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Background: The clinical burden of Clostridioides difficile infections (CDIs) remains substantial globally. This study aimed to investigate the ribotypes (RTs) and antimicrobial susceptibility of C. difficile isolates collected in Taiwan. Methods: C. difficile isolates were prospectively collected from four medical centers in Taiwan from 2019 to 2021. In a reference laboratory, in vitro susceptibility to clindamycin, moxifloxacin, metronidazole, vancomycin, fidaxomicin, and rifaximin were tested, and ribotyping was conducted to determine their genetic diversity. Results: A total of 568 C. difficile isolates were included. Metronidazole resistance was not observed, and the susceptibility rate of vancomycin was 99.5 %. Clindamycin showed poor activity against these isolates, with a resistance rate of 74.8 %. Fidaxomicin exhibited potent activity and 97.4 % of isolates were inhibited at 0.25 μg/mL. Rifaximin MIC90 increased from 0.015 μg/mL in 2019 to 0.03 μg/mL in 2020 and 2021. Of 40 RTs identified, two predominant RTs were RT 078/126 (78, 14 %) and 014/020 (76, 13 %). RT 017, traditional harboring truncated tcdA, accounted for 3 % (20 isolates) and there was no isolate belonging to RT 027. The proportions of RT 078 increased from 11.2 % in 2019 to 17.1 % in 2021, and the predominance of RT 078/126 was more evident in central Taiwan. Conclusions: Vancomycin, fidaxomicin, and metronidazole remain in vitro effective against clinical C. difficile isolates in Taiwan. The reservoirs and genetic relatedness of two major RTs with zoonotic potentials, RT 078/126 and 014/020, warrant further investigations.

Clostridioides (Clostridium) difficile is an enteric pathogen of several mammalian species including man, frequently involving nosocomial resurgence, following oral administration of broad-spectrum antibiotics, but also with human-to-human infection occurring, and neonatal pigs with zoonotic transmission. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. The neonatal pig is now recognized as a valuable model for human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24 h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (p > 0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3 h pi and 24 h pi (p > 0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3 h pi, and at 24 h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis. [ABSTRACT FROM AUTHOR]

Background: Clostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. Method: Dutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories (10.000 patient isolates), RT695 was found in only two patients with hospital-onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5C. difficile. Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. Conclusions: C. difficile was found in ∼20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

Clostridioides (Clostridium) difficile is a leading cause of infectious diarrhea in humans and production animals and can be found in a variety of environmental sources. The prevalence and diversity of multi-locus sequence type clade 5 strains of C. difficile in Australian production animals suggest Australia might be the ancestral home of this lineage of One Health importance. To better understand the role of the environment in the colonization of humans and animals in Australia, it is important to investigate these endemic sources. This study describes the prevalence, molecular epidemiology, and biogeographic distribution of C. difficile in soils of Western Australia. A total of 321 soil samples from remote geographical locations across the eight health regions of Western Australia were screened for C. difficile and isolates characterized by PCR ribotyping and toxin gene profiling. C. difficile was isolated from 31.15% of samples, with the highest prevalence in the Perth Metropolitan Health Region (49.25%, n = 33/67). Overall, 52 different strains [PCR ribotypes (RTs)] were identified, with 14 being novel, and 38% (38/100) of isolates being toxigenic, the most common of which was RT014/020. Five unique novel isolates showed characteristics similar to C. difficile clade 5. This is the first study of C. difficile isolated from soils in Australia. The high prevalence and heterogeneity of C. difficile strains recovered suggest that soils play a role in the survival and environmental dissemination of this organism, and potentially its transmission among native wildlife and production animals, and in community and hospital settings. [ABSTRACT FROM AUTHOR]

Abstract Background Clostridioides (Clostridium) difficile is an important infectious pathogen, which causes mild-to-severe gastrointestinal infections by creating resistant spores and producing toxins. Spores contaminated foods might be one of the most significant transmission ways of C. difficile-associated infections. This systematic review and meta-analysis study were conducted to investigate the prevalence of C. difficile in food. Methods Articles that published the prevalence of C. difficile in food in PubMed, Web of Science, and Scopus databases were retrieved using selected keywords between January 2009 and December 2019. Finally, 17,148 food samples from 60 studies from 20 countries were evaluated. Results The overall prevalence of C. difficile in various foods was 6.3%. The highest and lowest levels of C. difficile contamination were detected to seafood (10.3%) and side dishes (0.8%), respectively. The prevalence of C. difficile was 4% in cooked food, 6.2% in cooked chicken and 10% in cooked seafood. Conclusions There is still little known concerning the food-borne impact of C. difficile, but the reported contamination might pose a public health risk. Therefore, to improve the food safety and prevent contamination with C. difficile spores, it is necessary to observe hygienic issues during foods preparation, cooking and transfer.

Clostridioides (Clostridium) difficile is an enteric pathogen of several mammalian species including man, frequently involving nosocomial resurgence, following oral administration of broad-spectrum antibiotics, but also with human-to-human infection occurring, and neonatal pigs with zoonotic transmission. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. The neonatal pig is now recognized as a valuable model for human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24 h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (p > 0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3 h pi and 24 h pi (p > 0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3 h pi, and at 24 h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis.

