Clostridioides difficile and colorectal cancer: a dangerous liaison.

Many colorectal diseases depend on complex interactions between several pathophysiological factors, including the intestinal microbiota. In recent years, the widespread use of antibiotics has been recognized as a main cause of intestinal dysbiosis and a favouring factor for Clostridioides difficile infection. The latter, in addition, causes infectious diarrhoea, pseudomembranous colitis, and toxic megacolon by means of its toxins (A and, especially, B), is characterized by frequent relapses; thus, its persistence in a host may be long-lasting. Based on recent experimental evidence, here we analyse the possibility that, similarly to other bacteria, Clostridioides difficile may be considered a potential carcinogen for colorectal cancer.

Gut Microbiota and Clostridium difficile: What We Know and the New Frontiers.

Our digestive system, particularly our intestines, harbors a vast amount of microorganisms, whose genetic makeup is referred to as the microbiome. Clostridium difficile is a spore-forming Gram-positive bacterium, which can cause an infection whose symptoms range from asymptomatic colonization to fearsome complications such as the onset of toxic megacolon. The relationship between gut microbiota and Clostridium difficile infection has been studied from different perspectives. One of the proposed strategies is to be able to specifically identify which types of microbiota alterations are most at risk for the onset of CDI. In this article, we understood once again how crucial the role of the human microbiota is in health and especially how crucial it becomes, in the case of its alteration, for the individual's disease. Clostridium difficile infection is an emblematic example of how a normal and physiological composition of the human microbiome can play a very important role in immune defense against such a fearsome disease.

Acute Responses to Infectious and Inflammatory Disorders of the Gastrointestinal System.

Infectious and inflammatory disorders of the gastrointestinal system are life-threatening and occur frequently in hospitalized adults. Inflammatory and inflammation-related diseases of the gastrointestinal tract seen in the acutely and critically ill have numerous causes. In acute pancreatitis and toxic infections caused by the bacterium Clostridium difficile, where severe infections can develop, inflammation plays a causative and crucial role. Severe acute pancreatitis puts a patient at risk for infected necrosis, which can result in septicemia and shock. Similarly, patients treated with antibiotics are at risk for C difficile colitis, which can progress to toxic megacolon. These conditions require volume resuscitation and interventions supported by current evidence. Percutaneous or surgical interventions are often undertaken at a critical point in these illnesses. Patients who require surgery for these diagnoses present challenges for the interprofessional team. Inflammatory and infectious disorders often can lead to complications of systemic inflammatory response syndrome, sepsis, and multiorgan failure. New strategies are on the horizon to prevent the onset of and improve care for patients with severe acute pancreatitis, fulminant C difficile infection, and megacolon.

Bacterial Indole as a Multifunctional Regulator of Klebsiella oxytoca Complex Enterotoxicity.

Gastrointestinal microbes respond to biochemical metabolites that coordinate their behaviors. Here, we demonstrate that bacterial indole functions as a multifactorial mitigator of Klebsiella grimontii and Klebsiella oxytoca pathogenicity. These closely related microbes produce the enterotoxins tilimycin and tilivalline; cytotoxin-producing strains are the causative agent of antibiotic-associated hemorrhagic colitis and have been associated with necrotizing enterocolitis of premature infants. We demonstrate that carbohydrates induce cytotoxin synthesis while concurrently repressing indole biosynthesis. Conversely, indole represses cytotoxin production. In both cases, the alterations stemmed from differential transcription of npsA and npsB, key genes involved in tilimycin biosynthesis. Indole also enhances conversion of tilimycin to tilivalline, an indole analog with reduced cytotoxicity. In this context, we established that tilivalline, but not tilimycin, is a strong agonist of pregnane X receptor (PXR), a master regulator of xenobiotic detoxification and intestinal inflammation. Tilivalline binding upregulated PXR-responsive detoxifying genes and inhibited tubulin-directed toxicity. Bacterial indole, therefore, acts in a multifunctional manner to mitigate cytotoxicity by Klebsiella spp.: suppression of toxin production, enhanced conversion of tilimycin to tilivalline, and activation of PXR. IMPORTANCE The human gut harbors a complex community of microbes, including several species and strains that could be commensals or pathogens depending on context. The specific environmental conditions under which a resident microbe changes its relationship with a host and adopts pathogenic behaviors, in many cases, remain poorly understood. Here, we describe a novel communication network involving the regulation of K. grimontii and K. oxytoca enterotoxicity. Bacterial indole was identified as a central modulator of these colitogenic microbes by suppressing bacterial toxin (tilimycin) synthesis and converting tilimycin to tilivalline while simultaneously activating a host receptor, PXR, as a means of mitigating tissue cytotoxicity. On the other hand, fermentable carbohydrates were found to inhibit indole biosynthesis and enhance toxin production. This integrated network involving microbial, host, and metabolic factors provides a contextual framework to better understand K. oxytoca complex pathogenicity.

