terms of sensitivity, indicating that it may be useful for earlier warning of contamination of oysters by PST in the distinct coastal areas. To use the PSP-ELISA as a screening method prior to MBA, we finally set a screening level at 2 MU/g PSP-ELISA for oyster monitoring in Kumamoto prefecture. We confirmed that there were on samples exceeding the quarantine level (4 MU/g) in MBA among samples quantified as below the screening level by the PSP-ELISA. It was concluded that the use of PSP-ELISA could reduce the numbers of animals needed for MBA testing.

Comparative Effectiveness of Vancomycin and Metronidazole for the Prevention of Recurrence and Death in Patients With Clostridium difficile Infection.

PubMed

Stevens, Vanessa W; Nelson, Richard E; Schwab-Daugherty, Elyse M; Khader, Karim; Jones, Makoto M; Brown, Kevin A; Greene, Tom; Croft, Lindsay D; Neuhauser, Melinda; Glassman, Peter; Goetz, Matthew Bidwell; Samore, Matthew H; Rubin, Michael A

2017-04-01

Metronidazole hydrochloride has historically been considered first-line therapy for patients with mild to moderate Clostridium difficile infection (CDI) but is inferior to vancomycin hydrochloride for clinical cure. The choice of therapy may likewise have substantial consequences on other downstream outcomes, such as recurrence and mortality, although these secondary outcomes have been less studied. To evaluate the risk of recurrence and all-cause 30-day mortality among patients receiving metronidazole or vancomycin for the treatment of mild to moderate and severe CDI. This retrospective, propensity-matched cohort study evaluated patients treated for CDI, defined as a positive laboratory test result for the presence of C difficile toxins or toxin genes in a stool sample, in the US Department of Veterans Affairs health care system from January 1, 2005, through December 31, 2012. Data analysis was performed from February 7, 2015, through November 22, 2016. Treatment with vancomycin or metronidazole. The outcomes of interest in this study were CDI recurrence and all-cause 30-day mortality. Recurrence was defined as a second positive laboratory test result within 8 weeks of the initial CDI diagnosis. All-cause 30-day mortality was defined as death from any cause within 30 days of the initial CDI diagnosis. A total of 47 471 patients (mean [SD] age, 68.8 [13.3] years; 1947 women [4.1%] and 45 524 men [95.9%]) developed CDI, were treated with vancomycin or metronidazole, and met criteria for entry into the study. Of 47 147 eligible first treatment episodes, 2068 (4.4%) were with vancomycin. Those 2068 patients were matched to 8069 patients in the metronidazole group for a total of 10 137 included patients. Subcohorts were constructed that comprised 5452 patients with mild to moderate disease and 3130 patients with severe disease. There were no differences in the risk of recurrence between patients treated with vancomycin vs those treated with metronidazole in any of the disease severity cohorts. Among patients in the any severity cohort, those who were treated with vancomycin were less likely to die (adjusted relative risk, 0.86; 95% CI, 0.74 to 0.98; adjusted risk difference, -0.02; 95% CI, -0.03 to -0.01). No significant difference was found in the risk of mortality between treatment groups among patients with mild to moderate CDI, but vancomycin significantly reduced the risk of all-cause 30-day mortality among patients with severe CDI (adjusted relative risk, 0.79; 95% CI, 0.65 to 0.97; adjusted risk difference, -0.04; 95% CI, -0.07 to -0.01). Recurrence rates were similar among patients treated with vancomycin and metronidazole. However, the risk of 30-day mortality was significantly reduced among patients who received vancomycin. Our findings may further justify the use of vancomycin as initial therapy for severe CDI.

Management of Clostridium difficile Infection in Inflammatory Bowel Disease: Expert Review from the Clinical Practice Updates Committee of the AGA Institute.

PubMed

Khanna, Sahil; Shin, Andrea; Kelly, Ciarán P

2017-02-01

The purpose of this expert review is to synthesize the existing evidence on the management of Clostridium difficile infection in patients with underlying inflammatory bowel disease. The evidence reviewed in this article is a summation of relevant scientific publications, expert opinion statements, and current practice guidelines. This review is a summary of expert opinion in the field without a formal systematic review of evidence. Best Practice Advice 1: Clinicians should test patients who present with a flare of underlying inflammatory bowel disease for Clostridium difficile infection. Best Practice Advice 2: Clinicians should screen for recurrent C difficile infection if diarrhea or other symptoms of colitis persist or return after antibiotic treatment for C difficile infection. Best Practice Advice 3: Clinicians should consider treating C difficile infection in inflammatory bowel disease patients with vancomycin instead of metronidazole. Best Practice Advice 4: Clinicians strongly should consider hospitalization for close monitoring and aggressive management for inflammatory bowel disease patients with C difficile infection who have profuse diarrhea, severe abdominal pain, a markedly increased peripheral blood leukocyte count, or other evidence of sepsis. Best Practice Advice 5: Clinicians may postpone escalation of steroids and other immunosuppression agents during acute C difficile infection until therapy for C difficile infection has been initiated. However, the decision to withhold or continue immunosuppression in inflammatory bowel disease patients with C difficile infection should be individualized because there is insufficient existing robust literature on which to develop firm recommendations. Best Practice Advice 6: Clinicians should offer a referral for fecal microbiota transplantation to inflammatory bowel disease patients with recurrent C difficile infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Clostridium difficile phages: still difficult?

PubMed Central

Hargreaves, Katherine R.; Clokie, Martha R. J.

2014-01-01

Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

Cellular Uptake of the Clostridium perfringens Binary Iota-Toxin

PubMed Central

Blöcker, Dagmar; Behlke, Joachim; Aktories, Klaus; Barth, Holger

2001-01-01

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor. PMID:11292715

The association of hospital prevention processes and patient risk factors with the risk of Clostridium difficile infection: a population-based cohort study.

