Difficile Infection In Humans Research Articles

Precise quantification of bacterial strains after fecal microbiota transplantation delineates long-term engraftment and explains outcomes

Fecal microbiota transplantation (FMT) has been successfully applied to treat recurrent Clostridium difficile infection in humans, but a precise method to measure which bacterial strains stably engraft in recipients and evaluate their association with clinical outcomes is lacking. We assembled a collection of >1,000 different bacterial strains that were cultured from the fecal samples of 22 FMT donors and recipients. Using our strain collection combined with metagenomic sequencing data from the same samples, we developed a statistical approach named Strainer for the detection and tracking of bacterial strains from metagenomic sequencing data. We applied Strainer to evaluate a cohort of 13 FMT longitudinal clinical interventions and detected stable engraftment of 71% of donor microbiota strains in recipients up to 5 years post-FMT. We found that 80% of recipient gut bacterial strains pre-FMT were eliminated by FMT and that post-FMT the strains present persisted up to 5 years later, together with environmentally acquired strains. Quantification of donor bacterial strain engraftment in recipients independently explained (precision 100%, recall 95%) the clinical outcomes (relapse or success) after initial and repeat FMT. We report a compendium of bacterial species and strains that consistently engraft in recipients over time that could be used in defined live biotherapeutic products as an alternative to FMT. Our analytical framework and Strainer can be applied to systematically evaluate either FMT or defined live bacterial therapeutic studies by quantification of strain engraftment in recipients.

Protective Effect of Baicalin against Clostridioides difficile Infection in Mice.

This study investigated the prophylactic and therapeutic efficacies of baicalin (BC), a plant-derived flavone glycoside, in reducing the severity of Clostridioides difficile infection (CDI) in a mouse model. In the prophylactic trial, C57BL/6 mice were provided with BC (0, 11, and 22 mg/L in drinking water) from 12 days before C. difficile challenge through the end of the experiment, whereas BC administration started day 1 post challenge in the therapeutic trial. Both challenge and control groups were infected with 106 CFU/mL of hypervirulent C. difficile BAA 1803 spores or sterile PBS, and the clinical and diarrheal scores were recorded for 10 days post challenge. On day 2 post challenge, fecal and tissue samples were collected from mice prophylactically administered with BC for microbiome and histopathologic analysis. Both prophylactic and therapeutic supplementation of BC significantly reduced the severity of colonic lesions and improved CDI clinical progression and outcome compared with control (p < 0.05). Microbiome analysis revealed a significant increase in Gammaproteobacteria and reduction in the abundance of protective microbiota (Firmicutes) in antibiotic-treated and C. difficile-infected mice compared with controls (p < 0.05). However, baicalin supplementation favorably altered the microbiome composition, as revealed by an increased abundance in beneficial bacteria, especially Lachnospiraceae and Akkermansia. Our results warrant follow-up investigations on the use of BC as an adjunct to antibiotic therapy to control gut dysbiosis and reduce C. difficile infection in humans.

Clostridioides difficile carriage in animals and the associated changes in the host fecal microbiota

The relationship between the gut microbiota and Clostridioides difficile, and its role in the severity of C. difficile infection in humans is an area of active research. Intestinal carriage of toxigenic and non-toxigenic C. difficile strains, with and without clinical signs, is reported in animals, however few studies have looked at the risk factors associated with C. difficile carriage and the role of the host gut microbiota. Here, we isolated and characterized C. difficile strains from different animal species (predominantly canines (dogs), felines (cats), and equines (horses)) that were brought in for tertiary care at North Carolina State University Veterinary Hospital. C. difficile strains were characterized by toxin gene profiling, fluorescent PCR ribotyping, and antimicrobial susceptibility testing. 16S rRNA gene sequencing was done on animal feces to investigate the relationship between the presence of C. difficile and the gut microbiota in different hosts. Here, we show that C. difficile was recovered from 20.9% of samples (42/201), which included 33 canines, 2 felines, and 7 equines. Over 69% (29/42) of the isolates were toxigenic and belonged to 14 different ribotypes including ones known to cause CDI in humans. The presence of C. difficile results in a shift in the fecal microbial community structure in both canines and equines. Commensal Clostridium hiranonis was negatively associated with C. difficile in canines. Further experimentation showed a clear antagonistic relationship between the two strains in vitro, suggesting that commensal Clostridia might play a role in colonization resistance against C. difficile in different hosts.

Characterisation and Antibiotic Susceptibility Profile of Clostridioides (Clostridium) Difficile Isolated from Chicken Carcasses.