Teaching old mice new tricks: the utility of aged mouse models of C. difficile infection to study pathogenesis and rejuvenate immune response.

BACKGROUND: Clostridioides difficile is a serious problem for the aging population. Aged mouse model of C. difficile infection (CDI) has emerged as a valuable tool to evaluate the mechanism of aging in CDI. METHODS: We reviewed five published studies utilizing aged mice (7-28 months) for CDI model for findings that may advance our understanding of how aging influences outcome from CDI. RESULTS: Aged mouse models of CDI uniformly demonstrated more severe disease in the old compared to young mice. Diminished neutrophil recruitment to intestinal tissue in aged mice is the most consistent finding. Differences in innate and humoral immune responses were also observed. The effects of aging on the outcome of infection were reversed by pharmacologic or microbiota-targeted interventions. CONCLUSION: The aged mouse presents an important in vivo model to study CDI and elucidate the mechanisms underlying advanced age as an important risk factor for severe disease.

Probiotics in Pediatrics. A Review and Practical Guide.

The potential benefit of the administration of probiotics in children has been studied in many settings globally. Probiotics products contain viable micro-organisms that confer a health benefit on the host. Beneficial effects of selected probiotic strains for the management or prevention of selected pediatric conditions have been demonstrated. The purpose of this paper is to provide an overview of current available evidence on the efficacy of specific probiotics in selected conditions to guide pediatricians in decision-making on the therapeutic or prophylactic use of probiotic strains in children. Evidence to support the use of certain probiotics in selected pediatric conditions is often available. In addition, the administration of probiotics is associated with a low risk of adverse events and is generally well tolerated. The best documented efficacy of certain probiotics is for treatment of infectious gastroenteritis, and prevention of antibiotic-associated, Clostridioides difficile-associated and nosocomial diarrhea. Unfortunately, due to study heterogeneity and in some cases high risk of bias in published studies, a broad consensus is lacking for specific probiotic strains, doses and treatment regimens for some pediatric indications. The current available evidence thus limits the systematic administration of probiotics. The most recent meta-analyses and reviews highlight the need for more well-designed, properly powered, strain-specific and dedicated-dose response studies.

Large Clostridial Toxins: Mechanisms and Roles in Disease.

Large clostridial toxins (LCTs) are a family of bacterial exotoxins that infiltrate and destroy target cells. Members of the LCT family include Clostridioides difficile toxins TcdA and TcdB, Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL. Since the 19th century, LCT-secreting bacteria have been isolated from the blood, organs, and wounds of diseased individuals, and LCTs have been implicated as the primary virulence factors in a variety of infections, including C. difficile infection and some cases of wound-associated gas gangrene. Clostridia express and secrete LCTs in response to various physiological signals. LCTs invade host cells by binding specific cell surface receptors, ultimately leading to internalization into acidified vesicles. Acidic pH promotes conformational changes within LCTs, which culminates in translocation of the N-terminal glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol, leading first to cytopathic effects and later to cytotoxic effects. The focus of this review is on the role of LCTs in infection and disease, the mechanism of LCT intoxication, with emphasis on recent structural work and toxin subtyping analysis, and the genomic discovery and characterization of LCT homologues. We provide a comprehensive review of these topics and offer our perspective on emerging questions and future research directions for this enigmatic family of toxins.

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

New ribotype Clostridioides difficile from ST11 group revealed higher pathogenic ability than RT078.

Clostridioides difficile is the predominant antibiotic-associated enteropathogen associated with diarrhoea or pseudomembranous colitis in patients worldwide. Previously, we identified C. difficile RT078 isolates (CD21062) from elderly patients in China, including two new ribotype strains (CD10010 and CD12038) belonging to the ST11 group, and their genomic features were also investigated. This study compared sporulation, spore germination, toxin expression, flagellar characteristics, and adhesion among these strains in vitro and analysed their pathogenic ability in vivo using animal models. The results showed sporulation and spore germination did not significantly differ among the three C. difficile strains. CD10010 and CD12038 showed higher transcriptional levels of toxins until 48 h; thereafter, the transcriptional levels of toxins remained constant among RT078, CD10010, and CD12038. RT078 showed a loss of flagellum and its related genes, whereas CD12038 showed the highest motility in vitro. Both CD10010 and CD12038 initially showed flg phase OFF, and the flagellar switch reversed to phase ON after 48 h in swim agar. Flagellar proteins and toxins were both upregulated when flg phase OFF changed to flg phase ON status, enhancing their pathogenic ability. CD12038 showed the highest adhesion to Hep-2 cells. Histopathology and inflammation scores demonstrated that CD12038 caused the most severe tissue damage and infection in vivo. The new ribotype strains, particularly CD12038, exhibit higher pathogenic ability than the typical RT078 strain, both in vitro and in vivo. Therefore, more attention should be paid to this new C. difficile strain in epidemiological research; further studies are warranted.

