Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores.

BACKGROUND: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. MATERIALS & METHODS: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. RESULTS: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. CONCLUSION: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Convenient Protocol for Production and Purification of Clostridioides difficile Spores for Germination Studies.

Clostridioides difficile, an obligate anaerobic bacterium, causes infections leading to prolonged diarrhea. The bacterium produces dormant spores that can withstand an aerobic environment, resulting in easy environmental transfer. Here, we present a convenient sporulation and purification protocol that can be practiced in any lab setting using a portable anaerobic glove bag. This protocol also optimizes existing cell growth methods and presents a detailed trouble shooting guide. This protocol is a modification of those previously reported by Edwards and McBride (2016) and Shen et al. (2016).

Rapid in Vitro Assessment of Clostridioides difficile Inhibition by Probiotics Using Dielectrophoresis to Quantify Cell Structure Alterations.

Clostridioides difficile (C. difficile) infection (CDI) is the primary cause of nosocomial antibiotic-associated diarrhea, with high recurrence rates following initial antibiotic treatment regimens. Restoration of the host gut microbiome through probiotic therapy is under investigation to reduce recurrence. Current in vitro methods to assess C. difficile deactivation by probiotic microorganisms are based on C. difficile growth inhibition, but the cumbersome and time-consuming nature of the assay limits the number of assessed permutations. Phenotypic alterations to the C. difficile cellular structure upon interaction with probiotics can potentially enable rapid assessment of the inhibition without the need for extended culture. Because supernatants from cultures of commensal microbiota reflect the complex metabolite milieu that deactivates C. difficile, we explore coculture of C. difficile with an optimal dose of supernatants from probiotic culture to speed growth inhibition assays and enable correlation with alterations to its prolate ellipsoidal structure. Based on sensitivity of electrical polarizability to C. difficile cell shape and subcellular structure, we show that the inhibitory effect of Lactobacillus spp. supernatants on C. difficile can be determined based on the positive dielectrophoresis level within just 1 h of culture using a highly toxigenic strain and a clinical isolate, whereas optical and growth inhibition measurements require far greater culture time. We envision application of this in vitro coculture model, in conjunction with dielectrophoresis, to rapidly screen for potential probiotic combinations for the treatment of recurrent CDI.

Repurposing a platelet aggregation inhibitor ticagrelor as an antimicrobial against Clostridioides difficile.

Drug resistance in Clostridioides difficile becomes a public health concern worldwide, especially as the hypervirulent strains show decreased susceptibility to the first-line antibiotics for C. difficile treatment. Therefore, the simultaneous discovery and development of new compounds to fight this pathogen are urgently needed. In order to determinate new drugs active against C. difficile, we identified ticagrelor, utilized for the prevention of thrombotic events, as exhibiting potent growth-inhibitory activity against C. difficile. Whole-cell growth inhibition assays were performed and compared to vancomycin and metronidazole, followed by determining time-kill kinetics against C. difficile. Activities against biofilm formation and spore germination were also evaluated. Leakage analyses and electron microscopy were applied to confirm the disruption of membrane structure. Finally, ticagrelor's ability to synergize with vancomycin and metronidazole was determined using checkerboard assays. Our data showed that ticagrelor exerted activity with a MIC range of 20-40 µg/mL against C. difficile. This compound also exhibited an inhibitory effect on biofilm formation and spore germination. Additionally, ticagrelor did not interact with vancomycin nor metronidazole. Our findings revealed for the first time that ticagrelor could be further developed as a new antimicrobial agent for fighting against C. difficile.

Detection of mixed-strain infections by FACS and ultra-low input genome sequencing.

The epidemiological tracking of a bacterial outbreak may be jeopardized by the presence of multiple pathogenic strains in one patient. Nevertheless, this fact is not considered in most of the epidemiological studies and only one colony per patient is sequenced. On the other hand, the routine whole genome sequencing of many isolates from each patient would be costly and unnecessary, because the number of strains in a patient is never known a priori. In addition, the result would be biased by microbial culture conditions. Herein we propose an approach for detecting mixed-strain infection, providing C. difficile infection as an example. The cells of the target pathogenic species are collected from the bacterial suspension by the fluorescence activated cell sorting (FACS) and a shallow genome sequencing is performed. A modified sequencing library preparation protocol for low-input DNA samples can be used for low prevalence gut pathogens (< 0.1% of the total microbiome). This FACS-seq approach reduces diagnostics time (no culture is needed) and may promote discoveries of novel strains. Methodological details, possible issues and future directions for the sequencing of these natural pan-genomes are herein discussed.

Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.

Vegetative Cell and Spore Proteomes of Clostridioides difficile Show Finite Differences and Reveal Potential Protein Markers.

Clostridioides difficile-associated infection (CDI) is a health-care-associated infection caused, as the name suggests, by obligate anaerobic pathogen C. difficile and thus mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate into cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of C. difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic, and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative cell proteins CD1228, CD3301 and spore proteins CD2487, CD2434, and CD0684 are identified as potential protein markers for C. difficile infection.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.

Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.

OBJECTIVES: Ridinilazole (SMT19969) is a narrow-spectrum, non-absorbable antimicrobial with activity against Clostridium difficile undergoing clinical trials. The purpose of this study was to assess the pharmacological activity of ridinilazole and assess the effects on cell morphology. METHODS: Antibiotic killing curves were performed using the epidemic C. difficile ribotype 027 strain, R20291, using supra-MIC (4× and 40×) and sub-MIC (0.125×, 0.25× and 0.5×) concentrations of ridinilazole. Following exposure, C. difficile cells were collected for cfu counts, toxin A and B production, and morphological changes using scanning electron and fluorescence microscopy. Human intestinal cells (Caco-2) were co-incubated with ridinilazole-treated C. difficile growth medium to determine the effects on host inflammatory response (IL-8). RESULTS: Treatment at supra-MIC concentrations (4× and 40× MIC) of ridinilazole resulted in a significant reduction in vegetative cells over 72 h (4 log difference, P < 0.01) compared with controls without inducing spore formation. These results correlated with a 75% decrease in toxin A production (P < 0.05) and a 96% decrease in toxin B production (P < 0.05). At sub-MIC levels (0.5× MIC), toxin A production was reduced by 91% (P < 0.01) and toxin B production was reduced by 100% (P < 0.001), which resulted in a 74% reduction in IL-8 release compared with controls (P < 0.05). Sub-MIC (0.5×)-treated cells formed filamentous structures ∼10-fold longer than control cells. Following fluorescence labelling, the cell septum was not forming in sub-MIC-treated cells, yet the DNA was dividing. CONCLUSIONS: Ridinilazole had robust killing effects on C. difficile that significantly reduced toxin production and attenuated the inflammatory response. Ridinilazole also elicited significant cell division effects suggesting a potential mechanism of action.

Immunogenicity and protective efficacy of recombinant Clostridium difficile flagellar protein FliC.

Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630Δerm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.

Discovery and development of surotomycin for the treatment of Clostridium difficile.

The primary challenge for treating Clostridium difficile infections (CDI) is maintenance of clinical response after the end of treatment (sustained clinical response). Disease recurrence following a positive clinical response occurs in approximately 6-25 % of patients after the first episode and in up to 65 % for subsequent recurrences. Surotomycin, a novel cyclic lipopeptide antibiotic with a core derived by Streptomyces roseosporus fermentation, disrupts C. difficile cellular membrane activity in both logarithmic and stationary phases and minimally disturbs normal gastrointestinal microbiota because of its lack of activity against Gram-negative anaerobes and facultative anaerobes. Preclinical and clinical evidence indicate that surotomycin has low oral bioavailability, allowing gastrointestinal tract concentrations to greatly exceed its minimum inhibitory concentration for C. difficile. Surotomycin is well tolerated and effective in hamster models of CDI. Phase 2 clinical evidence suggests that surotomycin (250 mg twice daily) is an effective CDI treatment, with statistically lower recurrence rates than vancomycin.

Risk for Clostridium difficile infection after radical cystectomy for bladder cancer: Analysis of a contemporary series.

INTRODUCTION: This study seeks to evaluate the incidence and associated risk factors of Clostridium difficile infection (CDI) in patients undergoing radical cystectomy (RC) for bladder cancer. METHODS: We retrospectively reviewed a single institution׳s bladder cancer database including all patients who underwent RC between 2010 and 2013. CDI was diagnosed by detection of Clostridium difficile toxin B gene using polymerase chain reaction-based stool assay in patients with clinically significant diarrhea within 90 days of the index operation. A multivariable logistic regression model was used to identify demographics and perioperative factors associated with developing CDI. RESULTS: Of the 552 patients who underwent RC, postoperative CDI occurred in 49 patients (8.8%) with a median time to diagnosis after RC of 7 days (interquartile range: 5-19). Of the 122 readmissions for postoperative complications, 10% (n = 12) were related to CDI; 2 patients died of sepsis directly related to severe CDI. On multivariate logistic regression, the use of chronic antacid therapy (odds ratio = 1.9, 95% CI: 1.02-3.68, P = 0.04) and antibiotic exposure greater than 7 days (odds ratio = 2.2, 95% CI: 1.11-4.44, P = 0.02) were independently associated with developing CDI. The use of preoperative antibiotics for positive findings on urine culture within 30 days before surgery was not statistically significantly associated with development of CDI (P = 0.06). CONCLUSIONS: The development of CDI occurs in 8.8% of patients undergoing RC. Our study demonstrates that use of chronic antacid therapy and long duration of antimicrobial exposure are associated with development of CDI. Efforts focusing on minimizing antibiotic exposure in patients undergoing RC are needed, and perioperative antimicrobial prophylaxis guidelines should be followed.

Synthetic polymers active against Clostridium difficile vegetative cell growth and spore outgrowth.

Nylon-3 polymers (poly-ß-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe in vitro analysis of selected nylon-3 copolymers against Clostridium difficile, an important nosocomial pathogen that causes highly infectious diarrheal disease. The best polymers match the human host-defense peptide LL-37 in blocking vegetative cell growth and inhibiting spore outgrowth. The polymers and LL-37 were effective against both the epidemic 027 ribotype and the 012 ribotype. In contrast, neither vancomycin nor nisin inhibited outgrowth for the 012 ribotype. The best polymer was less hemolytic than LL-37. Overall, these findings suggest that nylon-3 copolymers may be useful for combatting C. difficle.

Initiation of sporulation in Clostridium difficile: a twist on the classic model.

The formation of dormant endospores is a complex morphological process that permits long-term survival in inhospitable environments for many Gram-positive bacteria. Sporulation for the anaerobic gastrointestinal pathogen Clostridium difficile is necessary for survival outside of the gastrointestinal tract of its host. While the developmental stages of spore formation are largely conserved among endospore-forming bacteria, the genus Clostridium appears to be missing a number of conserved regulators required for efficient sporulation in other spore-forming bacteria. Several recent studies have discovered novel mechanisms and distinct regulatory pathways that control the initiation of sporulation and early-sporulation-specific gene expression. These differences in regulating the decision to undergo sporulation reflects the unique ecological niche and environmental conditions that C. difficile inhabits and encounters within the mammalian host.

Clostridium difficile spore biology: sporulation, germination, and spore structural proteins.

Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of worldwide concern. Owing to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. Although important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and Bacillus subtilis at the level of sporulation, germination, and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed.

Identification and characterization of a gene cluster required for proper rod shape, cell division, and pathogenesis in Clostridium difficile.

Little is known about cell division in Clostridium difficile, a strict anaerobe that causes serious diarrheal diseases in people whose normal intestinal microbiome has been perturbed by treatment with broad-spectrum antibiotics. Here we identify and characterize a gene cluster encoding three cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, but MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of cyan fluorescent protein (CFP) to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. This application of CFP was possible because we discovered that O2-dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they might be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.

Culturing and maintaining Clostridium difficile in an anaerobic environment.

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

The Clostridium difficile exosporium cysteine (CdeC)-rich protein is required for exosporium morphogenesis and coat assembly.

Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ΔcdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ΔcdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.

Structural characterization of surface glycans from Clostridium difficile.

Whole-cell high-resolution magic angle spinning (HR-MAS) NMR was employed to survey the surface polysaccharides of a group of clinical and environmental isolates of Clostridium difficile. Results indicated that a highly conserved surface polysaccharide profile among all strains studied. Multiple additional peaks in the anomeric region were also observed which prompted further investigation. Structural characterization of the isolated surface polysaccharides from two strains confirmed the presence of the conserved water soluble polysaccharide originally described by Ganeshapillai et al. which was composed of a hexaglycosyl phosphate repeat consisting of [→6)-ß-D-Glcp-(1-3)-ß-D-GalpNAc-(1-4)-α-D-Glcp-(1-4)-[ß-D-Glcp(1-3]-ß-D-GalpNAc-(1-3)-α-D-Manp-(1-P→]. In addition, analysis of phenol soluble polysaccharides revealed a similarly conserved lipoteichoic acid (LTA) which could be detected on whole cells by HR-MAS NMR. Conventional NMR and mass spectrometry analysis indicated that the structure of this LTA consisted of the repeat unit [→6)-α-D-GlcpNAc-(1-3)-[→P-6]-α-D-GlcpNAc-(1-2)-D-GroA] where GroA is glyceric acid. The repeating units were linked by a phosphodiester bridge between C-6 of the two GlcNAc residues (6-P-6). A minor component consisted of GlcpN-(1-3) instead of GlcpNAc-(1-3) in the repeat unit. Through a 6-6 phosphodiester bridge this polymer was linked to →6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-1)-Gro, with glycerol (Gro) substituted by fatty acids. This is the first report of the utility of HR-MAS NMR in the examination of surface carbohydrates of Gram positive bacteria and identification of a novel LTA structure from Clostridium difficile.

Clostridium difficile carbohydrates: glucan in spores, PSII common antigen in cells, immunogenicity of PSII in swine and synthesis of a dual C. difficile-ETEC conjugate vaccine.

Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a D-glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester-phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin.