Genomic and vaccine preclinical studies reveal a novel mouse-adapted Helicobacter pylori model for the hpEastAsia genotype in Southeast Asia.

Introduction. Helicobacter pylori infection is a major global health concern, linked to the development of various gastrointestinal diseases, including gastric cancer. To study the pathogenesis of H. pylori and develop effective intervention strategies, appropriate animal pathogen models that closely mimic human infection are essential.Gap statement. This study focuses on the understudied hpEastAsia genotype in Southeast Asia, a region marked by a high H. pylori infection rate. No mouse-adapted model strains has been reported previously. Moreover, it recognizes the urgent requirement for vaccines in developing countries, where overuse of antimicrobials is fuelling the emergence of resistance.Aim. This study aims to establish a novel mouse-adapted H. pylori model specific to the hpEastAsia genotype prevalent in Southeast Asia, focusing on comparative genomic and histopathological analysis of pathogens coupled with vaccine preclinical studies.Methodology. We collected and sequenced the whole genome of clinical strains of H. pylori from infected patients in Vietnam and performed comparative genomic analyses of H. pylori strains in Southeast Asia. In parallel, we conducted preclinical studies to assess the pathogenicity of the mouse-adapted H. pylori strain and the protective effect of a new spore-vectored vaccine candidate on male Mlac:ICR mice and the host immune response in a female C57BL/6 mouse model.Results. Genome sequencing and comparison revealed unique and common genetic signatures, antimicrobial resistance genes and virulence factors in strains HP22 and HP34; and supported clarithromycin-resistant HP34 as a representation of the hpEastAsia genotype in Vietnam and Southeast Asia. HP34-infected mice exhibited gastric inflammation, epithelial erosion and dysplastic changes that closely resembled the pathology observed in human H. pylori infection. Furthermore, comprehensive immunological characterization demonstrated a robust host immune response, including both mucosal and systemic immune responses. Oral vaccination with candidate vaccine formulations elicited a significant reduction in bacterial colonization in the model.Conclusion. Our findings demonstrate the successful development of a novel mouse-adapted H. pylori model for the hpEastAsia genotype in Vietnam and Southeast Asia. Our research highlights the distinctive genotype and pathogenicity of clinical H. pylori strains in the region, laying the foundation for targeted interventions to address this global health burden.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Bacillus velezensis DSM 33864 reduces Clostridioides difficile colonization without disturbing commensal gut microbiota composition.

Up to 25% of the US population harbor Clostridioides difficile in the gut. Following antibiotic disruption of the gut microbiota, C. difficile can act as an opportunistic pathogen and induce potentially lethal infections. Consequently, reducing the colonization of C. difficile in at-risk populations is warranted, prompting us to identify and characterize a probiotic candidate specifically targeting C. difficile colonization. We identified Bacillus velezensis DSM 33864 as a promising strain to reduce C. difficile levels in vitro. We further investigated the effects of B. velezensis DSM 33864 in an assay including human fecal medium and in healthy or clindamycin-treated mouse models of C. difficile colonization. The addition of B. velezensis DSM 33864 to human fecal samples was shown to reduce the colonization of C. difficile in vitro. This was supported in vivo where orally administered B. velezensis DSM 33864 spores reduced C. difficile levels in clindamycin-treated mice. The commensal microbiota composition or post-antibiotic reconstitution was not impacted by B. velezensis DSM 33864 in human fecal samples, short-, or long-term administration in mice. In conclusion, oral administration of B. velezensis DSM 33864 specifically reduced C. difficile colonization in vitro and in vivo without adversely impacting the commensal gut microbiota composition.

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines.

BACKGROUND: Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR. MATERIALS AND METHODS: We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori. RESULTS: Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log. CONCLUSIONS: This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.

Heterologous Systemic Prime-Intranasal Boosting Using a Spore SARS-CoV-2 Vaccine Confers Mucosal Immunity and Cross-Reactive Antibodies in Mice as well as Protection in Hamsters.

Background: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are administered systemically and typically result in poor immunogenicity at the mucosa. As a result, vaccination is unable to reduce viral shedding and transmission, ultimately failing to prevent infection. One possible solution is that of boosting a systemic vaccine via the nasal route resulting in mucosal immunity. Here, we have evaluated the potential of bacterial spores as an intranasal boost. Method: Spores engineered to express SARS-CoV-2 antigens were administered as an intranasal boost following a prime with either recombinant Spike protein or the Oxford AZD1222 vaccine. Results: In mice, intranasal boosting following a prime of either Spike or vaccine produced antigen-specific sIgA at the mucosa together with the increased production of Th1 and Th2 cytokines. In a hamster model of infection, the clinical and virological outcomes resulting from a SARS-CoV-2 challenge were ameliorated. Wuhan-specific sIgA were shown to cross-react with Omicron antigens, suggesting that this strategy might offer protection against SARS-CoV-2 variants of concern. Conclusions: Despite being a genetically modified organism, the spore vaccine platform is attractive since it offers biological containment, the rapid and cost-efficient production of vaccines together with heat stability. As such, employed in a heterologous systemic prime-mucosal boost regimen, spore vaccines might have utility for current and future emerging diseases.

