Search Results (20)

The emergence of antimicrobial resistance (AMR) in Clostridium difficile (C. difficile), particularly to last-line antibiotics such as linezolid, represents a critical challenge in clinical settings. This study investigates the genomic epidemiology of linezolid-resistant C. difficile, focusing on the distribution and mutational patterns of the chloramphenicol–florfenicol resistance (cfr) gene and its association with multidrug resistance. We analyzed 514 clinical isolates (354 from NCBI Pathogen Detection, 160 from EnteroBase), revealing distinct prevalence patterns among cfr subtypes: cfr(C) was dominant (156/354 NCBI strains; 101/160 EnteroBase strains), whereas cfr(B) frequently harbored missense mutations (p.R247K, p.V294I, and less commonly p.A334T). The cfr(E) subtype was exclusively identified in ribotype 027 (RT027) strains. Notably, cfr(C) exhibited a strong association with RT017, correlating with a conserved 99 bp genomic deletion. Phylogenetic analysis linked cfr-carriage to predominant sequence types (ST1 in NCBI strains, ST37 in EnteroBase isolates). Furthermore, the co-occurrence of cfr with additional AMR genes conferred resistance to macrolides (erythromycin, azithromycin) and tetracyclines, indicating a convergent evolution toward multidrug resistance. These findings underscore the interplay between cfr mutations, hypervirulent ribotypes, and AMR dissemination, necessitating enhanced surveillance to mitigate the spread of resistant C. difficile lineages. Full article

Clostridioides difficile Infection (CDI) continues to be a major cause of antibiotic-associated diarrhea and pseudomembranous colitis, fueled in large measure by virulence factors TcdA and TcdB. These giant glucosyltransferase toxins interfere with host cytoskeletal integrity and inflammatory signaling by inhibiting Rho GTPase; however, the detailed structural dynamics, receptor selectivity, and subcellular trafficking mechanisms remain in part unspecified. This review integrates recent insights from cryo-electron microscopy (cryo-EM) and X-ray crystallography to describe the quaternary architecture of TcdA/B, emphasizing conformational changes key to pore formation and endosomal escape. We also examine the genomic heterogeneity of hypervirulent C. difficile strains (e.g., ribotype 027), correlating toxin gene polymorphisms (e.g., tcdC mutations) with increased toxin production and virulence. Mechanistic explanations of toxin-driven inflammasome activation and epithelial barrier dysfunction are situated within host immune evasion mechanisms, including microbiota-derived bile acid regulation of toxin stability. Subsequent innovative therapeutic strategies, encompassing the utilization of engineered neutralizing antibodies that specifically target the autoprocessing domain alongside structure-guided small-molecule inhibitors, are subjected to a rigorous evaluation. By integrating structural biology, systems-level omics, and clinical epidemiology, this review establishes a comprehensive framework for understanding C. difficile toxin pathogenesis and guiding next-generation precision antimicrobials. Full article

Background: Clostridioides difficile infection (CDI) remains a significant public health challenge in the United States, with limited treatment options currently available. Objectives: This study investigated the antimicrobial efficacy of a fungal-based fermentate derived from Aspergillus oryzae, cultivated in a proprietary food-grade medium, against toxigenic strains of C. difficile. Methods and Results: The ethyl acetate extract of A. oryzae fermentate (fungal extract) exhibited potent bactericidal activity, producing a significant zone of inhibition across all tested C. difficile strains, including hypervirulent Ribotype 027. Notably, 80% of the tested strains (four out of five) exhibited greater susceptibility to the fungal extract than to 5 µg vancomycin discs. Inner colony formation within the zone of inhibition was observed for all strains treated with vancomycin but only one strain was exposed to fungal extract. Time kill assays further confirmed the rapid bactericidal effect of the fungal extract, achieving complete C. difficile eradication within six hours. Mechanistic studies using scanning electron microscopy (SEM) and flow cytometry revealed that the fungal extract induced severe membrane disruption, leading to intracellular leakage and complete lysis. Flow cytometry analysis confirmed membrane depolarization and permeability loss on C. difficile cells. Conclusions: These findings highlight that the fungal extract of A. oryzae exhibits a promising antimicrobial activity against C. difficile. Future studies will focus on identifying its active components, evaluating its efficacy in vivo, and assessing its impact on gut microbiota to establish its potential clinical application in managing CDI. Full article

