Toxigenic Clostridium Difficile Strains Research Articles

The fulminant form of pseudomembranous colitis in a child with cystic fibrosis

Аnnotation. A long-term persistent infection of the lower respiratory tract with cystic fibrosis and, as a consequence, the need for regular use of antibacterial drugs inevitably leads to changes in the composition of the intestinal microflora. At the same time, the most dangerous are toxigenic strains of Clostridium difficile. In turn, excessive growth of C. Difficile leads to increased sensitivity of enterocytes to its toxins. The leading pathogenicity factors of C. Difficile are exotoxins A (TcdA), B (TcdB) and binary toxin. TcdA and TcdB are enterotoxins acting on intestinal enterocytes, which leads to inflammation and necrosis of the mucous membrane. The binary toxin forms a complex on the enterocyte membrane that penetrates the cytoplasm, disrupts the functioning of the cell, leads to its death, and also enhances the adhesion and colonization of C. Difficile. One of the most accessible methods for determining the presence of toxin-producing C. Difficile in feces is immunochromatographic examination. The frequency of such a complication in patients with MV is unknown, the literature presents isolated cases of such a complication in Russia. There is one known case of fulminant pseudomembranous colitis (lethal). A clinical case of fulminant pseudomembranous colitis with recovery is presented. Conclusion. Patients with cystic fibrosis are at risk for the development of this complication, especially patients with a history of intestinal resection against the background of meconium ileus. In about 5% of cases, there is a fulminant course of pseudomembarnous colitis. In this form, the duration of the disease can be several hours. Lethality in the lightning-fast course reaches 58-70%. This option presents the greatest difficulty in terms of diagnosis due to atypical clinical symptoms. In particular, the diarrheal syndrome may not be pronounced or absent at all. Such patients often show symptoms of an acute abdomen, which makes differential diagnosis difficult. This is confirmed in our clinical case. It is necessary to increase awareness and alertness of doctors regarding the risk of developing this formidable complication in patients with Сystic fibrosis

Optimization of cryoprotectants for freeze-dried potential probiotic Enterococcus faecalis and evaluation of its storage stability

Probiotic application remains a challenge since decades, as microorganisms lose their viability and functionality during the manufacture and storage of freeze-dried powders. Appropriate cryoprotectants are important in achieving particular probiotic preparation. Recently, Enterococcus spp. such as Enterococcus faecalis and E. faecium have been used as probiotics for human or animal health promotion. We aimed to determine a suitable protectant to protect the viability and pathogen inhibition potential of E. faecalis probiotic strains during freeze-drying and subsequent storage. E. faecalis probiotic strains were lyophilized in the presence of glucose, fructose, sucrose, skim milk, and distilled water. The lyophilized vials were stored at 4 °C for up to 30 days. Then, the cells from these vials were evaluated for their probiotic functionality. Among several protectants, skim milk exhibited highly protective capability to protect E. faecalis cell viability during freeze-drying and 30-day storage, with survival rates of E. faecalis strains ranging from 95.96 ± 0.20% to 103.42 ± 1.96% and 95.68 ± 0.72% to 102.95 ± 2.12%, respectively. The presence of skim milk could also effectively enhance the antimicrobial activity of all freeze-dried E. faecalis strains against toxigenic Clostridium difficile strains and maintain their activity after 30-day storage. Furthermore, hemolytic analysis demonstrated the safety of these freeze-dried E. faecalis strains. Overall, the results indicate that skim milk could be used as a suitable cryoprotective substance for freeze-dried E. faecalis probiotics, with potential applications in functional food, medicine, and feed industries.

Assessment of the Effect of Multidrug Resistance Clostridium difficile Clinical Strains on the Dynamics of Clostridium difficile Infection Rate at Pediatric Oncological Hospital

