Enterotoxin-producing Bacteroides fragilis (ETBF) Strains in Stool Samples Submitted for Testing ofClostridium difficile and its Toxins

Fifty faecal samples of patients suspected of having diarrhoea associated with Clostridium difficile were studied. Toxins of C. difficile were tested in vivo directly from the faecal sample using Toxin Detection Kits (Oxoid) to detect toxin A and primers for detection genes of Toxin A and B in a PCR test. The same samples were tested for B. fragilis enterotoxin gene directly from the faecal sample using special primers and a PCR test. Samples were inoculated onto selective media for C. difficile (CCCA) and B. fragilis (BBE) for isolation of bacteria.In vitro Toxin A of C. difficile in culture was tested using a C. difficile toxin A immunoassay (Oxoid, U.K. test and Toxin B of C. difficile was tested by using the McCoy cell line. C. difficile toxin A and B genes were determined in DNA of isolated strains using special primers and a PCR reaction. The enterotoxin production in B. fragilis strains was tested on the human carcinoma cell line HT29/C1. The presence of fragilysin gene was detected using a special pair of primers and a PCR reaction. Toxinogenic strains of C. difficile and enterotoxigenic Bacteroides fragilis (ETBF) strains were isolated from the same samples.     

Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.     

An in vitro culture model to study the dynamics of colonic microbiota in Syrian golden hamsters and their susceptibility to infection with Clostridium difficile

Clostridium difficile infections (CDI) are caused by colonization and growth of toxigenic strains of C. difficile in individuals whose intestinal microbiota has been perturbed, in most cases following antimicrobial therapy. Determination of the protective commensal gut community members could inform the development of treatments for CDI. Here, we utilized the lethal enterocolitis model in Syrian golden hamsters to analyze the microbiota disruption and recovery along a 20-day period following a single dose of clindamycin on day 0, inducing in vivo susceptibility to C. difficile infection. To determine susceptibility in vitro, spores of strain VPI 10463 were cultured with and without soluble hamster fecal filtrates and growth was quantified by quantitative PCR and toxin immunoassay. Fecal microbial population changes over time were tracked by 16S ribosomal RNA gene analysis via V4 sequencing and the PhyloChip assay. C. difficile culture growth and toxin production were inhibited by the presence of fecal extracts from untreated hamsters but not extracts collected 5 days post-administration of clindamycin. In vitro inhibition was re-established by day 15, which correlated with resistance of animals to lethal challenge. A substantial fecal microbiota shift in hamsters treated with antibiotics was observed, marked by significant changes across multiple phyla including Bacteroidetes and Proteobacteria. An incomplete return towards the baseline microbiome occurred by day 15 correlating with the inhibition of C. difficile growth in vitro and in vivo. These data suggest that soluble factors produced by the gut microbiota may be responsible for the suppression of C. difficile growth and toxin production.     

Spo0A Differentially Regulates Toxin Production in Evolutionarily Diverse Strains of Clostridium difficile

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.     

The Role of Glutamate Dehydrogenase (GDH) Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China

The incidence and severity of Clostridium difficile infection (CDI) in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH) test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0%) and specificity (92.8%), compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7%) and positive predictive value (97.2%), but low sensitivity (45.0%) and negative predictive value (88.3%), compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing.     

Comparison of VIDAS CDAB and CDA immunoassay for the detection of Clostridium difficile in a tcdA− tcdB+ C. difficile prevalent area

Enzyme immunoassays for TcdA and/or TcdB are widely used for diagnosis of C. difficile infection. This study compared the performance of the new VIDAS C. difficile Toxin A & B assay (CDAB) with that of the existing VIDAS C. difficile Toxin A II assay (CDA) in a tcdA⁻tcdB⁺ prevalent area. A total of 555 fecal samples were cultured and tested using CDAB and CDA. C. difficile was isolated in 150 samples and the concordance rate was 81.8% (454/555) between CDAB and CDA. PCR assays for tcdA and/or tcdB were used as a confirmatory test on C. difficile strains recovered from culture positive cases (n = 150) and on fecal specimens in culture negative/CDAB positive or equivocal cases (n = 27). The number of tcdA⁺tcdB⁺, tcdA⁻tcdB⁺, and tcdA⁻tcdB⁻ strains on culture positive isolates (n = 150) were 75 (50.0%), 41 (27.3%), and 34 (22.7%), respectively. PCR assays for tcdB gene alone in stool specimens (n = 27) showed positivity in five cases. The sensitivity of VIDAS CDAB was higher than that of VIDAS CDA (65.3% vs. 29.8%), by more than 2-fold. The specificity of CDAB was almost the same as CDA (93.8% vs. 94.5%). Toxigenic culture of C. difficile isolates in culture positive/VIDAS CDAB negative cases (n = 62) additionally detected 22 VIDAS CDAB positive and 9 VIDAS CDAB equivocal cases. The VIDAS CDAB assay detects more tcdA⁺tcdB⁺ strains (60% vs. 45.3%) and tcdA⁻tcdB⁺ strains (70.7% vs. 0%) compared with VIDAS CDA.     

The Acid-Dependent and Independent Effects of Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R,and Lacticaseibacillus rhamnosus CLR2 on Clostridioides difficile R20291

Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea which is life threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2 has demonstrated a strong inhibitory effect on the growth of several nosocomial C. difficile strains by production of antimicrobial metabolites during fermentation. Though there are several lactobacilli shown to inhibit C. difficile growth by processes relying on acidification, this probiotic has demonstrated potency for CDI prevention among hospitalized patients. Here, we describe the acid-dependent and independent mechanisms by which these strains impair the cytotoxicity of a hypervirulent strain, C. difficile R20291 (CD). These bacteria were co-cultured in a series of experiments under anaerobic conditions in glucose-rich and no-sugar medium to inhibit or stimulate CD toxin production, respectively. In glucose-rich medium, there was low CD toxin production, but sufficient amounts to cause cytotoxic damage to human fibroblast cells. In co-culture, there was acidification by the lactobacilli resulting in growth inhibition as well as ≥ 99% reduced toxin A and B production and no observable cytotoxicity. In the absence of glucose, CD produced much more toxin. In co-culture, the lactobacilli did not acidify the medium and CD growth was unaffected; yet, the amount of detected toxin A and B was decreased by 20% and 41%, respectively. Despite the high concentration of toxin, cells exposed to the supernatant from the co-culture were able to survive. These results suggest that in addition to known acid-dependent effects, the combination of L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 can interfere with CD pathogenesis without acidification: (1) reduced toxin A and B production and (2) toxin neutralization. This might explain the strain specificity of this probiotic in potently preventing C. difficile-associated diarrhea in antibiotic-treated patients compared with other probiotic formulae.

     

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

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

Surface-Layer Protein A (SlpA) Is a Major Contributor to Host-Cell Adherence of Clostridium difficile

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.     

Antimicrobial Agent-Associated Colitis and Diarrhea

Although antimicrobial agent-associated colitis has been recognized as a clinicopathologic entity for years, the cause of this disease has been determined only recently. Virtually all cases of pseudomembranous colitis and some cases of antimicrobial agent-associated nonspecific colitis or diarrhea have been shown to be caused by a toxin of Clostridium difficile. Methods for cultivating C difficile from feces and for detecting the toxin have been developed. Oral administration of vancomycin has proved to be effective for the treatment of C difficile-induced colitis, although isolated instances of relapse after treatment have been documented.The discovery of C difficile as a human intestinal pathogen has provided an explanation for some, but not all cases of antimicrobial agent-associated diarrhea. The epidemiology, pathogenesis and means of prevention of C difficile toxin-induced diarrhea remain to be determined.     

Diagnostic deficiencies of C. difficile infection among patients in a tertiary hospital in Saudi Arabia: A laboratory-based case series

Clostridioides difficile infection (CDI) has become a threatening public health problem in the developed world. In the kingdom of Saudi Arabia, prevalence of CDI is still unknown due to limited surveillance protocols and diagnostic resources. We used a two-step procedure to study and confirm C. difficile cases. We also studied toxin profiles of these isolates.Stool samples were collected from symptomatic patients and clinically suspected of CDI for almost 12 months. Isolates were confirmed by culture method followed by 16S rRNA sequencing. Multiplex PCR was performed for the identification of toxin A, toxin B and binary toxin genes and compared to Gene Expert results.Out of the 47 collected samples, 27 were successfully grown on culture media. 18 samples were confirmed as C. difficile by both culture and 16S rRNA sequencing. Interestingly, the rest of the isolates (9 species) belonged to different genera. Our results showed 95% of samples were positive for both toxin A and B (tcdA, tcdB) and all samples exhibited the toxin gene regulator tcdC. All samples were confirmed negative for the binary toxin gene ctdB and 11% of the isolates were positive for ctdA gene. Interestingly, one isolate harbored the binary toxin gene (cdtA⁺) and tested negative for both toxins A and B.We believe that combining the standard culture method with molecular techniques can make the detection of C. difficile more accurate.     

Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B

The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied. ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1–5 mM range) delay in the appearance of the cytopathogenic effect. With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h. Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction. The protective effect of ATP did not depend on its energy producing ability. Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells. ATP was protective even upon addition 10 min after the toxin binding step. In the presence of ATP, the toxin remained accessible to neutralization with antitoxin. In analogy with the P-site on diphtheria toxin, we postulate that C. difficile toxin B contains a polyphosphate-binding site. This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step.     

The P2Y6 Receptor Mediates Clostridium difficile Toxin-Induced CXCL8/IL-8 Production and Intestinal Epithelial Barrier Dysfunction

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y₆ receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y₆ receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y₆ inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y₆ receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y₆ receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y₆ receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.     

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ²⁸) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.     

Continuous Production of Clostridium tetani Toxin

The continuous production of Clostridium tetani toxin has been carried out in a 1-liter stirred culture vessel for as long as 65 days. Toxin production of approximately 120 flocculating units per ml was maintained with a dilution rate of 0.125 hr^-1, a temperature of 34 C, a pH of 7.4, and the addition to the medium of 0.1 g of potassium chloride per liter. The average minimal lethal intraperitoneal dose of the toxin in mice was approximately 10⁶ per ml.     

More on Clostridium difficile -associated Diarrhoea in Australia

To evaluate further the importance of Clostridium difficile as a cause of diarrhoea in Australia, 165 hospital laboratories were surveyed about 1995 using a self-administered questionnaire. Only 91 (55%) laboratories responded and only 39 of these (43%) had laboratory facilities to detect C. difficile routinely, 13 (33%) on request only and 19 (49%) on request or in certain situations. Culture was used by 26 laboratories (67%), 15 (36%) used tissue culture assays and 24 (62%) used commercially available toxin kits. C. difficile detection rates varied from <1 to 49 per 100 hospital beds. Isolation rates in some small hospitals (<100 beds) were surprisingly high (15 cases per 100 beds) indicating that C. difficile -associated diarrhoea is not just confined to large institutions. Hospital laboratories using culture and tissue culture cytotoxin assays had higher detection rates than laboratories relying only on toxin detection kits. Detection rates were correlated with antimicrobial usage figures for those hospitals which could provide usage data and with laboratories considered competent in detecting C. difficile. The correlation between incidence of C. difficile -associated diarrhoea and third generation cephalosporin usage was 0.87, while for gentamicin this figure was 0.37. The importance of C. difficile as a nosocomial pathogen is still generally underestimated in Australia. Significant diagnostic and financial benefits may follow improvements in detection techniques and restrictions in third generation cephalosporin usage.     

Evaluation of an Automated Rapid Diagnostic Test for Detection of Clostridium difficile

The Verigene Clostridium difficile Nucleic Acid Test (Verigene CDF Test) (Nanosphere, Northbrook, IL, USA) is a new multiplex qualitative polymerase chain reaction (PCR) test used to detect C. difficile toxin genes in fecal specimens. To evaluate the performance of the new method, we tested 69 fecal samples from patients with suspected C. difficile infection using the Verigene CDF test, an enzyme immunoassay (EIA) and PCR following anaerobic fecal culture. The sensitivity, specificity, and accuracy of the Verigene CDF test were 96.7% (29/30), 97.4% (38/39), and 97.1% (67/69) respectively, using PCR following fecal culture as a reference method. We also analyzed the potential clinical impact of the Verigene CDF test using chart reviews of the 69 patients with suspected C. difficile infection and found that 11 of the 69 patients were incorrectly diagnosed, and the Verigene CDF test would have led to them receiving more appropriate management including practice of treatment and contact precaution, although, of the 69 patients, there are two whose samples were incorrectly identified with the Verigene CDF test. The Verigene CDF test will have a positive impact on patient care.