Typing of Clostridium difficile causing diarrhoea in an orthopaedic ward.

In an outbreak of diarrhoeal disease in an orthopaedic ward Clostridium difficile was isolated from all six patients with diarrhoea. Attempts were made to type these isolates by means of antibiogram, detection of pre-formed enzymes, analysis of surface proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and plasmid profile analysis. This showed that a single strain (type E) indistinguishable by the four distinct methods of typing, was isolated from all six patients at some time during their episodes of diarrhoea. Relapse was caused by the acquisition of a new strain in two patients, and by re-emergence or reacquisition of the original strain in two patients. The immunochemical method was the most sensitive and discriminatory of the typing strategies adopted.     

Host factors are more important in predicting recurrent Clostridium difficile infection than ribotype and use of antibiotics

ObjectiveA frequent complication of Clostridium difficile infection (CDI) is recurrent disease. The aim of this study was to determine whether early recurrence risk was higher after infection with ribotype 027 (outbreak strain) compared with infection with endemic strain types of C. difficile.MethodsConsecutive patients diagnosed with CDI between May 2013 and March 2014 were included (outbreak strain, and non-outbreak strains). Patients who developed recurrent CDI within 30 days after completion of CDI treatment, were compared with patients without a recurrence. Medical charts were reviewed for demographic and clinical characteristics. General practitioners were contacted to complete data about the occurrence of recurrent CDI, and the use of medication after hospital discharge.ResultsIn total, 135 patients were at risk for the development of recurrent CDI; 74 patients were infected by ribotype 027, and 61 patients by other ribotypes. Thirty-nine patients (29%) developed recurrent CDI within 30 days after completion of CDI treatment. In multivariable analysis, age ≥70 years (HR 3.05, 95% CI 1.54–6.03), and a duration of CDI treatment ≥11 days (HR 1.92, 95% CI 1.00–3.69) were clearly associated with recurrence; infection with ribotype 027 showed a HR of 1.72 (95% CI 0.88–3.33).ConclusionDuring this outbreak of C. difficile in a tertiary care centre, age and a prolonged duration of CDI therapy (which is most likely a marker of underlying disease severity) were the main risk factors for recurrent CDI. This points to host factors as more important predictors for recurrent CDI than strain type or antibiotic use.     

Increased age and proton pump inhibitors are associated with severe Clostridium difficile infections in children

BackgroundClostridium difficile infection (CDI) is increasing in children. We aimed to compare the clinical characteristics between CDI and colonization and to identify the risk factors for severe diseases of CDI in children.MethodWe retrospectively reviewed 124 children (1–18 years old) from 2011 to 2018. CDI was defined as diarrhea (≥3 loose stool in the past 24 h) with confirmed toxigenic strain. Colonization was defined as presence of C. difficile without clinical symptoms. Severe diseases included ileus, acute kidney injury, gastrointestinal bleeding or mortality. Patients younger than 1 year old and coinfections with other enteric pathogens were excluded.ResultsAmong 124 patients with C. difficile identified, 49 of them fulfilled CDI definition and 75 had C. difficile colonization. Children with CDI were more likely to present with watery (74% vs. 1%, p < 0.01) and mucoid stool (25% vs. 7%, p < 0.01) and occult blood in stool (67% vs. 33%, p < 0.01) than children with colonization. In CDI cases, elevated age-adjusted creatinine (18% vs. 0%, p = 0.03) and hyponatremia (134 mEq/L vs. 137 mEq/L, p = 0.04) were found. Also, they had more complicated diseases (27% vs. 0%, p < 0.01). On multivariate analysis, age older than 4 years (adjusted odds ratio: 5.83; 95% confidence interval: 1.05–32.27) and proton pump inhibitor use (PPI) (adjusted odds ratio: 7.25; 95% confidence interval: 1.07–49.07) were the independent factors for severe diseases.ConclusionsWatery diarrhea, mucoid stool and occult blood in stool could differentiate CDI from colonization. Patients with increased age and previous PPI use were the independent risk factors for severe diseases in children.     