Relevance. At the children's oncological hospital guidelines to treat patients with several groups of antibiotics at the same time, which ensures the formation of multi-resistant strains of Clostridium difficile, which have a selective advantage for the Clostridium difficile infection developing, and also cause epidemics and /or in associating with an increase in the severity of Clostridium difficile – infection. Aims. Multidrug resistance Clostridium difficile strains and Clostridium difficile infection rate at pediatric oncological hospital. Results. An investigation of the Clostridium difficile resistance strains carried out among children at the children's oncological hospital. 143 toxigenic strains are resistant to moxifloxacin 72.41%, clindamycin 63.72%, rifampicin 35.54%, tetracycline 26.45%, tigecycline 11.42%, vancomycin 4.4%, metronidazole 3.9%. At the same time, the increase multidrug-resistant strains proportion note at the level of 3–4% per year. However, the rate of Clostridium difficile infection among children at the oncological hospital remained at the level of 0.4% to 3.1% with a downward trend. As a result, statistical calculations showed the absence of correlation between multidrug resistance and morbidity. Conclusions. Detection of multidrug-resistant microorganisms among toxigenic Clostridium difficile strains proves the need for further study of this problem in Russia and the advisability of monitoring Clostridium difficile infection rate and multidrug resistance Clostridium difficile strains at pediatric oncological hospitals.

Diarrhea associated with Clostridium difficile in the oncology clinic

Purpose of the study. To conduct typing of toxigenic strains of Clostridium difficile, obtained from oncological patients who are in hospital.Material and Methods. The study included 130 patients with Clostridium difficile – associated diarrhea. First, the presence of toxins A+B+ in the feces was determined by enzyme immunoassay on an automatic miniVidas analyzer from BioMieux SA (France). For screening using real-time PCR, 26 toxigenic Clostridium difficile were selected. For the study used the system GeneXpert company «Cepheid» (Sweden).Results. Of the 1287 patients with diarrhea who were treated in an oncologic hospital, 130 (10,1%) tested positive for toxin A+B+ Clostridium difficile. For the study using real-time PCR, 26 samples were taken. The average age of patients is 57 years. 10 (38,5%) toxigenic strains were identified as hypervirulent ribotype. 7/10 strains were isolated for 3 consecutive month. The average age of patients with hypervirulent strains was 45 years. Patients participating in the study, in most cases were from the surgical departments – 61,5%, from the therapeutic – 34,7%.Conclusion. The study showed that the prevalence of Clostridium difficile infection in our clinic is lower than in other countries. Although hypervirulent strains accounted for 1/3 of all typed Clostridium difficile, special attention should be paid to them. Delayed diagnosis, delay in the onset of specific therapy in cancer patients can lead to a deterioration in the quality of life and in some cases be fatal. PCR diagnostics can determine the hypervirulent ribotype and carry out epidemiological measures aimed at preventing local outbreaks in the hospital.

Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland

Two hundred and fifty-three non-duplicate toxigenic Clostridium difficile isolates, collected from February 2012 to December 2014, were evaluated for phenotypic resistance to ten antimicrobial drugs with the E-test gradient diffusion method. All strains of C. difficile were susceptible to metronidazole, vancomycin, and tigecycline. The metronidazole MIC values of the hyperepidemic PCR-ribotypes RT027 and RT176 were higher than those of non-epidemic PCR-ribotypes (p < 0.05, as evidenced by Mann-Whitney U test). In contrast, vancomycin susceptibility did not differ between hyperepidemic and non-epidemic strains, although the difference was almost significant (p = 0.065). Clostridium difficile RT027 and RT176 isolates could be assessed to five and four different susceptibility patterns, respectively, representing various combinations of resistance to different antimicrobial classes. A single point mutation (Thr82Ile) in the gyrA gene was detected in 11 (78.6%) of 14 isolates with high level of resistance to ciprofloxacin and moxifloxacin and four different types of single point mutations (Arg447Lys, Ser416Ala, Asp426Val, Asp426Asn) in the gyrB gene were detected in 4 strains, also with high level of resistance to ciprofloxacin and moxifloxacin. Four different point mutations were detected in the rpoB gene in 21 rifampicin-resistant strains of which one has not been reported previously, Gln489Leu. This study demonstrates the presence of multidrug-resistant C. difficile strains in Polish hospitals over the study period, irrespective of geographical location or reference level of the hospital.

Improved molecular typing of toxigenic Clostridium difficile strains affecting animal and human health

Clostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.

Evidence of transmission of Clostridium difficile in asymptomatic patients following admission screening in a tertiary care hospital.

