Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation

ABSTRACT

Antibiotic resistance is one of the world’s greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.     

A contemporary review of Clostridioides difficile infections in patients with haematologic diseases

Clostridioides (Clostridium) difficile (C. difficile) infection is one of the most common causes of increased morbidity and mortality. Approximately 500 000 C. difficile infections (CDIs) occur each year in the United States, and they result in more than 29 000 deaths. Patients with haematologic diseases are at a higher risk for this infection due to frequent hospitalization and exposure to treatment-associated risk factors. Whilst several currently available antimicrobial agents offer resolution, recurrence of infection remains a major concern. Recent advancement in deciphering C. difficile virulence mechanisms and identification of its allies in contributing to the infection has led to the development of alternative treatment strategies. Here, we will provide a contemporary discussion of how major risk factors in haematologic diseases, such as immunosuppression, chemoradiation, use of antibiotic, proton pump inhibitor and opioid, and deficiency in butyrate and antimicrobial peptides contribute to C. difficile infection. Next, we will highlight different approaches to control and mitigate this infection such as antibiotic stewardship and faecal microbiota transplantation. Finally, we will explore several emerging treatments such as use of pre- and probiotics, immunotherapy and microbiome-sparing agents.     

Challenges and opportunities in the management of Clostridium difficile infection

Clostridium difficile infection (CDI) is increasing in all regions of the world where sought. There is no gold standard for diagnosis of CDI, with available tests having limitations. Prevention of CDI will be seen with antibiotic stewardship, improved disinfection of hospitals and nursing homes, chemo- and immuno-prophylaxis and next generation probiotics. The important therapeutic agents are oral vancomycin and fidaxomicin with metronidazole being used only in mild cases or when oral therapy cannot be given. Current therapy of CDI for 10 days is associated with high rate of recurrence that may be prevented by prolonging initial therapy. Future treatment strategies will focus on drugs that inhibit C. difficile, reduce toxin activity and inflammation in the gut, and improve colonic flora diversity.     

老年难辨梭菌性结肠炎患者八例临床分析

目的　探讨老年患者使用广谱抗生素诱发难辨梭菌性结肠炎的临床特点、内镜下改变及防治措施。　方法　分析８例老年难辨梭菌性结肠炎患者的临床资料,对难辨梭菌分离、鉴定,并行细胞毒素测定。　结果　所用抗生素的种类繁多,在使用抗生素后３ 天至２周内发生腹泻,重者伴腹膜炎,４ 例行纤维结肠镜检查均见有小片至大斑片伪膜附着;治疗措施为停用原抗菌药物,应用万古霉素和（或）甲硝唑及加强支持疗法。８ 例中７例治愈,１例死亡。　结论　老年人特别是术后使用广谱抗生素者易诱发难辨梭菌性结肠炎     

Freeze-dried fecal samples are biologically active after long-lasting storage and suited to fecal microbiota transplantation in a preclinical murine model of Clostridioides difficile infection

ABSTRACT

Fecal microbiota transplantation is now recommended for treating recurrent forms of Clostridioides difficile infection. Recent studies have reported protocols using capsules of either frozen or freeze-dried stool allowing oral administration in in- and out-patient settings. However, a central question remains the viability, engraftment, and efficacy of the microbiome over time during storage life. This study shows that both the freeze-drying and freezing procedures for fecal samples allowed preserving viability, short-chain fatty acids concentration, and anti-Clostridioides difficile properties of microbiota without significant alteration after storage for 12 months. Fecal transplantation with freeze-dried microbiota allowed engraftment of microbiota leading to clearance of Clostridioides difficile infection in a preclinical murine model with a survival rate of 70% versus 53-60% in mice treated with frozen inocula, and 20% in the untreated group. Moreover, the freeze-dried powder can be used to fill oral hard capsules using a very low amount (0.5%) of glidant excipient, allowing oral formulation. Altogether, this study showed that freeze-dried inocula can be used for the treatment of Clostridioides difficile infection with long-lasting stability of the fecal microbiota. This formulation facilitates biobanking and allows the use of hard capsules, an essential step to simplify patient access to treatment.     

