Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers.

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.

Clostridium difficile ribotypes in humans and animals in Brazil

Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

BACKGROUND & AIMS: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. RESULTS: TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. CONCLUSIONS: TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Factors associated with Clostridium difficile diarrhea in a hospital in Beijing, China

Clostridium difficile is the most common cause of hospital-acquired diarrhea in patients treated with antibiotics, chemotherapeutic agents, and other drugs that alter the normal equilibrium of the intestinal flora. A better understanding of the risk factors for C. difficile-associated disease (CDAD) could be used to reduce the incidence of CDAD and the costs associated with its treatment. The aim of this study was to identify the risk factors for CDAD in a cohort of Chinese patients in a Beijing hospital. Medical charts of a total of 130 inpatients (62 males and 68 females) with hospital-acquired diarrhea (45 with CDAD; 85 without CDAD) were retrospectively reviewed. C. difficile toxins A and B were detected in fecal samples using enzyme-linked fluorescence assays. The drugs used by patients with and without CDAD before the onset of diarrhea were compared. Factors that differed significantly between the two groups by univariate analysis were analyzed by multivariate analysis using a logistic regression model. Multivariate analysis showed that cephalosporin treatment was associated with a significantly higher risk of CDAD in hospitalized patients, while treatment with glycopeptides was significantly associated with a reduction in CDAD (P<0.001 for cephalosporin; P=0.013 for glycopeptides). Our data confirmed previous findings that empirical treatment with cephalosporins is positively associated with CDAD compared to individuals using other CDAD-related drugs. Additionally, we showed that treatment with glycopeptides was negatively associated with CDAD, compared to individuals using other CDAD-related drugs.

Factores asociados a la infección para Clostridium difficile en un hospital universitario de Bogotá

Introducción La infección por Clostridium difficile, es una de las causas más frecuentes de diarrea nosocomial con una alta morbimortalidad, con un aumento exponencial en su incidencia, en Estados Unidos se duplicó, de 261 casos x 100.000 en 1993 pasó a 546 x 100.000 en 2003 2, y en Canadá se encontraron datos similares con un aumento de 4.5 veces, en 1991 de 35.6 casos x 100.000 a 156.3 casos por 100.000 en 2004 3 . Se han descrito varios factores asociados Materiales y Métodos Se trata de un estudio descriptivo de tipo serie de casos en el que se evaluaron pacientes con diagnóstico de infección por C. Difficile y los factores asociados en un Hospital Universitario entre febrero de 2010 hasta septiembre de 2011 Resultados Se recolectaron 31 pacientes la edad promedio fue de 58 años con un rango entre 18 y 93 años, de los cuales 19 (61%) fueron mujeres y 12 (39%) hombres. El factor asociado a la infección por C. Difficile más frecuentemente encontrado fue el uso de inhibidores de bomba de protones con 54.84% (n=17) .No se encontraron pacientes VIH positivos o con diagnóstico de enfermedad inflamatoria intestinal. Ningún paciente presentó complicaciones asociadas a la infección ni mortalidad alguna. Conclusión El factor asociado que más se presentó fue el uso de antimicrobianos en los quince dias previos al inicio del cuadro en el 74% de los pacientes lo que coincide con lo presentado en la literatura mundial.

Dietary-based gut flora modulation against Clostridium difficile onset

Clostridium difficile infection is a frequent complication of antibiotic therapy in hospitalised patients, which today is attracting more attention than ever and has led to its classification as a 'superbug'. Disruption of the composition of the intestinal microflora following antibiotic treatment is an important prerequisite for overgrowth of C. difficile and the subsequent development of an infection. Treatment options for antibiotic-associated diarrhoea and C. difficile-induced colitis include administration of specific antibiotics (e.g. vancomycin), which often leads to high relapse rates. More importantly, both the rate and severity of C. difficile-associated diseases are increasing, with new epidemic strains of C. difficile often implicated. For the prevention and treatment of antibiotic-associated diarrhoea and C. difficile infection, several probiotic bacteria such as selected strains of lactobacilli (especially Lactobacillus rhamnosus GG), Bifidobacterium longum, and Enterococcus faecium and the non-pathogenic yeast Saccharomyces boulardii have been used. Controlled trials indicate a benefit of S. boulardii and L. rhamnosus GG as therapeutic agents when used as adjuncts to antibiotics. However, the need for more well designed controlled trials with probiotics is explicit.

Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) 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 PAR(2) 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. PAR(2) 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 PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: is it due to synergies in probiotic mixtures or the specificity of single strains?

Probiotics are currently being investigated for prevention of infections caused by enteric pathogens. The aim of this in vitro study was to evaluate the influence of three single probiotics: Lactobacillus casei NCIMB 30185 (PXN 37), Lactobacillus acidophilus NCIMB 30184 (PXN 35), Bifidobacterium breve NCIMB 30180 (PXN 25) and a probiotic mixture containing the above strains plus twelve other strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera on the survival of Salmonella Typhimurium and Clostridium difficile using pH-controlled anaerobic batch cultures containing mixed fecal bacteria. Changes in relevant bacterial groups and effects of probiotic addition on survival of the two pathogens were assessed over 24 h. Quantitative analysis of bacterial populations revealed that there was a significant increase in lactobacilli and/or bifidobacteria numbers, depending on probiotic addition, compared with the control (no added probiotic). There was also a significant reduction in S. Typhimurium and C. difficile numbers in the presence of certain probiotics compared with controls. Of the probiotic treatments, two single strains namely L. casei NCIMB 30185 (PXN 37), and B. breve NCIMB 30180 (PXN 25) were the most potent in reducing the numbers of S. Typhimurium and C. difficile. In addition, the supplementation with probiotics into the systems influenced some fermentations parameters. Acetate was found in the largest concentrations in all vessels and lactate and formate were generally detected in higher amounts in vessels with probiotic addition compared to controls.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detection of A/B toxin and isolation of Clostridium difficile and Clostridium perfringens from foals