BackgroundClostridium difficile (CD) is the leading cause of infectious health-care associated diarrhea. However, little is known regarding CD carriage and transmission amongst asymptomatic colonizers. We evaluated carriage, characterized strains and examined epidemiologic linkages in asymptomatic colonized CD patients.MethodsRectal swabs from asymptomatic patients admitted to the general medicine ward from April 1-June 30 2012 were collected. PCR-confirmed CD colonies were ribotyped and characterized by Modified-Multi Locus Variable Number Tandem Repeat Analysis (MMLVA).Results1549-swabs were collected from 474-patients. Overall, 50/474(10.6%) were CD PCR-positive, 24/50 were colonized at admission, while 26/50 were first identified > = 72 hours after admission. Amongst the 50 CD PCR-positive patients, 90% were asymptomatically colonized and 80% of individuals carried toxigenic CD-strains, including ribotype-027 (5/45:11%). MMLVA revealed five-clusters involving 15-patients harboring toxigenic (4/5) and non-toxigenic CD strains (1/5). In two clusters, patients were CD positive on admission while in the other three clusters involving 10 patients, we observed CD transmission from asymptomatically colonized patients to 8 previously CD-negative patients.ConclusionsWe identified increasing rates of colonization during admission to medical wards. MMLVA typing effectively discriminated between strains and suggests that 20% of patients with CD colonization acquired their strain(s) from asymptomatically colonized individuals in hospital.

A study on clinical application of a novel reagent detecting toxigenic strains of Clostridium difficile

Objective: To evaluate the performances of a "ready-to-use" liquid chromogenic medium for detecting toxigenic strains of C. difficile from stool specimens, by comparing with the reference method of combined GeneXpert detection with MALDI-TOF MS identification. Methods: This study was a clinical application research. A total of 322 stool specimens from patients with diarrhea in Henan Provincial People's Hospital were detected by using this liquid chromogenic mediumor by combination of GeneXpert and MALDI-TOF MS identification after CCFA culture from March to November 2017. The reference method was combined GeneXpert detection with MALDI-TOF MS identification. Data were analyzed with kappa analysis by use of SPSS 13.0. In this study, the limit of detection and anti-interference capability of chromogenic medium were determined with standard strains. Results: This assay had a sensitivity of 92.86% (52/56), a specificity of 96.99% (258/266), a positive predictive value of 86.67% (52/60), and a negative predictive value of 98.47% (258/262). Furthermore, this assay was well consistent with the reference method (kappa=0.874, P<0.01). The limit of detection of this assay was 10(3) CFU/ml for toxigenic strain of ATCC9689, and there was no interference by non-toxigenic strain of ATCC43593 or Clostridium perfringens (ATCC13124) even.When the concentration of ATCC9689 was as low as 0.5 McFarland. Conclusions: It is worth expanding clinical research for this "ready-to-use" chromogenic medium and preparing for its popularization in primary medical institutions, due to its similar performance to the reference method for detecting toxigenic strains of C. difficile, simple operation and no need of anaerobic condition.

510. First Environmental Investigation of Toxigenic Clostridium difficile Strains in Texas Hospitals

Background Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients in the developed world and an emerging pathogen in developing countries due to increased use of broad-spectrum antibiotics worldwide. Spores of toxigenic C. difficile can survive and disseminate in any environs and act as sources for human colonization or infections. Although likely ubiquitous in any environs, the prevalence of C. difficile spores in the hospital environment of Texas hospitals is poorly understood. The objectives of the study are to isolate and characterize C. difficile from the hospital environs of three hospitals in three cities in Texas.MethodsAs part of a Texas hospital-wide surveillance effort, we collected shoe-bottom swabs samples from hospital employees, patients, and visitors inside three large hospital from three cities. Samples were analyzed for C. difficile using anaerobic enrichment culture and molecular methods. Suspected colonies from cycloserine cefoxitin fructose agar (CCFA) plates were identified by PCR (tcdA, tcdB, cdtA, cdtB, tpi) and genotyped using fluorescent PCR ribotyping.ResultsA total 229 of 1079 (21.2%) surface swab and 81 of 121 (66.9%) shoe swab samples were culture positive for toxigenic C. difficile (tcdA and tcdB). A total of 29 distinct ribotypes were identified from 166 C. difficile isolates tested. Predominant ribotypes were F106, F019, F014-020, F002, and F255. Interestingly, ribotype F027 was not a predominant strain among the swab samples. Each hospital had widely diverse strains. Shoes were the most contaminated item in all the hospitals.ConclusionWe identified a high prevalence of toxigenic C. difficile with diverse ribotypes from hospital environmental shoe-bottom swabs and high touch surface swabs in hospitals in three cities of Texas. Our findings suggest that patients might be at higher risk for C. difficile colonization or infection in these hospitals.Disclosures K. W. Garey, Merck & Co.: Grant Investigator, Grant recipient.

