Intestinal bacteria antagonistic to Clostridium difficile in mice

Overgrowth by Clostridium difficile has been reported in conventional mice injected intraperitoneally with ampicillin. In this study, we aimed to determine which types of indigenous intestinal bacteria were eliminated by ampicillin to allow overgrowth by C. difficile. C. difficile overgrowth was associated with a decrease in the numbers of lactobacilli, an increase in bacteroidaceae and a slight decrease in the frequency of isolation of fusiform-shaped bacteria (clostridia). C. difficile cytotoxin was detected in caeca from mice in which the numbers of C. difficile were greater than 10(5) per gram of faeces. Gnotobiotic mice were inoculated with various groups of intestinal anaerobes to determine which members of the indigenous flora would antagonize C. difficile. Gnotobiotic mice inoculated with three strains of lactobacilli, 37 strains of bacteroides or 46 strains of clostridia isolated from limited-flora mice were unable to eliminate C. difficile. C. difficile was eliminated, however, from the gastrointestinal tracts of gnotobiotic mice inoculated with whole faeces or chloroform-treated faeces from conventional mice or whole faeces from limited-flora mice containing only clostridia.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C.?difficile bacteriophage in hamsters during C.?difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in?vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in?vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C.?difficile host even during the infection process and highlights the importance of lysogeny of C.?difficile phages as an evolutionary adaptation for survival.     

酪酸梭菌与婴双歧杆菌对艰难梭菌体外生物拮抗作用的作用

用酪酸梭菌（Clostridium butyricum)和婴双歧杆菌（Bifidobacterium infantis)单独或联合艰难菌（Clostridium difficile)进行试管内的生物拮抗作用,将酪酸梭菌、婴儿双歧杆菌单独或酪酸梭菌和婴儿双歧杆菌联合分别与艰难梭菌以一定的比例等量混合后,接种于GAM液体培养基中进行厌氧培养。证明酪酸梭菌和婴儿双歧杆菌能明显抑制艰难梭菌的生长,并且两菌联合比各自单独培养时显示出更强的生物拮抗作用。     

Toxigenic C. difficile induced inflammatory marker expression by human intestinal epithelial cells is asymmetrical

Clostridium difficile infection of the intestinal epithelium and consequent pseudomembranous colitis is an important cause of morbidity and mortality. Pathogenesis has been ascribed exclusively to toxin production. Using in vitro models of human intestinal epithelial layers, we show that exposure to toxigenic C. difficile upregulates epithelial expression of IL-8 and ICAM-1, two molecules important in neutrophil chemoattraction and adhesion and subsequent inflammation. IL-8 production was also stimulated by toxin-containing supernatants. C difficile induced IL-8 release was inhibited by specific antiserum. Increased ICAM-1 expression only occurred after basolateral exposure to C. difficile while apical exposure had no effect on ICAM-1 expression. However, transepithelial electrical resistance was impaired by apical exposure to bacterial suspensions. We suggest that apical exposure to C. difficile induces changes in epithelial layer integrity which allows the bacteria and/or the toxin access to the basolateral compartment where pathogenic inflammatory mechanisms are activated.     

Inhibition of adhesion of Clostridium difficile to Caco-2 cells

Abstract For many microorganisms, including Clostridium difficile , mucosal association is an important factor influencing intestinal colonisation and subsequent infection. Inhibition of adhesion of C. difficile to intestinal mucosa could be a new promising strategy for prevention and treatment of antibiotic-associated diarrhoea. We investigated the possibilities of influencing the adhesion of C. difficile by xylitol and bovine colostrum.whey. Caco-2 cells and C. difficile cells were incubated with 1%, 5% and 10% solutions of xylitol and colostrum. Our study revealed that both xylitol and colostrum inhibited the adhesion of C. difficile to Caco-2 cells. Inhibition by xylitol was dose-dependent. When compared to the control, the count of adherent C. difficile decreased 3.4 times when treated with 1% xylitol, 12 times when 5% xylitol was applied, and 18.7 times when treated with 10% xylitol. The inhibition of adherence by colostrum was partially dose-dependent: 3.1 times in the case of 1%, and 5.5 times in the cases of 5% and 10% colostrum. Further experimental and clinical studies are needed for the application of xylitol and colostrum in the treatment and prophylaxis of pseudomembraneous colitis.     

Bile salts and glycine as cogerminants for Clostridium difficile spores

Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.     

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1β in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.     

Clostridium difficile in association with sporadic diarrhoea.