Hepatitis associated with Clostridium difficile in an ostrich chick

A live 19-day-old male ostrich chick was euthanized and necropsied. It was one of 12 chicks in a group in which 8 had died with history of anorexia, diarrhoea and weight loss. The birds had been treated with amikacin, piperacillin and enrofloxacin. Necropsy of the ostrich revealed dehydration, mild ascites and serous atrophy of fat around the heart. The liver had numerous yellow tan foci on the capsular surface as well as on the cut surface. Caecal contents were watery. Microscopic examination of the liver revealed multifocal necrosis of hepatocytes with infiltration of heterophils mixed with fibrin, few lymphocytes, and multinucleated giant cells. A Gram stain of the liver revealed a few gram-positive bacilli scattered within the necrotic foci. Clostridium difficile was isolated from the liver, and toxin A was detected by ELISA. A retrospective examination of approximately 1000 ostriches submitted during a seven year period to the laboratory system revealed seven cases of hepatitis due to Clostridium perfringens, two additional cases due to C. difficile and two cases due to C. sordelli.     

A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection

The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.     

Nonresident macrophages are absent from glomeruli in IgA nephropathy

The presence of nonresident cells of monocyte-macrophage lineage was investigated in 50 renal biopsies, including 20 from patients suffering from IgA nephropathy. Histochemical and immunological techniques were used to identify, respectively, nonspecific esterase and peroxidase activities and HLA class II molecules. Although intraglomerular phagocytes were observed in all control biopsies, small numbers were seen in only two samples from patients with IgA nephropathy. This feature is consistent with an impaired clearance of immune complexes, possibly leading to their deposition in the mesangium.     

Intrarectal Instillation of Clostridium difficile Toxin A Triggers Colonic Inflammation and Tissue Damage: Development of a Novel and Efficient Mouse Model of Clostridium difficile Toxin Exposure

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.     

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

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.     

Clostridium difficile carriage in elderly subjects and associated changes in the intestinal microbiota

Clostridium difficile is an important nosocomial pathogen associated particularly with diarrheal disease in elderly individuals in hospitals and long-term care facilities. We examined the carriage rate of Clostridium difficile by culture as a function of fecal microbiota composition in elderly subjects recruited from the community, including outpatient, short-term respite, and long-term hospital stay subjects. The carriage rate ranged from 1.6% (n = 123) for subjects in the community, to 9.5% (n = 43) in outpatient settings, and increasing to 21% (n = 151) for patients in short- or long-term care in hospital. The dominant 072 ribotype was carried by 43% (12/28) of subjects, while the hypervirulent strain R027 (B1/NAP1/027) was isolated from 3 subjects (11%), 2 of whom displayed C. difficile associated diarrhea (CDAD) symptoms at the time of sampling. Emerging ribotypes with enhanced virulence (078 and 018) were also isolated from two asymptomatic subjects. Pyrosequencing of rRNA gene amplicons was used to determine the composition of the fecal microbiota as a surrogate for the microbial population structure of the distal intestine. Asymptomatic subjects (n = 20) from whom C. difficile was isolated showed no dramatic difference at the phylum or family taxonomic level compared to those that were culture negative (n = 252). However, in contrast, a marked reduction in microbial diversity at genus level was observed in patients who had been diagnosed with CDAD at the time of sampling and from whom C. difficile R027 was isolated.     

The generation of life events in recurrent and non-recurrent depression

BACKGROUND: The stress generation hypothesis proposed by Hammen (1991) holds that depressed individuals generate stressful conditions for themselves, which lead to recurrence. The original test of this hypothesis compared dependent life events in women with recurrent depression to medical and normal controls. Two further research questions emerged from this work: (a) do individuals with a history of many depressive episodes generate more dependent life events than depressives with fewer episodes?; and (b) what is the aetiological relevance of any stress that may be generated? METHODS: The present research tested differences in dependent and independent events between depressed individuals who had experienced: (a) no previous major depressive episodes; (b) one previous episode; and (c) two or more previous episodes. We predicted that, based on the stress generation hypothesis, recurrent depressives would show more dependent events than people without a depression history, and that these generated stressors would be of aetiological importance for precipitating recurrence (i.e. severe events in the 3 months preceding recurrence). RESULTS: Recurrent depressives experienced significantly more total dependent events than first-onset depressives in the 12 months, but not the 3 months, preceding their episode. CONCLUSIONS: Although the findings supported the general premise of stress generation, the aetiological relevance of the generated stress for recurrence requires further study.     

