Effective Microorganisms (EM) Improve Internal Organ Morphology, Intestinal Morphometry and Serum Biochemical Activity in Japanese Quails under Clostridium perfringens Challenge.

The effect of effective microorganisms (EM) on internal organ morphology, intestinal morphometry, and serum biochemical activity in Japanese quails under Clostridium perfringens challenge was determined. After 30 days of EM addition, one group of quails was orally inoculated with Clostridium perfringens. The second group did not receive EM and was inoculated with C. perfringens. In the gut, EM supplementation reduced the number of lesions, enhanced gut health, and protected the mucosa from pathogenic bacteria. EM showed an anti-inflammatory effect and fewer necrotic lesions in villi. In the internal organs, EM showed a protective effect against a typical lesion of C. perfringens infection. Necrosis and degeneration of the hepatocytes, necrosis of bile ducts, and bile duct proliferation were more severe in the infected group without EM. Morphometric evaluation showed significantly higher villi in the jejunum after EM addition. A greater crypt depth was observed in the C. perfringens group. Biochemical analysis of the blood indicated lower cholesterol on the 12th day of the experiment and between-group differences in total protein, lactate dehydrogenase (LDH), and albumin levels in the EM group. Further studies are needed to improve EM activity against pathologic bacteria as a potential alternative to antibiotics and to develop future natural production systems.

Risk factors and intestinal microbiota: Clostridioides difficile infection in patients receiving enteral nutrition at Intensive Care Units.

BACKGROUND: Clostridioides difficile infection (CDI) is a leading cause of nosocomial diarrhea. Patients receiving enteral nutrition (EN) in the intensive care unit (ICU) are potentially at high risk of CDI. In the present study, we assessed the risk factors and intestinal microbiome of patients to better understand the occurrence and development of CDI. METHODS: Patients were screened for C. difficile every week after starting EN, and their clinical records were collected for risk factor identification. Fecal samples were analyzed using 16S rRNA sequencing to evaluate the intestinal microbiota. RESULTS: Overall incidence of CDI was 10.7% (18/168 patients). History of cerebral infarction was significantly associated with CDI occurrence (OR, 9.759; 95% CI, 2.140-44.498), and treatment with metronidazole was identified to be protective (OR, 0.287; 95% CI, 0.091-0.902). Patients with EN had lower bacterial richness and diversity, accompanied by a remarkable decrease in the abundance of Bacteroides, Prevotella_9, Ruminococcaceae, and Lachnospiraceae. Of these patients, acquisition of C. difficile resulted in a transient increase in microbial diversity, along with consistent alterations in the proportion of some bacterial taxa, especially Ruminococcaceae and Lachnospiraceae. Upon initiation of EN, patients who were positive for C. difficile later showed an enhanced load of Bacteroides, which was negatively correlated with the abundance of C. difficile when CDI developed. CONCLUSION: ICU patients receiving EN have a high prevalence of CDI and a fragile intestinal microbial environment. History of cerebral infarction and prior treatment with metronidazole are considered as vital risk and protective factors, respectively. We propose that the emergence of CDI could cause a protective alteration of the intestinal microbiota. Additionally, Bacteroides loads seem to be closely related to the occurrence and development of CDI.

Bacillus subtilis PB6 based probiotic supplementation plays a role in the recovery after the necrotic enteritis challenge.

In poultry production, birds are raised under intensive conditions, which can enable rapid spread of infections, with Clostridium perfringens-caused necrotic enteritis (NE) being one of the most devastating for the industry. The current investigation was conducted to evaluate the effectiveness of Bacillus subtilis PB6 probiotic supplementation on bird's post NE recovery, based on chicken performance, cecal microbiota composition, ileum histomorphometric measurements, and short-chain fatty acid production in the cecum of the birds that were challenged with NE mid-production. Birds were split into four groups, including a negative control, positive control challenged with C. perfringens, group supplemented with B. subtilis probiotic, and NE challenged birds supplemented with B. subtilis probiotic. Following NE challenge birds were allowed to reach the end of production time at 40 days, and samples were collected to estimate if probiotic supplementation resulted in better post-NE recovery. Intestinal lesion score across the duodenum, jejunum, and ileum indicated that at the end of production timeline NE challenged birds supplemented with B. subtilis probiotic had lower intestinal lesion scores compared to NE challenged birds without probiotic supplementation implying improved recovery. Probiotic supplementation improved performance of NE challenged birds only in the post-NE recovery stage. NE challenged birds had a significant increase in cecal propionic acid, which was not observed in NE challenged birds supplemented with B.subtilus. Both B. subtilis supplemented groups (challenged and unchanged) were characterized by a significant rise in cecal acetic and butyric acid. Our results demonstrate that B. subtilis supplementation can assist the birds in dealing with NE outbreak and long term recovery.