Intranasal Treatment of Ferrets with Inert Bacterial Spores Reduces Disease Caused by a Challenging H7N9 Avian Influenza Virus.

Background: Influenza is a respiratory infection that continues to present a major threat to human health, with ~500,000 deaths/year. Continued circulation of epidemic subtypes in humans and animals potentially increases the risk of future pandemics. Vaccination has failed to halt the evolution of this virus and next-generation prophylactic approaches are under development. Naked, "heat inactivated", or inert bacterial spores have been shown to protect against influenza in murine models. Methods: Ferrets were administered intranasal doses of inert bacterial spores (DSM 32444K) every 7 days for 4 weeks. Seven days after the last dose, the animals were challenged with avian H7N9 influenza A virus. Clinical signs of infection and viral shedding were monitored. Results: Clinical symptoms of infection were significantly reduced in animals dosed with DSM 32444K. The temporal kinetics of viral shedding was reduced but not prevented. Conclusion: Taken together, nasal dosing using heat-stable spores could provide a useful approach for influenza prophylaxis in both humans and animals.

Environmentally Acquired Bacillus and Their Role in C. difficile Colonization Resistance.

Clostridioides difficile is an environmentally acquired, anaerobic, spore-forming bacterium which ordinarily causes disease following antibiotic-mediated dysbiosis of the intestinal microbiota. Although much is understood regarding the life cycle of C. difficile, the fate of C. difficile spores upon ingestion remains unclear, and the underlying factors that predispose an individual to colonization and subsequent development of C. difficile infection (CDI) are not fully understood. Here, we show that Bacillus, a ubiquitous and environmentally acquired, spore-forming bacterium is associated with colonization resistance to C. difficile. Using animal models, we first provide evidence that animals housed under conditions that mimic reduced environmental exposure have an increased susceptibility to CDI, correlating with a loss in Bacillus. Lipopeptide micelles (~10 nm) produced by some Bacilli isolated from the gastro-intestinal (GI)-tract and shown to have potent inhibitory activity to C. difficile have recently been reported. We show here that these micelles, that we refer to as heterogenous lipopeptide lytic micelles (HELMs), act synergistically with components present in the small intestine to augment inhibitory activity against C. difficile. Finally, we show that provision of HELM-producing Bacillus to microbiota-depleted animals suppresses C. difficile colonization thereby demonstrating the significant role played by Bacillus in colonization resistance. In the wider context, our study further demonstrates the importance of environmental microbes on susceptibility to pathogen colonization.

Micellar Antibiotics of Bacillus.

Members of the Bacillus genus, particularly the "Bacillus subtilis group", are known to produce amphipathic lipopeptides with biosurfactant activity. This includes the surfactins, fengycins and iturins that have been associated with antibacterial, antifungal, and anti-viral properties. We have screened a large collection of Bacillus, isolated from human, animal, estuarine water and soil samples and found that the most potent lipopeptide producers are members of the species Bacillus velezensis. B. velezensis lipopeptides exhibited anti-bacterial activity which was localised on the surface of both vegetative cells and spores. Interestingly, lipopeptide micelles (6-10 nm diameter) were detectable in strains exhibiting the highest levels of activity. Micelles were stable (heat and gastric stable) and shown to entrap other antimicrobials produced by the host bacterium (exampled here was the dipeptide antibiotic chlorotetaine). Commercially acquired lipopeptides did not exhibit similar levels of inhibitory activity and we suspect that micelle formation may relate to the particular isomeric forms produced by individual bacteria. Using naturally produced micelle formulations we demonstrated that they could entrap antimicrobial compounds (e.g., clindamycin, vancomycin and resveratrol). Micellar incorporation of antibiotics increased activity. Bacillus is a prolific producer of antimicrobials, and this phenomenon could be exploited naturally to augment antimicrobial activity. From an applied perspective, the ability to readily produce Bacillus micelles and formulate with drugs enables a possible strategy for enhanced drug delivery.

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile.

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded ß-barrel fold with a neighbouring subdomain containing a five-stranded ß-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.

Impact of early-life events on the susceptibility to Clostridium difficile colonisation and infection in the offspring of the pig.

Clostridium difficile has been documented as a major cause of uncontrolled outbreaks of enteritis in neonatal pigs and antibiotic-associated infections in clinical settings. It belongs to the natural cohort of early colonisers of the gastrointestinal tract of pigs and can be detected in faeces up to two weeks post-partum. In older pigs, it often remains under the detection limit. Most neonatal pigs show no clinical signs of disease although C. difficile and its toxins can be detected at high levels in faeces. Increased mortality rates associated with C. difficile on pig farms are, so far, considered "spontaneous" and the predisposing factors are mostly not defined. The infection caused by C. difficile is multifactorial and it is likely that the repertoire of maternal factors, host physiology, the individually developing gut microbiota, co-infections and environmental stress define the conditions for disease development. In this addendum to our recently published work on CDI in neonatal piglets, we discuss the "early-life events" that influence C. difficile spread and infection in neonatal piglets.