Ribotype 027 Clostridium difficile infections with measurable stool toxin have increased lactoferrin and are associated with a higher mortality

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n?=?191) were positive by toxigenic culture and 61 % (n?=?129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 μg/g versus 106 μg/g stool; p?<?0.0001). Forty percent of patients (n?=?85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79 % vs. 50 %; p?<?0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 μg/g; p?=?0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18 %; p?=?0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR?=?1.1), intensive care unit treatment (OR?=?2.7), CI (OR?=?1.2), 027 CDI (OR?=?2.1), white blood cell count (OR?=?1.0), albumin level (OR?=?0.1), and stool toxin-positive 027 CDI (OR?=?2.5) were significantly associated with 100-day mortality (p?<?0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.     

First isolation of Clostridium difficile PCR ribotype 027 from a patient with severe persistent diarrhoea in Hungary

A recent Supplement to Clinical Microbiology and Infection entitled 'Infection control measures to limit the spread of C. difficile ' pointed out that the incidence of C. difficile -associated diarrhoea (CDAD) has been increasing worldwide, and stressed the importance of research in the fields of epidemiology and infection control [ 1 ]. Since 2003, one of the main causes of the increasing prevalence of CDAD has been claimed to be the emergence of PCR ribotype 027/NAP1, which has caused epidemics in North America, the UK, the Netherlands, Belgium and France. The presence of PCR ribotype 027 in Austria, Japan, Ireland, Germany and Switzerland has also been reported recently [ 2,3 ]. The majority of publications have emphasized that the presence of this strain is usually associated with more severe symptoms and signs than those associated with the other more common toxin-positive strains [ 4,5 ]. Whereas PCR ribotype 027 was present in the population earlier, the majority of the historic strains were fluoroquinolone sensitive [ 6 ]. The overuse of antibiotics such as fluoroquinolones may lead to the selection and emergence of resistant strains, and may contribute to the spread of PCR ribotype 027, which is usually resistant to erythromycin. Here, the Eastern European spread of C. difficile PCR ribotype 027 is reported.     

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin

A procedure was devised for routine examination of feces for Clostridium difficile with selective enrichment broth culture containing increased levels of carbohydrates and antibiotics to detect cytotoxin and volatile acids in broths inoculated with fecal samples. C. difficile was detected and identified with a rapidity comparable to that of conventional culture on selective cycloserine-cefoxitin fructose agar. Detection rates for C. difficile in inoculated broths (111/401 or 27%) were significantly higher than for culture on cycloserine-cefoxitin fructose agar (47/401 or 11%, P greater than 0.001). All fecal samples containing C. difficile and cytotoxin were correctly identified by the procedure. Isocaproic acid peak heights greater than 2 mm in selective enrichment broths inoculated with fecal samples indicated that C. difficile was present in the fecal sample examined. Of the positive specimens examined, 58% (64/111) produced peak heights greater than 10 mm. Peak heights less than 2 mm were not associated with C. difficile in the fecal sample. The investigated procedure provided a reliable alternative to the routine processing of feces for detecting C. difficile and associated cytotoxin in feces. Inoculated broths with isocaproic acid peak heights greater than 2 mm, after 24 to 48 h of incubation, and in which cytotoxin was detected, were subcultured to blood agar to obtain isolates of the organism as required. Broths which showed isocaproic acid peak heights less than 2 mm, and in which cytotoxin was not detected, were discarded as negative for C. difficile. The procedure was deemed potentially useful for epidemiological surveys of C. difficile.     

Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains.

Clostridium difficile, C. perfringens, and C. tertium are very often present simultaneously in the feces of conventional diarrheic young hares, whereas these three bacterial species are rarely encountered and never present simultaneously in the feces of healthy young hares. When a strain of each of the three bacterial species was monoassociated with axenic young hares, the appearance of pathological disorders was only observed in animals monoassociated with C. difficile, when the number of C. difficile exceeded 10(8) per g of fresh feces. When a strain of C. perfringens or a strain of C. tertium, or both, was associated with C. difficile, diarrhea and death occurred more rapidly than in hares monoassociated with C. difficile. C. difficile and C. perfringens became established more rapidly when disassociated than when monoassociated with axenic hares. The association of C. perfringens and C. tertium with axenic hares did not bring about any pathological disorders. It may be concluded that C. difficile is the causal agent of neonatal diarrhea in conventional and gnotobiotic young hares and that other strains of Clostridium enhance its pathogenic effect. C difficile alone or associated with C. perfringens or C. tertium does not play any pathogenic role in young rats, mice, or rabbits.