Characteristics of Clostridium difficile isolates and the burden of hospital-acquired Clostridium difficile infection in a tertiary teaching hospital in Chongqing, Southwest China.

BACKGROUND: Clostridium difficile infection (CDI), especially hospital-acquired Clostridium difficile infection (HA-CDI), continues to be a public health problem and has aroused great concern worldwide for years. This study aimed to elucidate the clinical and epidemiological features of HA-CDI and the characteristics of C.difficile isolates in Chongqing, Southwest China. METHODS: A case-control study was performed to identify the clinical incidence and risk factors of HA-CDI. C. difficile isolates were characterised by polymerase chain reaction (PCR) ribotyping, multilocus sequence typing (MLST), toxin gene detection and antimicrobial susceptibility testing. RESULTS: Of the 175 suspicious patients, a total of 122 patients with antibiotic-associated diarrhea (AAD) were included in the study; among them, 38 had HA-CDI. The incidence of AAD and HA-CDI was 0.58 and 0.18 per 1000 patient admissions, respectively. Chronic renal disease and cephalosporin use were independent risk factors for HA-CDI. Fifty-five strains were assigned into 16 sequence types (STs) and 15 ribotypes (RTs). ST2/RT449 (8, 14.5%) was the predominant genotype. Of the 38 toxigenic isolates, A + B + CDT- isolates accounted for most (34, 89.5%) and 1 A + B + CDT+ isolate emerged. No isolate was resistant to vancomycin, metronidazole or tigecycline, with A-B-CDT- being more resistant than A + B + CDT-. CONCLUSIONS: Different genotypes of C. difficile strains were witnessed in Chongqing, which hinted at the necessary surveillance of HA-CDI. Adequate awareness of patients at high risk of HA-CDI acquisition is advocated and cautious adoption of cephalosporins should be highlighted.

Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model.

Clostridium difficile is an opportunistic diarrhoeal pathogen, and C. difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone 1 . Several enteric pathogens, including C. difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut 2 . Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C. difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C. difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C. difficile fitness despite increased C. difficile toxin expression in the gut. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI.

Probiotics for prevention of Clostridium difficile infection.

PURPOSE OF REVIEW: Probiotics may prevent Clostridium difficile infection (CDI), a leading healthcare-associated infection in the United States. However, prior studies were limited by heterogeneity in products and patient populations. Recent clinical evidence and new approaches to probiotic development are reviewed. RECENT FINDINGS: Probiotic use may reduce incident CDI in high-risk populations by as much as 50%, though prior clinical trials have yielded conflicting results. Combining probiotics with prebiotics improves growth and engraftment in the host. Bacillus clausii and Lactobacillus reuteri secrete compounds that directly inhibit C. difficile. Organisms that produce secondary bile acids, such as Clostridium scindens, enhance C. difficile colonization resistance. Nontoxigenic C. difficile, which provides nutritional niche competition, may prevent CDI. Refinements to fecal microbiota transplantation (FMT) blur the line between probiotics and FMT. These include a quality-controlled stool product (RBX2660), purified Firmicutes spores (SER-109) and sterile fecal filtrate. Bacteriophages may treat CDI but have unknown safety and efficacy in humans. SUMMARY: There have been a number of advances in probiotics and our understanding of their role in prevention of CDI, but a number of important safety and efficacy questions remain. An improved understanding of the native microbiota structure and function will allow for continued development of rationally designed probiotic therapy to provide enhanced protection against CDI.

Revisión del papel de los probióticos en la patología gastrointestinal del adulto / Review of the role of probiotics in gastrointestinal diseases in adults

Antecedentes: Los probióticos pueden actuar como agentes biológicos que modifican la microbiota intestinal y ciertos perfiles de citoquinas, lo que puede conllevar una mejoría en ciertos procesos gastrointestinales. Objetivos: Realizar una revisión basada en la evidencia del papel de los probióticos en determinadas patologías gastrointestinales del adulto. Métodos de búsqueda: Revisión realizada utilizando los descriptores, filtros y límites adecuados en la base de datos PubMed (MEDLINE). Criterios de selección: Se han empleado los términos MeSH Probiotics [en el título] AND Gastrointestinal Diseases, con los siguientes límites o filtros: tipos de estudios: Systematic Reviews, Meta-Analysis, Guideline, Practice Guideline, Consensus Development Conference (y Consensus Development Conference NIH), Randomized Controlled Trial, Controlled Clinical Trial y Clinical Trial; edad: adultos (19 o más a˜nos); idioma: en inglés y español; en humanos, y que dispusieran, al menos, de un abstract. Recogida y análisis de datos: Se recuperaron los textos completos de todas las revisiones sistemáticas y metaanálisis directamente relacionados con el objetivo de la revisión, así como los ensayos clínicos aleatorizados de los estudios que se consideraron relevantes y de calidad para realizar esta revisión. Resultados principales: Determinados probióticos, diferentes para cada proceso, se han demostrado eficaces y beneficiosos en caso de diarrea aguda infecciosa, diarrea asociada a antibióticos, diarrea asociada a Clostridium difficile, pouchitis y en la erradicación de la infección por Helicobacter pylori. Conclusiones de los autores: Hay ciertas patologías gastrointestinales en las que se puede recomendar el uso de los probióticos, verdaderos agentes biológicos, y otras en las que no se ha demostrado beneficio (AU)

Background: Probiotics may act as biological agents that modify the intestinal microbiota and certain cytokine profiles, which can lead to an improvement in certain gastrointestinal diseases. Objectives: To conduct a review of the evidence of the role of probiotics in certain gastrointestinal diseases in adults. Search methods: Review conducted using appropriate descriptors, filters and limits in the PubMed database (MEDLINE). Selection criteria: The MeSH terms used were Probiotics [in the title] AND Gastrointestinal Diseases, with the following limits or filters: Types of study: Systematic Reviews, Meta-Analysis, Guideline, Practice Guideline, Consensus Development Conference (and Consensus Development Conference NIH), Randomized Controlled Trial, Controlled Clinical Trial and Clinical Trial; age: adults (19 or older); language: English and Spanish; in humans, and with at least one abstract. Data collection and analysis: Full texts of all the Systematic Reviews and meta-analyses directly related to the review’s objective were obtained, as well as the Randomised Controlled Trials of the studies that were considered relevant and of sufficient quality for this review. Main results: Certain probiotics, different for each process, have proven to be effective and beneficial in cases of acute infectious diarrhoea, antibiotic-associated diarrhoea, Clostridium difficile-associated diarrhoea, pouchitis and Helicobacter pylori infection eradication. Authors’ conclusions: Although some probiotics have not demonstrated any benefit, there are certain gastrointestinal diseases in which the use of probiotics, true biological agents, can be recommended (AU)

Amelioration of Clostridium difficile Infection in Mice by Dietary Supplementation With Indole-3-carbinol.

OBJECTIVE: To determine the therapeutic effects of dietary supplementation on Clostridium difficile infection (CDI). BACKGROUND: With limited treatment options, the rise of C. difficile-associated disease has spurred on the search for novel therapies. Recent data define a role for the aryl hydrocarbon receptor (AHR) and diet-derived AHR ligands in mucosal immunity. We investigated the efficacy of indole-3-carbinol (I3C), a dietary supplement, and AHR precursor ligand in a murine model of CDI. METHODS: C57BL/6 (B6), AHR, and AHR mice were placed on either grain-based or semipurified diets with or without I3C before and during CDI. Mice were followed clinically for a minimum of 6 days or euthanized between days 0 and 4 of inoculation for analysis of the inflammatory response and microbiota. RESULTS: B6 mice fed an AHR ligand-deficient, semipurified diet have significantly increased disease severity (P<0.001) and mortality (P < 0.001) compared with mice fed on diet containing I3C. The addition of I3C to the diet of AHR null mice had less of an impact than in AHR heterozygous littermates, although some protection was seen. Mice on semipurified I3C-diet had increased cecal Tregs, ILC3s, and γδ T cells and an increased neutrophilic response without increased inflammation or bacterial translocation compared with controls. CONCLUSIONS: I3C is a powerful treatment to reduce impact of CDI in mice. The findings indicate I3C may be acting through both AHR-dependent and -independent mechanisms in this model. Dietary supplementation with I3C is a potential new therapy for prevention and amelioration of C. difficile disease.

Heat Production and Energy Efficiency of Broilers Infected With Necrotic Enteritis.