Impact of Bacillus spp. spores and gentamicin on the gastrointestinal microbiota of suckling and newly weaned piglets.

Administrating antibiotics to young piglets may have short- and long-term consequences on the gut microbiota. We hypothesised that these consequences may be alleviated by concurrent probiotic administration. The study objective was to investigate the effect of administrating gentamicin and a mixture of Bacillus (B.) licheniformis, B. subtilis and B. amyloliquefaeceans spores on the gut microbiota of piglets pre- and post-weaning. Twenty-four sows and their litters were randomly allocated to four treatment groups receiving; a) Bacillus spore mixture (six B. subtilis, two B. amyloliquefaeceans, and one B. licheniformis) fed to sows and piglets (PRO); b) gentamicin (5 mg per day) administered to piglets on day 4, 5, and 6 of age (AB); c) Bacillus spore mixture fed to sows and piglets, and gentamicin to piglets (PRO+AB); or d) no administration of probiotics or antibiotics (CTRL). Faecal and digesta samples were collected repeatedly during the study. Selected samples were subjected to 16S rRNA gene sequencing, culture counts, and organic acid, biogenic amine and tissue gene expression analysis. Treatment had a significant effect on the faecal microbial community composition on day 28 and 42, and colonic community on day 28. Faecal species richness (observed and estimated) and Shannon index, and colonic species richness, were higher in AB compared to PRO piglets on day 28, and were not significantly different from day 42. PRO piglets had the highest faecal concentration of iso-butyric acid on day 7 and a higher butyric acid concentration compared to CTRL piglets. We conclude that gentamicin and Bacillus spores influence the gut microbial diversity of piglets, although administration of gentamicin did not result in dysbiosis as hypothesised.

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria.

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

Vaccines as alternatives to antibiotics for food producing animals. Part 1: challenges and needs.

Vaccines and other alternative products can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations, and are central to the future success of animal agriculture. To assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, part of a two-part series, synthesizes and expands on the expert panel discussions regarding opportunities, challenges and needs for the development of vaccines that may reduce the need for use of antibiotics in animals; new approaches and potential solutions will be discussed in part 2 of this series. Vaccines are widely used to prevent infections in food animals. Various studies have demonstrated that their animal agricultural use can lead to significant reductions in antibiotic consumption, making them promising alternatives to antibiotics. To be widely used in food producing animals, vaccines have to be safe, effective, easy to use, and cost-effective. Many current vaccines fall short in one or more of these respects. Scientific advancements may allow many of these limitations to be overcome, but progress is funding-dependent. Research will have to be prioritized to ensure scarce public resources are dedicated to areas of potentially greatest impact first, and private investments into vaccine development constantly compete with other investment opportunities. Although vaccines have the potential to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks, targeted research and development investments and concerted efforts by all affected are needed to realize that potential.

Vaccines as alternatives to antibiotics for food producing animals. Part 2: new approaches and potential solutions.

Vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. To assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the United States Department of Agriculture, with support from the World Organisation for Animal Health, organized the second International Symposium on Alternatives to Antibiotics. It focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. This article, the second part in a two-part series, highlights new approaches and potential solutions for the development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first part of this series. As discussed in part 1 of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. Promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral vaccine approaches, novel adjuvants, new delivery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. However, translating this research into commercial vaccines that effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public-private partnerships. Targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks.

Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guérin.

Tuberculosis (TB) is the most deadly infectious disease in existence, and the only available vaccine, Bacillus Calmette-Guérin (BCG), is almost a century old and poorly protective. The immunological complexity of TB, coupled with rising resistance to antimicrobial therapies, necessitates a pipeline of diverse novel vaccines. Here, we show that Bacillus subtilis spores can be coated with a fusion protein 1 ("FP1") consisting of Mycobacterium tuberculosis (Mtb) antigens Ag85B, ACR, and HBHA. The resultant vaccine, Spore-FP1, was tested in a murine low-dose Mtb aerosol challenge model. Mice were primed with subcutaneous BCG, followed by mucosal booster immunizations with Spore-FP1. We show that Spore-FP1 enhanced pulmonary control of Mtb, as evidenced by reduced bacterial burdens in the lungs. This was associated with elevated antigen-specific IgG and IgA titers in the serum and lung mucosal surface, respectively. Spore-FP1 immunization generated superior antigen-specific memory T-cell proliferation in both CD4+ and CD8+ compartments, alongside bolstered Th1-, Th17-, and Treg-type cytokine production, compared to BCG immunization alone. CD69+CD103+ tissue resident memory T-cells (Trm) were found within the lung parenchyma after mucosal immunization with Spore-FP1, confirming the advantages of mucosal delivery. Our data show that Spore-FP1 is a promising new TB vaccine that can successfully augment protection and immunogenicity in BCG-primed animals.

Biological Containment of Genetically Modified Bacillus subtilis.

Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes.IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment.

Inducible Expression of spo0A as a Universal Tool for Studying Sporulation in Clostridium difficile.

Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile.

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.