PubMed

Daneman, N; Guttmann, A; Wang, X; Ma, X; Gibson, D; Stukel, T A

2015-07-01

Clostridium difficile is the most common cause of healthcare-acquired infection; the real-world impacts of some proposed C. difficile prevention processes are unknown. We conducted a population-based retrospective cohort study of all patients admitted to acute care hospitals between April 2011 and March 2012 in Ontario, Canada. Hospital prevention practices were determined by survey of infection control programmes; responses were linked to patient-level risk factors and C. difficile outcomes in Ontario administrative databases. Multivariable generalised estimating equation (GEE) regression models were used to assess the impact of selected understudied hospital prevention processes on the patient-level risk of C. difficile infection, accounting for patient risk factors, baseline C. difficile rates and structural hospital characteristics. C. difficile infections complicated 2341 of 653 896 admissions (3.6 per 1000 admissions). Implementation of the selected C. difficile prevention practices was variable across the 159 hospitals with isolation of all patients at onset of diarrhoea reported by 43 (27%), auditing of antibiotic stewardship compliance by 26 (16%), auditing of cleaning practices by 115 (72%), on-site diagnostic testing by 74 (47%), vancomycin as first-line treatment by 24 (15%) and reporting rates to senior leadership by 52 (33%). None of these processes were associated with a significantly reduced risk of C. difficile after adjustment for baseline C. difficile rates, structural hospital characteristics and patient-level factors. Patient-level factors were strongly associated with C. difficile risk, including age, comorbidities, non-elective and medical admissions. In the largest study to date, selected hospital prevention strategies were not associated with a statistically significant reduction in patients' risk of C. difficile infection. These prevention strategies have either limited effectiveness or were ineffectively implemented during the study period. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

CodY-Dependent Regulation of Sporulation in Clostridium difficile.

PubMed

Nawrocki, Kathryn L; Edwards, Adrianne N; Daou, Nadine; Bouillaut, Laurent; McBride, Shonna M

2016-08-01

Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

CodY-Dependent Regulation of Sporulation in Clostridium difficile

PubMed Central

Nawrocki, Kathryn L.; Edwards, Adrianne N.; Daou, Nadine; Bouillaut, Laurent

2016-01-01

ABSTRACT Clostridium difficile must form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation in C. difficile remain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation in C. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation in C. difficile strains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation in C. difficile. The 027 codY mutant exhibited a greater increase in spore formation than the 012 codY mutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation in C. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation, opp and sinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation in C. difficile and that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked in C. difficile. IMPORTANCE This study sought to examine the relationship between nutrition and sporulation in C. difficile by examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator of C. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes of C. difficile. We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation in C. difficile and that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains. PMID:27246573

Underdiagnosis of Clostridium difficile across Europe: the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID).

PubMed

Davies, Kerrie A; Longshaw, Christopher M; Davis, Georgina L; Bouza, Emilio; Barbut, Frédéric; Barna, Zsuzsanna; Delmée, Michel; Fitzpatrick, Fidelma; Ivanova, Kate; Kuijper, Ed; Macovei, Ioana S; Mentula, Silja; Mastrantonio, Paola; von Müller, Lutz; Oleastro, Mónica; Petinaki, Efthymia; Pituch, Hanna; Norén, Torbjörn; Nováková, Elena; Nyč, Otakar; Rupnik, Maja; Schmid, Daniela; Wilcox, Mark H

2014-12-01

Variations in testing for Clostridium difficile infection can hinder patients' care, increase the risk of transmission, and skew epidemiological data. We aimed to measure the underdiagnosis of C difficile infection across Europe. We did a questionnaire-based study at 482 participating hospitals across 20 European countries. Hospitals were questioned about their methods and testing policy for C difficile infection during the periods September, 2011, to August, 2012, and September, 2012, to August, 2013. On one day in winter, 2012-13 (December, 2012, or January, 2013), and summer, 2013 (July or August), every hospital sent all diarrhoeal samples submitted to their microbiology laboratory to a national coordinating laboratory for standardised testing of C difficile infection. Our primary outcome measures were the rates of testing for and cases of C difficile infection per 10 000 patient bed-days. Results of local and national C difficile infection testing were compared with each other. If the result was positive at the national laboratory but negative at the local hospital, the result was classified as undiagnosed C difficile infection. We compared differences in proportions with the Mann-Whitney test, or McNemar's test if data were matched. During the study period, participating hospitals reported a mean of 65·8 tests (country range 4·6-223·3) for C difficile infection per 10 000 patient-bed days and a mean of 7·0 cases (country range 0·7-28·7) of C difficile infection per 10 000 patient-bed days. Only two-fifths of hospitals reported using optimum methods for testing of C difficile infection (defined by European guidelines), although the number of participating hospitals using optimum methods increased during the study period, from 152 (32%) of 468 in 2011-12 to 205 (48%) of 428 in 2012-13. Across all 482 European hospitals on the two sampling days, 148 (23%) of 641 samples positive for C difficile infection (as determined by the national laboratory) were not diagnosed by participating hospitals because of an absence of clinical suspicion, equating to about 74 missed diagnoses per day. A wide variety of testing strategies for C difficile infection are used across Europe. Absence of clinical suspicion and suboptimum laboratory diagnostic methods mean that an estimated 40 000 inpatients with C difficile infection are potentially undiagnosed every year in 482 European hospitals. Astellas Pharmaceuticals Europe. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prevalence of Clostridium difficile infection in acute care hospitals, long-term care facilities, and outpatient clinics: Is Clostridium difficile infection underdiagnosed in long-term care facility patients?

PubMed

Krishna, Amar; Pervaiz, Amina; Lephart, Paul; Tarabishy, Noor; Varakantam, Swapna; Kotecha, Aditya; Awali, Reda A; Kaye, Keith S; Chopra, Teena

2017-10-01

Clostridium difficile infection is a common cause of diarrhea in long-term care facility (LTCF) patients. The high prevalence of C difficile infection in LTCFs noted in our study calls for a critical need to educate LTCF staff to send diarrheal stool for C difficile testing to identify more cases and prevent transmission. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin.

PubMed Central

Little, S F; Leppla, S H; Cora, E

1988-01-01

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity. Images PMID:3384478

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts

PubMed Central

Liu, Dan; Wang, Qianqian; Ruan, Zengliang; He, Qian; Zhang, Liming

2015-01-01

Background Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. Results We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases. This is the first description of degenerative disease-associated genes in jellyfish. Conclusion We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species. PMID:26551022

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts.

PubMed

Liu, Guoyan; Zhou, Yonghong; Liu, Dan; Wang, Qianqian; Ruan, Zengliang; He, Qian; Zhang, Liming

2015-01-01

Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington's, Alzheimer's and Parkinson's diseases. This is the first description of degenerative disease-associated genes in jellyfish. We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species.

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones.

PubMed

Srinivasan, Usha; Bala, Aveenash; Jao, Shu-chuan; Starke, David W; Jordan, T William; Mieyal, John J

2006-07-25

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.

Regulation of Toxin Production in Clostridium perfringens

PubMed Central

Ohtani, Kaori; Shimizu, Tohru

2016-01-01

The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

Evaluation of a chromogenic culture medium for isolation of Clostridium difficile within 24 hours.