Necrotic enteritis (NE) in poultry is the most important bacterial disease in terms of economic losses. The present study was conducted to evaluate the effect of an experimental challenge with necrotic enteritis on respiration and heat production in birds pretreated with dietary acylated starch or antibiotics (AB) zinc bacitracin (50 mg/kg) plus salinomycin (60 mg/kg). In total, 48 1-day-old Ross 308 male broilers were assigned to floor pens until day 10. On day 11, birds were randomly placed into 16 calorimetric chambers with four replicates of three birds per treatment. Treatments were: control, AB, acetylated high-amylose maize starch (SA), or butyrylated high-amylose maize starch (SB). Birds were NE challenged by inoculation with 5000 sporulated oocysts each of Eimeria maxima and Eimeria acervulina and 2500 sporulated oocysts of Eimeria brunetti on day 9 and Clostridium perfringens (3.8 × 10(8) colony-forming units) on day 14. The results showed that heat production (HP), respiratory quotient (RQ), heat increment, weight gain (WG), feed intake (FI), and livability (LV) of birds fed control, SA, and SB diets were lower than birds fed AB at 19 and 42 hr postinoculation (P < 0.05). At 65 hr postchallenge, increased FI and WG of birds were observed, indicating recovery from NE. During the entire period, from day 14 to day 17, birds fed control, SA, and SB had lower WG, FI, HP, RQ, metabolizable energy intake (MEI), and metabolizable energy (P < 0.01) than those fed AB. The data demonstrate that Eimeria sp. and C. perfringens challenge reduces growth performance, HP, RQ, metabolizable energy, and MEI of birds fed control, SA, and SB but not AB diets.

Dietary acylated starch improves performance and gut health in necrotic enteritis challenged broilers.

Resistant starch has been reported to act as a protective agent against pathogenic organisms in the gut and to encourage the proliferation of beneficial organisms. This study examined the efficacy of acetylated high amylose maize starch (SA) and butyralated high-amylose maize starch (SB) in reducing the severity of necrotic enteritis (NE) in broilers under experimental challenge. A total of 720 one-day-old male Ross 308 chicks were assigned to 48 floor pens with a 2 × 4 factorial arrangement of treatments. Factors were a) challenge: no or yes; and b) feed additive: control, antibiotics (AB), SA, or SB. Birds were challenged with Eimeria and C. perfringens according to a previously reported protocol. On d 24 and 35, challenged birds had lower (P < 0.001) livability (LV), weight gain (WG), and feed intake (FI) compared to unchallenged birds. Challenged birds fed SA and SB had higher FI and WG at d 24 and 35 (P < 0.05) compared to birds fed the control diet, while being significantly lower than those fed AB. Unchallenged birds fed SA or SB had higher FI at d 24 and 35 compared to those fed the control diet (P < 0.05). Birds fed SB had increased (P < 0.001) jejunal villus height/crypt depth (VH:CD) ratios at d 15, increased ileal (P < 0.001) and caecal (P < 0.001) butyrate levels at d 15 and 24, and decreased (P < 0.01) caecal pH at d 15. Birds fed SA had increased (P < 0.001) ileal acetate content at d 24 and decreased (P < 0.01) caecal pH at d 15. These results demonstrated that dietary acylated starch improved WG in birds challenged with necrotic enteritis. Depending on the acid used, starch acylation also offers a degree of specificity in short chain fatty acid (SCFA) delivery to the lower intestinal tract which improves gut health.

Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis.

Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota.

Emergence in gnotobiotic mice of nontoxinogenic clones of Clostridium difficile from a toxinogenic one.

In previous studies, we showed that diet composition or Saccharomyces boulardii ingestion could protect gnotobiotic mice against lethal Clostridium difficile infection. Using an original method, we detected nontoxinogenic clones from feces of protected mice challenged with a toxinogenic clone of C. difficile. These clones became established at the same level as the toxinogenic one after about 30 days. In these protected mice bearing nontoxinogenic clones, no enterotoxin production could be detected and cytotoxin titers were highly reduced. These nontoxinogenic clones were genetically stable because nontoxinogenic clones and clones that produce intermediate levels of toxins in vivo did not revert to toxin production, even after repeated culture in vitro. Furthermore, the nontoxinogenic clones were shown to arise from a single toxinogenic clone and were identical to that clone in metabolic patterns and antibiotic sensitivity tests. When mice fed a nonprotective diet were challenged with a nontoxinogenic or intermediate clone, they remained healthy and no toxin production could be detected in their feces. Moreover, these mice were protected against further infections with toxinogenic strains of C. difficile, and a strong antagonism between nontoxinogenic and toxinogenic clones was observed.

Relapsing Clostridium difficile enterocolitis cured by rectal infusion of normal faeces.

Repeated recurrence of Clostridium difficile-associated enterocolitis is uncommon but troublesome for the afflicted patient. The patient described here received vancomycin treatment several times but always had a relapse of C. difficile enterocolitis 2-3 weeks after discontinuation of treatment. She did not form serum antibodies to C. difficile cytotoxin (toxin B). Rectal infusion of enemas prepared from fresh faeces resulted in final cure.