IntroductionClostridioides (Clostridium) difficile is a Gram+, anaerobic, spore-forming, rod-shaped bacterium that can produce toxins, and it is mainly because its virulence is attributed. The objective of this study was to evaluate the presence of C. difficile and hyper virulent ribotypes in chicken carcasses and the antibiotic susceptibility of isolated strains.Material and MethodsC. difficile was isolated from chicken carcasses by microbiological methods, its ribotypes were identified by means of PCR, the toxin production ability was defined by ELISA, and the susceptibility of the isolates to selected antibiotics was determined by minimum inhibitory concentration evaluator strips.ResultsThe bacterium was isolated from 69 out of 185 (37.3%) examined chicken carcass samples, and six out of the 69 (8.7%) isolates were identified as ribotype 027. All isolates were susceptible to amoxicillin-clavulanic acid (100.0%), vancomycin (97.1%), metronidazole (88.4%), and tetracycline (95.7%), whereas they were resistant to cefotaxime (97.1%) and imipenem (89.9%).ConclusionThe results of this study demonstrate the presence of toxigenic C. difficile isolates such as ribotype 027 (one of the most common causes of C. difficile infection in humans) in chicken carcasses. Although there is no case for stating that C. difficile is a food-borne pathogen, the presence of C. difficile in chicken may be considered to be a potential risk to consumers.

Characterization of Clostridioides difficile strains isolated from manure and digestate in five agricultural biogas plants

Clostridioides difficile strains were isolated from manure and digestate samples from five biogas plants in France. The objective of this study was to characterize these isolates using PCR ribotyping, wgMLST, a multiplex PCR targeting genes encoding for the main virulence factors, i.e. tcdA, tcdB, cdtA and cdtB, and antimicrobial susceptibility assays. The 54 strains characterized were all positive for tcdA and tcdB and 83% (45/54) were positive for the binary toxin genes. PCR ribotypes 126 (59%) and 078 (37%) were predominant, and wgMLST analysis of 18 isolates showed close proximity of strains within a single biogas plant. Samples from the biogas plant supplied with cattle and poultry manure displayed the largest variety in PCR ribotypes. The in vitro activities of nine antimicrobial agents were determined. All the strains were susceptible to vancomycin and metronidazole, which are currently considered first-line treatments for C. difficile infection in humans. All the strains were resistant to clindamycin. The results of this study show that a high percentage of C. difficile strains present in the French biogas plants investigated are toxigenic strains from PCR ribotypes also commonly found in humans.

Dissemination of Clostridium difficile spores between environment and households: Dog paws and shoes.

Clostridium difficile is an anaerobic, spore-forming bacterium that causes intestinal infections. Although C.difficile is still predominantly considered as a nosocomial pathogen, there has been an increase in the number of community-associated infections. Since C.difficile is ubiquitous and can be isolated from nearly any environment, one of the possibilities for community acquisition could be exposure to spores in the domestic environment. The aim of this study was to evaluate the presence of C.difficile spores on shoes, slippers and on dog paws and to explore the importance of these surfaces as vectors for the dissemination of C.difficile in a domestic environment. Overall, C.difficile was present in 14 (70%) of 20 households and in 31 of 90 (34%) collected samples. Shoes and slippers had the highest positivity rates, 19 of 44 (43%) and 6 of 21 (28%), respectively, followed by dog paws 6 of 25 (24%). Thirteen C.difficilePCR ribotypes were identified with half of the isolates belonging to ribotype 014/020, which is the predominant type circulating in human population and is also commonly found in the environment (e.g. soil and water) in Slovenia. In three households, identical PCR ribotypes were found on dog paws, shoes and slippers. To understand the fine-scale genetic relatedness of these isolates, we sequenced the genomes. Low level of single nucleotide variant (SNV) differences between isolates from the same households, consistent with a recent transmission from a common source, were seen for isolates of PCR ribotype 014/020 but not for PCR ribotype 010. Our results suggest that shoe soles and dog paws could serve for the dissemination of C.difficile spores between households and environment and could contribute to community-relevant sources for C.difficile infection in humans.

More than 50% of Clostridium difficile Isolates from Pet Dogs in Flagstaff, USA, Carry Toxigenic Genotypes.

Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans.