PubMed

Perry, John D; Asir, Kerry; Halimi, Diane; Orenga, Sylvain; Dale, Joanne; Payne, Michelle; Carlton, Ruth; Evans, Jim; Gould, F Kate

2010-11-01

Rapid and effective methods for the isolation of Clostridium difficile from stool samples are desirable to obtain isolates for typing or to facilitate accurate diagnosis of C. difficile-associated diarrhea. We report on the evaluation of a prototype chromogenic medium (ID C. difficile prototype [IDCd]) for isolation of C. difficile. The chromogenic medium was compared using (i) 368 untreated stool samples that were also inoculated onto CLO medium, (ii) 339 stool samples that were subjected to alcohol shock and also inoculated onto five distinct selective agars, and (iii) standardized suspensions of 10 C. difficile ribotypes (untreated and alcohol treated) that were also inoculated onto five distinct selective agars. Two hundred thirty-six isolates of C. difficile were recovered from 368 untreated stool samples, and all but 1 of these strains (99.6%) were recovered on IDCd within 24 h, whereas 74.6% of isolates were recovered on CLO medium after 48 h. Of 339 alcohol-treated stool samples cultured onto IDCd and five other selective agars, C. difficile was recovered from 218 samples using a combination of all media. The use of IDCd allowed recovery of 96.3% of isolates within 24 h, whereas 51 to 83% of isolates were recovered within 24 h using the five other media. Finally, when they were challenged with pure cultures, all 10 ribotypes of C. difficile generated higher colony counts on IDCd irrespective of alcohol pretreatment or duration of incubation. We conclude that IDCd is an effective medium for isolation of C. difficile from stool samples within 24 h.

Asymptomatic Carriers Contribute to Nosocomial Clostridium difficile Infection: A Cohort Study of 4508 Patients.

PubMed

Blixt, Thomas; Gradel, Kim Oren; Homann, Christian; Seidelin, Jakob Benedict; Schønning, Kristian; Lester, Anne; Houlind, Jette; Stangerup, Marie; Gottlieb, Magnus; Knudsen, Jenny Dahl

2017-04-01

Nosocomial infections with Clostridium difficile present a considerable problem despite numerous attempts by health care workers to reduce risk of transmission. Asymptomatic carriers of C difficile can spread their infection to other patients. We investigated the effects of asymptomatic carriers on nosocomial C difficile infections. We performed a population-based prospective cohort study at 2 university hospitals in Denmark, screening all patients for toxigenic C difficile in the intestine upon admittance, from October 1, 2012, to January 31, 2013. Screening results were blinded to patients, staff, and researchers. Patients were followed during their hospital stay by daily registration of wards and patient rooms. The primary outcomes were rate of C difficile infection in exposed and unexposed patients and factors associated with transmission. C difficile infection was detected in 2.6% of patients not exposed to carriers and in 4.6% of patients exposed to asymptomatic carriers at the ward level (odds ratio for infection if exposed to carrier, 1.79; 95% confidence interval, 1.16-2.76). Amount of exposure correlated with risk of C difficile infection, from 2.2% in the lowest quartile to 4.2% in the highest quartile of exposed patients (P = .026). Combining the load of exposure to carriers and length of stay seemed to have an additive effect on the risk of contracting C difficile. In a population-based prospective cohort study in Denmark, we found that asymptomatic carriers of toxigenic C difficile in hospitals increase risk of infection in other patients. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Evaluation of a Chromogenic Culture Medium for Isolation of Clostridium difficile within 24 Hours ▿

PubMed Central

Perry, John D.; Asir, Kerry; Halimi, Diane; Orenga, Sylvain; Dale, Joanne; Payne, Michelle; Carlton, Ruth; Evans, Jim; Gould, F. Kate

2010-01-01

Rapid and effective methods for the isolation of Clostridium difficile from stool samples are desirable to obtain isolates for typing or to facilitate accurate diagnosis of C. difficile-associated diarrhea. We report on the evaluation of a prototype chromogenic medium (ID C. difficile prototype [IDCd]) for isolation of C. difficile. The chromogenic medium was compared using (i) 368 untreated stool samples that were also inoculated onto CLO medium, (ii) 339 stool samples that were subjected to alcohol shock and also inoculated onto five distinct selective agars, and (iii) standardized suspensions of 10 C. difficile ribotypes (untreated and alcohol treated) that were also inoculated onto five distinct selective agars. Two hundred thirty-six isolates of C. difficile were recovered from 368 untreated stool samples, and all but 1 of these strains (99.6%) were recovered on IDCd within 24 h, whereas 74.6% of isolates were recovered on CLO medium after 48 h. Of 339 alcohol-treated stool samples cultured onto IDCd and five other selective agars, C. difficile was recovered from 218 samples using a combination of all media. The use of IDCd allowed recovery of 96.3% of isolates within 24 h, whereas 51 to 83% of isolates were recovered within 24 h using the five other media. Finally, when they were challenged with pure cultures, all 10 ribotypes of C. difficile generated higher colony counts on IDCd irrespective of alcohol pretreatment or duration of incubation. We conclude that IDCd is an effective medium for isolation of C. difficile from stool samples within 24 h. PMID:20739493

Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

PubMed Central

Edwards, Adrianne N.; Nawrocki, Kathryn L.

2014-01-01

The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. PMID:25069979

Evaluation of a commercial latex test for Clostridium difficile for reactivity with C. difficile and cross-reactions with other bacteria.

PubMed Central

Miles, B L; Siders, J A; Allen, S D

1988-01-01

Seventy-eight species of bacteria (739 isolates) were tested for reactivity with a commercial latex test for Clostridium difficile. All noncytotoxic as well as cytotoxic strains of C. difficile reacted positively. Immuno-specific cross-reactions were found only with C. sporogenes, proteolytic C. botulinum, and Peptostreptococcus anaerobius. PMID:3235677

Efficacy of cleaning products for C. difficile: environmental strategies to reduce the spread of Clostridium difficile-associated diarrhea in geriatric rehabilitation.

PubMed

Macleod-Glover, Nora; Sadowski, Cheryl

2010-05-01

To review the evidence for the efficacy of products used for environmental or hand cleaning on the rates of Clostridium difficile-associated diarrhea (CDAD). MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews were searched for articles pertinent to the efficacy of cleaning products against C. difficile or studies with outcomes related to rates of CDAD. Evidence was level II. Minimizing the incidence of CDAD in geriatric rehabilitation units is essential to achieving the goals of increasing patient function and independence for discharge into the community. Attention to environmental control of C. difficile and its spores by health care workers and patient visitors is an important secondary prevention strategy. Chlorine-releasing agents are more effective than detergents for killing spores produced by C. difficile. No level I evidence is available to determine if the use of chlorine-releasing agents has an effect on rates of CDAD. Hand-washing is currently the recommended strategy for reducing transmission of C. difficile. Alcohol gels do not inactivate C. difficile spores; however, increased use of alcohol hand gel has not been associated with higher rates of CDAD.