Factors Related to Outcomes of Fecal Microbiota Transplantation in Patients with Clostridioides difficile Infection

Background/AimsThe aim of this study was to evaluate factors related to outcomes of fecal microbiota transplantation (FMT) in patients with Clostridioides difficile infection (CDI) and viability of frozen stock for FMT.MethodsClinical data of patients who had received FMT for CDI were prospectively collected. Next-generation 16S rRNA gene sequencing of bacteria was performed from donors’ and recipients’ stool. Colony-forming units (CFUs) of cultures from frozen stock solutions for FMT were measured at 0, 4, 8, 12, 24, 48 weeks after preparation of the solutions.ResultsIn total, 25 FMT procedures were performed in 20 cases (14 fresh and 11 frozen FMT). Forty-five percent of cases involved fulminant CDI. The overall success rate was 55% after the 1st FMT and 75% after the 2nd FMT. The success rate was significantly higher in partially treated CDI than in refractory CDI (100% vs 71.4%; p=0.001). In successful cases only, the decrease in alpha-diversity in the recipient stool microbiomes was recovered after FMT to a level similar to that in donor stools. There was a significant difference in the microbiome composition in pre-FMT recipients’ stool between successful and failed cases (p=0.001). The CFUs of frozen solution for FMT did not decrease for 48 weeks in both aerobic and anaerobic cultures.ConclusionsFMT is highly effective in partially treated CDI but not in refractory CDI. The microbiome differs between failed and successful cases. Frozen stock for FMT is viable up to 48 weeks.     

Bile salt metabolism is not the only factor contributing to Clostridioides (Clostridium) difficile disease severity in the murine model of disease

ABSTRACT

Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.     

Lactobacillus gasseri OLL2716 as a probiotic in clarithromycin-resistant Helicobacter pylori infection

BACKGROUNDS AND AIM: Clarithromycin (CAM)-resistant Helicobacter pylori sometimes offers serious problems with eradication by antibiotics. The aim of this study was to determine whether a probiotic can be an alternative therapy in CAM-resistant Hp infection. METHODS: The effects of Lactobacillus gasseri (strain OLL2716) on the growth of CAM-susceptible and CAM-resistant H. pylori and interleukin (IL)-8 production provoked by these strains were examined by in vitro experiments. Moreover, mice were infected with these CAM-susceptible or CAM-resistant H. pylori, and were treated with CAM or L. gasseri. RESULTS: In vitro experiments demonstrated that L. gasseri inhibited the growth of H. pylori and suppressed H. pylori-associated IL-8 production. Such effects were noted in CAM-resistant and CAM-susceptible H. pylori. Similarly, in an in vivo model of H. pylori infection, H. pylori colonization was significantly decreased by L. gasseri. CONCLUSION: Therefore, L. gasseri was found to act as a probiotic in CAM-resistant H. pylori infection.     

Lactobacillus reuteri reduces bone loss in older women with low bone mineral density: a randomized,placebo‐controlled,double‐blind,clinical trial

Background

The importance of the gut microbiome for bone metabolism in mice has recently been demonstrated, but no studies are available in humans. Lactobacillus reuteri ATCCPTA 6475 (L. reuteri 6475) has been reported to increase bone mineral density (BMD ) in mice but its effect on the human skeleton is unknown. The objective of this trial was to investigate if L. reuteri 6475 affects bone loss in older women with low BMD .

Methods

In this double‐blind, placebo‐controlled study, women from the population who were 75 to 80 years old and had low BMD were randomized to orally receive 10¹⁰ colony‐forming units of L. reuteri 6475 daily or placebo. The predefined primary end‐point was relative change after 12 months in tibia total volumetric BMD (vBMD ).

Results

Ninety women were included and 70 completed the study. L. reuteri 6475 reduced loss of total vBMD compared to placebo both in the intention‐to‐treat (ITT ) analysis [?0.83% (95% confidence interval [CI ], ?1.47 to ?0.19%) vs. ?1.85% (95% CI , ?2.64 to ?1.07%); mean difference 1.02% (95% CI , 0.02–2.03)] and per protocol analysis [?0.93% (95% CI , ?1.45 to ?0.40) vs. ?1.86% (95% CI , ?2.35 to ?1.36); mean difference 0.93% (95% CI , 0.21–1.65)]. In general, similar but smaller effects were observed in the secondary bone variable outcomes, but these differences did not reach statistical significance in the ITT population. Adverse events did not differ between groups.

Conclusions

Supplementation with L. reuteri 6475 should be further explored as a novel approach to prevent age‐associated bone loss and osteoporosis.