Potentially hypervirulent Clostridium difficile PCR ribotype 078 lineage isolates in pigs and possible implications for humans in Taiwan

Clostridium difficile is a human and animal pathogen. Recently, the incidence of community-acquired C. difficile infection has increased, and many studies have indicated that C. difficile might be food-borne. The correlation between C. difficile infection in humans and in animals has been a topic of debate. The objective of this study was to determine the genetic relatedness of C. difficile from human and pigs in Taiwan. We investigated the molecular epidemiology of C. difficile in healthy humans and pigs from 2011 to 2015. The isolation rate of C. difficile from pigs in 13 commercial farms was 49% (100/204), and a high proportion of hypervirulent (C. difficile carrying tcdA, tcdB, and cdtA/B genes and a 39-bp deletion in the tcdC gene) ribotype 078 lineage isolates (90%, 90/100; including 078, 126, 127, and 066-like isolates) were identified. In addition, the C. difficile ribotype 127 isolates from pigs typically exhibited moxifloxacin resistance (37/43; 86%). In healthy humans, the isolation rate was 4.3% (3/69), and all healthy human isolates were non-toxigenic. In particular, we compared the porcine isolates with two patient strains (ribotype 127) obtained from two hospitals in central Taiwan. The multilocus variable number tandem repeat analysis revealed a high genetic relatedness between ribotype 127 from patients and pigs. This study indicated that isolates of the ribotype 078 lineage, and especially ribotype 127, were widely distributed in pig farms and showed a high frequency of moxifloxacin resistance. The closely related ribotype 127 from patients and pigs may have had a common origin or low diversity. In conclusion, C. difficile ribotype 127 is a noteworthy pathogen in pigs and poses a potential public health threat.

A modified R-type bacteriocin specifically targeting Clostridium difficile prevents colonization of mice without affecting gut microbiota diversity.

ABSTRACTClostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin (“diffocin”) from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota.

Carriage of Clostridium difficile in free-living South American coati (Nasua nasua) in Brazil

The objective of this study was to isolate and characterize Clostridium difficile strains in stool samples from a wild urban mammal, a South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped, and stool samples were collected. C. difficile was isolated from three (6.5%) sampled animals, two strains were toxigenic (A+B+CDT−, PCR ribotype 014/020 and 106) and one was non toxigenic (A−B−CDT−, PCR ribotype 053). The present work confirms that ring-tailed coati (N. nasua) could harbor C. difficile strains, including those PCR ribotypes commonly reported in C. difficile infection in humans.

Prevalence and molecular characterization of Clostridium difficile isolated from European Barn Swallows (Hirundo rustica) during migration

BackgroundClostridium difficile is an important bacterial pathogen of humans and a variety of animal species. Birds, especially migratory passerine species, can play a role in the spread of many pathogens, including Clostridium difficile. Barn Swallows (Hirundo rustica) nest in close proximity to human habitats and their biology is closely associated with cattle farming. Therefore, we hypothesized that Barn Swallows can be the reservoir of Clostridium difficile.ResultsBarn Swallows (n = 175) were captured on their autumn migration across Europe to sub-Saharan Africa. Droppings were collected from juvenile (n = 152) and adult birds (n = 23). Overall prevalence of Clostridium difficile was 4% (7/175); 4.6% (7/152) in juvenile birds and 0/23 in adults. Clostridium difficile ribotypes 078, 002 and 014 were identified, which are commonly found in farm animals and humans. Three new Clostridium difficile ribotypes were also identified: SB3, SB159 and SB166, one of which was toxigenic, harbouring genes for toxins A and B.ConclusionsResults of this study indicate that Barn Swallows might play a role in national and international dissemination of Clostridium difficile and could serve as a source for human and animal infection. Clostridium difficile ribotype 078 was identified, which has been reported as an emerging cause of community-associated Clostridium difficile infection in humans. Based on this and other studies, however, it is more likely that Barn Swallows have a more indicative than perpetuating role in Clostridium difficile epidemiology.

Correlation between virulence gene expression and proton pump inhibitors and ambient pH in Clostridium difficile: results of an in vitro study.

Proton pump inhibitors (PPIs) are associated with the development of Clostridium difficile infection in humans. Though it is assumed that PPIs mediate this effect through gastric acid suppression, there has been little investigation into whether PPIs, or ambient pH, might directly affect the expression of C. difficile toxin genes. In the present study, C. difficile ribotypes 001, 027 and 078 obtained from human subjects were grown under anaerobic conditions prepared at pHs of 5, 7.3 and 9. Matched trios were exposed to 100 µM and 200 µM of omeprazole along with PPI untreated controls. Custom designed reverse transcription quantitative PCR hydrolysis probes were used to assess C. difficile gene expression for toxins A (tcdA), B (tcdB) and binary toxin (cdtB), as well as their positive regulators (tcdR and cdtR), using rrsA, which encodes 16S rRNA, as a constitutively expressed reference gene. tcdC and codY, negative regulators of toxin expression, were also assessed. Basic pH resulted in greater expression of tcdA, and with PPI exposure a 120-fold higher expression was noted with ribotype 001. tcdB and cdtB expressions were much less responsive to pH or PPIs, though a clear response to acidic pH and PPI exposure was observed in ribotype 027. tcdC and codY expressions were largely unaffected, except with ribotype 027; low pH and PPIs resulted in their greater expression, though to a lesser degree than with toxin genes and their positive regulators. Non-neutral pH and PPI exposure appear to have an effect on C. difficile, one that has a net effect towards toxin gene expression.