Evaluation of a novel molecular assay to diagnose toxigenic strains of Clostridium difficile

The objective of this study was to evaluate the Amplidiag C. difficile+027® assay, a new molecular method that detects toxin B gene in stool samples and identifies the hypervirulent 027 strain, to diagnose Clostridium difficile infections. The assay was compared to the reference method i.e. toxigenic culture. Amplidiag C. difficile+027® assay was prospectively evaluated from 309 diarrheal stool specimens of patients suspected of C. difficile infection. Forty-five (14.6%) stools were positive by toxigenic culture and 11 (3.6%) stools gave discordant results with the molecular method. PR027 was not recovered during the study. After resolving the discrepant results, the sensitivity, specificity, positive and negative predictive values of Amplidiag C. difficile+027® assay were 91.1% [CI 95% 77.9–97.1], 99.6% [CI 95% 97.6–100], 97.6% [CI 95% 85.9–99.9] and 98.5% [CI 95% 96–99.5], respectively compared to toxigenic culture. This assay is sensitive compared to the toxigenic culture.

High Prevalence of Toxigenic and Nontoxigenic Clostridium difficile Strains in Malaysia.

Accumulating evidence shows a high prevalence of Clostridium difficile in Southeast Asia associated with a range of clinical presentations. However, severe infections are rarely reported. We investigated C. difficile infection (CDI) across four hospitals in Kuala Lumpur and Kota Bharu, Malaysia. Enzyme immunoassays for glutamate dehydrogenase (GDH) and toxin A or B were performed on diarrheal stool specimens collected from patients in 2015 and 2016. Specimens were also cultured and isolates of C. difficile characterized by PCR ribotyping and detection of toxin genes. In total, 437 specimens were collected and fecal toxin was detected in 3.0%. A further 16.2% of specimens were GDH positive and toxin negative. After culture, toxigenic strains were isolated from 10.3% and nontoxigenic strains from 12.4% of specimens. The most prevalent PCR ribotypes (RTs) were RT 017 (20.0%) and RT 043 (10.0%). The high prevalence of RT 017 and nontoxigenic strains in Malaysia and in neighboring Thailand and Indonesia suggests that they localize to the region of Southeast Asia, with an implication that they may mediate the burden of CDI in the region.

Asymptomatic and yet C. difficile-toxin positive? Prevalence and risk factors of carriers of toxigenic Clostridium difficile among geriatric in-patients.

Background Clostridium difficile infections (CDI) are the most frequent cause of diarrhoea in hospitals. Geriatric patients are more often affected by the condition, by a relapse and complications. Therefore, a crucial question is how often colonization with toxigenic Clostridium difficile strains occurs in elderly patients without diarrhoea and whether there is a “risk pattern” of colonized patients that can be defined by geriatric assessment. Furthermore, the probability for those asymptomatic carriers to develop a symptomatic infection over time has not been sufficiently explored.MethodsWe performed a cohort study design to assess the association of clinical variables with Clostridium difficile colonization. The first stool sample of 262 consecutive asymptomatic patients admitted to a geriatric unit was tested for toxigenic Clostridium difficile using PCR (GeneXpert, Cepheid). A comprehensive geriatric assessment (CGA) including Barthel Index, Mini Mental State Examination (MMSE) and hand grip-strength was performed. In addition, Charlson Comorbidity Index, body mass index, number and length of previous hospital stays, previous treatment with antibiotics, institutionalization, primary diagnoses and medication were recorded and evaluated as possible risk factors of colonization by means of binary logistic regression. Secondly, we explored the association of C. difficile colonization with subsequent development of CDI during hospital stay.ResultsAt admission, 43 (16.4%) patients tested positive for toxin B by PCR. Seven (16.3%) of these colonized patients developed clinical CDI during hospital stay, compared to one out of 219 patients with negative or invalid PCR testing (Odds ratio 12,3; Fisher’s exact test: p = 0.000). Overall, 7 out of 8 (87.5%) CDI patients had been colonized at admission. Risk factors of colonization with C. difficile were a history of CDI, previous antibiotic treatment and hospital stays. The parameters of the CGA were not significantly associated with colonization.ConclusionColonization with toxigenic Clostridium difficile strains occurs frequently in asymptomatic patients admitted to a geriatric unit. Previous CDI, antibiotic exposure and hospital stay, but not clinical variables such as CGA, are the main factors associated with asymptomatic Clostridium difficile carriage. Colonization is a crucial risk factor for subsequent development of symptomatic CDI.

Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type

Recent surveys indicate that the majority of toxigenic Clostridium difficile strains isolated in European hospitals belonged to PCR-ribotypes (RTs) different from RT 027 or RT 078. Among these types, RT 018 has been reported in Italy and, more recently, in Korea and Japan. In Italy, strains RT 018 have become predominant in the early 2000s, whereas the majority of strains isolated before were RT 126, a type belonging to the same lineage as the RT 078. In this study, we have found that Italian strains RT 018 are resistant to erythromycin, clindamycin, moxifloxacin and rifampicin. Rifampicin resistance is rarely observed in strains RT 018 from other countries and in Italian strains RT 078 and RT 126, therefore the decennial use of rifamycin antibiotics in Italy may be one of the driving factors for the spread of RT 018 in our country. The strains RT 018 examined showed a significant higher adhesion to Caco-2 cells compared to strains RT 078 and RT 126. Furthermore, strains RT 018 became predominant in in vitro competition assays with strains RT 078 or RT 126. If maintained in vivo, these characteristics could lead to a rapid colonization of the intestine by strains RT 018. Under the conditions used, isolates RT 018 produced significantly higher toxins levels compared to strains RT 078 and RT 126, while heat-resistant CFUs production seems to be strain-dependent. Robust toxin production and enhanced sporulation could in part explain the high diffusion and interpatient transmissibility observed for strains RT 018 in the hospital environment. In conclusion, the characteristics observed in the Italian isolates RT 018 seem to contribute in conferring an adaptive advantage to these strains, allowing their successful spread in our country.

Contamination of Australian newborn calf carcasses at slaughter with Clostridium difficile

In North America and Europe, reports of a genetic overlap between toxigenic strains of Clostridium difficile isolated from humans, livestock and retail meat suggest that food-borne transmission may be occurring. We investigated the prevalence, concentration and genetic diversity of C. difficile on the carcasses (n = 300) and in the faeces (n = 30) of neonatal veal calves at three abattoirs in Australia in 2013. Selective culture (both direct and enrichment) was performed, and all isolates were characterized by PCR for the toxin genes tcdA, tcdB and cdtA/B and by PCR ribotyping. Prevalence of C. difficile was 25.3% (76/300) on carcasses and 60.0% (18/30) in faeces. Multiple PCR ribotypes (RT) were detected, with four binary toxin-positive RTs accounting for 70.3% (71/101) of isolates; 127 (A+, B+, CDT+, 32.7%), 288 (A–, B–, CDT+, 28.7%), 033 (A–, B–, CDT+, 6.9%) and 126 (A+, B+, CDT+, 2.0%). Viable counts of a subset of samples revealed detectable numbers of C. difficile in 66.7% (10/15) of faecal samples (range 2.0 × 103 to 2.3 × 106 CFU/mL, median count 2.5 × 104 CFU/mL) and in 16.7% (25/150) of carcase samples (range 3 to 33 CFU/cm2, median count 7 CFU/cm2). These data further confirm that Australian neonatal veal calf carcasses are contaminated with potentially significant strains of C. difficile at slaughter.

Ecological effect of ceftazidime/avibactam on the normal human intestinal microbiota