     

A novel probiotic,Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period

ABSTRACT

Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456 maintained higher viability at lower pH conditions compared to other tested strains. LBJ 456 also altered pathogen adhesion to LS 174T monolayers and demonstrated contact-dependent and independent inhibition of pathogen growth. Genome analyses further revealed possible genetic elements involved in host attachment and pathogen inhibition. Importantly, we show that ingestion of Lactobacillus johnsonii 456 over a one week yogurt course leads to persistent viable bacteria detectable even beyond the period of initial ingestion, unlike many other previously described probiotic species of lactic acid bacteria.     

Clostridioides difficile infection in liver cirrhosis patients: A population-based study in United States

BACKGROUNDClostridioides (formerly Clostridium) difficile infection (CDI) is an increasingly frequent cause of morbidity and mortality in hospitalized patients. Multiple risk factors are documented in the literature that includes, but are not limited to, antibiotics use, advanced age, and gastric acid suppression. Several epidemiological studies have reported an increased incidence of CDI in advanced liver disease patients. Some have also demonstrated a higher prevalence of nosocomial infections in cirrhotic patients. AIMTo use a large nationwide database, we sought to determine CDI’s risk among liver cirrhosis patients in the United States.METHODSWe queried a commercial database (Explorys Inc^TM, Cleveland, OH, United States), and obtained an aggregate of electronic health record data from 26 major integrated United States healthcare systems comprising 360 hospitals in the United States from 2018 to 2021. Diagnoses were organized into the Systematized Nomenclature of Medicine Clinical Terms (SNOMED–CT) hierarchy. Statistical analysis for the multivariable model was performed using Statistical Package for Social Sciences (SPSS version 25, IBM Corp^TM). For all analyses, a two-sided P value of < 0.05 was considered statistically significant.RESULTSThere were a total of 19387760 patients in the database who were above 20 years of age between the years 2018-2021. Of those, 133400 were diagnosed with liver cirrhosis. The prevalence of CDI amongst the liver cirrhosis population was 134.93 per 100.000 vs 19.06 per 100.000 in non-cirrhotic patients (P < 0.0001). The multivariate analysis model uncovered that cirrhotic patients were more likely to develop CDI (OR: 1.857; 95%CI: 1.665-2.113, P < 0.0001) compared to those without any prior history of liver cirrhosis. CONCLUSIONIn this large database study, we uncovered that cirrhotic patients have a significantly higher CDI prevalence than those without cirrhosis. Liver cirrhosis may be an independent risk factor for CDI. Further prospective studies are needed to clarify this possible risk association that may lead to the implementation of screening methods in this high-risk population.     

Sentinel surveillance and epidemiology of Clostridioides difficile in Denmark, 2016 to 2019

BackgroundSince 2008, Danish national surveillance of Clostridioides difficile has focused on binary toxin-positive strains in order to monitor epidemic types such as PCR ribotype (RT) 027 and 078. Additional surveillance is needed to provide a more unbiased representation of all strains from the clinical reservoir.AimSetting up a new sentinel surveillance scheme for an improved understanding of type distribution relative to time, geography and epidemiology, here presenting data from 2016 to 2019.MethodsFor 2─4 weeks in spring and autumn each year between 2016 and 2019, all 10 Danish Departments of Clinical Microbiology collected faecal samples containing toxigenic C. difficile. Isolates were typed at the national reference laboratory at Statens Serum Institut. The typing method in 2016–17 used tandem-repeat-sequence typing, while the typing method in 2018–19 was whole genome sequencing.ResultsDuring the study period, the sentinel surveillance scheme included ca 14–15% of all Danish cases of C. difficile infections. Binary toxin-negative strains accounted for 75% and 16 of the 20 most prevalent types. The most common sequence types (ST) were ST2/13 (RT014/020) (19.5%), ST1 (RT027) (10.8%), ST11 (RT078) (6.7%), ST8 (RT002) (6.6%) and ST6 (RT005/117) (5.1%). The data also highlighted geographical differences, mostly related to ST1 and temporal decline of ST1 (p = 0.0008) and the increase of ST103 (p = 0.002), ST17 (p = 0.004) and ST37 (p = 0.003), the latter three binary toxin-negative.ConclusionSentinel surveillance allowed nationwide monitoring of geographical differences and temporal changes in C. difficile infections in Denmark, including emerging types, regardless of binary toxin status.     

Immunological mechanisms of fecal microbiota transplantation in recurrent Clostridioides difficile infection

Fecal microbiota transplantation (FMT) is a successful method for treating recurrent Clostridioides difficile (C. difficile) infection (rCDI) with around 90% efficacy. Due to the relative simplicity of this approach, it is being widely used and currently, thousands of patients have been treated with FMT worldwide. Nonetheless, the mechanisms underlying its effects are just beginning to be understood. Data indicate that FMT effectiveness is due to a combination of microbiological direct mechanisms against C. difficile, but also through indirect mechanisms including the production of microbiota-derived metabolites as secondary bile acids and short chain fatty acids. Moreover, the modulation of the strong inflammatory response triggered by C. difficile after FMT seems to rely on a pivotal role of regulatory T cells, which would be responsible for the reduction of several cells and soluble inflammatory mediators, ensuing normalization of the intestinal mucosal immune system. In this minireview, we analyze recent advances in these immunological aspects associated with the efficacy of FMT.     

Overview of current detection methods and microRNA potential in Clostridioides difficile infection screening

Clostridioides difficile (formerly called Clostridium difficile,C.difficile) infection (CDI)is listed as an urgent threat on the 2019 antibiotic resistance threats report in the United States by the Centers for Disease Control and Prevention.Early detection and appropriate disease management appear to be essential.Meanwhile,although the majority of cases are hospital-acquired CDI,community-acquired CDI cases are also on the rise,and this vulnerability is not limited to immunocompromised patients...     

The outcomes of Clostridioides difficile infection in patients with diverticular disease: a nationwide analysis

Abstract

Background: Clostridioides difficile infection (CDI) is one of the most common healthcare-associated infections. It contributes to significant morbidity and mortality among hospitalized patients in the United States. Prior studies suggest worse outcomes of CDI in patients with diverticulitis and increased risk for recurrent CDI. We conducted this study to evaluate the outcomes of CDI in patients with diverticular disease from a nationwide data sample (2012–2015).

Methods: The National Inpatient Sample (NIS) database between January 2012 and September 2015 was queried for CDI admissions using the International Classification of Diseases, Ninth Edition, Clinical Modification [ICD-9-CM] codes 008.45, 562.11, 562.10, 562.12, and 562.13 for diagnoses of CDI and diverticular disease.

Results: The study included 1,327,595 patients who were admitted between 2012 and 2105 for CDI. Out of all of the patients, 84,170 (6.34%) had a concurrent diagnosis of diverticular disease. After adjusting for confounding variables, the in-hospital mortality was lower [odd ratio (OR): 0.48, 95% CI: 0.44–0.52, p?<?.001] for patients with diverticular disease. The length of stay (LOS) was longer [10.5 versus 9.3?days, p?<?.001] and mean cost of hospitalization was significantly higher in patients without a history of diverticular disease.

Discussion: In a nationwide population study, admissions with CDI, patients with a concurrent diagnosis of diverticular disease had lower in-hospital mortality. The observed results are different from prior studies and might be attributed to a higher burden of normal flora in those patients and increased use of antibiotic stewardship program across many hospitals nationwide.     

Clostridium difficile infection in patients with inflammatory bowel disease: a case control study

Objective: Characterization of predisposing factors for Clostridium difficile infection recurrence (rCDI) and outcome in inflammatory bowel disease (IBD) patients.

Methods: Clinical characteristics of 167 inflammatory bowel disease patients with Clostridium difficile infection (IBD-CDI cohort) treated in Helsinki University Central Hospital were gathered. Medical history of the last three months preceding a toxin positive CDI test was recorded. Parameters, including ribotype of C. difficile, mortality and recurrence were compared with age and gender-matched C. difficile patients (CDI cohort).

Results: No difference was found in rCDI between IBD-CDI and CDI cohorts. As compared with IBD subtypes, rCDI was least common among patients with Crohn’s disease. The use of immunosuppressant therapy was higher in IBD patients with two or more CDI episodes. C. difficile ribotype 027 increased the rates for rCDI in IBD patients but not in non-IBD-CDI patients. The prevalence of 027 ribotype and mortality rates did not differ significantly among the cohorts. None of the IBD patients underwent colectomy upon CDI.

Conclusion: IBD patients are not more susceptible for rCDI than non-IBD patients. Predisposing factors for rCDI among IBD patients are associated with immunosuppressant treatments, colon affecting IBD and CDI caused by ribotype 027. CDI does not worsen the prognosis of IBD patients.