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of Three Enzyme Immunoassays for Diagnosis of Clostridium difficile-Associated Diarrhea in Foals

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identifikation des Gruppe I Introns CDISt1 in offenen Leserastern des Clostridium difficile Genoms und dessen Charakterisierung als chimäres genetisches Element

Im tcdA-Gen des Clostridium difficile Stammes C34 wurde eine Insertion mit einer Größe von 1975 bp lokalisiert. Der als CdISt1 bezeichneten Insertion konnten charakteristische Merkmale von Gruppe I Introns und von Insertionselementen zugewiesen werden. Dem im 5’ Bereich gelegenen Anteil ließen sich die Intron-spezifischen Eigenschaften zuordnen, im 3’ Anteil wurden zwei offene Leseraster gefunden, die hohe Homologien zu Transposasen der IS605 Familie hatten. Funktionelle Analysen belegten die Spleißaktivität des chimären Ribozymes. CdISt1 konnte in mehren Kopien in allen untersuchten C. difficile Stämmen nachgewiesen werden. In anderen clostridialen Spezies konnte das Gruppe I Intron bislang nicht vorgefunden werden. Der Integrationsort in C. difficile war in allen untersuchten Fällen immer ein offenes Leseraster. Bislang waren Gruppe I Introns noch nie in bakteriellen offenen Leserastern beschrieben worden. Es kann angenommen werden, dass der chimäre Aufbau des Ribozymes die Integration in bakterielle offene Leseraster ermöglicht. Dabei wäre für die Spleißaktivität der Gruppe I Intron Anteil maßgeblich, die Mobilität würde über den IS Element Anteil vermittelt. Im Rahmen der Dissertationsarbeit konnten erste experimentelle Hinweise erbracht werden, dass das chimäre Ribozym an der evolution clostridialer Proteine beteiligt sein kann, wovon seinen Wirt C. difficile entsprechend profitieren würde.An insertion of 1975 bp is situated in the tcdA-gene of Clostridium difficile strain C34. The insertion was designated as CdISt1 and it had characteristics of group I introns and insertion elements. The group I characteristcs could be found in the 5’ area of the genetic element, in the 3’ area two open reading frames were located with high homologies to transposases of the IS605 family. Functional studies could proof the splicing activity of the ribozyme. CdISt1 could be found in several copies in all C. difficile strains examined so far. It was absent in other examined clostridial species. In all cases, the integration site in C. difficile was an open reading frame. Up to now, group I introns never were discovered in bacterial open reading frames. It can be assumed that the chimeric characteristics of the ribozyme permit an integration in bacterial open reading frames. The group I intron part would be responsible of the splicing activity, the IS element part could mediate the mobility of the genetic element. First experimental evidences point to a possible involvement of the chimeric ribozyme in the evolution of clostridial proteins, so the host C. difficile could benefit from its presence.

Clostridium difficile: Entdeckung und Charakterisierung der autokatalytischen Prozessierung von Toxin B

Clostridium difficile, der Auslöser der nosokomialen Antibiotika-assoziierten Durchfälle und der Pseudomembranösen Kolitis, besitzt zwei Hauptvirulenzfaktoren: die Toxine A und B. In vorangegangenen Veröffentlichungen wurde gezeigt, dass Toxin B durch einen zytosolischen Faktor der eukaryotischen Zielzelle während des Aufnahmeweges in die Zelle gespalten wird. Nur die N-terminale katalytische Domäne erreicht das Zytosol. Hierbei wurde davon ausgegangen, dass eine Protease der Zielzelle die Spaltung katalysiert. In dieser Arbeit konnte gezeigt werden, dass die Spaltung von Toxin B ein intramolekularer Prozess ist, der zytosolisches Inositolphosphat der Zielzelle als Kofaktor zur Aktivierung der intrinsischen Protease benötigt. Die Freisetzung der katalytischen Domäne durch Inositolphosphat-induzierte Spaltung ist nicht nur das Prinzip des Clostridium difficile Toxin B sondern auch des Toxin A, als auch des alpha Toxin von Clostridium novyi und das Letale Toxin von Clostridium sordellii. Der kovalente Inhibitor von Aspartatproteasen 1,2-epoxy-3-(p-nitrophenoxy)propan (EPNP), wurde dazu verwendet die intrinsische Protease von Toxin B zu blockieren und ermöglichte die Identifikation des katalytischen Zentrums. EPNP modifiziertes Toxin B verliert die intrinsische Proteaseaktivität und Zytotoxizität, aber wenn es direkt in das Zytosol der Wirtszelle injiziert ist, bleibt die Toxizität erhalten. Diese ist damit der erste Bericht eines bakteriellen Toxins, das eukaryotische Signale zur induzierten Autoproteolyse nutzt, um seine katalytisch-toxische Domäne in das Zytosol der Zielzelle freizusetzen. Durch diese Ergebnisse kann das Modell der Toxin-Prozessierung nun um einen weiteren entscheidenden Schritt vervollständigt werden.