Difficile Challenge Research Articles

Gut microbiome changes in mouse, Mongolian gerbil, and hamster models following Clostridioides difficile challenge.

Clostridioides difficile infection (CDI), as well as its etiology and pathogenesis, have been extensively investigated. However, the absence of suitable CDI animal models that reflect CDI symptoms and the associated gut microbiome changes in humans has limited research progress in this field. Thus, we aimed to investigate whether Mongolian gerbils, which present a range of human pathological conditions, can been used in studies on CDI. Methods: In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. The results obtained showed that C. difficile colonized the gastrointestinal tracts of the three rodents, and after the C. difficile challenge, C57BL/6J mice did not manifest CDI symptoms and their intestines showed no significant pathological changes. However, the hamsters showed explosive intestinal bleeding and inflammation and the Mongolian gerbils presented diarrhea as well as increased infiltration of inflammatory cells, mucus secretion, and epithelial cell shedding in their intestinal tissue. Further, intestinal microbiome analysis revealed significant differences with respect to intestinal flora abundance and diversity. Specifically, after C. difficile challenge, the Firmicutes/Bacteroidetes ratio decreased for C57BL/6J mice, but increased significantly for Mongolian gerbils and hamsters. Furthermore, the abundance of Proteobacteria increased in all three models, especially in hamsters, while that of Verrucomicrobia only increased significantly in C57BL/6J mice and Mongolian gerbils. Our results also indicated that differences in the relative abundances of Lactobacillaceae and Akkermansia were primarily responsible for the observed differences in response to C. difficile challenge. Based on the observed responses to C. difficile challenge, we concluded for the first time that the Mongolian gerbil could be used as an animal model for CDI. Additionally, the taxa identified in this study may be used as biomarkers for further studies on CDI and to improve understanding regarding changes in gut microbiome in CDI-related diseases.

Modeling the effects of prebiotic interventions on luminal and mucosa-associated gut microbiota without and with Clostridium difficile challenge in vitro.

Prebiotics can modulate the gut microbial community composition and function for improved (gut) health and increase resilience against infections. In vitro models of the gut facilitate the study of intervention effects on the gut microbial community relevant to health. The mucosa-associated gut microbiota, which thrives in close contact with the host plays a pivotal role in colonization resistance and health. Therefore, we here introduce the Mi-screen, an experimental approach implementing a 96-well plate equipped with a mucus agar layer for the additional culturing of mucosa-associated microbiota in vitro. In this study, we screened the effects of 2'-Fucosyllactose (2'-FL), fructooligosaccharides (FOS), and inulin within a complex microbiota without and with infection with the C. difficile strains ATCC 43599 (Ribotype 001) or ATCC BAA-1870 (Ribotype 027). We analyzed the microbial community composition and short-chain fatty acid levels after 48 h of incubation. The inclusion of an additional substrate and surface in the form of the mucus agar layer allowed us to culture a microbial richness ranging between 100-160 in Chao index, with Shannon indices of 5-6 across culture conditions, indicative of a microbial diversity of physiological relevance. The mucus agar layer stimulated the growth of characteristic mucosa-associated bacteria such as Roseburia inulinovorans. The prebiotic interventions affected luminal and mucosal microbial communities cultured in vitro and stimulated short-chain fatty acid production. FOS, inulin and 2'-FL promoted the growth of Bifidobacterium adolescentis within the mucosa-associated microbiota cultured in vitro. When spiking the untreated conditions with pathogenic C. difficile, the strains thrived within the luminal and the mucosal sample types, whereas prebiotic treatments exhibited inhibitory effects on C. difficile growth and prevented colonization. In conclusion, the Mi-screen facilitates the screening of luminal and mucosa-associated gut microbial community dynamics in vitro and therefore fills an important gap in the field of in vitro modeling.

Oral teicoplanin administration suppresses recurrence of Clostridioides difficile infection: Proof of concept

