Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity

ABSTRACT Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficile in vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficile in vivo. However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro. In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

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Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01401-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01401-20

Author(s):

Hannah C. Harris ◽

Emma L. Best ◽

Charmaine Normington ◽

Nathalie Saint-Lu ◽

Frédérique Sablier-Gallis ◽

...

Keyword(s):

Healthy Subjects ◽

Toxin Production ◽

Antibiotic Exposure ◽

Anaerobic Incubation ◽

Colonization Resistance ◽

Fecal Samples ◽

Content Type ◽

Clostridioides Difficile ◽

Gastrointestinal Pathogens

ABSTRACTA healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

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2′FL and LNnT Exert Antipathogenic Effects against C. difficile ATCC 9689 In Vitro, Coinciding with Increased Levels of Bifidobacteriaceae and/or Secondary Bile Acids

Pathogens ◽

10.3390/pathogens10080927 ◽

2021 ◽

Vol 10 (8) ◽

pp. 927

Author(s):

Louise Kristine Vigsnaes ◽

Jonas Ghyselinck ◽

Pieter Van den Van den Abbeele ◽

Bruce McConnell ◽

Frédéric Moens ◽

...

Keyword(s):

Antimicrobial Effect ◽

Secondary Bile Acid ◽

Clostridioides Difficile ◽

Life Threatening ◽

Intermediate Time ◽

Indigenous Microbiota ◽

Hospital Acquired ◽

Gut Microbial Community ◽

Secondary Bile Acids

Clostridioides difficile (formerly Clostridium difficile) infection (CDI) is one of the most common hospital-acquired infections, which is often triggered by a dysbiosed indigenous gut microbiota (e.g., upon antibiotic therapy). Symptoms can be as severe as life-threatening colitis. The current study assessed the antipathogenic potential of human milk oligosaccharides (HMOs), i.e., 2′-O-fucosyllactose (2′FL), lacto-N-neotetraose (LNnT), and a combination thereof (MIX), against C. difficile ATCC 9689 using in vitro gut models that allowed the evaluation of both direct and, upon microbiota modulation, indirect effects. During a first 48 h fecal batch study, dysbiosis and CDI were induced by dilution of the fecal inoculum. For each of the three donors tested, C. difficile levels strongly decreased (with >4 log CFU/mL) upon treatment with 2′FL, LNnT and MIX versus untreated blanks, coinciding with increased acetate/Bifidobacteriaceae levels. Interindividual differences among donors at an intermediate time point suggested that the antimicrobial effect was microbiota-mediated rather than being a direct effect of the HMOs. During a subsequent 11 week study with the PathogutTM model (specific application of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®)), dysbiosis and CDI were induced by clindamycin (CLI) treatment. Vancomycin (VNC) treatment cured CDI, but the further dysbiosis of the indigenous microbiota likely contributed to CDI recurrence. Upon co-supplementation with VNC, both 2′FL and MIX boosted microbial activity (acetate and to lesser extent propionate/butyrate). Moreover, 2′FL avoided CDI recurrence, potentially because of increased secondary bile acid production. Overall, while not elucidating the exact antipathogenic mechanisms-of-action, the current study highlights the potential of HMOs to combat CDI recurrence, help the gut microbial community recover after antibiotic treatment, and hence counteract the adverse effects of antibiotic therapies.

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Efficacy of a novel lantibiotic, CMB001, against MRSA

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab040 ◽

2021 ◽

Author(s):

Jerzy Karczewski ◽

Christine M Brown ◽

Yukari Maezato ◽

Stephen P Krasucki ◽

Stephen J Streatfield

Keyword(s):

Antibacterial Activity ◽

Pharmacokinetic Analysis ◽

Maximum Tolerable Dose ◽

In Vivo Efficacy ◽

Clostridioides Difficile ◽

Alternative Treatment Option ◽

Significant Damage ◽

Tolerable Dose

Abstract Objectives To evaluate the efficacy of a novel lantibiotic, CMB001, against MRSA biofilms in vitro and in an in vivo experimental model of bacterial infection. Methods Antibacterial activity of CMB001 was measured in vitro after its exposure to whole blood or to platelet-poor plasma. In vitro efficacy of CMB001 against a Staphylococcus aureus biofilm was studied using scanning electron microscopy. The maximum tolerable dose in mice was determined and a preliminary pharmacokinetic analysis for CMB001 was performed in mice. In vivo efficacy was evaluated in a neutropenic mouse thigh model of infection. Results CMB001 maintained its antibacterial activity in the presence of blood or plasma for up to 24 h at 37°C. CMB001 efficiently killed S. aureus within the biofilm by causing significant damage to the bacterial cell wall. The maximum tolerable dose in mice was established to be 10 mg/kg and could be increased to 30 mg/kg in mice pretreated with antihistamines. In neutropenic mice infected with MRSA, treatment with CMB001 reduced the bacterial burden with an efficacy equivalent to that of vancomycin. Conclusions CMB001 offers potential as an alternative treatment option to combat MRSA. It will be of interest to evaluate the in vivo efficacy of CMB001 against infections caused by other pathogens, including Clostridioides difficile and Acinetobacter baumannii, and to expand its pharmacokinetic/pharmacodynamic parameters and safety profile.

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Antimicrobial-resistant Bacteroides fragilis in Thailand and their inhibitory effect in vitro on the growth of Clostridioides difficile

Anaerobe ◽

10.1016/j.anaerobe.2021.102505 ◽

2021 ◽

pp. 102505

Author(s):

Korakrit Imwattana ◽

Pattarachai Kiratisin ◽

Thomas V. Riley

Keyword(s):

Bacteroides Fragilis ◽

Inhibitory Effect ◽

Clostridioides Difficile

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An aniline-substituted bile salt analog protects both mice and hamsters from multiple Clostridioides difficile strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01435-21 ◽

2021 ◽

Author(s):

Jacqueline R. Phan ◽

Dung M. Do ◽

Minh Chau Truong ◽

Connie Ngo ◽

Julian H. Phan ◽

...

Keyword(s):

Bile Salt ◽

Spore Germination ◽

Dependent Manner ◽

Germination Inhibitors ◽

New Methods ◽

Clostridioides Difficile ◽

Antibiotic Associated Diarrhea ◽

Careful Screening ◽

Strain Dependent

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

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Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01431-20 ◽

2021 ◽

Author(s):

Noah Budi ◽

Jared J. Godfrey ◽

Nasia Safdar ◽

Sanjay K. Shukla ◽

Warren E. Rose

Keyword(s):

Limit Of Detection ◽

Disease Relapse ◽

Vegetative Cells ◽

Spore Form ◽

Safety And Efficacy ◽

Healthcare Environment ◽

Clostridioides Difficile ◽

Spore Shedding ◽

Dormant Spore

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

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How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽

10.1128/mbio.01559-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Nicolas Kint ◽

Carolina Alves Feliciano ◽

Maria C. Martins ◽

Claire Morvan ◽

Susana F. Fernandes ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Anaerobic Bacteria ◽

Growth Defect ◽

Strictly Anaerobic ◽

Low Oxygen ◽

Air Exposure ◽

Vegetative Cells ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

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688. In Vitro Activity of Eravacycline, a New Tetracycline Analog, and Comparators Against the Six Most Commonly Isolated Ribotypes of Clostridioides difficile

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.756 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S312-S313 ◽

Cited By ~ 2

Author(s):

Eugenie Basseres ◽

Julie Miranda ◽

Anne J Gonzales-Luna ◽

Travis J Carlson ◽

Tasnuva Rashid ◽

...

Keyword(s):

Geometric Mean ◽

Vitro Activity ◽

In Vitro Activity ◽

Large Collection ◽

In Vitro Susceptibility ◽

Clostridioides Difficile ◽

Clostridioides Difficile Infection ◽

Abdominal Infections ◽

Insight Into

Abstract Background Eravacycline is a novel, tetracycline class antibacterial indicated for the treatment of complicated intra-abdominal infections in adults. In clinical trials, patients given eravacycline had a low likelihood of developing Clostridioides difficile infection (CDI). We hypothesized this was likely due, in part, to the in vitro susceptibility of eravacycline to C. difficile. The purpose of this study was to test the in vitro susceptibility of eravacycline vs. comparators on contemporary clinical isolates representing common ribotypes, including isolates with decreased susceptibility to metronidazole and vancomycin. Methods Two hundred and thirty-four isolates from our biobank were selected from the six most common ribotypes (F001, F002, F014-020, F027, F106, and F255). Minimum inhibitory concentrations (MIC) at 24 hours were measured according to CLSI guidelines for eravacycline, vancomycin, metronidazole and fidaxomicin. MICs results were tabulated and are presented as the geometric mean by ribotype. Results Geometric MIC results are shown in Table 1. Eravacycline was the most potent antimicrobial tested followed by fidaxomicin, metronidazole, and vancomycin. Results were consistent amongst all ribotypes, including isolates with reduced susceptibility to vancomycin and metronidazole. Conclusion Eravacycline displayed potent in vitro activity against a large collection of clinical C. difficile isolates. These data provide insight into why patients given eravacycline had a low likelihood of developing CDI and support further research to better understand the use of eravacycline to prevent or potentially treat patients with CDI. Disclosures All authors: No reported disclosures.

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Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa062 ◽

2020 ◽

Vol 75 (6) ◽

pp. 1458-1465

Author(s):

C H Chilton ◽

G S Crowther ◽

C Miossec ◽

J de Gunzburg ◽

A Andremont ◽

...

Keyword(s):

Limit Of Detection ◽

Toxin Production ◽

Human Gut ◽

Antibiotic Resistant ◽

Delayed Onset ◽

Clostridioides Difficile ◽

Clostridioides Difficile Infection ◽

Clinical Benefits ◽

Bacteroides Fragilis Group

Abstract Background Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. Objectives We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. Methods C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. Results DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). Conclusions DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

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