scholarly journals FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291

2021 ◽  
Vol 12 ◽  
Author(s):  
Duolong Zhu ◽  
Shaohui Wang ◽  
Xingmin Sun
Keyword(s):  
Bacterial Colonization ◽  
Toxin Production ◽  
Toxin Gene ◽  
Clostridioides Difficile ◽  
Significant Difference ◽  
Assembly Factor ◽  
Carbon Storage Regulator ◽  
Transcription Suppression ◽  
Post Transcriptional Regulation

Clostridioides difficile flagellin FliC is associated with toxin gene expression, bacterial colonization, and virulence, and is also involved in pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated in C. difficile remains unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor (FliW), flagellin Hag (FliC homolog), and Carbon storage regulator A (CsrA), which is referred to as partner-switching mechanism “FliW-CsrA-Hag.” In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that fliW deletion, csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion mutant) dramatically decreased FliC production, but not fliC gene transcription. Suppression of fliC translation by csrA overexpression can be relieved mostly when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Our data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation. In light of “FliW-CsrA-Hag” switch coregulation mechanism reported in B. subtilis, our data also suggest that “FliW-CsrA-fliC/FliC” can regulate many facets of C. difficile R20291 pathogenicity. These findings further aid us in understanding the virulence regulation in C. difficile.

scholarly journals FliW and CsrA govern flagellin (FliC) synthesis and play pleiotropic roles in virulence and physiology of Clostridioides difficile R20291

2021 ◽  
Author(s):  
Duolong Zhu ◽  
Shaohui Wang ◽  
Xingmin Sun
Keyword(s):  
Gene Regulation ◽  
Bacterial Colonization ◽  
Toxin Production ◽  
Toxin Gene ◽  
Flic Gene ◽  
Clostridioides Difficile ◽  
Significant Difference ◽  
Assembly Factor ◽  
Carbon Storage Regulator

Clostridioides difficile is a Gram-positive, spore-forming, and toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. In C. difficile, the expression of flagellar genes is coupled to toxin gene regulation and bacterial colonization and virulence. The flagellin FliC is responsible for pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated is unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor FliW, Flagellin Hag (FliC homolog), and CsrA (Carbon storage regulator A), which is referred to as partner-switching mechanism "FliW-CsrA-Hag". In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that both fliW deletion or csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion) dramatically decreased FliC production, however, fliC gene transcription was unaffected. While suppression of fliC translation by csrA overexpression was mostly relieved when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Taken together, these data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation, which seems similar to the "FliW-CsrA-Hag" switch in B. subtilis. Our data also suggest that "FliW-CsrA-fliC/FliC" can regulate many facets of C. difficile R20291 pathogenicity.

scholarly journals Temporal Variations in Patterns of Clostridioides difficile Strain Diversity and Antibiotic Resistance in Thailand

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 714
Author(s):  
Supapit Wongkuna ◽  
Tavan Janvilisri ◽  
Matthew Phanchana ◽  
Phurt Harnvoravongchai ◽  
Amornrat Aroonnual ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Susceptibility ◽  
Reference Strain ◽  
Toxin Production ◽  
Temporal Variations ◽  
Toxin Gene ◽  
Data Sets ◽  
Clostridioides Difficile ◽  
Life Threatening ◽  
Susceptibility Profiles