ObjectivesTeicoplanin is a potential antimicrobial candidate for Clostridioides difficile infection (CDI) treatment. However, the therapeutic potential of teicoplanin against severe CDI has not been clinically proven. In the present study, we investigated the efficacy of oral teicoplanin administration against severe CDI and the recurrence of severe CDI after teicoplanin treatment in a mouse model. MethodsA lethal CDI mouse model was established by colonizing the mice with C. difficile ATCC® 43255; they were orally administered teicoplanin (128 mg/kg/d) or vancomycin (160 mg/kg/d) for 10 d, 24 h after C. difficile spore challenge, and physiological and biological responses were monitored for 20 d after the initial antibiotic treatment. We also performed the in vitro time-kill assay and determined minimum inhibitory concentration (MIC), post-antibiotic effect, and toxin production with antibiotic exposure. ResultsThe therapeutic response (survival rates, body weight change, clinical sickness score grading, C. difficile load, and toxin titer in feces) of oral teicoplanin administration was comparable to that of oral vancomycin administration in the lethal CDI mouse model. Moreover, teicoplanin treatment suppressed the re-onset of diarrhea and re-increase in toxin titer 10 d after treatment compared with that by vancomycin treatment. In in vitro experiments, teicoplanin exhibited time-dependent antibacterial activity and possessed lower MIC and longer post-antibiotic effect than vancomycin against C. difficile. C. difficile toxin production was numerically lower with teicoplanin exposure than with vancomycin exposure. ConclusionsThe results obtained from the present basic experiments could suggest that teicoplanin is a potential antibiotic for the treatment of severe CDI with recurrence-prevention activity.

Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo.

The Centers for Disease Control and Prevention estimate that Clostridioides difficile (C. difficile) causes half a million infections (CDI) annually and is a major cause of total infectious disease death in the United States, causing inflammation of the colon and potentially deadly diarrhea. We recently reported the isolation of ADS024, a Bacillus velezensis (B. velezensis) strain, which demonstrated direct in vitro bactericidal activity against C. difficile, with minimal collateral impact on other members of the gut microbiota. In this study, we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models. To investigate the in vivo efficacy of B. velezensis ADS024 in protecting against CDI challenge in mouse models. To mimic disruption of the gut microbiota, the mice were exposed to vancomycin prior to dosing with ADS024. For the mouse single-dose study, the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h, 8 h, and 24 h after a single oral dose of 5 × 108 colony-forming units (CFU)/mouse of freshly grown ADS024. The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024, and a group that was not pre-dosed with vancomycin and received a single dose of ADS024. The ADS024 colonies [assessed by quantitative polymerase chain reaction (qPCR) using ADS024-specific primers] were counted on agar plates. For the 28-d miniature swine study, qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5 × 109 CFU for 28 consecutive days, followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota. Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI: Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice, and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024. C. difficile challenge was performed 24 h after the start of ADS024 exposure. To model the human distal colon, an anerobic fecal fermentation system was used. MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment. To assess the potential of ADS024 to be a source of antibiotic resistance, its susceptibility to 18 different antibiotics was tested. In a mouse model of CDI challenge, single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C. difficile pathogen-induced disease. ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine. In a 28-d repeat-dose study in miniature swine, ADS024 was not detected in fecal samples using plating and qPCR methods. Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota, similarly to the in vivo studies performed in miniature swine. Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested, while in silico testing revealed a low potential for off-target activity or virulence and antibiotic-resistance mechanisms. Our findings, demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models, support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.

Targeted activation of IL-22 producing innate lymphoid cells enhances host resistance against Clostridioides difficileinfection

Abstract Clostridioides difficile, the most common hospital acquired pathogen in the United States, infects the gastrointestinal tract following perturbation of the intestinal microbiota causing debilitating, potentially fatal colitis. Primary C. difficile infection has a high recurrence rate following antibiotic treatment, emphasizing the need to develop alternative treatment strategies to reduce the burden on the healthcare system. Innate lymphoid cells (ILCs) are necessary for acute host defense following C. difficile infection and represent a promising therapeutic target to harness the patient’s innate immune defenses to limit disease. Here, using a murine model of C. difficile infection, we report that oral administration of Resiquimod (R848), a TLR-7 agonist previously reported to activate type-3 ILCs (ILC3s), protects mice from lethal C. difficile challenge. R848 treatment did not alter intestinal microbial communities or impact C. difficile burden. R848 treatment conveyed protection via activation of the host immune system in a TLR-7 dependent manner. R848-mediated protection was independent of T and B cells as well as type-I IFN signaling. Mice lacking ILCs, however, did not respond to R848 treatment and remained susceptible to infection, identifying these cells as the key targets of R848 activity. Last, R848 treatment robustly induced ILC3s in the large intestine to produce IL-22 and genetic ablation of IL-22 abrogated R848-mediated protection from C. difficile infection. Combined, these data identify an immunostimulatory molecule that activates the IL-22 producing ILC3s to support host defense against C. difficile infection without causing further dysbiosis of the intestinal microbiota. NIH R01AI158830, R21AI164385

Effect of Doxycycline in Decreasing the Severity of Clostridioides difficile Infection in Mice.

Background: Doxycycline possesses antibacterial activity against Clostridioides difficile and anti-inflammatory effects. Materials and Methods: The influence of doxycycline on the development of CDI was studied in an established animal model of CDI using C57BL/6 mice. Results: Mice intraperitoneally administered doxycycline had higher cecum weight (1.3 ± 0.1 vs. 0.5 ± 0.1 g; p < 0.001) and less body weight reduction (0.7 ± 0.5 g vs. −17.4 ± 0.2 g; p < 0.001) than untreated mice infected with C. difficile. Oral doxycycline, metronidazole, or vancomycin therapy resulted in less body weight reduction in mice with CDI than in untreated mice (1.1 ± 0.1 g, 1.3 ± 0.2 g, 1.2 ± 0.1 g, vs. 2.9 ± 0.3 g; p < 0.001). Doxycycline therapy led to lower expression levels of inflammatory cytokines, such as macrophage inflammatory protein-2 (0.4 ± 0.1 vs. 2.9 ± 1.3, p = 0.02), and higher levels of zonula occludens-1 (1.2 ± 0.1 vs. 0.8 ± 0.1, p = 0.02) in colonic tissues than in untreated mice. Conclusions: Concurrent intraperitoneal administration of doxycycline and oral C. difficile challenge does not aggravate the disease severity of CDI, and oral doxycycline may be a potential therapeutic option for CDI.

Use of a Clostridioides difficile Murine Immunization and Challenge Model to Evaluate Single and Combination Vaccine Adjuvants Consisting of Alum and NKT Cell-Activating Ligands.

Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts.

Lacticaseibacillus casei Strain T21 Attenuates Clostridioides difficile Infection in a Murine Model Through Reduction of Inflammation and Gut Dysbiosis With Decreased Toxin Lethality and Enhanced Mucin Production.

Clostridioides difficile is a major cause of diarrhea in patients with antibiotic administration. Lacticaseibacillus casei T21, isolated from a human gastric biopsy, was tested in a murine C. difficile infection (CDI) model and colonic epithelial cells (Caco-2 and HT-29). Daily administration of L. casei T21 [1 × 108 colony forming units (CFU)/dose] for 4 days starting at 1 day before C. difficile challenge attenuated CDI as demonstrated by a reduction in mortality rate, weight loss, diarrhea, gut leakage, gut dysbiosis, intestinal pathology changes, and levels of pro-inflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α, macrophage inflammatory protein 2 (MIP-2), and keratinocyte chemoattractant (KC)] in the intestinal tissue and serum. Conditioned media from L. casei T21 exerted biological activities that fight against C. difficile as demonstrated in colonic epithelial cells by the following: (i) suppression of gene expression and production of IL-8, an important chemokine involved in C. difficile pathogenesis, (ii) reduction in the expression of SLC11A1 (solute carrier family 11 member 1) and HuR (human antigen R), important genes for the lethality of C. difficile toxin B, (iii) augmentation of intestinal integrity, and (iv) up-regulation of MUC2, a mucosal protective gene. These results supported the therapeutic potential of L. casei T21 for CDI and the need for further study on the intervention capabilities of CDI.

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.

NKT cells induce IgG1 class switch against C. difficile carbohydrate antigens and enhance immunity against a live pathogen challenge

Abstract All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier and induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG3 titers were evident in sera from immunized mice, but the inclusion of α-GC led to additional isotype switch and production of IgG1. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18−/− mice were not protected by the PSII/α-GC immunization modality. These results indicate that α-GC-driven NKT cells stimulate a protective IgG1 response directed at the C. difficile PSII antigen. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop a C. difficile vaccine.

Neutralization of macrophage migration inhibitory factor improves host survival after Clostridium difficile infection

Clostridium difficile is an important cause of nosocomial diarrhea in the western world. Toxins (A, B, and binary toxins) generated by C. difficile bacteria damage intestinal epithelial cells. Hallmarks of host response to C. difficile infection (CDI) include upregulation of inflammatory mediators and tissue infiltration by immune cells. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that is known to enhance the host immune response to infectious pathogens. Additionally, MIF can adversely impact host survival to numerous infections. The role of MIF in the pathogenesis of CDI remains poorly understood. Here, we show that patients with CDI had significantly higher circulating MIF compared to patients who had diarrhea but tested negative for C. difficile (non-CDI controls). Similarly, in a mouse model, C. difficile challenge significantly increased levels of plasma and tissue MIF. Antibody-mediated depletion of MIF decreased C. difficile-induced inflammatory responses, clinical disease, and mortality. Together, these results uncover a potential role for MIF in exacerbating CDI and suggest that use of anti-MIF antibodies may represent a therapeutic strategy to curb host inflammatory responses and improve disease outcomes in CDI.

