Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

Identification of Functional Spo0A Residues Critical for Sporulation in Clostridioides difficile.

Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.

Multi-omics investigation of Clostridioides difficile-colonized patients reveals pathogen and commensal correlates of C. difficile pathogenesis.

Clostridioides difficile infection (CDI) imposes a substantial burden on the health care system in the United States. Understanding the biological basis for the spectrum of C. difficile-related disease manifestations is imperative to improving treatment and prevention of CDI. Here, we investigate the correlates of asymptomatic C. difficile colonization using a multi-omics approach. We compared the fecal microbiome and metabolome profiles of patients with CDI versus asymptomatically colonized patients, integrating clinical and pathogen factors into our analysis. We found that CDI patients were more likely to be colonized by strains with the binary toxin (CDT) locus or strains of ribotype 027, which are often hypervirulent. We find that microbiomes of asymptomatically colonized patients are significantly enriched for species in the class Clostridia relative to those of symptomatic patients. Relative to CDI microbiomes, asymptomatically colonized patient microbiomes were enriched with sucrose degradation pathways encoded by commensal Clostridia, in addition to glycoside hydrolases putatively involved in starch and sucrose degradation. Fecal metabolomics corroborates the carbohydrate degradation signature: we identify carbohydrate compounds enriched in asymptomatically colonized patients relative to CDI patients. Further, we reveal that across C. difficile isolates, the carbohydrates sucrose, rhamnose, and lactulose do not serve as robust growth substrates in vitro, consistent with their enriched detection in our metagenomic and metabolite profiling of asymptomatically colonized individuals. We conclude that pathogen genetic variation may be strongly related to disease outcome. More interestingly, we hypothesize that in asymptomatically colonized individuals, carbohydrate metabolism by other commensal Clostridia may prevent CDI by inhibiting C. difficile proliferation. These insights into C. difficile colonization and putative commensal competition suggest novel avenues to develop probiotic or prebiotic therapeutics against CDI.

In Vitro and In Vivo Anti-Clostridioides difficile Effect of a Probiotic Bacillus amyloliquefaciens Strain.

Clostridioides difficile infection (CDI) is a significant cause of hospital-acquired and antibiotic-mediated intestinal diseases and is a growing global public health concern. Overuse of antibiotics and their effect on normal intestinal flora has increased the incidence and severity of infections. Thus, the development of new, effective, and safe treatment options is a high priority. Here, we report a new probiotic strain, Bacillus amyloliquefaciens (BA PMC-80), and its in vitro/in vivo anti-C. difficile effect as a prospective novel candidate for replacing conventional antibiotics. BA PMC-80 showed a significant anti-C. difficile effect in coculture assay, and its cell-free supernatant (CFS) also exhibited a considerable anti-C. difficile effect with an 89.06 µg/ml 50% minimal inhibitory concentration (MIC) in broth microdilution assay. The CFS was stable and equally functional under different pHs, heat, and proteinase treatments. It also exhibited a high sensitivity against current antibiotics and no toxicity in subchronic toxicity testing in hamsters. Finally, BA PMC-80 showed a moderate effect in a hamster CDI model with reduced infection severity and delayed death. However, further studies are required to optimize the treatment condition of the hamster CDI model for better efficacy and identify the antimicrobial compound produced by BA PMC-80.

Gut microbiota features associated with Clostridioides difficile colonization in dairy calves.

Diarrheal disease, a major cause of morbidity and mortality in dairy calves, is strongly associated with the health and composition of the gut microbiota. Clostridioides difficile is an opportunistic pathogen that proliferates and can produce enterotoxins when the host experiences gut dysbiosis. However, even asymptomatic colonization with C. difficile can be associated with differing degrees of microbiota disruption in a range of species, including people, swine, and dogs. Little is known about the interaction between C. difficile and the gut microbiota in dairy calves. In this study, we sought to define microbial features associated with C. difficile colonization in pre-weaned dairy calves less than 2 weeks of age. We characterized the fecal microbiota of 80 calves from 23 different farms using 16S rRNA sequencing and compared the microbiota of C. difficile-positive (n = 24) and C. difficile-negative calves (n = 56). Farm appeared to be the greatest source of variability in the gut microbiota. When controlling for calf age, diet, and farm location, there was no significant difference in Shannon alpha diversity (P = 0.50) or in weighted UniFrac beta diversity (P = 0.19) between C. difficile-positive and-negative calves. However, there was a significant difference in beta diversity as assessed using Bray-Curtiss diversity (P = 0.0077), and C. difficile-positive calves had significantly increased levels of Ruminococcus (gnavus group) (Adj. P = 0.052), Lachnoclostridium (Adj. P = 0.060), Butyricicoccus (Adj. P = 0.060), and Clostridium sensu stricto 2 compared to C. difficile-negative calves. Additionally, C. difficile-positive calves had fewer microbial co-occurrences than C. difficile-negative calves, indicating reduced bacterial synergies. Thus, while C. difficile colonization alone is not associated with dysbiosis and is therefore unlikely to result in an increased likelihood of diarrhea in dairy calves, it may be associated with a more disrupted microbiota.

Production of p-cresol by Decarboxylation of p-HPA by All Five Lineages of Clostridioides difficile Provides a Growth Advantage.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.

Pathobionts: mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile.

Pathobionts are opportunistic microbes that emerge as a result of perturbations in the healthy microbiome due to complex interactions of various genetic, exposomal, microbial, and host factors that lead to their selection and expansion. Their proliferations can aggravate inflammatory manifestations, trigger autoimmune diseases, and lead to severe life-threatening conditions. Current surge in microbiome research is unwinding these complex interplays between disease development and protection against pathobionts. This review summarizes the current knowledge of pathobiont emergence with a focus on Clostridioides difficile and the recent findings on the roles of immune cells such as iTreg cells, Th17 cells, innate lymphoid cells, and cytokines in protection against pathobionts. The review calls for adoption of innovative tools and cutting-edge technologies in clinical diagnostics and therapeutics to provide insights in identification and quantification of pathobionts.

Changes in antibiotic consumption, AMR and Clostridioides difficile infections in a large tertiary-care center following the implementation of institution-specific guidelines for antimicrobial therapy: A nine-year interrupted time series study.

BACKGROUND: Institution-specific guidelines (ISGs) within the framework of antimicrobial stewardship programs offer locally tailored decision support taking into account local pathogen and resistance epidemiology as well as national and international guidelines. OBJECTIVES: To assess the impact of ISGs for antimicrobial therapy on antibiotic consumption and subsequent changes in resistance rates and Clostridioides difficile infections (CDIs). METHODS: The study was conducted at the Leipzig University Hospital, a 1,451-bed tertiary-care medical center, and covered the years 2012 to 2020. Since 2014, ISGs were provided to optimize empirical therapies, appropriate diagnostics, and antimicrobial prophylaxis. We used interrupted time series analysis (ITSA) and simple linear regression to analyze changes in antimicrobial consumption, resistance and CDIs. RESULTS: Over the study period, 1,672,200 defined daily doses (DDD) of antibiotics were dispensed, and 85,645 bacterial isolates as well as 2,576 positive C. difficile cultures were collected. Total antimicrobial consumption decreased by 14% from 2012 to 2020, without clear impact of the deployment of ISGs. However, implementation of ISGs was associated with significant decreases in the use of substances that were rarely recommended (e.g., fluoroquinolones). Over the whole study period, we observed declining resistance rates to most antibiotic classes of up to 25% in Enterobacterales, staphylococci, and Pseudomonas aeruginosa. Switching from ceftriaxone to cefotaxime was associated with reduced resistance to third-generation cephalosporins. The number of CDI cases fell by 65%, from 501 in 2012 to 174 in 2020. CONCLUSIONS: Well-implemented ISGs can have a significant, immediate, and lasting impact on the prescription behavior. ISGs might thereby contribute to reduce resistance rates and CDI incidences in the hospital setting.

Updating changes in human gut microbial communities associated with Clostridioides difficile infection.

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea, a worldwide public health problem. Different factors can promote the progression of C. difficile infection (CDI), mainly altered intestinal microbiota composition. Microbial species belonging to different domains (i.e., bacteria, archaea, eukaryotes, and even viruses) are synergistically and antagonistically associated with CDI. This review was aimed at updating changes regarding CDI-related human microbiota composition using recent data and an integral approach that included the different microorganism domains. The three domains of life contribute to intestinal microbiota homeostasis at different levels in which relationships among microorganisms could explain the wide range of clinical manifestations. A holistic understanding of intestinal ecosystem functioning will facilitate identifying new predictive factors for infection and developing better treatment and new diagnostic tools, thereby reducing this disease's morbidity and mortality.

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence.

Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile.

Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.

Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA.

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool: A cross-sectional proof-of-concept study.

Clostridioides difficile infection (CDI) is an important infectious cause of antibiotic-associated diarrhea, with significant morbidity and mortality. Current diagnostic algorithms are based on identifying toxin by enzyme immunoassay (EIA) and toxin gene by real-time polymerase chain reaction (PCR) in patients with diarrhea. EIA's sensitivity is poor, and PCR, although highly sensitive and specific, cannot differentiate infection from colonization. An ideal test that incorporates microbial factors, host factors, and host-microbe interaction might characterize true infection, and assess prognosis and recurrence. The study of volatile organic compounds (VOCs) has the potential to be an ideal diagnostic test. The presence of VOCs accounts for the characteristic odor of stool in CDI but their presence in breath and plasma has not been studied yet. A cross-sectional proof-of-concept study analyzing VOCs using selected ion flow tube mass spectrometry (SIFT-MS) was done on breath, stool, and plasma of patients with clinical features and positive PCR for CDI (cases) and compared with patients with clinical features but a negative PCR (control). Our results showed that VOC patterns in breath, stool, and plasma, had good accuracy [area under the receiver operating characteristic curve (ROC) 93%, 86%, and 91%, respectively] for identifying patients with CDI.

Caloric restriction disrupts the microbiota and colonization resistance.

Diet is a major factor that shapes the gut microbiome1, but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.

High-throughput screening identifies a novel natural product-inspired scaffold capable of inhibiting Clostridioides difficile in vitro.

Clostridioides difficile is an enteric pathogen responsible for causing debilitating diarrhea, mostly in hospitalized patients. The bacterium exploits on microbial dysbiosis induced by the use of antibiotics to establish infection that ranges from mild watery diarrhea to pseudomembranous colitis. The increased prevalence of the disease accompanied by exacerbated comorbidity and the paucity of anticlostridial drugs that can tackle recurrence entails novel therapeutic options. Here, we report new lead molecules with potent anticlostridial activity from the AnalytiCon NATx library featuring natural product-inspired or natural product-derived small molecules. A high-throughput whole-cell-based screening of 5000 synthetic compounds from the AnalytiCon NATx library helped us identify 10 compounds capable of inhibiting the pathogen. Out of these 10 hits, we found 3 compounds with potent activity against C. difficile (MIC = 0.5-2 µg/ml). Interestingly, these compounds had minimal to no effect on the indigenous intestinal microbial species tested, unlike the standard-of-care antibiotics vancomycin and fidaxomicin. Further in vitro investigation revealed that the compounds were nontoxic to Caco-2 cell line. Given their potent anticlostridial activity, natural product-inspired scaffolds may suggest potential avenues that can address the unmet needs in preventing C. difficile mediated disease.

Metabolic Labeling of Clostridioides difficile Proteins.

The introduction of stable isotopes in vivo via metabolic labeling approaches (SILAC or 15N-labeling) allows, after combination of differentially treated labeled and unlabeled cells or protein extracts, for correction of protein quantification errors implemented during elaborated sample preparation workflows. The SILAC-based approach uses heavy arginine and lysine to incorporate the label into bacterial strains and cell lines, whereas 15N-metabolic labeling is achieved by cultivation in 15N-salt containing media. In case of Clostridioides difficile, the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism makes metabolic labeling challenging. Here, a step-by-step guideline for the metabolic labeling of C. difficile is described, which combines cultivation in liquid 15N-substituted medium followed by cultivation steps on solid 15N-substituted medium. The described procedure results in a label incorporation rate higher than 97%. Cells prepared by the following method can be used as standard for relative quantification approaches of, e.g., the membrane or surface proteome of C. difficile.

A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores.

BACKGROUND: Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile. METHODS: Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. RESULTS: Deletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. CONCLUSIONS: We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

Signal Versus Noise: How to Analyze the Microbiome and Make Progress on Antimicrobial Resistance.

Antimicrobial resistance has become a worldwide medical challenge [1], so impactful that vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have entered the common vernacular. We have attempted to reduce the selective pressure through antimicrobial stewardship, curtail the spread by identifying and isolating carriers and individuals with symptomatic infection, and treat antibiotic-resistant organisms (AROs) by developing novel antimicrobials. Despite these extraordinary measures, the challenge of AROs continues to grow. The gut microbiome, the ecosystem of microbes (ie, the microbiota) and metabolites present upon and within all humans, is an emerging target for both the risk for colonization and defense against infection with AROs. Here, informed from experiences and successes with understanding the role of the microbiome in mediating risk of Clostridioides difficile infection (CDI), we (1) review our understanding of the risk from ARO acquisition; (2) review our current understanding of the gut microbiome's ability to resist colonization with AROs; (3) describe how experimental model systems can test these initial, global insights to arrive at more granular, mechanistic ones; and (4) suggest a path forward to make further progress in the field.

The prevalence of Clostridioides difficile on farms, in abattoirs and in retail foods in Ireland.

An increasing proportion of Clostridioides difficile infections (CDI) are community acquired. This study tested farm, abattoir and retail food samples for C. difficile, using peer reviewed culture and molecular methods. The contamination rate on beef, sheep and broiler farms ranged from 2/30 (7%) to 25/30 (83%) in faeces, soil and water samples, while concentrations ranged from 2.9 log10 cfu/ml to 8.4 log10 cfu/g. The prevalence and associated counts were much lower in abattoir samples. Although 26/60 were C. difficile positive by enrichment and PCR, only 6 samples yielded counts by direct plating (1.1 log10 cfu/cm2 to 5.1 log10 cfu/g). At retail, 9/240 samples were C. difficile positive, including corned beef (1), spinach leaves (2), iceberg lettuce, little gem lettuce, wild rocket, coleslaw, whole milk yogurt and cottage cheese (1 sample each), with counts of up to 6.8 log10 cfu/g. The tcdA, tcdB, cdtA, cdtB, tcdC and tcdR genes were detected in 41%, 99.2%, 33.6%, 32%, 46.7% and 31.1%, respectively, of the 122 C. difficile isolates obtained. It was concluded that although the prevalence of C. difficile decreased along the food chain, retail foods were still heavily contaminated. This pathogen may therefore be foodborne, perhaps necessitating dietary advice for potentially vulnerable patients.

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile.

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.