Impact of terminal cleaning in rooms previously occupied by patients with healthcare-associated infections.

Healthcare associated infections (HAIs) are costly but preventable. A limited understanding of the effects of environmental cleaning on the riskiest HAI associated pathogens is a current challenge in HAI prevention. This project aimed to quantify the effects of terminal hospital cleaning practices on HAI pathogens via environmental sampling in three hospitals located throughout the United States. Surfaces were swabbed from 36 occupied patient rooms with a laboratory-confirmed, hospital- or community-acquired infection of at least one of the four pathogens of interest (i.e., Acinetobacter baumannii (A. baumannii), methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis/faecium (VRE), and Clostridioides difficile (C. difficile)). Six nonporous, high touch surfaces (i.e., chair handrail, bed handrail, nurse call button, desk surface, bathroom counter near the sink, and a grab bar near the toilet) were sampled in each room for Adenosine Triphosphate (ATP) and the four pathogens of interest before and after terminal cleaning. The four pathogens of interest were detected on surfaces before and after terminal cleaning, but their levels were generally reduced. Overall, C. difficile was confirmed on the desk (n = 2), while MRSA (n = 24) and VRE (n = 25) were confirmed on all surface types before terminal cleaning. After cleaning, only MRSA (n = 6) on bed handrail, chair handrail, and nurse call button and VRE (n = 5) on bathroom sink, bed handrail, nurse call button, toilet grab bar, and C. difficile (n = 1) were confirmed. At 2 of the 3 hospitals, pathogens were generally reduced by >99% during terminal cleaning. One hospital showed that VRE increased after terminal cleaning, MRSA was reduced by 73% on the nurse call button, and VRE was reduced by only 50% on the bathroom sink. ATP detections did not correlate with any pathogen concentration. This study highlights the importance of terminal cleaning and indicates room for improvement in cleaning practices to reduce surface contamination throughout hospital rooms.

Encapsulation protocol for fecal microbiota transplantation.

Introduction: Clostridioides difficile infections (CDI) continue to pose a challenge for clinicians. Fecal microbiota transplantation (FMT) is an effective treatment option in CDI. Furthermore, recent and ongoing studies suggest potential benefits of FMT in other diseases as well. Methods: We would like to present a novel protocol for encapsulation of lyophilized fecal material. Our method provides with better compliance as well as improved flexibility, storage and safety. Results: FMT was conducted in 28 patients with an overall success rate of 82,14% using apsules containing lyophilized stool. 16 of patients were given capsules with lessened bacteria counts. The success rate in this group was 93,75%. Discussion: The results highlight the still unanswered questions about the mechanism of action and contribute to a wider use of FMT in the clinical praxis and in research.

PhosphoLipidome Alteration Induced by Clostridioides difficile Toxin B in Enteric Glial Cells.

Clostridioides difficile (C. difficile) is responsible for a spectrum of nosocomial/antibiotic-associated gastrointestinal diseases that are increasing in global incidence and mortality rates. The C. difficile pathogenesis is due to toxin A and B (TcdA/TcdB), both causing cytopathic and cytotoxic effects and inflammation. Recently, we demonstrated that TcdB induces cytopathic and cytotoxic (apoptosis and necrosis) effects in enteric glial cells (EGCs) in a dose/time-dependent manner and described the underlying signaling. Despite the role played by lipids in host processes activated by pathogens, to counter infection and/or induce cell death, to date no studies have investigated lipid changes induced by TcdB/TcdA. Here, we evaluated the modification of lipid composition in our in vitro model of TcdB infection. Apoptosis, cell cycle, cell viability, and lipidomic profiles were evaluated in EGCs treated for 24 h with two concentrations of TcdB (0.1 ng/mL; 10 ng/mL). In EGCs treated with the highest concentration of TcdB, not only an increased content of total lipids was observed, but also lipidome changes, allowing the separation of TcdB-treated cells and controls into different clusters. The statistical analyses also allowed us to ascertain which lipid classes and lipid molecular species determine the clusterization. Changes in lipid species containing inositol as polar head and plasmalogen phosphatidylethanolamine emerged as key indicators of altered lipid metabolism in TcdB-treated EGCs. These results not only provide a picture of the phospholipid profile changes but also give information regarding the lipid metabolism pathways altered by TcdB, and this might represent an important step for developing strategies against C. difficile infection.

Spores of Clostridioides difficile are toxin delivery vehicles.

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.

Fecal microbiota composition is a better predictor of recurrent Clostridioides difficile infection than clinical factors in a prospective, multicentre cohort study.

INTRODUCTION: Clostridioides difficile infection (CDI) is the most common cause of antibiotic-associated diarrhoea. Fidaxomicin and fecal microbiota transplantation (FMT) are effective, but expensive therapies to treat recurrent CDI (reCDI). Our objective was to develop a prediction model for reCDI based on the gut microbiota composition and clinical characteristics, to identify patients who could benefit from early treatment with fidaxomicin or FMT. METHODS: Multicentre, prospective, observational study in adult patients diagnosed with a primary episode of CDI. Fecal samples and clinical data were collected prior to, and after 5 days of CDI treatment. Follow-up duration was 8 weeks. Microbiota composition was analysed by IS-pro, a bacterial profiling technique based on phylum- and species-specific differences in the 16-23 S interspace regions of ribosomal DNA. Bayesian additive regression trees (BART) and adaptive group-regularized logistic ridge regression (AGRR) were used to construct prediction models for reCDI. RESULTS: 209 patients were included, of which 25% developed reCDI. Variables related to microbiota composition provided better prediction of reCDI and were preferentially selected over clinical factors in joint prediction models. Bacteroidetes abundance and diversity after start of CDI treatment, and the increase in Proteobacteria diversity relative to baseline, were the most robust predictors of reCDI. The sensitivity and specificity of a BART model including these factors were 95% and 78%, but these dropped to 67% and 62% in out-of-sample prediction. CONCLUSION: Early microbiota response to CDI treatment is a better predictor of reCDI than clinical prognostic factors, but not yet sufficient enough to predict reCDI in daily practice.

The state of play of rodent models for the study of Clostridioides difficile infection.

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhoea and is responsible for a spectrum of diseases characterized by high levels of recurrence and morbidity. In some cases, complications can lead to death. Currently, several types of animal models have been developed to study various aspects of C. difficile infection (CDI), such as colonization, virulence, transmission and recurrence. These models have also been used to test the role of environmental conditions, such as diet, age and microbiome that modulate infection outcome, and to evaluate several therapeutic strategies. Different rodent models have been used successfully, such as the hamster model and the gnotobiotic and conventional mouse models. These models can be applied to study either the initial CDI infectious process or recurrences. The applications of existing rodent models and their advantages and disadvantages are discussed here.

Insoluble polysaccharides produced in plant cell cultures protect from Clostridioides difficile colitis.

Clostridioides difficile infection (CDI) poses a significant health threat due to high recurrence rates. Antimicrobial agents are commonly used to manage CDI-related diarrhoea; however, by aggravating intestinal dysbiosis, antibiotics enable C. difficile spores germination and production of toxins, the main virulence factors. Therefore, the binding of exotoxins using adsorbents represents an attractive alternative medication for the prevention and treatment of relapses. In this study, we provided evidence that the natural insoluble polysaccharides, named ABR119, extracted by plant cell cultures, effectively trap C. difficile toxins. In our experiments, ABR119 exhibited no cytotoxicity in vitro and was safely administered in vivo. In the animal model of C. difficile-associated colitis, ABR119 (50 mg/kg body weight) significantly reduced the colonic myeloperoxidase activity and severity of inflammation, preventing body weight loss. These effects were not evident when we treated animals with wheat bran polysaccharides. We did not detect bacterial killing effects of ABR119 against C. difficile nor against bacterial species of the normal gut microbiota. Moreover, ABR119 did not interfere in vitro with the antimicrobial activities of most clinically used antibiotics. In summary, ABR119 holds promise for treating and preventing C. difficile colitis by trapping the bacterial toxins, warranting further studies to assess the ABR119 potential in human infections caused by C. difficile.

RelQ-mediated alarmone signalling regulates growth, stress-induced biofilm formation and spore accumulation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signalling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sublethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and spore accumulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. RESULTS: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. CONCLUSIONS: This proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches. Video Abstract.

Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

Fit-for-purpose heterodivalent single-domain antibody for gastrointestinal targeting of toxin B from Clostridium difficile.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

Effectiveness of C. Difficile Prevention Framework Depends on How Measures Are Implemented.

According to this study.

Clostridioides difficile-mucus interactions encompass shifts in gene expression, metabolism, and biofilm formation.

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides difficile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter the viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and the predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight flexibility in metabolism that may influence pathogenesis. IMPORTANCE: Clostridioides difficile results in upward of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise, and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand the dynamics of C. difficile-mucus interactions in the context of the human gut.

[Analysis of Clostridioides difficile infection characteristics and risk factors in patients hospitalized for diarrhea in 3 university hospitals in a mid-south city of China].

OBJECTIVE: To investigate the characteristics of Clostridioides difficile infection (CDI) in patients hospitalized for diarrhea and analyze the risk factors for CDI. METHODS: Stool samples were collected from 306 patients with diarrhea hospitalized in 3 university hospitals in a mid-south city of China from October to December, 2020. C. difficile was isolated by anaerobic culture, and qRT-PCR was used to detect the expressions of toxin A (tcdA) and B (tcdB) genes and the binary toxin genes (cdtA and cdtB). Multilocus sequence typing (MLST) was performed for the isolated strains without contaminating strains as confirmed by 16S rDNA sequencing. Etest strips were used to determine the drug resistance profiles of the isolated strains, and the risk factors of CDI in the patients were analyzed. RESULTS: CDI was detected in 25 (8.17%) out of the 306 patients. All the patients tested positive for tcdA and tcdB but negative for the binary toxin genes. Seven noncontaminated C. difficile strains with 5 ST types were isolated, including 3 ST54 strains and one strain of ST129, ST98, ST53, and ST631 types each, all belonging to clade 1 and sensitive to metronidazole and vancomycin. Hospitalization within the past 6 months (OR= 3.675; 95% CI: 1.405-9.612), use of PPIs (OR=7.107; 95% CI: 2.575-19.613), antibiotics for ≥1 week (OR=7.306; 95% CI: 2.274-23.472), non-steroidal anti-inflammatory drugs (OR=4.754; 95% CI: 1.504-15.031) in the past month, and gastrointestinal disorders (OR=5.050; 95% CI: 1.826-13.968) were all risk factors for CDI in the patients hospitalized for diarrhea. CONCLUSION: The CDI rate remains low in the hospitalized patients with diarrhea in the investigated hospitals, but early precaution measures are recommended when exposure to the risk factors is reported to reduce the risk of CDI in the hospitalized patients.

Differences in enteric pathogens and intestinal microbiota between diarrheic weaned piglets and healthy penmates.

Postweaning diarrhea (PWD) is a multifactorial disease caused by different aetiological agents, like viruses or bacteria and where the role of the microbiota remains unclear. The aim of this study was to assess differences between healthy and diarrheic weaned pigs concerning the prevalence of pathogens and changes in the intestinal microbiota. Eighteen farms with PWD were selected and 277 fecal samples were collected (152 diarrheic vs 125 healthy). Presence of Rotavirus A (RVA), B (RVB), C (RVC) and Porcine Epidemic Diarrhea Virus (PEDV), virulence factors of Escherichia coli and Clostridioides difficile were analyzed by PCR. Finally, the microbiota composition was also study by 16 S rRNA sequencing on 148 samples (102 diarrheic vs 46 healthy). RVA (53.95 % vs 36 %, p=0.04) and RVB (49.67 % vs 28.8 %, p<0.001) were more frequent in diarrheic animals. Furthermore, RVA viral load was higher in diseased animals. VT2 toxin was significantly associated with diarrhea, whereas other virulence factors were not. Presence of C. difficile and PEDV was almost negligible. Regarding microbiota changes, Fusobacteriota phylum was more frequent in diarrheic samples and Ruminococcaceae family in healthy penmates. During the first week postweaning, Enterobacteriace and Campylobacteria were enriched in animals presenting diarrhea. Furthermore, Lactobacillus was detected in those individuals with no RVA infection. In conclusion, RVA seems to play a primary role in PWD. Classic E. coli virulence factors were not associated with diarrhea, indicating the need for revising their implication in disease. Moreover, Lactobacillus was found frequently in animals negative for RVA, suggesting some protective effect.

Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Insight into the Mechanism of Lysogeny Control of phiCDKH01 Bacteriophage Infecting Clinical Isolate of Clostridioides difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

Comparative evaluation of the STANDARD M10 and Xpert C. difficile assays for detection of toxigenic Clostridioides difficile in stool specimens.

This study compared the performance of two commercial molecular assays, the STANDARD M10 Clostridioides difficile assay (M10) and the Xpert C. difficile assay (Xpert), for detecting toxigenic C. difficile in stool specimens. A total of 487 consecutive stool specimens submitted for routine C. difficile testing between June and November 2023 were included. Following routine testing using C. DIFF QUIK CHEK COMPLETE (QCC), M10 and Xpert were tested in parallel, alongside toxigenic culture (reference standard). Additionally, two-step algorithms, using QCC on the first step and either M10 or Xpert on the second step, were assessed. Both M10 and Xpert demonstrated a sensitivity and negative predictive value (NPV) of 100%. M10 exhibited significantly higher specificity and positive predictive value (PPV; 91.9% and 64.2%, respectively) than Xpert (90.3% and 59.8%, respectively). Both two-step algorithms showed a sensitivity and NPV of 98.4% and 99.8%, respectively. The specificity and PPV of the two-step algorithm using M10 (95.2% and 75.0%, respectively) were slightly higher than those of the one using Xpert (94.8% and 73.2%, respectively), without statistical significance. Receiver operating characteristic curve analysis, assessing the predictive ability of cycle threshold (Ct) values for the detection of free toxin, exhibited an area under the curve of 0.825 for M10 and 0.843 for Xpert. This indicates the utility of Ct values as predictors for the detection of free toxin in both assays. In conclusion, M10 proves to be an effective diagnostic tool with performance comparable to Xpert, whether utilized independently or as part of a two-step algorithm.

Cyclic diguanylate differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in Clostridioides difficile.

Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.