Pigeons are known for their capacity to harbor and spread several zoonotic agents. Studies have suggested that pigeons are also relevant disseminators of multidrug-resistant strains. In this study, pigeons surrounding a veterinary hospital were sampled and tested for the presence of pathogenic Escherichia coli, Salmonella spp., Staphylococcus spp., and Clostridioides (Clostridium) difficile. E. coli isolates from 19 (40.4%) pigeons tested positive for the E. coli heat-stable enterotoxin 1 (EAST1)-encoding gene. The intimin-encoding gene (eae) of enteropathogenic E. coli (EPEC) was found in one isolate (2.1%). Salmonella spp. were found in nine (19.1%) pigeons, all from the first capture event (P < 000.1). S. Typhimurium and S. Heidelberg were isolated from six and three pigeons, respectively. Enterobacterial repetitive intergenic consensus (ERIC-PCR) of the Salmonella spp. isolates suggested that eight of the nine strains had a high genetic similarity, supporting the hypothesis of an outbreak of salmonellosis in these pigeons. Twenty (42.5%) staphylococcal isolates were recovered from 18 (38.3%) pigeons. Eight different species were detected, with S. xylosus being the most frequent. Two (4.3%) C. difficile strains were isolated. Three isolates, one each of S. Typhimurium, S. aureus, and C. difficile, were classified as multidrug-resistant strains. The present research suggested that pigeons residing in urban areas can act as reservoirs and disseminators of pathogenic bacteria, including nosocomial pathogens, such as diarrheagenic E. coli and multidrug-resistant Staphylococcus spp., C. difficile, and Salmonella spp. [ABSTRACT FROM AUTHOR]

Aims To investigate the prevalence, molecular type, and antimicrobial susceptibility of Clostridioides difficile in the environment in Vietnam, where little is known about C. difficile. Methods and results Samples of pig faeces, soils from pig farms, potatoes, and the hospital environment were cultured for C. difficile. Isolates were identified and typed by polymerase chain reaction (PCR) ribotyping. The overall prevalence of C. difficile contamination was 24.5% (68/278). Clostridioides difficile was detected mainly in soils from pig farms and hospital soils, with 70%–100% prevalence. Clostridioides difficile was isolated from 3.4% of pig faecal samples and 5% of potato surfaces. The four most prevalent ribotypes (RTs) were RTs 001, 009, 038, and QX574. All isolates were susceptible to metronidazole, fidaxomicin, vancomycin, and amoxicillin/clavulanate, while resistance to erythromycin, tetracycline, and moxifloxacin was common in toxigenic strains. Clostridioides difficile RTs 001A+B+CDT– and 038A–B–CDT– were predominantly multidrug resistant. Conclusions Environmental sources of C. difficile are important to consider in the epidemiology of C. difficile infection in Vietnam, however, contaminated soils are likely to be the most important source of C. difficile. This poses additional challenges to controlling infections in healthcare settings. [ABSTRACT FROM AUTHOR]

This is the first report to discover Clostridiodes difficile (C. difficile) ribotype RT126 and RT598 (both ribotypes belong to RT078-lineage) in a river water system in southern Taiwan. Fluoroquinolone resistance was also found. The connection between clinical isolates and those from the environment needs further investigation.

Background: Clostridioides (Clostridium) difficile is an important infectious pathogen, which causes mild-to-severe gastrointestinal infections by creating resistant spores and producing toxins. Spores contaminated foods might be one of the most significant transmission ways of C. difficile-associated infections. This systematic review and meta-analysis study were conducted to investigate the prevalence of C. difficile in food. Methods: Articles that published the prevalence of C. difficile in food in PubMed, Web of Science, and Scopus databases were retrieved using selected keywords between January 2009 and December 2019. Finally, 17,148 food samples from 60 studies from 20 countries were evaluated. Results: The overall prevalence of C. difficile in various foods was 6.3%. The highest and lowest levels of C. difficile contamination were detected to seafood (10.3%) and side dishes (0.8%), respectively. The prevalence of C. difficile was 4% in cooked food, 6.2% in cooked chicken and 10% in cooked seafood. Conclusions: There is still little known concerning the food-borne impact of C. difficile, but the reported contamination might pose a public health risk. Therefore, to improve the food safety and prevent contamination with C. difficile spores, it is necessary to observe hygienic issues during foods preparation, cooking and transfer. [ABSTRACT FROM AUTHOR]

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In adults, Clostridioides difficile infections are associated with alterations of the intestinal bacterial populations. Although preterm neonates (PN) are frequently colonized by C. difficile, limited data are available regarding the relationship between C. difficile and the intestinal microbiota of this specific population. Therefore, we studied the intestinal microbiota of PN from two multicenter cohorts using high-throughput sequencing of the bacterial 16S rRNA gene. Our results showed that alpha diversity was significantly higher in children colonized by C. difficile than those without colonization. Beta diversity significantly differed between the groups. In multivariate analysis, C. difficile colonization was significantly associated with the absence of postnatal antibiotherapy and higher gestational age. Taxa belonging to the Lachnospiraceae, Enterobacteriaceae, Oscillospiraceae families and Veillonella sp. were positively associated with C. difficile colonization, whereas Bacteroidales and Bifidobacterium breve were negatively associated with C. difficile colonization. After adjustment for covariables, Clostridioides, Rothia, Bifidobacterium, Veillonella, Eisenbergiella genera and Enterobacterales were more abundant in the gut microbiota of colonized children. There was no significant association between C. difficile colonization and necrotizing enterocolitis in PN. Our results suggest that C. difficile colonization in PN is related to the establishment of physiological microbiota. [ABSTRACT FROM AUTHOR]