A total of 154 patients admitted to an infectious diseases unit were included in a year''s prospective survey of sporadic diarrhoeal disease. Stools from 19 of them yielded Clostridium difficile, generally on more than one occasion. Twelve of these patients were assessed as having a severe or moderately severe gastrointestinal illness: Cl difficile was the only pathogen isolated from 10 of them, and two had an associated salmonella infection. Seven had had a recent course of antibiotics, but five had not taken antibiotics. Faeces from seven patients with moderate or mild gastrointestinal illness yielded Cl difficile, and two of these patients also had an associated salmonella infection. Two patients in this group had no antibiotic history. From these findings, the occurrence of C difficile in faeces could not be described as antibiotic-associated. Faecal Cl difficile cytotoxin was detected in only six patients, and generally at low levels. In such patients a more relevant pathogenic index might take account of the numbers of Cl difficile present and of their toxigenic potential.     

Prevention of Clostridium difficile-induced experimental pseudomembranous colitis by Saccharomyces boulardii: a scanning electron microscopic and microbiological study

The ability of Saccharomyces boulardii to protect mice against intestinal pathology caused by toxinogenic Clostridium difficile was studied. Different regions of the intestine of experimental mice were prepared for observation by scanning electron microscopy or homogenized for C. difficile enumeration and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The test group was treated for 6 d with an S. boulardii suspension in drinking water and challenged with C. difficule on day 4. The three control groups were: axenic mice, mice treated with only S. boulardii and mice only challenged with C. difficile. The results showed that: (i) 70% of the mice infected by C. difficile survived when treated with S. boulardii; (ii) the C. difficile-induced lesions on the small and large intestinal mucosa were absent or markedly less severe in S. boulardii-treated mice; and (iii) there was no decrease in the number of C. difficile but rather a reduction in the amount of toxins A and B in S. boulardii-treated mice.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

Zero prevalence of Clostridium difficile in wild passerine birds in Europe

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

Clostridium difficile-associated diarrhea from a general hospital in Argentina

Clostridium difficile is responsible for 15-25% of all cases of antibiotic associated diarrhea. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Although the clinical and financial impact of nosocomial C. difficile infection is believed to be significant, only limited information is available on the importance of C. difficile as a cause of diarrhea in Argentina. The aim of the study was to evaluate the impact and diagnosis methods of CDAD from symptomatic patients in a general hospital from Argentina. Consecutive diarrheal stool samples from symptomatic patients from a General Hospital in Argentina were screened for toxigenic C. difficile between April 2000 and April 2001. Toxins were detected in stools by the Premier Cytoclone A+B EIA. Each specimen was examined for toxigenic C. difficile strains by culture. From 104 specimens, 40 (38.5%) [32 of 87 patients (36.8%)] were positive and 64 (61.5%) [55 of 87 patients (63.2%)] were negative by stool toxin assay and/or toxigenic culture. In 11 of 40 positives samples C. difficile toxins were detected only by toxigenic culture. Five (15.6%) patients presented with symptomatic recurrences. Toxin-negative strains were not isolated. This data indicates that the high prevalence of toxigenic strains of C. difficile is of concern in routine diagnostic testing for C. difficile toxins in our study population. Detection of toxins in stools by EIA, coupled with testing strains for toxigenicity only in those cases in which direct toxin assay produces negative results, may be a satisfactory strategy. CDAD is an emerging nosocomial problem in our hospital. It will be necessary to evaluate the epidemiology and measures to control nosocomial spread.     

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Abstract Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile . A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated λCd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone λCd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against λCd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.     

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city,Brazil

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.     

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

Protective Role of Commensals against Clostridium difficile Infection via an IL-1β-Mediated Positive-Feedback Loop

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of C. difficile after antibiotic treatment, but little is known about how they affect host immunity during C. difficile infection. In this article, we report that C. difficile infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1β-mediated positive-feedback loop. Mice lacking ASC, an essential mediator of IL-1β and IL-18 processing and secretion, were highly susceptible to C. difficile infection. ASC(-/-) mice exhibited enhanced translocation of commensals to multiple organs after C. difficile infection. Notably, ASC(-/-) mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to C. difficile infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1β and CXCL1 but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1β secretion by neutrophils. Mice lacking IL-1β were deficient in inducing CXCL1 secretion, suggesting that IL-1β is the dominant inducer of ASC-mediated CXCL1 production during C. difficile infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1β/NLRP3/ASC-mediated positive-feedback mechanism that is important for host survival and clearance of translocated commensals during C. difficile infection.