Pre-colonization with the commensal fungus Candida albicans reduces murine susceptibility to Clostridium difficile infection

ABSTRACTClostridium difficile is a major nosocomial pathogen responsible for close to half a million infections and 27,000 deaths annually in the U.S. Preceding antibiotic treatment is a major risk factor for C. difficile infection (CDI) leading to recognition that commensal microbes play a key role in resistance to CDI. Current antibiotic treatment of CDI is only partially successful due to a high rate of relapse. As a result, there is interest in understanding the effects of microbes on CDI susceptibility to support treatment of patients with probiotic microbes or entire microbial communities (e.g., fecal microbiota transplantation). The results reported here demonstrate that colonization with the human commensal fungus Candida albicans protects against lethal CDI in a murine model. Colonization with C. albicans did not increase the colonization resistance of the host. Rather, our findings showed that one effect of C. albicans colonization was to enhance a protective immune response. Mice pre-colonized with C. albicans expressed higher levels of IL-17A in infected tissue following C. difficile challenge compared to mice that were not colonized with C. albicans. Administration of cytokine IL-17A was demonstrated to be protective against lethal murine CDI in mice not colonized with C. albicans. C. albicans colonization was associated with changes in the abundance of some bacterial components of the gut microbiota. Therefore, C. albicans colonization altered the gut ecosystem, enhancing survival after C. difficile challenge. These findings demonstrate a new, beneficial role for C. albicans gut colonization.

IL-27/IL-27 Receptor Signaling Provides Protection in Clostridium difficile-Induced Colitis.

Clostridium difficile is a leading cause of nosocomial infection. The role of cytokine interleukin-27 (IL-27) in the immunopathology of C. difficile infection (CDI) remains unknown. The production of IL-27 was determined in human and murine CDI. Furthermore, wild-type (WT) and IL-27 receptor-deficient (WSX-1-/-) mice were treated with an antibiotic mixture and infected with C. difficile to investigate the effects of IL-27 on host response to CDI. IL-27 production was elevated during CDI in humans and mice. Infected WSX-1-/- mice experienced increased weight loss, enhanced colonic histology damage, less C. difficile clearance, and decreased survival compared to WT controls during CDI. IL-27 administration reduced CDI-associated mortality and tissue pathology with improved C. difficile clearance in WT mice after C. difficile challenge. Mechanistically, IL-27-mediated host defense against CDI was associated with downregulation of IL-17A and IL-23, but upregulation of IL-10 and interferon-gamma during CDI. Our data suggest a previously unrecognized role for IL-27 in the pathogenesis of CDI, potentially providing new insight for IL-27-mediated protection against C. difficile-induced pathology.

Stability and efficacy of frozen and lyophilized fecal microbiota transplant (FMT) product in a mouse model of Clostridium difficile infection (CDI)

Freezing donor fecal microbiota has simplified fecal microbiota transplantation (FMT) in the treatment of recurrent C. difficile infection (CDI). However, the optimal storage time for the frozen FMT products remains unknown. Using an established murine model of CDI, stability and efficacy of frozen and lyophilized FMT product was studied at time points from 2 months to 15 months. DNA was extracted from fecal samples from the mice with identification of specific bacterial species by real-time quantitative PCR (qPCR). FMT product stability and efficacy were measured by occurrence of diarrhea in the challenged mice together with stability of the microbiota composition. The results were analyzed and compared by SAS statistical software. All mice treated with only C. difficile developed diarrhea within 72 h. Mice treated with frozen (n = 5/group), lyophilized (n = 5/group) products stored for ≤ 7-month or fresh FMT product (n = 22) were protected from post C. difficile challenge diarrhea. There was no difference between frozen and lyophilized products (n = 5/group) stored for ≤ 7 months 95% CI 1.00 (0.38–2.64) and 1.00 (0.38–2.64), respectively. Prevention if CDI by frozen and lyophilized product was not different for storage of 9-, 11- and 15-months. qPCR results demonstrated there were no significant quantitative change in Bacteroides and Clostridium species during any of the storage times (P > 0.05). In the present study, frozen and lyophilized FMT products were stored up to 7 months without losing microbiota composition and therapeutic efficacy. The animal model described may be useful to study stability of human microbiota designed for FMT.

Immunization with Recombinant TcdB-Encapsulated Nanocomplex Induces Protection against Clostridium difficile Challenge in a Mouse Model.