Background/Objectives: Clostridioides difficile infection (CDI) represents a significant public health challenge globally, driven by its increasing prevalence, hypervirulent strains like ribotype 027 (RT027), and growing antibiotic resistance. This study aimed to evaluate the prevalence of RT027 and analyze molecular markers of vancomycin and metronidazole resistance in stool samples from CDI patients hospitalized in Poland between 2017 and 2019. Methods: A total of 200 stool samples from confirmed CDI cases were analyzed for the presence of RT027, vanA (vancomycin resistance), and nim (metronidazole resistance) genes. DNA was extracted, and a polymerase chain reaction (PCR) was conducted using specific primers. Statistical associations between RT027 and resistance genes were evaluated using chi-square tests and logistic regression. Results: RT027 was detected in 14% of samples. The vanA gene, indicative of vancomycin resistance, was found in 52.5% of samples, while the nim gene, associated with metronidazole resistance, was present in 1.5% of cases. Co-occurrence of RT027 with vanA was not statistically significant. The study revealed no significant association between RT027 and vanA. Also, no significant association was observed between RT027 and nim due to the latter’s low prevalence. Conclusions: This study highlights a concerning prevalence of vanA among CDI cases, indicating widespread vancomycin resistance and challenging current treatment guidelines. While RT027 prevalence was moderate, no significant associations with vancomycin or metronidazole resistance were observed. These findings emphasize the need for molecular surveillance and improved antimicrobial stewardship to manage CDI effectively. Full article

Clostridioides difficile is a complex of anaerobic bacteria responsible for the epidemics of post-antibiotic diarrhea as one of the examples of CDI (Clostridioides difficile infection). As many as 70% of cases concern hospitalized patients, particularly those in intensive care units. Ribotyping is one of the most common methods for differentiating bacterial strains. The purpose of this work was to show the effectiveness of the gel electrophoresis-based PCR ribotyping method and the Webribo database for typing C. difficile isolates, including the hypervirulent 027 ribotype. DNA samples extracted from 69 C. difficile strains with previously marked genotypes were included in this study. PCR was performed using 16S–23S primers, and capillary gel electrophoresis was performed on the Applied Biosystem 3130xl Genetic Analyzer. The Webribo database was applied for ribotype assignment. Out of 69 samples, 48 belonged to already known ribotypes, 13 represented new ribotypes and 8 was indicated as similar to the existing ones, having some differences. Capillary gel electrophoresis-based PCR is an effective method for the differentiation of C. difficile ribotypes and can be recognized as a very useful tool in epidemiological studies, while the Webribo database is a useful and an accessible database for a quick analysis of C. difficile ribotypes. Full article

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3′)-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven “hypervirulent” RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential. Full article

Clostridioides difficile remains an important public health threat, globally. Since the emergence of the hypervirulent strain, ribotype 027, new strains have been reported to cause C. difficile infection (CDI) with poor health outcomes, including ribotypes 014/020, 017, 056, 106, and 078/126. These strains differ in their geographic distribution, genetic makeup, virulence factors, and antimicrobial susceptibility profiles, which can affect their ability to cause disease and respond to treatment. As such, understanding C. difficile epidemiology is increasingly important to allow for effective prevention measures. Despite the heightened epidemiological surveillance of C. difficile over the past two decades, it remains challenging to accurately estimate the burden and international epidemiological trends given the lack of concerted global effort for surveillance, especially in low- and middle-income countries. This review summarizes the changing epidemiology of C. difficile based on available data within the last decade, highlights the pertinent ribotypes from a global perspective, and discusses evolving treatments for CDI. Full article

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can occur in cases were the individual fails to re-establish a protective microflora. Bacteriophages and their lysins are currently being explored as treatment options due to their specificity, which minimizes the disruption to the other members of the gut microflora that are protective. The feasibility of employing recombinant endolysins to target the vegetative form of C. difficile has been demonstrated in animal models. In this study, we cloned and expressed the enzyme active domain of LysCD6356 and confirmed its ability to lyse the vegetative forms of a diverse range of clinical isolates of C. difficile, which included members of the hypervirulent 027 ribotype. Lytic activity was adversely affected by calcium, which is naturally found in the gut and is released from the spore upon germination. Our results suggests that a strategy in which the triggering of spore germination is separated in time from the application of the lysin could be developed as a strategy to reduce the risk of relapsing C. difficile infections. Full article