In adults, Clostridioides difficile infections are associated with alterations of the intestinal bacterial populations. Although preterm neonates (PN) are frequently colonized by C. difficile, limited data are available regarding the relationship between C. difficile and the intestinal microbiota of this specific population. Therefore, we studied the intestinal microbiota of PN from two multicenter cohorts using high-throughput sequencing of the bacterial 16S rRNA gene. Our results showed that alpha diversity was significantly higher in children colonized by C. difficile than those without colonization. Beta diversity significantly differed between the groups. In multivariate analysis, C. difficile colonization was significantly associated with the absence of postnatal antibiotherapy and higher gestational age. Taxa belonging to the Lachnospiraceae, Enterobacteriaceae, Oscillospiraceae families and Veillonella sp. were positively associated with C. difficile colonization, whereas Bacteroidales and Bifidobacterium breve were negatively associated with C. difficile colonization. After adjustment for covariables, Clostridioides, Rothia, Bifidobacterium, Veillonella, Eisenbergiella genera and Enterobacterales were more abundant in the gut microbiota of colonized children. There was no significant association between C. difficile colonization and necrotizing enterocolitis in PN. Our results suggest that C. difficile colonization in PN is related to the establishment of physiological microbiota.

Clostridioides difficile is often found in animals and their environment. However, not much has been reported on veterinary clinics environment in terms of the spore load, prevalence and PCR ribotype diversity. The aim of this study was to assess the prevalence of C. difficile on shoe soles of veterinarians, veterinary support staff and veterinary students at the Veterinary Faculty campus. Altogether, 50 shoe sole swabs were collected, and the positivity rates ranged from 86.7% in swabs from veterinarians to 100% in swabs from support staff and students. Non‐toxigenic and toxigenic strains representing toxinotypes 0, IV and XIX were isolated and distributed into 17 different PCR ribotypes, most common being 010, 014/020, SLO002 and 009. PCR ribotype 010 was the most prevalent and isolated from shoe soles sampled in 6/7 areas. Students' shoes had highest ribotype diversity (15/17 PCR ribotypes) but showed a low overlap with ribotype isolated from vets and support staff shoes. Veterinary students are likely the main vectors of C. difficile spores transmissions among veterinary teaching clinics and the hospital. [ABSTRACT FROM AUTHOR]

Introduction: Clostridioides (Clostridium) difficile infection, the leading cause of healthcare-associated diarrhea, represents a significant burden on global healthcare systems. Despite being a global issue, information on C. difficile from a global perspective is lacking. The aim of this study is to model the global phylogeography of clinical C. difficile. Methods: Using samples collected from the MODIFY I and II studies (NCT01241552, NCT01513239), we performed whole-genome sequencing of 1501 clinical isolates including 37 novel sequence types (STs), representing the largest worldwide collection to date. Results: Our data showed ribotypes, multi-locus sequence typing clades, and whole-genome phylogeny were in good accordance. The clinical C. difficile genome was found to be more conserved than previously reported (61% core genes), and modest recombination rates of 1.4–5.0 were observed across clades. We observed a significant continent distribution preference among five C. difficile clades (Benjamini-Hochberg corrected Fisher's exact test P < 0.01); moreover, weak association between geographic and genetic distance among ribotypes suggested sources beyond healthcare-related transmission. Markedly different trends of antibiotic susceptibility among lineages and regions were identified, and three novel mutations (in pyridoxamine 5′-phosphate oxidase family protein: Tyr130Ser, Tyr130Cys, and a promoter SNP) associated with metronidazole-reduced susceptibility were discovered on a nim-related gene and its promotor by genome-wide association study. Toxin gene polymorphisms were shown to vary within and between prevalent ribotypes, and novel severe mutations were found on the tcdC toxin regulator protein. Conclusion: Our systematic characterization of a global set of clinical trial C. difficile isolates from infected individuals demonstrated the complexity of the genetic makeup of this pathogenic organism. The geographic variability of clades, variability in toxin genes, and mutations associated with antibiotic susceptibility indicate a highly complex interaction of C. difficile between host and environment. This dataset will provide a useful resource for validation of findings and future research of C. difficile. [ABSTRACT FROM AUTHOR]

Objective(s): Clostridioides (Clostridium) difficile infection as a healthcare-associated infection can cause life-threatening infectious diarrhea in hospitalized patients. The aim of this study was to investigate the toxin profiles and antimicrobial resistance patterns of C. difficile isolates obtained from hospitalized patients in Shiraz, Iran.Materials and Methods: This study was performed on 45 toxigenic C. difficile isolates. Determination of toxin profiles was done using polymerase chain reaction method. Antimicrobial susceptibility to vancomycin, metronidazole, clindamycin, tetracycline, moxifloxacin, and chloramphenicol was determined by the agar dilution method. The genes encoding antibiotic resistance were detected by the standard procedures.Results: The most frequent toxin profile was tcdA+, tcdB+, cdtAˉ, cdtBˉ (82.2%), and only one isolate harboured all toxin associated genes (tcdA+, tcdB+, cdtA+, cdtB+) (2.2%). The genes encoding CDT (binary toxin) were also found in six (13.3%) isolates. Resistance to tetracycline, clindamycin and moxifloxacin was observed in 66.7%, 60% and 42.2% of the isolates, respectively. None of the strains showed resistance to other antibiotics. The distribution of the ermB gene (the gene encoding resistance to clindamycin) was 57.8% and the tetM and tetW genes (the genes encoding resistance to tetracycline) were found in 62.2% and 13.3% of the isolates, respectively. The substitutions Thr82 to Ile in GyrA and Asp426 to Asn in GyrB were seen in moxifloxacin resistant isolates.Conclusion: Our data contributes to the present understanding of virulence and resistance traits amongst the isolates. Infection control strategies should be implemented carefully in order to curb the dissemination of C. difficile strains in hospital.