Correlation between virulence gene expression and proton pump inhibitors and ambient pH in Clostridium difficile: results of an in vitro study

Proton pump inhibitors (PPIs) are associated with the development of Clostridium difficile infection in humans. Though it is assumed that PPIs mediate this effect through gastric acid suppression, there has been little investigation into whether PPIs, or ambient pH, might directly affect the expression of C. difficile toxin genes. In the present study, C. difficile ribotypes 001, 027 and 078 obtained from human subjects were grown under anaerobic conditions prepared at pHs of 5, 7.3 and 9. Matched trios were exposed to 100 µM and 200 µM of omeprazole along with PPI untreated controls. Custom designed reverse transcription quantitative PCR hydrolysis probes were used to assess C. difficile gene expression for toxins A (tcdA), B (tcdB) and binary toxin (cdtB), as well as their positive regulators (tcdR and cdtR), using rrsA, which encodes 16S rRNA, as a constitutively expressed reference gene. tcdC and codY, negative regulators of toxin expression, were also assessed. Basic pH resulted in greater expression of tcdA, and with PPI exposure a 120-fold higher expression was noted with ribotype 001. tcdB and cdtB expressions were much less responsive to pH or PPIs, though a clear response to acidic pH and PPI exposure was observed in ribotype 027. tcdC and codY expressions were largely unaffected, except with ribotype 027; low pH and PPIs resulted in their greater expression, though to a lesser degree than with toxin genes and their positive regulators. Non-neutral pH and PPI exposure appear to have an effect on C. difficile, one that has a net effect towards toxin gene expression.

Prevalence and Genotypic Characterization of Clostridium difficile From Ruminants in Switzerland

Clostridium difficile is an emerging enteric pathogen of humans and animals with a known main reservoir in the intestinal tract of various warm-blooded animals. This study was carried out to evaluate the prevalence of C. difficile in 150 rectal swab samples collected from cattle and goats in Switzerland. The overall prevalence of C. difficile was 6.6%. The isolates belonged to the PCR ribotypes 033, 066, 070, 003, 001 and 137. In addition, the occurrence of C. difficile in faecal samples collected from farm ground was also evaluated and C. difficile was detected in 21% (7/30) of these samples. These isolates belonged to ribotypes 033, 066, 014 and 137. Several isolates pertained to ribotypes known to cause Clostridium difficile infections in humans.

Macro and Micro Diversity of Clostridium difficile Isolates from Diverse Sources and Geographical Locations

Clostridium difficile has emerged rapidly as the leading cause of antibiotic-associated diarrheal disease, with the temporal and geographical appearance of dominant PCR ribotypes such as 017, 027 and 078. Despite this continued threat, we have a poor understanding of how or why particular variants emerge and the sources of strains that dominate different human populations. We have undertaken a breadth genotyping study using multilocus sequence typing (MLST) analysis of 385 C. difficile strains from diverse sources by host (human, animal and food), geographical locations (North America, Europe and Australia) and PCR ribotypes. Results identified 18 novel sequence types (STs) and 3 new allele sequences and confirmed the presence of five distinct clonal lineages generally associated with outbreaks of C. difficile infection in humans. Strains of animal and food origin were found of both ST-1 and ST-11 that are frequently associated with human disease. An in depth MLST analysis of the evolutionary distant ST-11/PCR ribotype 078 clonal lineage revealed that ST-11 can be found in alternative but closely related PCR ribotypes and PCR ribotype 078 alleles contain mutations generating novel STs. PCR ribotype 027 and 017 lineages may consist of two divergent subclades. Furthermore evidence of microdiversity was present within the heterogeneous clade 1. This study helps to define the evolutionary origin of dominant C. difficile lineages and demonstrates that C. difficile is continuing to evolve in concert with human activity.

The Ecology and Pathobiology of Clostridium difficile Infections: An Interdisciplinary Challenge

Clostridium difficile is a well recognized pathogen of humans and animals. Although C. difficile was first identified over 70 years ago, much remains unknown in regards to the primary source of human acquisition and its pathobiology. These deficits in our knowledge have been intensified by dramatic increases in both the frequency and severity of disease in humans over the last decade. The changes in C. difficile epidemiology might be due to the emergence of a hypervirulent stain of C. difficile, ageing of the population, altered risk of developing infection with newer medications, and/or increased exposure to C. difficile outside of hospitals. In recent years, there have been numerous reports documenting C. difficile contamination of various foods, and reports of similarities between strains that infect animals and strains that infect humans as well. The purposes of this review are to highlight the many challenges to diagnosing, treating, and preventing C. difficile infection in humans, and to stress that collaboration between human and veterinary researchers is needed to control this pathogen.

Low occurrence of Clostridium difficile in fecal samples of healthy calves and pigs at slaughter and in minced meat in Switzerland.

Clostridium difficile is a cause of diarrhea and colitis in humans. The increase of incidence and severity of C. difficile infections in humans in past years is due, at least in part, to the emergence of more virulent strains (PCR ribotypes 027 and 078). Recent studies describe the occurrence of hypervirulent strains in ground meat products. Therefore, food animals and food need to be assessed for their possible role as vectors of C. difficile to humans. In this pilot study, fecal samples of 204 calves and 165 pigs, as well as 46 minced meat products were investigated to determine the occurrence of C. difficile in farm animals at slaughter and in ground meat products at the retail level in Switzerland. C. difficile was isolated from only one fecal sample of a calf. All samples from pigs and ground meat were negative. Further characterization revealed that the isolated strain harbored genes for toxins A and B as well as binary toxin, and belonged to the ribotype 078. Based on these results, low occurrence of C. difficile in farm animals at slaughter and retail ground meat in Switzerland is postulated. However, further studies are necessary to confirm these preliminary data and to assess future trends.

Detection and Enumeration ofClostridium difficileSpores in Retail Beef and Pork

Recent studies have identified Clostridium difficile in food animals and retail meat, and concern has been raised about the potential for food to act as a source of C. difficile infection in humans. Previous studies of retail meat have relied on enrichment culture alone, thereby preventing any assessment of the level of contamination in meat. This study evaluated the prevalence of C. difficile contamination of retail ground beef and ground pork in Canada. Ground beef and ground pork were purchased from retail outlets in four Canadian provinces. Quantitative and enrichment culture was performed. Clostridium difficile was isolated from 28/230 (12%) samples overall: 14/115 (12%) ground beef samples and 14/115 (12%) ground pork samples (P = 1.0). For ground beef, 10/14 samples (71%) were positive by enrichment culture only. Of the 4 ground beef samples that were positive by direct culture, 20 spores/g were present in 2 while 120 and 240 spores/g were present in 1 each. For ground pork, 10/14 (71%) samples were positive by enrichment culture only. Of the 4 ground pork samples that were positive by direct culture, 20 spores/g were present in 3 while 60 spores/g were present in 1. Ribotype 078 predominated, consistent with some previous studies of C. difficile in food animals. Ribotype 027/North American pulsotype 1 was also identified in both retail beef and pork. This study has identified relatively common contamination of retail ground beef and pork with C. difficile spores; however, the levels of contamination were very low.

Clostridium difficile: a survey of fecal carriage in cats in a veterinary medical teaching hospital.

Fecal samples collected from 245 cats over a 6-month period were analyzed for the presence of Clostridium difficile. After culture on selective media, isolates were identified by a latex agglutination test, and the presence of toxin A and toxin B gene sequences was determined by polymerase chain reaction. Clostridium difficile was isolated from 23 (9.4%) of the cats, and 34.8% of that group were colonized with toxigenic strains. All of the cats colonized with toxigenic C. difficile had > or = 1 of the risk factors (antibiotic use, antineoplastic therapy, immunosuppressive virus infection) associated with C. difficile infection in humans. Clostridium difficile was not found in any of the cats from a clinically healthy outpatient group of cats examined from the same hospital nor in cats from a specific-pathogen-free research colony on the same campus tested during the same time period. The data obtained in this study confirm the presence of C. difficile in cats at a veterinary teaching hospital. DNA fingerprinting analysis of these isolates allowed separation of the strains into 5 groups. Type 4 strain found in 7 cats was also recovered from the floor drain in the same hospital, suggesting a possible source of infection. Whether the organism is of clinical significance in diarrheal diseases of cats remains to be determined.

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans.