Clostridium difficile testing algorithms: What is practical and feasible?

There has been renewed interest in the laboratory diagnosis of Clostridium difficile infections due in large measure to the increase in both numbers and severity of cases of this disease. For the past two decades, enzyme-immunoassays (EIAs) for the detection of first C. difficile toxin A and then toxins A and B have been the most widely used diagnostic test for diagnosis of C. difficile infections. Recently this diagnostic approach has been called into question by the recognition that a screening test which detects glutamate dehydrogenase, a cell wall antigen of C. difficile, was significantly more sensitive than toxins A and B EIAs making it an effective screening test for C. difficile infection. Although sensitive, GDH lacks specificity and so if this test was utilized, a confirmatory test to differentiate false positives from true positives was needed. Studies to date have used cytotoxin neutralization or toxigenic culture as confirmatory tests but both of these have their limitations. A testing algorithm using rapid immunochromatographic devices for detection of GDH and toxins A and B as screening tests will give an accurate test result in approximately 90% of specimens within one hour when using cytotoxin neutralization as a reference method. For the other 10% of specimens, a third test would be needed in the algorithm. This test could be cytotoxin neutralization, toxigenic culture, or PCR for toxin or toxin operon genes.     

Identification of Toxemia in Patients with Clostridium difficile Infection

Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.     

Computational modeling of the gut microbiota reveals putative metabolic mechanisms of recurrent Clostridioides difficile infection

Approximately 30% of patients who have Clostridioides difficile infection (CDI) will suffer at least one incident of reinfection. While the underlying causes of CDI recurrence are poorly understood, interactions between C. difficile and commensal gut bacteria are thought to play an important role. In this study, an in silico pipeline was used to process 16S rRNA gene amplicon sequence data of 225 stool samples from 93 CDI patients into sample-specific models of bacterial community metabolism. Clustered metabolite production rates generated from post-diagnosis samples generated a high Enterobacteriaceae abundance cluster containing disproportionately large numbers of recurrent samples and patients. This cluster was predicted to have significantly reduced capabilities for secondary bile acid synthesis but elevated capabilities for aromatic amino acid catabolism. When applied to 16S sequence data of 40 samples from fecal microbiota transplantation (FMT) patients suffering from recurrent CDI and their stool donors, the community modeling method generated a high Enterobacteriaceae abundance cluster with a disproportionate large number of pre-FMT samples. This cluster also was predicted to exhibit reduced secondary bile acid synthesis and elevated aromatic amino acid catabolism. Collectively, these in silico predictions suggest that Enterobacteriaceae may create a gut environment favorable for C. difficile spore germination and/or toxin synthesis.     

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

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

ICU-Onset Clostridium difficile Infection in a University Hospital in China: A Prospective Cohort Study

A prospective study was conducted to investigate the incidence, clinical profiles and outcome of ICU-onset CDI in a 50-bed medical ICU at a university hospital in China. Stools were collected from patients who developed ICU-onset diarrhea and was screened for tcdA (toxin A gene) and tcdB (toxin B gene) by PCR. CDI cases were compared with the ICU-onset non-CDI diarrhea cases for demographics, comorbidities, potential risk factors, major laboratory findings and outcomes. Stool samples from CDI cases were subjected to C. difficile culture and C. difficile isolates were screened for tcdA, tcdB and the binary toxin genes (cdtA and cdtB) using multiplex PCR. Strain typing of toxigenic C. difficile isolates was performed using multilocus sequence typing. There were 1,277 patients in the ICU during the study period and 124 (9.7%) developed ICU-onset diarrhea, of which 31 patients had CDI. The incidence of ICU-onset CDI was 25.2 cases per 10,000 ICU days. ICU-onset CDI cases had similar features with ICU-onset non-CDI diarrhea cases including the use of proton pump inhibitors and antibacterial agents. The crude mortality rate of ICU-onset CDI was 22.6%, but the attributable mortality rate of ICU-onset CDI was only 3.2% here. Toxigenic C. difficile isolates were recovered from 28 out of the 31 patients with CDI. cdtA and cdtB were found in two strains. Seventeen STs including 11 new STs were identified. All of the 11 new STs were single-locus variants of known STs and the 17 STs identified here could be clustered into 3 clades. The incidence of ICU-onset CDI here is similar to those in Europe and North America, suggesting that CDI is likely to be a common problem in China. Toxigenic C. difficile here belonged to a variety of STs, which may represent a significant clonal expansion rather than the true clonal diversity.     

Molecular Epidemiology of Clostridium difficile Infection in a Large Teaching Hospital in Thailand

Clostridium difficile infection (CDI) is a leading cause of healthcare-associated morbidity and mortality worldwide. In Thailand, CDI exhibits low recurrence and mortality and its molecular epidemiology is unknown. CDI surveillance was conducted in a tertiary facility (Siriraj Hospital, Bangkok). A total of 53 toxigenic C. difficile strains from Thai patients were analyzed by multi-locus sequence typing (MLST), PCR ribotyping, and pulse-field gel electrophoresis (PFGE). The mean age of the cohort was 64 years and 62.3% were female; 37.7% of patients were exposed to > two antibiotics prior to a diagnosis of CDI, with beta-lactams the most commonly used drug (56.3%). Metronidazole was used most commonly (77.5%; success rate 83.9%), and non-responders were treated with vancomycin (success rate 100%). None of the isolates carried binary toxin genes. Most isolates (98.2–100%) were susceptible to metronidazole, vancomycin, tigecycline and daptomycin. There were 11 sequence types (STs), 13 ribotypes (RTs) and four PFGE types. Six previously identified STs (ST12, ST13, ST14, ST33, ST41 and ST45) and five novel STs unique to Thailand (ST66, ST67, ST68, ST69 and ST70) were identified. PCR RTs UK 017 (ST45) (45.3%) and UK 014/020 (ST33) (24.5%) were the most common. High concordance was observed between the MLST and ribotyping results (p<0.001). C. difficile isolates from Thai patients were highly susceptible to standard antimicrobial agents. In conclusion, the five STs indicate the high genetic diversity and unique polymorphisms in Thailand. Moreover, the emergence of antimicrobial resistance to vancomycin warranted continuous surveillance to prevent further spread of the toxigenic C. difficile isolates.     

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model

BackgroundClostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model.MethodsC57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.ResultsC. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet.ConclusionsLow protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.     

Characterisation and carriage ratio of Clostridium difficile strains isolated from a community-dwelling elderly population in the United Kingdom

Background

Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.

Methods

Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier''s CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.

Results

Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.

Conclusions

To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.     

Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

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

An Integrated Metabolomic and Microbiome Analysis Identified Specific Gut Microbiota Associated with Fecal Cholesterol and Coprostanol in Clostridium difficile Infection

Clostridium difficile infection (CDI) is characterized by dysbiosis of the intestinal microbiota and a profound derangement in the fecal metabolome. However, the contribution of specific gut microbes to fecal metabolites in C. difficile-associated gut microbiome remains poorly understood. Using gas-chromatography mass spectrometry (GC-MS) and 16S rRNA deep sequencing, we analyzed the metabolome and microbiome of fecal samples obtained longitudinally from subjects with Clostridium difficile infection (n = 7) and healthy controls (n = 6). From 155 fecal metabolites, we identified two sterol metabolites at >95% match to cholesterol and coprostanol that significantly discriminated C. difficile-associated gut microbiome from healthy microbiota. By correlating the levels of cholesterol and coprostanol in fecal extracts with 2,395 bacterial operational taxonomic units (OTUs) determined by 16S rRNA sequencing, we identified 63 OTUs associated with high levels of coprostanol and 2 OTUs correlated with low coprostanol levels. Using indicator species analysis (ISA), 31 of the 63 coprostanol-associated bacteria correlated with health, and two Veillonella species were associated with low coprostanol levels that correlated strongly with CDI. These 65 bacterial taxa could be clustered into 12 sub-communities, with each community containing a consortium of organisms that co-occurred with one another. Our studies identified 63 human gut microbes associated with cholesterol-reducing activities. Given the importance of gut bacteria in reducing and eliminating cholesterol from the GI tract, these results support the recent finding that gut microbiome may play an important role in host lipid metabolism.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.     