ABSTRACT Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC’s ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC’s self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores. IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly’s molecular bases may be essential to developing new therapies against C. difficile infection.

Hernández et al. studied the fecal microbiota associated with I C. difficile i infection and I Akkermansia i to be predictive for the presence of a I C. difficile i infection and highlight that co-infection with other pathogenic agents are to be considered in treatment. Dayananda and Wilcox review the effect of co-infecting or colonizing on the infection of I C. difficile i strains to identify potential exploitable mechanisms to prevent I C. difficile i infection. Keywords: Clostridioides (Clostridium) difficile; toxins A and B Clostridium difficile; binary toxin CDT; Clostridium difficile infection (CDI); anaerobic metabolism EN Clostridioides (Clostridium) difficile toxins A and B Clostridium difficile binary toxin CDT Clostridium difficile infection (CDI) anaerobic metabolism 1 3 3 04/11/22 20220406 NES 220406 Introduction I Clostridioides i (formerly I Clostridium i ) I difficile i is an anaerobic, spore-forming bacterium, widely distributed in soil, water, animals and the gut of healthy humans (Hall and O'Toole, [4]; al Saif and Brazier, [1]; Lawson et al., [5]). Clostridioides (Clostridium) difficile, toxins A and B Clostridium difficile, binary toxin CDT, Clostridium difficile infection (CDI), anaerobic metabolism. [Extracted from the article]

Clostridioides (Clostridium) difficile causes asymptomatic colonization or diseases with different clinical severity in children. Asymptomatic colonization occurs mainly in children ≤ 2 years old. Clinically significant C. difficile produce at least one of the three major toxins: toxin A, toxin B or binary toxin and frequently are associated with CDI in children > 2 years. Some of the main risk factors for C. difficile infection (CDI) are irrational antibiotic treatment; prolonged hospital stay, concomitant diseases and reduced patient’s immunity. In the last few decades an increasing number of hospital and community acquired CDIs have been reported worldwide, both adult patients and children. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is the leading cause of antibiotic-associated nosocomial diarrhea, but extra-intestinal manifestations are rare. We describe the first documented case of bacteraemia with pacemaker pocket and lead infection with the toxigenic C. difficile ribotype 014 with a lack of abdominal symptoms. The patient underwent pacemaker extraction and treatment with intravenous and oral vancomycin. Genotyping and molecular subtyping revealed clonality between pacemaker and intestinal isolates. This case illustrates the risk of intravascular device infections due to C. difficile. Even asymptomatic C. difficile colonization might pose a risk for prosthetic material infection. [ABSTRACT FROM AUTHOR]

Clostridioides (Clostridium) difficile infection is implicated as a major cause of antibiotic-associated diarrhea in hospitals worldwide. Probiotics, especially lactic acid bacteria, are the most frequently used alternative treatment. This study aims to identify potential probiotic enterococci strains that act against C. difficile strains and exert a protective effect on colon adenocarcinoma cells (HT-29 cells). To this end, nine Enterococcus strains isolated from the feces of breast-fed infants were investigated. They were identified as E. faecalis by 16s rRNA sequencing and MALDI-TOF. The probiotic properties including their viabilities in simulated gastrointestinal condition, cell adhesion ability, and their safety were evaluated. All strains exhibited more tolerance toward both pepsin and bile salts and adhered more tightly to HT-29 cells compared with the reference probiotic strain Lactobacillus plantarum ATCC 14917. Polymerase chain reaction (PCR) results exhibited that six of nine strains carried at least one virulence determinant gene; however, none exhibited virulence phenotypes or carried transferable antibiotic resistance genes. These strains did not infect Galleria mellonella when compared to pathogenic E. faecalis strain (p < 0.05). Moreover, their antibacterial activities against C. difficile were examined using agar well-diffusion, spore production, and germination tests. The six safe strains inhibited spore germination (100 – 98.20% ± 2.17%) and sporulation, particularly in C. difficile ATCC 630 treated with E. faecalis PK 1302. Furthermore, immunofluorescence assay showed that the cytopathic effects of C. difficile of HT-29 cells were reduced by the treatment with the cell-free supernatant of E. faecalis strains. These strains prevented rounding of HT-29 cells and preserved the F-actin microstructure and tight junctions between adjacent cells, which indicated their ability to reduce the clostridial cytopathic effects. Thus, the study identified six E. faecalis isolates that have anti-C. difficile activity. These could be promising probiotics with potential applications in the prevention of C. difficile colonization and treatment of C. difficile infection.