The aim of this study was to characterize C. difficile isolates from the farm, abattoir, and retail outlets in Ireland in terms of ribotype and antibiotic resistance (vancomycin, erythromycin, metronidazole, moxifloxacin, clindamycin, and rifampicin) using PCR and E-test methods, respectively. The most common ribotype in all stages of the food chain (including retail foods) was 078 and a variant (RT078/4). Less commonly reported (014/0, 002/1, 049, and 205) and novel (RT530, 547, and 683) ribotypes were also detected, but at lower frequencies. Approximately 72% (26/36 tested) of the isolates tested were resistant to at least one antibiotic, with the majority of these (65%; 17/26) displaying a multi-drug (three to five antibiotics) resistant phenotype. It was concluded that ribotype 078, a hypervirulent strain commonly associated with C. difficile infection (CDI) in Ireland, was the most frequent ribotype along the food chain, resistance to clinically important antibiotics was common in C. difficile food chain isolates, and there was no relationship between ribotype and antibiotic resistance profile. Full article

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium. The clinical features of C. difficile infections (CDIs) can vary, ranging from the asymptomatic carriage and mild self-limiting diarrhoea to severe and sometimes fatal pseudomembranous colitis. C. difficile infections (CDIs) are associated with disruption of the gut microbiota caused by antimicrobial agents. The infections are predominantly hospital-acquired, but in the last decades, the CDI patterns have changed. Their prevalence increased, and the proportion of community-acquired CDIs has also increased. This can be associated with the appearance of hypervirulent epidemic isolates of ribotype 027. The COVID-19 pandemic and the associated antibiotic overuse could additionally change the patterns of infections. Treatment of CDIs is a challenge, with only three appropriate antibiotics for use. The wide distribution of C. difficile spores in hospital environments, chronic persistence in some individuals, especially children, and the recent detection of C. difficile in domestic pets can furthermore worsen the situation. “Superbugs” are microorganisms that are both highly virulent and resistant to antibiotics. The aim of this review article is to characterise C. difficile as a new member of the “superbug” family. Due to its worldwide spread, the lack of many treatment options and the high rates of both recurrence and mortality, C. difficile has emerged as a major concern for the healthcare system. Full article

The recent discovery of the same Clostridioides difficile ribotypes associated with human infection in a broad range of environments, animals and foods, coupled with an ever-increasing rate of community-acquired infections, suggests this pathogen may be foodborne. The objective of this review was to examine the evidence supporting this hypothesis. A review of the literature found that forty-three different ribotypes, including six hypervirulent strains, have been detected in meat and vegetable food products, all of which carry the genes encoding pathogenesis. Of these, nine ribotypes (002, 003, 012, 014, 027, 029, 070, 078 and 126) have been isolated from patients with confirmed community-associated C. difficile infection (CDI). A meta-analysis of this data suggested there is a higher risk of exposure to all ribotypes when consuming shellfish or pork, with the latter being the main foodborne route for ribotypes 027 and 078, the hypervirulent strains that cause most human illnesses. Managing the risk of foodborne CDI is difficult as there are multiple routes of transmission from the farming and processing environment to humans. Moreover, the endospores are resistant to most physical and chemical treatments. The most effective current strategy is, therefore, to limit the use of broad-spectrum antibiotics while advising potentially vulnerable patients to avoid high-risk foods such as shellfish and pork. Full article

Despite an increased incidence of Clostridioides difficile infections, data on the reservoirs and dissemination routes of this bacterium are limited. This study examined the prevalence and characteristics of C. difficile isolates in spinach fields. C. difficile was detected in 2/60 (3.3%) of spinach and 6/60 (10%) of soil samples using culture-based techniques. Whole genome sequencing (WGS) analysis identified the spinach isolates as belonging to the hypervirulent clade 5, sequence type (ST) 11, ribotypes (RT) 078 and 126 and carried the genes encoding toxins A, B and CDT. The soil isolates belonged to clade 1 with different toxigenic ST/RT (ST19/RT614, ST12/RT003, ST46/RT087, ST16/RT050, ST49/RT014/0) strains and one non-toxigenic ST79/RT511 strain. Antimicrobial resistance to erythromycin (one spinach isolate), rifampicin (two soil isolates), clindamycin (one soil isolate), both moxifloxacin and rifampicin (one soil isolate), and multi-drug resistance to erythromycin, vancomycin and rifampicin (two soil isolates) were observed using the E test, although a broader range of resistance genes were detected using WGS. Although the sample size was limited, our results demonstrate the presence of C. difficile in horticulture and provide further evidence that there are multiple sources and dissemination routes for these bacteria. Full article

Clostridioides difficile is an important health care-associated pathogen. The aim of this study was to analyze the antibiotic susceptibility of C. difficile isolates from feces of patients from 13 hospitals in Silesia, Poland. The incidence of CDI per 100.000 people in Silesia in 2018–2019 was higher than the average in Poland (39.3–38.7 vs. 30.2–29.5, respectively). The incidence doubled from 26.4 in 2020 to 55.1 in 2021. Two hundred and thirty stool samples tested positive for GDH (glutamate dehydrogenase) and toxins were cultured anaerobically for C. difficile. The isolates were characterized, typed, and tested for susceptibility to 11 antibiotics by E-test (EUCAST, 2021). The genes of toxins A/B and binary were detected by mPCR. Of 215 isolates, 166 (77.2%) were classified as RT 027 and 6 (2.8%) as related RT 176. Resistance to ciprofloxacin (96.7%), moxifloxacin (79.1%), imipenem (78.1%), penicillin (67%), and rifampicin (40.5%) was found. The ermB gene was detected in 79 (36.7%) strains. Multidrug resistance (MDR) was confirmed in 50 (23.3%) strains of RT 027 (94%). We concluded that a high prevalence of MDR among hypervirulent RT 027/176 C. difficile was found in the Silesian region of Poland, emphasizing the need to enhance regional infection control on CDI and antibiotic stewardships. Full article

The US Centers for Disease Control and Prevention (CDC) lists Clostridioides difficile as an urgent bacterial threat. Yet, only two drugs, vancomycin and fidaxomicin, are approved by the FDA for the treatment of C. difficile infections as of this writing, while the global pipeline of new drugs is sparse at best. Thus, there is a clear and urgent need for new antibiotics against that organism. Herein, we disclose that AJ-024, a nitroimidazole derivative of a 26-membered thiopeptide, is a promising anti-C. difficile lead compound. Despite their unique mode of action, thiopeptides remain largely unexploited as anti-infective agents. AJ-024 combines potent in vitro activity against various strains of C. difficile with a noteworthy safety profile and desirable pharmacokinetic properties. Its time-kill kinetics against a hypervirulent C. difficile ribotype 027 and in vivo (mouse) efficacy compare favorably to vancomycin, and they define AJ-024 as a valuable platform for the development of new anti-C. difficile antibiotics. Full article

Background: Clostridioides (Clostridium) difficile is the most common nosocomial pathogen and antibiotic-related diarrhea in health-care facilities. Over the last few years, there was an increase in the incidence rate of C. difficile infection cases in Slovakia. In this study, the phenotypic (toxigenicity, antimicrobial susceptibility) and genotypic (PCR ribotypes, genes for binary toxins) patterns of C. difficile isolates from patients with CDI were analyzed, from July to August 2016, taken from hospitals in the Horne Povazie region of northern Slovakia. The aim of the study was also to identify hypervirulent strains (e.g., the presence of RT027 or RT176). Methods: The retrospective analysis of biological samples suspected of CDI were analyzed by GDH, anaerobic culture, enzyme immunoassay on toxins A/B, multiplex “real-time” PCR and PCR capillary-based electrophoresis ribotyping, and by MALDI TOF MS. Results: C. difficile isolates (n = 44) were identified by PCR ribotyping, which revealed five different ribotypes (RT001, 011, 017, 081, 176). The presence of hypervirulent RT027 was not identified. The C. difficile isolates (RT001, 011, 081, 176) were susceptible to metronidazole and vancomycin. One isolate RT017 had reduced susceptibility to vancomycin. A statistically significant difference between the most prevalent PCR ribotypes, RT001 and RT176, regarding variables such as albumin, CRP, creatinine, the length of hospitalization (p = 0.175), and glomerular filtration (p = 0.05) was not found. Conclusion: The results of PCR capillary-based electrophoresis ribotyping in the studied samples showed a high prevalence of RT176 and 001. Full article