Clostridioides difficile has been recognized as a life-threatening pathogen that causes enteric diseases, including antibiotic-associated diarrhea and pseudomembranous colitis. The severity of C. difficile infection (CDI) correlates with toxin production and antibiotic resistance of C. difficile. In Thailand, the data addressing ribotypes, toxigenic, and antimicrobial susceptibility profiles of this pathogen are scarce and some of these data sets are limited. In this study, two groups of C. difficile isolates in Thailand, including 50 isolates collected from 2006 to 2009 (THA group) and 26 isolates collected from 2010 to 2012 (THB group), were compared for toxin genes and ribotyping profiles. The production of toxins A and B were determined on the basis of toxin gene profiles. In addition, minimum inhibitory concentration of eight antibiotics were examined for all 76 C. difficile isolates. The isolates of the THA group were categorized into 27 A−B+CDT− (54%) and 23 A-B-CDT- (46%), while the THB isolates were classified into five toxigenic profiles, including six A+B+CDT+ (23%), two A+B+CDT− (8%), five A−B+CDT+ (19%), seven A−B+CDT− (27%), and six A−B−CDT− (23%). By visually comparing them to the references, only five ribotypes were identified among THA isolates, while 15 ribotypes were identified within THB isolates. Ribotype 017 was the most common in both groups. Interestingly, 18 unknown ribotyping patterns were identified. Among eight tcdA-positive isolates, three isolates showed significantly greater levels of toxin A than the reference strain. The levels of toxin B in 3 of 47 tcdB-positive isolates were significantly higher than that of the reference strain. Based on the antimicrobial susceptibility test, metronidazole showed potent efficiency against most isolates in both groups. However, high MIC values of cefoxitin (MICs 256 μg/mL) and chloramphenicol (MICs ≥ 64 μg/mL) were observed with most of the isolates. The other five antibiotics exhibited diverse MIC values among two groups of isolates. This work provides evidence of temporal changes in both C. difficile strains and patterns of antimicrobial resistance in Thailand.

scholarly journals RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Content Type ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

scholarly journals 2435. The Impact of Treatment Strategy and Toxin Status on Outcomes of Patients with Clostridioides difficile Infections

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S842-S842
Author(s):  
Daniel Friedman ◽  
Karen Zurek ◽  
Leyla Asadi ◽  
Mao-Cheng Lee ◽  
Holly Hoang
Keyword(s):  
Treatment Guidelines ◽  
Retrospective Chart Review ◽  
Toxin Production ◽  
Toxin Gene ◽  
Composite Outcome ◽  
Related Complication ◽  
Clostridioides Difficile ◽  
Increased Risk ◽  
Treatment Escalation ◽  
The Impact

Abstract Background Clostridioides difficile infection (CDI) is an important cause of morbidity and mortality and management continues to evolve. For laboratories that diagnose by detection of toxin gene, it is unclear whether reporting toxin production is additive to patient care. Furthermore, is there still a role for metronidazole (MNZ) given treatment guidelines now recommend vancomycin (VAN) as first-line therapy for non-severe cases? We analyzed cases of CDI in our hospital to assess outcomes of patients on MNZ vs. VAN and with or without toxin production. Methods A retrospective chart review of inpatients with CDI (based on detection of C. difficile toxin gene by PCR) was conducted between November 2017 and August 2018. Comparison of demographics and outcomes was performed in a) cases that were toxin-positive by enzyme immunoassay vs. negative and b) non-severe cases initially managed with MNZ vs. VAN. Results 76 patients were included (46 toxin-positive, 30 toxin-negative). Toxin-positive patients were older (mean age 77 vs. 62, p = 0.002) but had similar disease severity and initial treatment. A CDI recurrence occurred in 22% vs 0% in the toxin-positive cases (p = 0.006). Any CDI-related complication occurred in 23% of toxin-negative and 35% of toxin-positive cases (ns). After adjusting for toxin-status, age, and severity, the odds ratio of the composite outcome of any complication with toxin-positive CDI was not significant (OR 1.45 95% CI 0.45 -4.6, p = 0.52). There were 37 (49%) patients with non-severe CDI (27 MNZ, 10 VAN). Patients treated with VAN had higher stooling/day (6.3 vs 4.4, p = 0.04) and heart rate (p = 0.02). Initial MNZ use was associated with treatment escalation in 48% of cases compared with 10% in those treated with VAN alone (p = 0.03). CDI-associated mortality was higher in the VAN group (2/10 vs 0/27, p = 0.017). The rate of other complications was not significantly different. Conclusion Although no difference in the composite outcome of any CDI-related complication was detected between toxin positive vs negative patients, toxin-positivity may predict patients at risk for subsequent recurrence. Patients with non-severe CDI did not have increased risk of complications when managed with MNZ; however, they were more likely to require treatment escalation. Disclosures All authors: No reported disclosures.

scholarly journals RstA is a Major Regulator ofClostridioides difficileToxin Production and Motility

2018 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Binding Domain ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes,tcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multi-species chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside of the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. In addition, the ability for RstA to bind DNA and repress toxin production requires the species-specific domain predicted to respond to small quorum-sensing peptides. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct sensing mechanism.

scholarly journals 2398. Effect of Eosinopenia and Binary Toxin on Clostridioides difficile Infection Clinical Outcomes

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S828-S828
Author(s):  
Travis J Carlson ◽  
Bradley T Endres ◽  
Julie Le Pham ◽  
Anne J Gonzales-Luna ◽  
Faris S Alnezary ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Outcomes ◽  
Eosinophil Count ◽  
Toxin Production ◽  
Albumin Level ◽  
Binary Toxin ◽  
Inpatient Mortality ◽  
Healthcare Facilities ◽  
Clostridioides Difficile

Abstract Background The ability of Clostridioides difficile to cause clinical disease in humans is dependent on toxin production. Significantly fewer eosinophils are seen in the peripheral blood of mice infected with a binary toxin positive (CDT+) C. difficile strain. Furthermore, the presence of CDT and eosinopenia have separately been associated with increased mortality in humans with C. difficile infection (CDI). We hypothesized that CDI due to a CDT+ C. difficile strain accompanied by peripheral eosinopenia would be associated with higher odds of inpatient mortality. Methods This multicenter, retrospective cohort study included all patients ≥ 18 years of age with toxigenic CDI in which specimen ribotype data were available as part of our ongoing surveillance study. The cohort was stratified by eosinophil count (0.0 cells/μL vs. > 0.0 cells/μL). The primary outcome was inpatient mortality. A logistic regression model was developed modeling inpatient mortality as a function of the available patient covariates. All P-values were from 2-sided tests, and results were deemed statistically significant at P < 0.05. Results A total of 688 patients from 13 institutions in six cities were included. Of those, 132 had a baseline eosinophil count of 0.0 cells/µL and 556 had a baseline eosinophil count > 0.0 cells/µL. While the odds of inpatient mortality were higher among patients with eosinopenia and those infected with a CDT+ ribotype, the combination of these variables remained an independent predictor of inpatient mortality after adjusting for CCI score, WBC count, and serum albumin level (OR, 7.84; 95% CI, 1.85–33.20; P = 0.005). Conclusion This is the first attempt to study the in vivo relationship between CDT presence, human immune response, and CDI clinical outcome. We identified an association between CDT presence with concomitant eosinopenia and worsened CDI outcomes. Healthcare facilities should consider identifying this important subset of patients at the time of CDI diagnosis. Future CDI drug development might benefit from targeting C. difficile properties that impair host immune response, which may in turn decrease adverse clinical outcomes associated with this disease. Disclosures All authors: No reported disclosures.

scholarly journals Strain-Dependent RstA Regulation of Clostridioides difficile Toxin Production and Sporulation

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Ellen G. Krall ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Gastrointestinal Tract ◽  
Toxin Production ◽  
Spore Formation ◽  
Toxin Gene ◽  
Disease Symptoms ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Toxin Gene Expression ◽  
Strain Dependent

ABSTRACT The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm. To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm. In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits. IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm. Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

scholarly journals Alimentary and Pharmaceutical Approach to Natural Antimicrobials against Clostridioides difficile Gastrointestinal Infection

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1124
Author(s):  
Miguel Tortajada-Girbés ◽  
Alejandro Rivas ◽  
Manuel Hernández ◽  
Ana González ◽  
Maria A. Ferrús ◽  
...  
Keyword(s):  
Toxin Production ◽  
Gastrointestinal Infection ◽  
Natural Antimicrobials ◽  
Gastrointestinal Microbiome ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Successful Control ◽  
Marine Bioactives

Incidence of Clostridioides difficile infection (CDI) has been increasing in recent decades due to different factors, namely (i) extended use of broad-spectrum antibiotics, (ii) transmission within asymptomatic and susceptible patients, and (iii) unbalanced gastrointestinal microbiome and collateral diseases that favor C. difficile gastrointestinal domination and toxin production. Although antibiotic therapies have resulted in successful control of CDI in the last 20 years, the development of novel strategies is urged in order to combat the capability of C. difficile to generate and acquire resistance to conventional treatments and its consequent proliferation. In this regard, vegetable and marine bioactives have emerged as alternative and effective molecules to fight against this concerning pathogen. The present review examines the effectiveness of natural antimicrobials from vegetable and algae origin that have been used experimentally in in vitro and in vivo settings to prevent and combat CDI. The aim of the present work is to contribute to accurately describe the prospective use of emerging antimicrobials as future nutraceuticals and preventive therapies, namely (i) as dietary supplement to prevent CDI and reduce CDI recurrence by means of microbiota modulation and (ii) administering them complementarily to other treatments requiring antibiotics to prevent C. difficile gut invasion and infection progression.

scholarly journals The mechanisms of in vivo commensal control of Clostridioides difficile virulence

2020 ◽  
Author(s):  
BP Girinathan ◽  
N DiBenedetto ◽  
J Worley ◽  
J Peltier ◽  
R Lavin ◽  
...  
Keyword(s):  
Stress Responses ◽  
Toxin Production ◽  
Protective Effects ◽  
Clostridioides Difficile ◽  
Lethal Infection ◽  
Cellular Machinery ◽  
Clostridium Bifermentans ◽  
Gnotobiotic Mice ◽  
Acid Fermenter

AbstractWe define multiple mechanisms by which commensals protect against or worsen Clostridioides difficile infection. Using a systems-level approach we show how two species of Clostridia with distinct metabolic capabilities modulate the pathogen’s virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Clostridium bifermentans survived infection, while colonization with the butyrate-producer, Clostridium sardiniense, more rapidly succumbed. Systematic in vivo analyses revealed how each commensal altered the pathogen’s carbon source metabolism, cellular machinery, stress responses, and toxin production. Protective effects were replicated in infected conventional mice receiving C. bifermentans as an oral bacteriotherapeutic that prevented lethal infection. Leveraging a systematic and organism-level approach to host-commensal-pathogen interactions in vivo, we lay the groundwork for mechanistically-informed therapies to treat and prevent this disease.

scholarly journals Epidemic Ribotypes Of Clostridium (Now Clostridioides) Difficile Are Likely To Be More Virulent Than Non-Epidemic Ribotypes In Animal Models

2019 ◽  
Author(s):  
John C. Vitucci ◽  
Mark Pulse ◽  
Leslie Tabor-Simecka ◽  
Jerry W. Simecka
Keyword(s):  
Animal Models ◽  
Toxin Production ◽  
Intestinal Epithelial ◽  
Toxin B ◽  
Golden Syrian Hamster ◽  
Clostridioides Difficile ◽  
Conflicting Evidence ◽  
Epithelial Cell Lines

Abstract Background: Clostridioides difficile infections have become more frequently diagnosed and associated with greater disease severity, which has resulted in an increase burden on the healthcare system. These increases are attributed to the increased prevalence of hypervirulent strains encompassing select ribotypes. These epidemic ribotypes were characterized as hypervirulent due to higher in vitro spore and toxin production, as well as increased incidence, severity and mortality within patients. However, it is unclear whether epidemic ribotypes are truly more virulent than non-epidemic ribotypes in vivo. Furthermore, there is conflicting evidence about the ability of a strain’s in vitro phenotype to be predictive of their in vivo virulence. The goals of the current studies were to determine if epidemic ribotypes are more virulent than other ribotypes in animal models, and whether the in vitro virulence phenotype of an isolate or ribotype predict in vivo virulence. Results. To determine if epidemic strains were truly more virulent than other non-epidemic strains, the in vivo virulence of thirteen C. difficile isolates (7 non-epidemic and 6 epidemic ribotype isolates) were determined in murine (C57BL/6 mice) and hamster (golden Syrian hamster) models of C. difficile infections. The isolates of epidemic ribotype of C. difficile were found to be more virulent in both the murine and hamster models than non-epidemic isolates. In particular, the group of epidemic ribotypes of C. difficile had lower LD 50 values in hamsters. The increased severity of disease was associated with higher levels of Toxin A and Toxin B production found in fecal samples, but not numbers of organisms recovered. The isolates were further characterized for their in vitro virulence phenotypes, e.g. toxin production, growth rates, spore formation and adherence of spores to intestinal epithelial cell lines. Although there were higher levels of toxins produced and greater adherence for the group of epidemic ribotypes, the in vitro profiles of individual isolates were not always predictive of their in vivo virulence. Conclusions. Overall, the group of epidemic ribotypes of C. difficile were more virulent in vivo despite individual isolates having similar phenotypes to the non-epidemic isolates in vitro .

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