Abstract Background Community acquired Clostridioides (Clostridium) difficile infection (CA-CDI) is a significant health problem in human and veterinary medicine. Animals are often considered as potential reservoirs for CA-CDI. In Europe, family farming is the most predominant farming operation, with a complex interaction between animals and the community. Therefore, it is pertinent to evaluate transmission patterns of C. difficile on such prominent European farming model. Fecal samples from calves (n = 2442) were collected biweekly over a period of one year on 20 mid-size family dairy farms. Environmental samples (n = 475) were collected in a three month interval. Clostridioides difficile was detected using qPCR in 243 fecal samples (243/2442); positive samples were then quantified. Association between prevalence/load of C. difficile and age of the calves was estimated with logistic regression model. Most common C. difficile isolate from calves (n = 76) and the environment (n = 14) was C. difficile ribotype 033, which was further analyzed using multilocus variable-number tandem-repeat analysis (MLVA) to assess intra- and between-farm relatedness. Results Clostridioides difficile was detected in feces of calves less than 24 h old. Results showed a non-linear statistically significant decrease in shedding load of C. difficile with age (P

Clostridioides (also known as Clostridium) difficile is a Gram-positive anaerobic, spore producing bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are inadequate, expensive, and limited, and thus inexpensive drug treatments for C. difficile infection (CDI) with improved efficacy and specificity are urgently needed. To improve the solubility of our cationic amphiphilic 1,1′-binaphthylpeptidomimetics developed earlier that showed promise in an in vivo murine CDI model we have synthesized related compounds with an N-arytriazole or N-naphthyltriazole moiety instead of the 1,1′-biphenyl or 1,1′-binaphthyl moiety. This modification was made to increase the polarity and thus water solubility of the overall peptidomimetics, while maintaining the aromatic character. The dicationic N-naphthyltriazole derivative 40 was identified as a C. difficile-selective antibacterial with MIC values of 8 µg/mL against C. difficile strains ATCC 700057 and 132 (both ribotype 027). This compound displayed increased water solubility and reduced hemolytic activity (32 µg/mL) in an in vitro hemolysis assay and reduced cytotoxicity (CC50 32 µg/mL against HEK293 cells) relative to lead compound 2. Compound 40 exhibited mild efficacy (with 80% survival observed after 24 h compared to the DMSO control of 40%) in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.

Clostridioides difficile is an important nosocomial pathogen increasingly observed in the community and in different non-human reservoirs. The epidemiology and transmissibility of C. difficile has been studied using a variety of typing methods, including more recently developed whole-genome sequence (WGS) analysis that is becoming used routinely for bacterial typing worldwide. Here we review the schemes for WGS-based typing methods available for C. difficile and their applications in the field of human C. difficile infection (CDI). The two main approaches to discover genomic variations are single nucleotide variant (SNV) analysis and methods based on gene-by-gene comparisons (frequently called core genome or whole genome MLST, cgMLST, or wgMLST). SNV analysis currently provides the ultimate resolution, however, typing nomenclature and standardized methodology are missing. On the other hand, gene-by-gene approaches allow portability and standardized nomenclature, and are therefore becoming increasingly popular in bacterial epidemiology and outbreak investigation. Two commercial software packages (BioNumerics and Ridom SeqSphere+) and an open source database (EnteroBase) for allele and sequence type determination for C. difficile are currently available. Proof-of-concept WGS studies have already enabled advances in the investigation of the population structure of C. difficile species, microevolution within the epidemic strains, intercontinental transmission over time and in tracking of transmission events. WGS of clinical C. difficile isolates demonstrated a considerable genetic diversity suggesting diverse reservoirs for CDI. WGS was also shown to aid in resolving relapses and reinfections in recurrent CDI and has potential for use as a tool for assessing hospital infection prevention and control performance.

Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios.

Regulation of bacterial motility to maximize nutrient acquisition or minimize exposure to harmful substances plays an important role in microbial proliferation and host colonization. The technical difficulties of performing high-resolution live microscopy on anaerobes have hindered mechanistic studies of motility in Clostridioides (formerly Clostridium) difficile. Here, we present a widely applicable protocol for live cell imaging of anaerobic bacteria that has allowed us to characterize C. difficile swimming at the single-cell level. This accessible method for anaerobic live cell microscopy enables inquiry into previously inaccessible aspects of C. difficile physiology and behavior. We present the first report that vegetative C. difficile are capable of regulated motility in the presence of different nutrients. We demonstrate that the epidemic C. difficile strain R20291 exhibits regulated motility in the presence of multiple nutrient sources by modulating its swimming velocity. This is a powerful illustration of the ability of single-cell studies to explain population-wide phenomena such as dispersal through the environment. • Live anaerobic bacteria can be imaged on a standard microscope. • Clostridium difficile modulates its swimming velocity in response to nutrient availability. • The gastric mucus component N-acetylneuraminic acid regulates C. difficile motility. [ABSTRACT FROM AUTHOR]

Abstract Clostridioides (formerly Clostridium) difficile is a Gram-positive anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are vastly inadequate, expensive and limited; this results in an exorbitant medical and financial burden. New, inexpensive chemotherapeutic treatments for C. difficile infection with improved efficacy are urgently needed. A streamlined synthetic pathway was developed to allow access to 38 novel mono- and di-cationic biaryl 1,2,3-triazolyl peptidomimetics with increased synthetic efficiency, aqueous solubility and enhanced antibacterial efficacy. The monocationic arginine derivative 28 was identified as a potent, Gram-positive selective antibacterial with MIC values of 4 μg/mL against methicillin-resistant Staphylococcus aureus and 8 μg/mL against C. difficile. Furthermore, the dicationic bis-triazole analogue 50 was found to exhibit broad-spectrum activity with substantial Gram-negative efficacy against Acinetobacter baumannii (8 μg/mL), Pseudomonas aeruginosa (8 μg/mL) and Klebsiella pneumoniae (16 μg/mL); additionally, compound 50 displayed reduced haemolytic activity (<13%) in an in vitro haemolysis assay. Membrane-disruption assays were conducted on selected derivatives to confirm the membrane-active mechanism of action inherent to the synthesized amphiphilic compounds. A comparative solubility assay was developed and utilized to optimize the aqueous solubility of the compounds for in vivo studies. The biaryl peptidomimetics 28 and 67 were found to exhibit significant efficacy in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease. Graphical abstract Image 1 Highlights • Improved synthetic access to antibacterial biaryl peptidomimetic amphiphiles. • Modular synthesis of 38 cationic biaryl 1,2,3-triazolyl peptidomimetics. • Broad-spectrum or Gram-positive selective antibacterial activity achieved. • Significant in vivo efficacy in a murine C. difficile infection model. [ABSTRACT FROM AUTHOR]

Disturbance in gut microbiota is crucial for the development of Clostridioides difficile infection (CDI). Different mechanisms through which gut microbiota influences C. difficile colonization are known. However, C. difficile could also affect gut microbiota balance as previously demonstrated by cultivation of fecal microbiota in C. difficile conditioned medium. In current study, the interactions of C. difficile cells with gut microbiota were addressed. Three different strains (ribotypes 027, 014/020, and 010) were co-cultivated with two types of fecal microbiota (healthy and dysbiotic) using in vitro batch model. While all strains showed higher sporulation frequency in the presence of dysbiotic fecal microbiota, the growth was strain dependent. C. difficile either proliferated to comparable levels in the presence of dysbiotic and healthy fecal microbiota or grew better in co-culture with dysbiotic microbiota. In co-cultures with any C. difficile strain fecal microbiota showed decreased richness and diversity. Dysbiotic fecal microbiota was more affected after co-culture with C. difficile than healthy microbiota. Altogether, 62 OTUs were significantly changed in co-cultures of dysbiotic microbiota/C. difficile and 45 OTUs in co-cultures of healthy microbiota/C. difficile. However, the majority of significantly changed OTUs in both types of microbiota belonged to the phylum Firmicutes with Lachnospiraceae and Ruminococcaceae origin.

Disturbance in gut microbiota is crucial for the development of Clostridioides difficile infection (CDI). Different mechanisms through which gut microbiota influences C. difficile colonization are known. However, C. difficile could also affect gut microbiota balance as previously demonstrated by cultivation of fecal microbiota in C. difficile conditioned medium. In current study, the interactions of C. difficile cells with gut microbiota were addressed. Three different strains (ribotypes 027, 014/020, and 010) were co-cultivated with two types of fecal microbiota (healthy and dysbiotic) using in vitro batch model. While all strains showed higher sporulation frequency in the presence of dysbiotic fecal microbiota, the growth was strain dependent. C. difficile either proliferated to comparable levels in the presence of dysbiotic and healthy fecal microbiota or grew better in co-culture with dysbiotic microbiota. In co-cultures with any C. difficile strain fecal microbiota showed decreased richness and diversity. Dysbiotic fecal microbiota was more affected after co-culture with C. difficile than healthy microbiota. Altogether, 62 OTUs were significantly changed in co-cultures of dysbiotic microbiota/C. difficile and 45 OTUs in co-cultures of healthy microbiota/C. difficile. However, the majority of significantly changed OTUs in both types of microbiota belonged to the phylum Firmicutes with Lachnospiraceae and Ruminococcaceae origin. [ABSTRACT FROM AUTHOR]