Clostridium difficile is considered to be one of the major cause of infectious diarrhea in healthcare systems worldwide. Symptoms of C. difficile infection are caused largely by the production of two cytotoxins: toxin A (TcdA) and toxin B (TcdB). Vaccine development is considered desirable as it would decrease the mounting medical costs and mortality associated with C. difficile infections. Biodegradable nanoparticles composed of poly-γ-glutamic acid (γ-PGA) and chitosan have proven to be a safe and effective antigen delivery system for many viral vaccines. However, few studies have used this efficient antigen carrier for bacterial vaccine development. In this study, we eliminated the toxin activity domain of toxin B by constructing a recombinant protein rTcdB consists of residues 1852-2363 of TcdB receptor binding domain. The rTcdB was encapsulated in nanoparticles composed of γ-PGA and chitosan. Three rounds of intraperitoneal vaccination led to high anti-TcdB antibody responses and afforded mice full protection mice from lethal dose of C. difficile spore challenge. Protection was associated with high levels of toxin-neutralizing antibodies, and the rTcdB-encapsulated NPs elicited a longer-lasting antibody titers than antigen with the conventional adjuvant, aluminum hydroxide. Significant reductions in the level of proinflammatory cytokines and chemokines were observed in vaccinated mouse. These results suggested that polymeric nanocomplex-based vaccine design can be useful in developing vaccine against C. difficile infections.

Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Clostridium difficile Infection.

Clostridium difficile is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by C. difficile-associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in C. difficile. We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against C. difficile challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low anti-pilin antibody titers and no protection upon C. difficile challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of C. difficile infection, are needed to determine if an anti-T4P vaccine would be protective against C. difficile infection.

Effect of age on Clostridium difficile infection in a murine model of infection

Clostridium difficile, a Gram-positive, spore-forming bacillus, is the leading cause of nosocomial antibiotic-associated diarrhea in the U.S. A major risk factor for developing C. difficile infection (CDI) is advanced age (>65 years), which also accounts for 93% of CDI-related deaths. It is unknown why CDI becomes more severe with age. The objective of this study was to develop a CDI mouse model to study the effects of aging on the pathogenesis of CDI. To achieve this goal we compared CDI in aged (12 months) versus young (2 months) C57BL/6J mice. Six days after C. difficile spore challenge, clinical symptoms (e.g. wet tail, hunching, dehydration) were exhibited by a greater proportion of aged mice compared to young mice (p=0.08). Aged mice experienced greater weight loss than younger animals (p=0.013) and shed greater numbers of bacteria into the feces (p=0.023). Not surprisingly, aged mice suffered a higher cumulative mortality (44%) compared to the young (20%). Histological examination of cecum and colon tissue revealed the severity of inflammation and mucosal damage was greater in aged mice than younger animals (p=0.004). Because mortality is typically associated with lower anti-toxin antibody titers, splenic B cells (CD45+CD19+) numbers were measured by flow cytometry. In comparison to young mice, the proportion of splenic B cells was significantly reduced in the aged group (p=0.006). These results demonstrate that the consequences of CDI are more severe in aged mice and associated with B cell changes. Utilizing our model will help to advance our understanding of age-related changes on host immune defenses against CDI, which is crucial for designing new prophylactic and treatment strategies to reduce morbidity and mortality in older adults.

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2–3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI.

Equine hyperimmune serum protects mice againstClostridium difficilespore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 107 C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.

Preliminary Investigation on the Effect of &amp;lt;i&amp;gt;Lactobacillus&amp;lt;/i&amp;gt; and Epidermal Growth Factor on Tight Junction Proteins in Experimental Clostridium &amp;lt;i&amp;gt;difficile&amp;lt;/i&amp;gt; Infection

Clostridium difficile associated disease (CDAD) is the most common hospital acquired infection, due to exposure to various drugs. C. difficile toxins influence barrier function in intestinal epithelium. Biotherapeutic approaches, employing probiotic and epidermal growth factor (EGF) could help in barrier protein protection and aid in CDAD management. A preliminary investigation on the effect of Lactobacillus acidophilus and EGF on tight junction proteins in experimentally induced C. difficile infection was done. BALB/mice were divided into 5 groups. Group 1 was comprised of healthy controls, whereas animals in Groups 2 - 5 were sub-divided into 3 subgroups (a, b and c) each. Animals in Groups 2 - 5 received C. difficile inoculum either on day 1 (Group 2) or after pretreatment with ampicillin (Group 3), cyclosporine (Group 4) or lansoprazole (Group 5). Additionally animals in subgroups “b” and “c” also received L. acidophilus and EGF inocula respectively after C. difficile challenge. All animals were investigated for the presence of tight junction proteins (occludin, α-actinin and zonula occludens) in their colonic segments. Data were analyzed using the SPSS version 10 software. These three proteins were present in significantly less (P L. acidophilus and EGF after challenge with ampicillin, cyclosporine and lansoprazole, suggesting their role in protecting intestinal barrier function.