Ceftazidime/avibactam is a new combination of the antibiotic ceftazidime with the novel, non-β-lactam β-lactamase inhibitor avibactam. The purpose of the present study was to investigate the effect of ceftazidime/avibactam on the human intestinal microbiota following intravenous (i.v.) administration. Twelve healthy volunteers received ceftazidime/avibactam by i.v. infusion (2000mg ceftazidime and 500mg avibactam) given over 2h every 8h on Days 1–6 (inclusive) and a single dose on Day 7. Faecal samples were collected on Day–1 (pre-dose), during administration on Days 2, 5 and 7 and post-dose on Days 9, 14 and 21. Samples were cultured on non-selective and selective media. The number of Escherichia coli and other enterobacteria decreased significantly during administration of ceftazidime/avibactam, whereas the number of enterococci increased. Lactobacilli, bifidobacteria, clostridia and Bacteroides decreased significantly during ceftazidime/avibactam administration. The effects on lactobacilli, bifidobacteria and Bacteroides were similar in the 12 volunteers, whilst clostridia showed different ecological patterns among the volunteers. Toxigenic Clostridium difficile strains were detected in five volunteers during the study. In four of the volunteers, loose stools were reported as adverse events. Plasma samples were collected on Days −1, 2, 5 and 7. Ceftazidime and avibactam concentrations in plasma (ceftazidime 0–224.2mg/L of plasma and avibactam 0–70.5mg/L of plasma) and faeces (ceftazidime 0–468.2mg/kg of faeces and avibactam 0–146.0mg/kg of faeces) were found by bioassay. New colonising resistant clostridia were found in five volunteers and lactobacilli were found in three volunteers.

Infections caused by Clostridium difficile bacterium

Toxigenic strains of Clostridium difficile are the major cause of intestinal tract diseases of hospitalized patients. For a long time, Clostridium difficile was considered to produce both types of toxins (A+/B+ strain). However, the investigations conducted in the last ten years point to the existence of clinically significant isolates which produce only toxin B, i.e. toxin A negative. Toxigenic strains Clostridium difficile are present in our country so this bacterium sort has to be considered in differential causal diagnosis of diarrhea syndrome. Considering that it can cause a difficult form of the disease, it is an obligation to establish the presence of some toxins in stool samples of patients and/or production of some toxins in liquid culture of isolates.

Detection of toxigenic Clostridium difficile strains isolated from meat and meat products in Iran

Detection of toxigenic Clostridium difficile strains isolated from meat and meat products in Iran

A search for Clostridium difficile ribotypes 027 and 078 in Brazil

Toxigenic strains of Clostridium difficile may be disseminating. Here we prospectively screened patients with nosocomial diarrhoea in two hospitals in Brazil. To identify C. difficile polymerase chain reaction ribotypes 027/078 strains, we used high resolution melting and multiplex polymerase chain reaction. Among 116 screened patients, 11 were positive for C. difficile. The polymerase chain reaction ribotypes 027/078 strains were not identified in this study.

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins.

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.

Immunsuppressive Behandlung als Risikofaktor für das Auftreten einer Clostridium-difficile-Infektion (CDI)

Toxigenic Clostridium difficile strains are known as the most common infectious cause of antibiotic-associated intestinal disease and nosocomial diarrhoea. The increased incidence of hypervirulent strains gives rise to worldwide concern. In particular, courses with multiple recurrences are observed in the presence of immunosuppression. In this retrospective controlled observational study we aimed to determine immunosuppression as an independent risk factor for symptomatic CDI and to identify characteristics and differences of immunocompromised patients with respect to disease severity, disease progression, intestinal manifestations, recurrence rates and other factors. We compared symptoms and clinical features of 55 immunosuppressed patients with confirmed CDI with those of 50 patients without immunosuppressive medication (mean age 59.3 years ± 16.2 vs. 69.2 years ± 15.0) who were treated at the Departments of Internal Medicine I and IV of the University Hospital Halle (Saale), Germany, between 2006 and 2009. Multivariate analysis using binary logistic regression was performed for a control group of 105 patients without CDI. In this group, there were 62 patients without evidence of immunosuppression and 43 immunosuppressed patients (mean age 66.9 years ± 12.4 vs. 56.0 years ± 13.7). The clinical courses of the two groups differed considerably. Immunosuppressed patients were significantly more frequently colonised with Clostridium difficile without clinically detectable manifestation or only mild clinical symptoms not requiring therapy (22 vs. 2 %, p = 0.003), while there were similar numbers of moderate (46 vs. 52 %, p = 0.503) but less severe CDI cases (27 vs. 40 %, p = 0.167). Relapses were observed more frequently in the group of immunosuppressed patients (15 vs. 6 %, p = 0.153). Multivariate analysis using logistic regression identified immunosuppression as an independent risk factor for CDI (OR = 2.75), in addition to prior antibiotic therapy (OR = 10.15) and PPI intake (OR = 2.93). We conclude that immunosuppression has to be regarded as an independent risk factor for CDI. Immunosuppressive treatment increases the risk of colonisation and infection with Clostridium difficile and leads to a higher relapse rate in patients with CDI.