Clostridioides (Clostridium) difficile causes antimicrobial-associated diarrhoea, however, presentations may range from asymptomatic carriage to severe diarrhoea, life-threatening toxic megacolon and even death. Reports on C. difficile infection (CDI) in Vietnam remain limited. The objectives of this study were to evaluate the epidemiology, molecular characteristics, and antimicrobial susceptibility of C. difficile isolated from adults with diarrhoea in Vietnam. Diarrhoeal stool samples from adult patients aged ≥17 years old were collected at Thai Binh General Hospital in northern Vietnam between March 1, 2021 and February 28, 2022. All samples were transported to The University of Western Australia, Perth, Western Australia for C. difficile culture, toxin gene profiling, PCR ribotyping and antimicrobial susceptibility testing. A total of 205 stool samples were collected from patients aged from 17 to 101 years old. The overall prevalence of C. difficile was 15.1% (31/205) with the recovery of toxigenic and non-toxigenic isolates 9.8% (20/205) and 6.3% (13/205), respectively. Thus 33 isolates were recovered comprising 18 known ribotypes (RTs) and one novel RT (two samples contained two different RTs in each sample). The most prevalent strains were RT 012 (five strains) and RTs 014/020, 017 and QX 070 three strains each. All C. difficile were susceptible to amoxicillin/clavulanate, fidaxomicin, metronidazole, moxifloxacin and vancomycin, while resistance to varying degrees was seen to clindamycin, erythromycin, tetracycline and rifaximin, 78.8% (26/33), 51.5% (17/33), 27.3% (9/33) and 6.1% (2/33), respectively. The prevalence of multidrug resistance was 27.3% (9/33) and multidrug resistance was most common in toxigenic RT 012 and non-toxigenic RT 038 strains. The prevalence of C. difficile in adults with diarrhoea and multidrug resistance in C. difficile isolates was relatively high. A clinical assessment to differentiate between CDI/disease and colonisation is required. • The prevalence of C. difficile in adults with diarrhoea in Vietnam was high (15.1%). • C. difficile ribotype 012 was the most prevalent strain. • Most C. difficile strains were resistant to clindamycin (78.8%). • Multidrug resistance, resistance to >3 classes of antimicrobial, was common (27.3%). [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is one of the greatest threats to animal and public health. Clostridioides (prev. Clostridium) difficile is a major burden to healthcare and a relevant AMR gene reservoir. Despite the known importance of AMR in C. difficile epidemiology and treatment, antimicrobial susceptibility testing for this pathogen is still based on the determination of the minimal inhibitory concentration (MIC) by the agar dilution method, which is technically demanding and labor-intensive. In this study, the disk diffusion method was used to evaluate the susceptibility of C. difficile to erythromycin, rifampicin, and tetracycline. A total of 155 isolates isolated between 2011 and 2022 from humans and animals in Brazil were simultaneously tested using the disk diffusion method and the epsilometer test (Etest) for these three antimicrobials on Brucella blood agar supplemented with vitamin K and hemin. The results suggest that disk diffusion can be an interesting routine tool to identify erythromycin- and rifampicin - resistant C. difficile isolates (≥20 mm cut-off) and wild type (WT) strains (≥28 mm). However, the disk diffusion protocol tested in this study does not seem suitable for tetracycline because of the common misclassification of resistant strains. • Antimicrobial resistance is of importance in C. difficile epidemiology. • Minimal inhibitory concentration (MIC) is technically demanding and labor-intensive. • Disk diffusion method correlated with MIC values for erythromycin and rifampicin. • Disk diffusion method lead to misclassification of tetracycline-resistant strains. • Clade 5 isolates were more likely to be resistant to these three antimicrobials. [ABSTRACT FROM AUTHOR]

Clostridioides (Clostridium) difficile infection (CDI) places a burden on healthcare facilities worldwide. Most research studies have been concentrated in high-income countries in North America, Europe, Asia and Australia, where C. difficile is the leading cause of diarrhoea associated with antimicrobial use. This narrative review summarises African CDI studies, focussing on reports published in the last 20 years. Although relatively sparse, the data suggest that CDI is an important cause of diarrhoea on the continent. African CDI patient populations are often younger than in European and North American settings, probably due to the high prevalence of co-morbid conditions such as tuberculosis, particularly in sub-Saharan Africa. Strain typing data are rare and where reported generally limited to single sites and institutions. Despite challenges, including a lack of facilities and awareness, there is a need for further investigation to more accurately determine the true burden of disease caused by C. difficile in Africa. • Clostridioides difficile infection (CDI) is a cause of diarrhoea in Africa that is often overlooked. • African CDI patient populations tend to be younger than those in high-income nations. • Tuberculosis is an important under-investigated co-morbidity. • Ribotypes (RT)027 and RT078 are not commonly reported in African studies. • Surveillance programs and identification of strain reservoirs are necessary. [ABSTRACT FROM AUTHOR]

Clostridioides (also known as Clostridium) difficile is a Gram-positive anaerobic, spore producing bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are inadequate, expensive, and limited, and thus inexpensive drug treatments for C. difficile infection (CDI) with improved efficacy and specificity are urgently needed. To improve the solubility of our cationic amphiphilic 1,1′-binaphthylpeptidomimetics developed earlier that showed promise in an in vivo murine CDI model we have synthesized related compounds with an N-arytriazole or N-naphthyltriazole moiety instead of the 1,1′-biphenyl or 1,1′-binaphthyl moiety. This modification was made to increase the polarity and thus water solubility of the overall peptidomimetics, while maintaining the aromatic character. The dicationic N-naphthyltriazole derivative 40 was identified as a C. difficile-selective antibacterial with MIC values of 8 µg/mL against C. difficile strains ATCC 700057 and 132 (both ribotype 027). This compound displayed increased water solubility and reduced hemolytic activity (32 µg/mL) in an in vitro hemolysis assay and reduced cytotoxicity (CC50 32 µg/mL against HEK293 cells) relative to lead compound 2. Compound 40 exhibited mild efficacy (with 80% survival observed after 24 h compared to the DMSO control of 40%) in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacillus which is the leading cause of health-care-associated infective diarrhea. The rising incidence of antibiotic resistance in pathogens such as C. difficile makes researches on alternative antibacterial products very important, especially those exploring natural products like propolis. Brazilian Red Propolis, found in the Northeast region of Brazil, is composed by products from regional plants that have the antimicrobial properties. This study aimed to evaluate the in vitro activity of Brazilian Red Propolis (BRP) against C. difficile strains in planktonic and biofilm forms. The susceptibility of four strains of C. difficile to BRP was analyzed by broth microdilution method and vancomycin was included as control drug. BRP-exposed C. difficile cells were evaluated by scanning electron microscopy (SEM). Then, the effects of BRP on growing and mature C. difficile biofilms were also evaluated. BRP minimum inhibitory concentration was 625 μg/mL against all tested strains, while vancomycin MIC range was 0.5–2 μg/mL. SEM showed the loss of homogeneity in bacterial cell wall and cell fragmentation, after BRP-exposure. BRP, at MIC, reduced (P < 0.05) the biomass, matrix proteins and matrix carbohydrates of growing biofilms, and, at 8xMIC, reduced (P < 0.05) the biomass and matrix proteins of mature biofilms. The present study demonstrated that BRP inhibits planktonic growth, damages cell wall, decreases biofilm growth and harms mature biofilms of C. difficile. [Display omitted] • Brazilian Red Propolis (BRP) have the antimicrobial activity at C. difficile strains. • BRP inhibits planktonic growth and cause damages cell wall of C. difficile strains. • BRP decreases biofilm growth and reduces mature biofilms in vitro of C. difficile. [ABSTRACT FROM AUTHOR]

• Tigecycline (TGC) monotherapy failure in severe C. difficile infection (CDI) may occur. • We enrolled 110 patients in a cohort study, failure with TGC occurred in 37.3%. • Patients with failure had shorter symptom durations and higher ICU admittance rates. • Pulmonary disease, ileus, parenteral nutrition, TGC therapy length were failure predictors. • Severe CDI cases with higher risk for TGC failure might be identified by some factors. Our aim was to analyze characteristics of treatment failure with intravenous tigecycline monotherapy among adults with severe Clostridioides (Clostridium) difficile infection (CDI). A single-centre observational cohort study was performed between 2014 and 2018. Data were collected by charts review, diagnosis and severity were determined by ESCMID guidelines. Primary outcome was treatment failure, secondary outcomes were in-hospital mortality, relapse, colectomy, and complication rates. Independent predictors of failure were identified using logistic regression. Altogether 110 patients were included, failure occurred in 37.3%. Patients with failure frequently had chronic heart and pulmonary co-morbidities, peritonitis, higher CRP levels, ICU admittance rates and need for total parenteral nutrition and vasopressors. Mostly, CDI-specific mortality and complications contributed to failure. Relapse rates were similar. Chronic pulmonary disease, ileus, total parenteral nutrition, and duration of tigecycline therapy were predictors of failure. We conclude that severe CDI cases with higher risk for tigecycline monotherapy failure might be identified by contributing factors. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA + B + isolates with known ribotypes (RTs). Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene. Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene. The present study demonstrates that the majority of clinical tcdA + B + isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/toxinotype 0 and the RT 126/toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence. • Toxinotypes 0 and V are the most frequent type among tcdA + B + C. difficile isolates. • RT 001/0 and the RT 126/V are the most common types among Iranian isolates. • The majority of tcdA + B + clinical C. difficile isolates harbor tcdC genetic variants. [ABSTRACT FROM AUTHOR]

Clostridioides (Clostridium) difficile is the leading cause of healthcare-associated infectious diarrhea in the developed world. Retrospective studies have shown a lower incidence of C. difficile infection (CDI) in Japan than in Europe or North America. Prospective studies are needed to determine if this is due lack of testing for C. difficile or a true difference in CDI epidemiology. A prospective cohort study of CDI was conducted from May 2014 to May 2015 at 12 medical facilities (20 wards) in Japan. Patients with at least three diarrheal bowel movements (Bristol stool grade 6–7) in the preceding 24 h were enrolled. CDI was defined by positive result on enzyme immunoassay for toxins A/B, nucleic acid amplification test for the toxin B gene or toxigenic culture. C. difficile isolates were subjected to PCR-ribotyping (RT), slpA -sequence typing (slpA -ST), and antimicrobial susceptibility testing. The overall incidence of CDI was 7.4/10,000 patient-days (PD). The incidence was highest in the five ICU wards (22.2 CDI/10,000 PD; range: 13.9–75.5/10,000 PD). The testing frequency and CDI incidence rate were highly correlated (R2 = 0.91). Of the 146 isolates, RT018/018″ was dominant (29%), followed by types 014 (23%), 002 (12%), and 369 (11%). Among the 15 non-ICU wards, two had high CDI incidence rates (13.0 and 15.9 CDI/10,000 PD), with clusters of RT018/ slpA -ST smz-02 and 018"/smz-01, respectively. Three non-RT027 or 078 binary toxin-positive isolates were found. All RT018/018" isolates were resistant to moxifloxacin, gatifloxacin, clindamycin, and erythromycin. This study identified a higher CDI incidence in Japanese hospitals than previously reported by actively identifying and testing patients with clinically significant diarrhea. This suggests numerous patients with CDI are being overlooked due to inadequate diagnostic testing in Japan. • A prospective and multi-center study of CDI was conducted in Japan. • The incidence rate (7.4/10,000 patient-days) of CDI was high. • The incidence was highest in five ICU wards (22.2 CDI/10,000 patient-days). • PCR-ribotypes 018, 014, 002, and 369 were predominant. [ABSTRACT FROM AUTHOR]