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Murine Intrarectal Instillation of Purified Recombinant Clostridioides difficile Toxins Enables Mechanistic Studies of Pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474-4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Burden and risk factors for inappropriate Clostridioides Difficile infection testing among hospitalized patients.

BACKGROUND: The purpose of this study was to identify the burden and risk factors for inappropriate Clostridioides difficile infection (CDI) testing. METHODS: This was a retrospective cohort study among adults hospitalized between 2010 and 2019. Inappropriate CDI testing was defined as a formed stool specimen, an order within 7 days of a previously negative test, or an order within 24 hours of laxative administration. RESULTS: A total of 51,302 CDI orders were placed for 29,840 unique patients. 59% were appropriate and 41% were inappropriate. An additional 24% of the appropriate orders never resulted. Risk factors for inappropriate testing included orders placed by a nurse practitioner, orders placed by high-ordering providers, specific hospital units, fever, and leukocytosis. CONCLUSIONS: Nearly half of all CDI orders were inappropriate among hospitalized patients, and an additional 24% of test results never returned. Provider- and patient-level risk factors included type of provider, specific hospital units, and signs of sepsis.

Clostridioides difficile infections: Epidemiology, correlations and treatment in a Lebanese cohort with use of ATLAS scoring.

INTRODUCTION: The objectives of the present study were to investigate epidemiology, correlations, severity, and therapeutic response of Clostridioides difficile infections in a Lebanese tertiary care hospital. METHODOLOGY: In this retrospective cohort study, patients having at least one positive Clostridioides difficile test (antigen glutamate dehydrogenase/GDH with toxins, or PCR) were studied. RESULTS: Among 58 patients, 20 (34.5%) and 53 (91.4%) had positive antigen GDH and toxins, respectively. PCR was performed in 25 (43.1%) patients without any positive ribotype 027. Fifteen (25.9%) patients were immunocompromised, 35 (60.3%) patients received antibiotics prior to the infection and 34 (58.6%) on proton pump inhibitors. Fifty-four (93%) patients had a resolution of their symptoms after a mean period of 4.2 days of treatment. Twenty-two (38%) participants were treated with oral vancomycin, 11 (19%) with intravenous metronidazole and 23 (39.6%) with both antibiotics. Resolution of symptoms was significantly more rapid with monotherapy (p = 0.007) with no significant difference between vancomycin and metronidazole (p = 0.413). A positive correlation was found between ATLAS score and delay to symptoms resolution (r = 0.553; p < 0.001; N = 54), as well as between ATLAS score and prevalence of complications (p = 0.003). CONCLUSIONS: History of treatment with antibiotics, proton pump inhibitors, and hospital admission during the previous year were prevalent among our patient cohort. Rates of symptomatic resolution were similar with monotherapy and dual therapy.

Comparative genomics of Clostridioides difficile toxinotypes identifies module-based toxin gene evolution.

Clostridioides difficile is a common cause of nosocomial diarrhoea. Toxins TcdA and TcdB are considered to be the main virulence factors and are encoded by the PaLoc region, while the binary toxin encoded in the CdtLoc region also contributes to pathogenicity. Variant toxinotypes reflect the genetic diversity of a key toxin-encoding 19 kb genetic element (the PaLoc). Here, we present analysis of a comprehensive collection of all known major C. difficile toxinotypes to address the evolutionary relationships of the toxin gene variants, the mechanisms underlying the origin and development of variability in toxin genes and the PaLoc, and the relationship between structure and function in TcdB variants. The structure of both toxin genes is modular, composed of interspersed blocks of sequences corresponding to functional domains and having different evolutionary histories, as shown by the distribution of mutations along the toxin genes and by incongruences of domain phylogenies compared to overall C. difficile cluster organization. In TcdB protein, four mutation patterns could be differentiated, which correlated very well with the type of TcdB cytopathic effect (CPE) on cultured cells. Mapping these mutations to the three-dimensional structure of the TcdB showed that the majority of the variation occurs in surface residues and that point mutation at residue 449 in alpha helix 16 differentiated strains with different types of CPE. In contrast to the PaLoc, phylogenetic trees of the CdtLoc were more consistent with the core genome phylogenies, but there were clues that CdtLoc can also be exchanged between strains.

[Fecal microbiota transplants in the treatment of pseudomembranous colitis (1958-2013): priority of discovery and thought styles in the academic literature]. / Transplantes de microbiota fecal para tratamento da colite pseudomembranosa (1958-2013): prioridade de descoberta e estilos de pensamento na literatura acadêmica.

In 1958, Eiseman and contributors published the first scientific paper reporting the use of fecal microbiota transplant for treating pseudomembranous colitis. The relevance of this innovative paper was only acknowledged in 1990. The academic literature on the theme is characterized by a narrative that has undergone successive revisions. We suggest that such revisions were based on claims of priority of scientific discoveries, as described by Merton. The revival of fecal microbiota transplants is interpreted as a process of genesis of a scientific fact, as defined by Fleck: there is a switch from a thought style based on the use of antibiotics to treat infectious diseases to another that accepts the ecological relations between hosts, vectors and parasites.

Em 1958, Eiseman e colaboradores publicaram o primeiro artigo científico relatando o uso de transplante de microbiota fecal para tratar casos graves de colite pseudomembranosa. A relevância desse trabalho inovador só foi reconhecida em 1990. A literatura acadêmica sobre o tema caracteriza-se por sucessivas reconstruções. Sugerimos que tais reconstruções foram orientadas por questões de atribuição de prioridade de descoberta científica nos termos propostos por Merton. A retomada do uso de transplantes de microbiota fecal é interpretada como processo de gênese de um fato científico, conforme Fleck: ocorre a mudança de um estilo de pensamento baseado no uso de antibióticos no tratamento de doenças infecciosas para outro que considera as relações ecológicas entre hospedeiros, vetores e agentes etiológicos de doenças.

Changes in blood biomarkers in Arabian horses with Clostridium difficile-induced enterocolitis.

Clostridium difficile (CD) is considered a major health care problem both in developing and developed countries; frequently reported to be associated with enterocolitis and diarrhea in horses and other animals. In this study, we examined acute phase response (APR), cytokines response, neopterin (NP) procalcitonin (PCT) production and oxidative stress condition in horses and foals with C. difficile-induced enterocolitis (CDIE) and evaluated the effectiveness of these parameters as biomarkers for the disease. A total of 407 Arabian horses in 35 stables were examined between January 2017 to December 2018. Only 24 out of 407 horses showed two or more signs of CDIE. The blood level of serum amyloid A (SAA), haptoglobin (HP), proinflammatory cytokines (TNF-α, IL-6 and IL1-ß), serum malondialdehyde (MDA), PCT and NPT in horses with CDIE were higher than in healthy horses. Nevertheless, the levels of nitric oxide (NO), superoxide dismutase (SOD) and total antioxidant concentration (TAC) were considerably lower in diseased horses compared to those that were healthy. The ROC curves for eleven selected blood parameters, both in healthy horses and horses with CDIE demonstrated that all examined blood markers had significant levels of differentiation between CDIE cases and healthy controls (AUC > 87.5). The data in this study suggest that the evaluation of acute-phase proteins, cytokines, PCT, NPT, and oxidative stress biomarkers may well be used as a tool for diagnosis and assessment of CDIE and in disease pathogenesis in Arabian horses.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Transplantes de microbiota fecal para tratamento da colite pseudomembranosa (1958-2013): prioridade de descoberta e estilos de pensamento na literatura acadêmica / Fecal microbiota transplants in the treatment of pseudomembranous colitis (1958-2013): priority of discovery and thought styles in the academic literature

Resumo Em 1958, Eiseman e colaboradores publicaram o primeiro artigo científico relatando o uso de transplante de microbiota fecal para tratar casos graves de colite pseudomembranosa. A relevância desse trabalho inovador só foi reconhecida em 1990. A literatura acadêmica sobre o tema caracteriza-se por sucessivas reconstruções. Sugerimos que tais reconstruções foram orientadas por questões de atribuição de prioridade de descoberta científica nos termos propostos por Merton. A retomada do uso de transplantes de microbiota fecal é interpretada como processo de gênese de um fato científico, conforme Fleck: ocorre a mudança de um estilo de pensamento baseado no uso de antibióticos no tratamento de doenças infecciosas para outro que considera as relações ecológicas entre hospedeiros, vetores e agentes etiológicos de doenças.

Abstract In 1958, Eiseman and contributors published the first scientific paper reporting the use of fecal microbiota transplant for treating pseudomembranous colitis. The relevance of this innovative paper was only acknowledged in 1990. The academic literature on the theme is characterized by a narrative that has undergone successive revisions. We suggest that such revisions were based on claims of priority of scientific discoveries, as described by Merton. The revival of fecal microbiota transplants is interpreted as a process of genesis of a scientific fact, as defined by Fleck: there is a switch from a thought style based on the use of antibiotics to treat infectious diseases to another that accepts the ecological relations between hosts, vectors and parasites.