Dissemination of Clostridium difficile in food and the environment: Significant sources of C. difficile community-acquired infection?

PubMed

Warriner, K; Xu, C; Habash, M; Sultan, S; Weese, S J

2017-03-01

Clostridium difficile is a significant pathogen with over 300 000 cases reported in North America annually. Previously, it was thought that C. difficile was primarily a clinically associated infection. However, through the use of whole genome sequencing it has been revealed that the majority of cases are community acquired. The source of community-acquired C. difficile infections (CDI) is open to debate with foodborne being one route considered. Clostridium difficile fits the criteria of a foodborne pathogen with respect to being commonly encountered in a diverse range of foods that includes meat, seafood and fresh produce. However, no foodborne illness outbreaks have been directly linked to C. difficile there is also no conclusive evidence that its spores can germinate in food matrices. This does not exclude food as a potential vehicle but it is likely that the pathogen is also acquired through zoonosis and the environment. The most significant factor that defines susceptibility to CDI is the host microbiome and functioning immune system. In this respect, effective control can be exercised by reducing the environmental burden of C. difficile along with boosting the host defences against the virulent enteric pathogen. © 2016 The Society for Applied Microbiology.

Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms

PubMed Central

Koludarov, Ivan; Jackson, Timothy NW; op den Brouw, Bianca; Dobson, James; Dashevsky, Daniel; Clemente, Christofer J.; Stockdale, Edward J.; Cochran, Chip; Debono, Jordan; Stephens, Carson; Panagides, Nadya; Li, Bin; Roy Manchadi, Mary-Louise; Violette, Aude; Fourmy, Rudy; Hendrikx, Iwan; Nouwens, Amanda; Clements, Judith; Martelli, Paolo; Kwok, Hang Fai; Fry, Bryan G.

2017-01-01

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds. PMID:28783084

Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms.

PubMed

Koludarov, Ivan; Jackson, Timothy Nw; Brouw, Bianca Op den; Dobson, James; Dashevsky, Daniel; Arbuckle, Kevin; Clemente, Christofer J; Stockdale, Edward J; Cochran, Chip; Debono, Jordan; Stephens, Carson; Panagides, Nadya; Li, Bin; Manchadi, Mary-Louise Roy; Violette, Aude; Fourmy, Rudy; Hendrikx, Iwan; Nouwens, Amanda; Clements, Judith; Martelli, Paolo; Kwok, Hang Fai; Fry, Bryan G

2017-08-06

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma , Lanthanotus , and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds.

Mechanical stimulation of skeletal muscle increases prostaglandin F2(alpha) synthesis and cyclooxygenase activity by a pertussis toxin sensitive mechanism

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, Janet; Solerssi, Rosa; Chromiak, Joseph

1992-01-01

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increases the production of prostaglandin F(sub 2(alpha)), an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical activity, the activity of cyclooxygenase, a regulatory enzyme in prostaglandin synthesis, was increased 82% (P is less than .005), and this increase was maintained for at least 24 h. Kinetic analysis of the stretch-activated cyclooxygenase indicated a two to three-fold decrease in the enzyme's K(sub m) with no change in V(sub max). The stretch-induced increase in enzymatic activity was not inhibited by cycloheximide, was independent of cellular electrical activity (tetrodotoxin-insensitive), but was prevented by the G protein inhibitor pertussis toxin. Pertussis toxin also inhibited the stretch-induced increases in PGF(sub 2(alpha)) production, and cell growth. It is concluded that stretch of skeletal muscle increases the synthesis of the anabolic modulator PGF(sub 2(alpha)) by a G protein-dependent process which involves activation of cyclooxygenase by a posttranslational mechanism.

New Developments in Vaccines, Inhibitors of Anthrax Toxins, and Antibiotic Therapeutics for Bacillus anthracis

PubMed Central

Beierlein, J.M.; Anderson, A.C.

2013-01-01

Bacillus anthracis, the causative agent responsible for anthrax infections, poses a significant biodefense threat. There is a high mortality rate associated with untreated anthrax infections; specifically, inhalation anthrax is a particularly virulent form of infection with mortality rates close to 100%, even with aggressive treatment. Currently, a vaccine is not available to the general public and few antibiotics have been approved by the FDA for the treatment of inhalation anthrax. With the threat of natural or engineered bacterial resistance to antibiotics and the limited population for whom the current drugs are approved, there is a clear need for more effective treatments against this deadly infection. A comprehensive review of current research in drug discovery is presented in this article, including efforts to improve the purity and stability of vaccines, design inhibitors targeting the anthrax toxins, and identify inhibitors of novel enzyme targets. High resolution structural information for the anthrax toxins and several essential metabolic enzymes has played a significant role in aiding the structure-based design of potent and selective antibiotics. PMID:22050756

Fecal microbiota transplantation in children with recurrent Clostridium difficile infection.

PubMed

Pierog, Anne; Mencin, Ali; Reilly, Norelle Rizkalla

2014-11-01

Clostridium difficile eradication using fecal microbiota transplantation (FMT) has been successful in adults but little information is available in pediatrics. We report 6 pediatric patients with refractory C. difficile cured by FMT with no recurrences to date. Our results demonstrate that FMT can be an effective treatment for refractory C. difficile infection in pediatrics. Long-term safety and efficacy need to be studied.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

PubMed

Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

2016-02-01

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure, Function and Evolution of Clostridium botulinum C2 and C3 Toxins: Insight to Poultry and Veterinary Vaccines.

PubMed

Chellapandi, Paulchamy; Prisilla, Arokiyasamy

2017-01-01

Clostridium botulinum group III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced by this organism are the most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis to the development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxin whereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B. Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships with them are often determined by using evolutionary constraints as valuable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems in eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cellular Uptake of Clostridium botulinum C2 Toxin Requires Acid Sphingomyelinase Activity.

PubMed

Nagahama, Masahiro; Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko

2017-04-01

Clostridium botulinum C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca 2+ influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca 2+ ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca 2+ Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection. Copyright © 2017 American Society for Microbiology.

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa)

PubMed Central

Ponce, Dalia; Brinkman, Diane L.; Potriquet, Jeremy; Mulvenna, Jason

2016-01-01

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms. PMID:27058558

The Burden of Clostridium difficile after Cervical Spine Surgery.

PubMed

Guzman, Javier Z; Skovrlj, Branko; Rothenberg, Edward S; Lu, Young; McAnany, Steven; Cho, Samuel K; Hecht, Andrew C; Qureshi, Sheeraz A

2016-06-01

Study Design Retrospective database analysis. Objective The purpose of this study is to investigate incidence, comorbidities, and impact on health care resources of Clostridium difficile infection after cervical spine surgery. Methods A total of 1,602,130 cervical spine surgeries from the Nationwide Inpatient Sample database from 2002 to 2011 were included. Patients were included for study based on International Classification of Diseases Ninth Revision, Clinical Modification procedural codes for cervical spine surgery for degenerative spine diagnoses. Baseline patient characteristics were determined. Multivariable analyses assessed factors associated with increased incidence of C. difficile and risk of mortality. Results Incidence of C. difficile infection in postoperative cervical spine surgery hospitalizations is 0.08%, significantly increased since 2002 (p < 0.0001). The odds of postoperative C. difficile infection were significantly increased in patients with comorbidities such as congestive heart failure, renal failure, and perivascular disease. Circumferential cervical fusion (odds ratio [OR] = 2.93, p < 0.0001) increased the likelihood of developing C. difficile infection after degenerative cervical spine surgery. C. difficile infection after cervical spine surgery results in extended length of stay (p < 0.0001) and increased hospital costs (p < 0.0001). Mortality rate in patients who develop C. difficile after cervical spine surgery is nearly 8% versus 0.19% otherwise (p < 0.0001). Moreover, multivariate analysis revealed C. difficile to be a significant predictor of inpatient mortality (OR = 3.99, p < 0.0001). Conclusions C. difficile increases the risk of in-hospital mortality and costs approximately $6,830,695 per year to manage in patients undergoing elective cervical spine surgery. Patients with comorbidities such as renal failure or congestive heart failure have increased probability of developing infection after surgery. Accepted antibiotic guidelines in this population must be followed to decrease the risk of developing postoperative C. difficile colitis.

An Economic Analysis of Strategies to Control Clostridium Difficile Transmission and Infection Using an Agent-Based Simulation Model.

PubMed

Nelson, Richard E; Jones, Makoto; Leecaster, Molly; Samore, Matthew H; Ray, William; Huttner, Angela; Huttner, Benedikt; Khader, Karim; Stevens, Vanessa W; Gerding, Dale; Schweizer, Marin L; Rubin, Michael A

2016-01-01

A number of strategies exist to reduce Clostridium difficile (C. difficile) transmission. We conducted an economic evaluation of "bundling" these strategies together. We constructed an agent-based computer simulation of nosocomial C. difficile transmission and infection in a hospital setting. This model included the following components: interactions between patients and health care workers; room contamination via C. difficile shedding; C. difficile hand carriage and removal via hand hygiene; patient acquisition of C. difficile via contact with contaminated rooms or health care workers; and patient antimicrobial use. Six interventions were introduced alone and "bundled" together: (a) aggressive C. difficile testing; (b) empiric isolation and treatment of symptomatic patients; (c) improved adherence to hand hygiene and (d) contact precautions; (e) improved use of soap and water for hand hygiene; and (f) improved environmental cleaning. Our analysis compared these interventions using values representing 3 different scenarios: (1) base-case (BASE) values that reflect typical hospital practice, (2) intervention (INT) values that represent implementation of hospital-wide efforts to reduce C. diff transmission, and (3) optimal (OPT) values representing the highest expected results from strong adherence to the interventions. Cost parameters for each intervention were obtained from published literature. We performed our analyses assuming low, normal, and high C. difficile importation prevalence and transmissibility of C. difficile. INT levels of the "bundled" intervention were cost-effective at a willingness-to-pay threshold of $100,000/quality-adjusted life-year in all importation prevalence and transmissibility scenarios. OPT levels of intervention were cost-effective for normal and high importation prevalence and transmissibility scenarios. When analyzed separately, hand hygiene compliance, environmental decontamination, and empiric isolation and treatment were the interventions that had the greatest impact on both cost and effectiveness. A combination of available interventions to prevent CDI is likely to be cost-effective but the cost-effectiveness varies for different levels of intensity of the interventions depending on epidemiological conditions such as C. difficile importation prevalence and transmissibility.

Recovery of the gut microbiome following fecal microbiota transplantation.

PubMed

Seekatz, Anna M; Aas, Johannes; Gessert, Charles E; Rubin, Timothy A; Saman, Daniel M; Bakken, Johan S; Young, Vincent B

2014-06-17

Clostridium difficile infection is one of the most common health care-associated infections, and up to 40% of patients suffer from recurrence of disease following standard antibiotic therapy. Recently, fecal microbiota transplantation (FMT) has been successfully used to treat recurrent C. difficile infection. It is hypothesized that FMT aids in recovery of a microbiota capable of colonization resistance to C. difficile. However, it is not fully understood how this occurs. Here we investigated changes in the fecal microbiota structure following FMT in patients with recurrent C. difficile infection, and imputed a hypothetical functional profile based on the 16S rRNA profile using a predictive metagenomic tool. Increased relative abundance of Bacteroidetes and decreased abundance of Proteobacteria were observed following FMT. The fecal microbiota of recipients following transplantation was more diverse and more similar to the donor profile than the microbiota prior to transplantation. Additionally, we observed differences in the imputed metagenomic profile. In particular, amino acid transport systems were overrepresented in samples collected prior to transplantation. These results suggest that functional changes accompany microbial structural changes following this therapy. Further identification of the specific community members and functions that promote colonization resistance may aid in the development of improved treatment methods for C. difficile infection. Within the last decade, Clostridium difficile infection has surpassed other bacterial infections to become the leading cause of nosocomial infections. Antibiotic use, which disrupts the gut microbiota and its capability in providing colonization resistance against C. difficile, is a known risk factor in C. difficile infection. In particular, recurrent C. difficile remains difficult to treat with standard antibiotic therapy. Fecal microbiota transplantation (FMT) has provided a successful treatment method for some patients with recurrent C. difficile infection, but its mechanism and long-term effects remain unknown. Our results provide insight into the structural and potential metabolic changes that occur following FMT, which may aid in the development of new treatment methods for C. difficile infection. Copyright © 2014 Seekatz et al.

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905

PubMed Central

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-01-01

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene (mcyB) and key photosynthesis genes (psaB, psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation. PMID:28513574

Transcriptional and Physiological Responses to Nutrient Loading on Toxin Formation and Photosynthesis in Microcystis Aeruginosa FACHB-905.

PubMed

Peng, Guotao; Lin, Sijie; Fan, Zhengqiu; Wang, Xiangrong

2017-05-17

An important goal of understanding harmful algae blooms is to determine how environmental factors affect the growth and toxin formation of toxin-producing species. In this study, we investigated the transcriptional responses of toxin formation gene ( mcyB ) and key photosynthesis genes ( psaB , psbD and rbcL) of Microcystis aeruginosa FACHB-905 in different nutrient loading conditions using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Three physio-biochemical parameters (malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)) were also evaluated to provide insight into the physiological responses of Microcystis cells. We observed an upregulation of mcyB gene in nutrient-deficient conditions, especially in nitrogen (N) limitation condition, and the transcript abundance declined after the nutrient were resupplied. Differently, high transcription levels were seen in phosphorus (P) deficient treatments for key photosynthesis genes throughout the culture period, while those in N-deficient cells varied with time, suggesting an adaptive regulation of Microsystis cells to nutrient stress. Increased contents of antioxidant enzymes (SOD and GSH) were seen in both N and P-deficient conditions, suggesting the presence of excess amount of free radical generation caused by nutrient stress. The amount of SOD and GSH continued to increase even after the nutrient was reintroduced and a strong correlation was seen between the MDA and enzyme activities, indicating the robust effort of rebalancing the redox system in Microcystis cells. Based on these transcriptional and physiological responses of M. aeruginosa to nutrient loading, these results could provide more insight into Microcystis blooms management and toxin formation regulation.

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices

PubMed Central

Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard

2015-01-01

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. PMID:25979885

Environmental Contamination in Households of Patients with Recurrent Clostridium difficile Infection

PubMed Central

Bobr, Aleh; Kuskowski, Michael A.; Johnston, Brian D.; Sadowsky, Michael J.; Khoruts, Alexander

2016-01-01

Recurrent Clostridium difficile infection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). Although C. difficile spores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence of C. difficile. Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptive C. difficile isolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping, tcdC gene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detected C. difficile on ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P = 0.025). The most common C. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%). C. difficile was detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multiple C. difficile-positive samples had a single genotype present each. In conclusion, C. difficile was found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective. PMID:26921425

The housefly Musca domestica as a mechanical vector of Clostridium difficile.

PubMed

Davies, M P; Anderson, M; Hilton, A C

2016-11-01

Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/- 66.9 for vegetative cell suspension and 288.2 +/- 83.2 for spore suspension). After 1h, this had reduced (21.2 +/- 11.4 for vegetative cell suspension and 19.9 +/- 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/- 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/- 0.58. C. difficile could be recovered from fly excreta for up to 96h. This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Risk factors for Clostridium difficile infection in HIV-infected patients.

PubMed

Imlay, Hannah; Kaul, Daniel; Rao, Krishna

2016-01-01

Clostridium difficile infection is a healthcare-associated infection resulting in significant morbidity. Although immunosuppression is associated with Clostridium difficile infection acquisition and adverse outcomes, the epidemiology of Clostridium difficile infection in HIV-infected patients has been little studied in the era of antiretroviral therapy. This study identifies the risk factors for acquisition of Clostridium difficile infection in HIV-infected patients. A retrospective, propensity score-matched case-control study design was employed, with patients selected from our institution's outpatient HIV clinic. Clostridium difficile infection cases were defined as having positive stool testing plus an appropriate clinical presentation. The propensity score was generated via multiple logistic regression from year of HIV diagnosis, age at first contact, duration of follow-up, gender, and initial CD4 count. The 46 cases included were matched to a total of 180 controls. Prior antibiotic treatment was a significant predictor of Clostridium difficile infection (odds ratio: 13, 95% confidence interval: 3.49-48.8, p  < .001) as was number of hospital admissions in the preceding year (odds ratio: 4.02, confidence interval: 1.81-8.94, p  < .001). Having both proton pump inhibitor use and CD4 count <200 cells/µL significantly increased odds of Clostridium difficile infection in the multivariable model (odds ratio: 15.17, confidence interval: 1.31-175.9, p  = .021). As in the general population, frequent hospitalizations and exposure to antimicrobials are independent predictors of Clostridium difficile infection acquisition in patients with HIV. Additionally, low CD4 count and proton pump inhibitor use are new potentially modifiable variables that can be targeted for prevention of Clostridium difficile infection in future interventional studies.

Real-Time Electronic Tracking of Diarrheal Episodes and Laxative Therapy Enables Verification of Clostridium difficile Clinical Testing Criteria and Reduction of Clostridium difficile Infection Rates.

PubMed

Truong, Cynthia Y; Gombar, Saurabh; Wilson, Richard; Sundararajan, Gopalakrishnan; Tekic, Natasa; Holubar, Marisa; Shepard, John; Madison, Alexandra; Tompkins, Lucy; Shah, Neil; Deresinski, Stan; Schroeder, Lee F; Banaei, Niaz

2017-05-01

Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [≥3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days ( P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days ( P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days ( P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates. Copyright © 2017 American Society for Microbiology.

Mortality and Clostridium difficile infection in an Australian setting.

PubMed

Mitchell, Brett G; Gardner, Anne; Hiller, Janet E

2013-10-01

To quantify the risk of death associated with Clostridium difficile infection, in an Australian tertiary hospital. Two reviews examining Clostridium difficile infection and mortality indicate that Clostridium difficile infection is associated with increased mortality in hospitalized patients. Studies investigating the mortality of Clostridium difficile infection in settings outside of Europe and North America are required, so that the epidemiology of Clostridium difficile infection in these regions can be understood and appropriate prevention strategies made. An observational non-concurrent cohort study design was used. Data from all persons who had (exposed) and a matched sample of persons who did not have Clostridium difficile infection, for the calendar years 2007-2010, were analysed. The risk of dying within 30, 60, 90 and 180 days was compared using the two groups. Kaplan-Meier survival analysis and conditional logistic regression models were applied to the data to examine time to death and mortality risk adjusted for comorbidities using the Charlson Comorbidity Index. One hundred and fifty-eight cases of infection were identified. A statistically significant difference in all-cause mortality was identified between exposed and non-exposed groups at 60 and 180 days. In a conditional regression model, mortality in the exposed group was significantly higher at 180 days. In this Australian study, Clostridium difficile infection was associated with increased mortality. In doing so, it highlights the need for nurses to immediately instigate contact precautions for persons suspected of having Clostridium difficile infection and to facilitate a timely faecal collection for testing. Our findings support ongoing surveillance of Clostridium difficile infection and associated prevention and control activities. © 2013 Blackwell Publishing Ltd.

Household Transmission of Clostridium difficile to Family Members and Domestic Pets.

PubMed

Loo, Vivian G; Brassard, Paul; Miller, Mark A

2016-11-01

OBJECTIVE To determine the risk of Clostridium difficile transmission from index cases with C. difficile infection (CDI) to their household contacts and domestic pets. DESIGN A prospective study from April 2011 to June 2013. SETTING Patients with CDI from Canadian tertiary care centers. PARTICIPANTS Patients with CDI, their household human contacts, and pets. METHODS Epidemiologic information and stool or rectal swabs were collected from participants at enrollment and monthly for up to 4 months. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates. Probable transmission was defined as the conversion of a C. difficile culture-negative contact to C. difficile culture-positive contact with a PFGE pattern indistinguishable or closely related to the index case. Possible transmission was defined as a contact with a positive C. difficile culture at baseline with a strain indistinguishable or closely related to the index case. RESULTS A total of 51 patients with CDI participated in this study; 67 human contacts and 15 pet contacts were included. Overall, 9 human contacts (13.4%) were C. difficile culture positive; 1 contact (1.5%) developed CDI; and 8 contacts were asymptomatic. Of 67 human contacts, probable transmission occurred in 1 human contact (1.5%) and possible transmission occurred in 5 human contacts (7.5%). Of 15 pet contacts, probable transmission occurred in 3 (20%) and possible transmission occurred in 1 (6.7%). CONCLUSIONS There was a high proportion of C. difficile culture positivity at 13.4% among human contacts and asymptomatic carriage of domestic pets reached 26.7%. These results suggest that household transmission of C. difficile may be a source of community-associated cases. Infect Control Hosp Epidemiol 2016;1-7.

The effect of bezlotoxumab for prevention of recurrent Clostridium difficile infection (CDI) in Japanese patients.

PubMed

Mikamo, Hiroshige; Aoyama, Norihiro; Sawata, Miyuki; Fujimoto, Go; Dorr, Mary Beth; Yoshinari, Tomoko

2018-02-01

Recurrent Clostridium difficile infection is considered as a significant health care burden. The global study (MODIFY II) of antibody treatment (bezlotoxumab) for the prevention of recurrent C. difficile infection includes Japanese patients (95 subjects); The aim of this subgroup analysis is to report the data obtained from Japanese patients. Patients with C. difficile infection receiving standard of care antibiotic treatment and a single infusion of bezlotoxumab 10 mg/kg, actoxumab 10 mg/kg + bezlotoxumab 10 mg/kg or placebo. Recurrent C. difficile infection through Week 12 was evaluated. In the Full Analysis Set (93 subjects), 91% were older than 65 years of age and 93% were hospitalized at the time of study entry. The standard of care antibiotic for C. difficile infection was metronidazole for 57 subjects and vancomycin for 36 subjects. The recurrent C. difficile infection rate was 46% in the placebo, 21% in the bezlotoxumab (p = 0.0197) and 28% in the actoxumab + bezlotoxumab group. No additive recurrent C. difficile infection-reducing effect with the addition of actoxumab was demonstrated. There were no events representing safety concern in bezlotoxumab. Among 54 clinical isolates of C. difficile as a baseline culture in Japanese patients, the common ribotypes were 052 (28%), 018 (19%), 002 (15%) and 369 (9%). It showed distinctly different distribution from that in the United States and Europe. The superior effect of bezlotoxumab 10 mg/kg in the prevention of recurrent C. difficile infection suggests that the agent will be useful in the rapidly aging Japanese society. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ursodeoxycholic Acid Inhibits Clostridium difficile Spore Germination and Vegetative Growth, and Prevents the Recurrence of Ileal Pouchitis Associated With the Infection.

PubMed

Weingarden, Alexa R; Chen, Chi; Zhang, Ningning; Graiziger, Carolyn T; Dosa, Peter I; Steer, Clifford J; Shaughnessy, Megan K; Johnson, James R; Sadowsky, Michael J; Khoruts, Alexander

2016-09-01

To test whether ursodeoxycholic acid (UDCA) is inhibitory to Clostridium difficile and can be used in the treatment of C. difficile-associated ileal pouchitis. The restoration of secondary bile metabolism may be the key mechanism for fecal microbiota transplantation (FMT) in treating recurrent C. difficile infections (RCDI). Therefore, it is possible that exogenous administration of inhibitory bile acids may be used directly as nonantibiotic therapeutics for this indication. The need for such a treatment alternative is especially significant in patients with refractory C. difficile-associated pouchitis, where the efficacy of FMT may be limited. We measured the ability of UDCA to suppress the germination and the vegetative growth of 11 clinical isolate strains of C. difficile from patients treated with FMT for RCDI. In addition, we used oral UDCA to treat a patient with RCDI pouchitis that proved refractory to multiple antibiotic treatments and FMT. UDCA was found to be inhibitory to the germination and the vegetative growth of all C. difficile strains tested. Fecal concentrations of UDCA from the patient with RCDI pouchitis exceeded levels necessary to inhibit the germination and the growth of C. difficile in vitro. The patient has remained infection free for over 10 months after the initiation of UDCA. UDCA can be considered as a therapeutic option in patients with C. difficile-associated pouchitis. Further studies need to be conducted to define the optimal dose and duration of such a treatment. In addition, bile acid derivatives inhibitory to C. difficile that are able to achieve high intracolonic concentrations may be developed as therapeutics for RCDI colitis.

Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.

2008-07-15

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity againstmore » ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.« less

The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides

PubMed Central

Huang, Chen; Morlighem, Jean-Étienne RL; Zhou, Hefeng; Lima, Érica P; Gomes, Paula B; Cai, Jing; Lou, Inchio; Pérez, Carlos D; Lee, Simon Ming; Rádis-Baptista, Gandhi

2016-01-01

Abstract Protopalythoa is a zoanthid that, together with thousands of predominantly marine species, such as hydra, jellyfish, and sea anemones, composes the oldest eumetazoan phylum, i.e., the Cnidaria. Some of these species, such as sea wasps and sea anemones, are highly venomous organisms that can produce deadly toxins for preying, for defense or for territorial disputes. Despite the fact that hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, practically nothing is known about the toxin repertoire in zoanthids. Here, based on a transcriptome analysis of the zoanthid Protopalythoa variabilis, numerous predicted polypeptides with canonical venom protein features are identified. These polypeptides comprise putative proteins from different toxin families: neurotoxic peptides, hemostatic and hemorrhagic toxins, membrane-active (pore-forming) proteins, protease inhibitors, mixed-function venom enzymes, and venom auxiliary proteins. The synthesis and functional analysis of two of these predicted toxin products, one related to the ShK/Aurelin family and the other to a recently discovered anthozoan toxin, displayed potent in vivo neurotoxicity that impaired swimming in larval zebrafish. Altogether, the complex array of venom-related transcripts that are identified in P. variabilis, some of which are first reported in Cnidaria, provides novel insight into the toxin distribution among species and might contribute to the understanding of composition and evolution of venom polypeptides in toxiferous animals. PMID:27566758

Prevalence and risk factors of Clostridium difficile infection in patients hospitalized for flare of inflammatory bowel disease: a retrospective assessment.

PubMed

Regnault, Helene; Bourrier, Anne; Lalande, Valerie; Nion-Larmurier, Isabelle; Sokol, Harry; Seksik, Philippe; Barbut, Frederic; Cosnes, Jacques; Beaugerie, Laurent

2014-12-01

Recent studies have identified a high frequency of Clostridium difficile infections in patients with active inflammatory bowel disease. To retrospectively assess the determinants and results of Clostridium difficile testing upon the admission of patients hospitalized with active inflammatory bowel disease in a tertiary care centre and to determine the predicting factors of Clostridium difficile infections. We reviewed all admissions from January 2008 and December 2010 for inflammatory bowel disease flare-ups. A toxigenic culture and a stool cytotoxicity assay were performed for all patients tested for Clostridium difficile. Out of 813 consecutive stays, Clostridium difficile diagnostic assays have been performed in 59% of inpatients. The independent predictive factors for the testing were IBD (ulcerative colitis: OR 2.0, 95% CI 1.5-2.9; p<0.0001) and colonic involvement at admission (OR 2.2, 95% CI 1.5-3.1, p<0.0001). Clostridium difficile infection was present in 7.0% of the inpatients who underwent testing. In a multivariate analysis, the only independent predictor was the intake of nonsteroidal anti-inflammatory drugs within the two months before admission (OR 3.8, 95% CI 1.2-12.3; p=0.02). Clostridium difficile infection is frequently associated with active inflammatory bowel disease. Our study suggests that a recent intake of nonsteroidal anti-inflammatory drugs is a risk factor for inflammatory bowel disease -associated Clostridium difficile infection. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Clostridium difficile contamination of health care workers' hands and its potential contribution to the spread of infection: Review of the literature.

PubMed

Jullian-Desayes, Ingrid; Landelle, Caroline; Mallaret, Marie-Reine; Brun-Buisson, Christian; Barbut, Frédéric

2017-01-01

Clostridium difficile infection (CDI) can be transmitted from patient to patient by the hands of health care workers (HCWs); however, the relative importance of this route in the spread of C difficile in the hospital is currently unknown. Our aim was to review studies examining HCWs' hand carriage and its potential role in CDI transmission. First, English-speaking references addressing HCWs' hand sampling obtained from the PubMed database were reviewed. Second, C difficile outbreaks definitely or probably implicating HCWs were retrieved from the Outbreak Database Web site (www.outbreak-database.com). Finally, cases of C difficile occurring in HCWs after contact with an infected patient were retrieved from PubMed. A total of 11 studies dealing with HCWs' hand carriage were selected and reviewed. Between 0% and 59% of HCWs' hands were found contaminated with C difficile after caring for a patient with CDI. There were several differences between studies regarding site of hands sampling, timing after contact, and bacteriologic methods. Only 2 C difficile outbreaks implicating HCWs and 6 series of cases of transmission from patients to HCWs have been reported. This review shows that HCWs' hands could play an important role in the transmission of C difficile. Hand hygiene and reduction of environmental contamination are essential to control C difficile transmission. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile.

PubMed

Edwards, Adrianne N; Nawrocki, Kathryn L; McBride, Shonna M

2014-10-01

The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fecal Microbiome Among Nursing Home Residents with Advanced Dementia and Clostridium difficile.

PubMed

Araos, Rafael; Andreatos, Nikolaos; Ugalde, Juan; Mitchell, Susan; Mylonakis, Eleftherios; D'Agata, Erika M C

2018-06-01

Patients colonized with toxinogenic strains of Clostridium difficile have an increased risk of subsequent infection. Given the potential role of the gut microbiome in increasing the risk of C. difficile colonization, we assessed the diversity and composition of the gut microbiota among long-term care facility (LTCF) residents with advanced dementia colonized with C. difficile. Retrospective analysis of rectal samples collected during a prospective observational study. Thirty-five nursing homes in Boston, Massachusetts. Eighty-seven LTCF residents with advanced dementia. Operational taxonomic units were identified using 16S rRNA sequencing. Samples positive for C. difficile were matched to negative controls in a 1:3 ratio and assessed for differences in alpha diversity, beta diversity, and differentially abundant features. Clostridium difficile sequence variants were identified among 7/87 (8.04%) residents. No patient had evidence of C. difficile infection. Demographic characteristics and antimicrobial exposure were similar between the seven cases and 21 controls. The overall biodiversity among cases and controls was reduced with a median Shannon index of 3.2 (interquartile range 2.7-3.9), with no statistically significant differences between groups. The bacterial community structure was significantly different among residents with C. difficile colonization versus those without and included a predominance of Akkermansia spp., Dermabacter spp., Romboutsia spp., Meiothermus spp., Peptoclostridium spp., and Ruminococcaceae UGC 009. LTCF residents with advanced dementia have substantial dysbiosis of their gut microbiome. Specific taxa characterized C. difficile colonization status.

Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu3+-Doped Oxide Nanoparticles

PubMed Central

Türkcan, Silvan; Masson, Jean-Baptiste; Casanova, Didier; Mialon, Geneviève; Gacoin, Thierry; Boilot, Jean-Pierre; Popoff, Michel R.; Alexandrou, Antigoni

2012-01-01

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm2. Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm2/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family. PMID:22677383

[GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

PubMed

Markina, O V; Alekseeva, L P; Telesmanich, N R; Chemisova, O S; Akulova, M V; Markin, N V

2011-05-01

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

A Putative Gene Cluster from a Lyngbya wollei Bloom that Encodes Paralytic Shellfish Toxin Biosynthesis

PubMed Central

Mihali, Troco K.; Carmichael, Wayne W.; Neilan, Brett A.

2011-01-01

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds. PMID:21347365