Diagnostic trends in Clostridium difficile detection in Finnish microbiology laboratories

Due to increased interest directed to Clostridium difficile-associated infections, a questionnaire survey of laboratory diagnostics of toxin-producing C. difficile was conducted in Finland in June 2006. Different aspects pertaining to C. difficile diagnosis, such as requests and criteria used for testing, methods used for its detection, yearly changes in diagnostics since 1996, and the total number of investigations positive for C. difficile in 2005, were asked in the questionnaire, which was sent to 32 clinical microbiology laboratories, including all hospital-affiliated and the relevant private clinical microbiology laboratories in Finland. The situation was updated by phone and email correspondence in September 2008. In June 2006, 28 (88%) laboratories responded to the questionnaire survey; 24 of them reported routinely testing requested stool specimens for C. difficile. Main laboratory methods included toxin detection (21/24; 88%) and/or anaerobic culture (19/24; 79%). In June 2006, 18 (86%) of the 21 laboratories detecting toxins directly from feces, from the isolate, or both used methods for both toxin A (TcdA) and B (TcdB), whereas only one laboratory did so in 1996. By September 2008, all of the 23 laboratories performing diagnostics for C. difficile used methods for both TcdA and TcdB. In 2006, the number of specimens processed per 100,000 population varied remarkably between different hospital districts. In conclusion, culturing C. difficile is common and there has been a favorable shift in toxin detection practice in Finnish clinical microbiology laboratories. However, the variability in diagnostic activity reported in 2006 creates a challenge for national monitoring of the epidemiology of C. difficile and related diseases.     

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

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

C. difficile infection (CDI) in a long-term acute care facility (LTAC)

BackgroundC. difficile infection (CDI) is a common nosocomial infection in hospitals and impacts increased hospital cost and length of stay. Since scant information is available about the incidence and prevalence of CDI in Long-term acute care hospitals (LTACs), we therefore studied this at one local facility.MethodsDemographic and other data, and a fresh stool sample were obtained from all new LTAC admissions not carrying a prior diagnosis of CDI during the study period (July 23 to August 22, 2007). A GDH test for C. difficile antigen was performed. All initially positive stools were tested for toxins A and B and a sample was frozen for culture and typing. All antigen-negative patients were monitored for the development of diarrhea during the course of their LTAC hospitalization and, if clinically indicated, a sample was sent for toxins A and B testing and if positive, a stool sample was frozen and stored for culture and typing. Therapy of CDI was noted.Results36 patients were admitted during the study period. 4 of 31 (12.9%) of patients tested were antigen (+) on admission of which 2 (6.5%) were asymptomatic carriers and 2 (6.5%) had unsuspected active disease, including one with the BI epidemic strain. In follow-up, 20/36 (55.5%) developed diarrhea of which an additional 5 (13.8%) patients had developed CDI (average, hospital day 38) in the hospital. Therapy was instituted with vancomycin in 5/7 patients and metronidazole in 2/7 patients. During that quarter, the rate of nosocomial acquired CDI was 3.12 per 1000 patient days.ConclusionsC. difficile carriage and unsuspected clinical CDI occurs, including with the BI epidemic strain disease, in an important minority of patients, which may act as a reservoir for spread. New strategies for detection and prevention of CDI are needed.     

Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis.     

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

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

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.     

Molecular Characterization of Clostridium difficile Isolates from Human Subjects and the Environment

Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients.