scholarly journals 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 ◽  
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.

scholarly journals Strain-dependent inhibition of Clostridioides difficile by commensal Clostridia encoding the bile acid inducible (bai) operon

2020 ◽  
Author(s):  
A.D. Reed ◽  
M.A. Nethery ◽  
A. Stewart ◽  
R. Barrangou ◽  
C.M. Theriot
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Culture System ◽  
Dependent Manner ◽  
Secondary Bile Acid ◽  
Clostridioides Difficile ◽  
The Relationship ◽  
Strain Dependent ◽  
Secondary Bile Acids

AbstractClostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that encode the bile acid inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the ability of four commensal Clostridia encoding the bai operon (C. scindens VPI 12708, C. scindens ATCC 35704, C. hiranonis, and C. hylemonae) to convert CA to DCA in vitro, and if the amount of DCA produced was sufficient to inhibit growth of a clinically relevant C. difficile strain. We also investigated the competitive relationship between these commensals and C. difficile using an in vitro co-culture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain-dependent, correlated with the production of ∼2 mM DCA, and increased expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro co-culture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important.ImportanceCommensal Clostridia encoding the bai operon such as C. scindens have been associated with protection against CDI, however the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that encode the bai operon effect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro co-culture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.

Identification of unique bile acid-metabolizing bacteria from the microbiome of centenarians

2020 ◽  
Author(s):  
Kenya Honda ◽  
Yuko Sato ◽  
Koji Atarashi ◽  
Damian Plichta ◽  
Yasumichi Arai ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Lithocholic Acid ◽  
Multidrug Resistant ◽  
Bile Acid Metabolism ◽  
Critical Determinant ◽  
Secondary Bile Acid ◽  
Clostridioides Difficile ◽  
Vancomycin Resistant ◽  
Secondary Bile Acids

Abstract Centenarians, or individuals who have lived more than a century, represent the ultimate model of successful longevity associated with decreased susceptibility to ageing-associated illness and chronic inflammation. The gut microbiota is considered to be a critical determinant of human health and longevity. Here we show that centenarians (average 107 yo) have a distinct gut microbiome enriched in microbes capable of generating unique secondary bile acids, including iso-, 3-oxo-, and isoallo-lithocholic acid (LCA), as compared to elderly (85-89 yo) and young (21-55 yo) controls. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from a centenarian’s faecal microbiota, we identified Parabacteroides merdae and Odoribacteraceae strains as effective producers of isoalloLCA. Furthermore, we generated and tested mutant strains of P. merdae to show that the enzymes 5α-reductase (5AR) and 3β-hydroxysteroid dehydrogenase (3βHSDH) were responsible for isoalloLCA production. This secondary bile acid derivative exerted the most potent antimicrobial effects among the tested bile acid compounds against gram-positive (but not gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and vancomycin-resistant Enterococcus faecium. These findings suggest that specific bile acid metabolism may be involved in reducing the risk of pathobiont infection, thereby potentially contributing to longevity.

scholarly journals Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Casey M. Theriot ◽  
Alison A. Bowman ◽  
Vincent B. Young
Keyword(s):  
Bile Acid ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Bile Acids ◽  
Large Intestine ◽  
Spore Germination ◽  
Secondary Bile Acid ◽  
Content Type ◽  
Secondary Bile Acids

ABSTRACT Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine. It is hypothesized that the depletion of microbial members responsible for converting primary bile acids into secondary bile acids reduces resistance to Clostridium difficile colonization. To date, inhibition of C. difficile growth by secondary bile acids has only been shown in vitro. Using targeted bile acid metabolomics, we sought to define the physiologically relevant concentrations of primary and secondary bile acids present in the murine small and large intestinal tracts and how these impact C. difficile dynamics. We treated mice with a variety of antibiotics to create distinct microbial and metabolic (bile acid) environments and directly tested their ability to support or inhibit C. difficile spore germination and outgrowth ex vivo. Susceptibility to C. difficile in the large intestine was observed only after specific broad-spectrum antibiotic treatment (cefoperazone, clindamycin, and vancomycin) and was accompanied by a significant loss of secondary bile acids (deoxycholate, lithocholate, ursodeoxycholate, hyodeoxycholate, and ω-muricholate). These changes were correlated to the loss of specific microbiota community members, the Lachnospiraceae and Ruminococcaceae families. Additionally, physiological concentrations of secondary bile acids present during C. difficile resistance were able to inhibit spore germination and outgrowth in vitro. Interestingly, we observed that C. difficile spore germination and outgrowth were supported constantly in murine small intestinal content regardless of antibiotic perturbation, suggesting that targeting growth of C. difficile will prove most important for future therapeutics and that antibiotic-related changes are organ specific. Understanding how the gut microbiota regulates bile acids throughout the intestine will aid the development of future therapies for C. difficile infection and other metabolically relevant disorders such as obesity and diabetes. IMPORTANCE Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine.

scholarly journals Strain-Dependent Inhibition of Clostridioides difficile by Commensal Clostridia Carrying the Bile Acid-Inducible (bai) Operon

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
A. D. Reed ◽  
M. A. Nethery ◽  
A. Stewart ◽  
R. Barrangou ◽  
C. M. Theriot
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Dependent Manner ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Coculture System ◽  
The Relationship ◽  
Strain Dependent ◽  
Secondary Bile Acids

ABSTRACT Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain. We also investigated the competitive relationships between these commensals and C. difficile using an in vitro coculture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of ∼2 mM DCA, and increased the expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important. IMPORTANCE Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.

scholarly journals 2567. Effect of Broad vs. Narrow-Spectrum Clostridioides difficile Treatment on Human Stool Bile Acid Composition Over Time

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S891-S891
Author(s):  
Cheleste M Thorpe ◽  
Xi Qian ◽  
Karin Yanagi ◽  
Anne Kane ◽  
Nicholas Alden ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Acid Composition ◽  
Critical Factor ◽  
Acid Synthesis ◽  
Secondary Bile Acid ◽  
Narrow Spectrum ◽  
Commensal Flora ◽  
Clostridioides Difficile ◽  
Secondary Bile Acids

Abstract Background Secondary bile acid production by a diverse commensal flora may be a critical factor in preventing recurrence of Clostridioides difficile infection (CDI). Key enzymes involved are bacterial-encoded bile salt hydrolases (BSHs), felt to be “gatekeepers” to secondary bile acid synthesis. Ridinilazole, a novel narrow-spectrum drug for CDI, demonstrated superior sustained clinical response compared with vancomycin in Phase 2. Longitudinal sampling during this trial allowed for assessment of metabolites differentially present in stools during/after therapy with either broad or narrow-spectrum anti-CDI agent. Previous work characterizing subject’s fecal microbiota in this trial showed that unlike vancomycin, ridinilazole has little effect on commensal flora during and after therapy. We hypothesized that ridinilazole’s microbiota-preserving effect is associated with lack of accumulation of conjugated primary bile acids and/or reaccumulation/persistence of secondary bile acids over the course of CDI treatment, when compared with vancomycin-treated subjects. Furthermore, we hypothesized that we would observe correlations between bile acid profiles and predicted BSH gene abundances. Methods Sequential stool samples were obtained from 44 subjects treated with either ridinilazole or vancomycin (22 in each arm), ranging from time of CDI diagnosis, at end-of-therapy, and up to 40 days after diagnosis. Bile acids were quantitated by liquid chromatography-mass spectrometry. Using the PICRUSt algorithm, metagenomic predictions of BSH gene abundances were performed. Results Stool bile acid compositions differed between ridinilazole-treated and vancomycin-treated subjects at end-of-therapy. In vancomycin-treated subjects, stool composition became dominated by conjugated primary bile acids and decreased levels of secondary bile acids compared with baseline; the ratio of stool conjugated bile acids to secondary bile acids significantly predicted treatment arm. This ratio was also associated with predicted BSH gene abundance in ridinilazole-treated subjects. Conclusion Microbiota-preserving CDI treatment with ridinilazole preserves bile acid composition, which may decrease likelihood of recurrence. Disclosures All authors: No reported disclosures.

scholarly journals 1197. Inhibitory Effect of Ursodeoxycholic Acid on Clostridioides difficile Growth

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S621-S621
Author(s):  
Faris Alnezary ◽  
M Jahangir Alam ◽  
Masaad Almutairi ◽  
Saad Fallatah ◽  
Khurshida Begum ◽  
...  
Keyword(s):  
Ursodeoxycholic Acid ◽  
Galleria Mellonella ◽  
Inhibitory Effect ◽  
Secondary Bile Acid ◽  
In Vivo Experiments ◽  
Clostridioides Difficile ◽  
Colony Forming Units ◽  
Germination And Growth

Abstract Background Ursodeoxycholic acid (UDCA), a secondary bile acid, inhibits germination and growth of Clostridioides difficile in vitro, but the results from in vivo experiments have been conflicting. We evaluated the effects of UDCA on C. difficile in vitro and in a wax moth, Galleria mellonella model. Methods The in vitro growth and germination effects of UDCA on C. difficile were assessed with increased concentration of UDCA (0.001, 0.01, 0.05, and 0.1%). To assess treatment effects of UDCA, C. difficile spores (approximately 1x10^6-8 colony forming units (CFU)) were force fed to G. mellonella larvae treated with UDCA (50 mg/kg/day) 24 hours prior to C. difficile inoculation. Forty G. mellonella larvae were used for each experiment, which was repeated with two distinct strains (R20291 and CD196). Larvae were housed at 37°C and monitored for the next five days for mortality. Results In vitro experiment demonstrated inhibition of C. difficile growth at 0.1% concentration (P < 0.001 vs control). Larvae treated with UDCA had a numerically higher survival rate (60% / 24/40) compared to controls (40% / 16/40) but the results were not statistically significant (p=0.14). Identical rates of survival were observed in the control arms for both strains (40%) and similar in the treatment arms (R20291: 70%; CD 196: 50%). Conclusion Overall, UDCA shows inhibitory effect of growth and germination of C. difficile in vitro. However, in our G. mellonella model, a single dose of UDCA given prior to infection did not prevent CDI. Further dose dependent, and multiday studies investigating the role of UDCA in CDI is needed to better understand this in vitro / in vivo paradox. Disclosures Kevin W. Garey, PharMD, MS, FASHP, Merck & Co. (Grant/Research Support, Scientific Research Study Investigator)

THE ACTIVITY OF ESSENTIAL OILS OBTAINED FROM SPECIES AND INTERSPECIES HYBRIDS OF THE Mentha GENUS AGAINST SELECTED PLANT PATHOGENIC BACTERIA

2018 ◽  
Vol 17 (6) ◽  
pp. 167-174 ◽  
Author(s):  
Małgorzata Schollenberger ◽  
Tomasz M. Staniek ◽  
Elżbieta Paduch-Cichal ◽  
Beata Dasiewicz ◽  
Agnieszka Gadomska-Gajadhur ◽  
...  
Keyword(s):  
Essential Oils ◽  
Pseudomonas Syringae ◽  
Pathogenic Bacteria ◽  
Antimicrobial Effect ◽  
Mentha Piperita ◽  
Mentha Spicata ◽  
Plant Essential Oils ◽  
Plant Pathogenic Bacteria ◽  
Interspecies Hybrids

Plant essential oils of six aromatic herb species and interspecies hybrids of the family Lamiaceae – chocolate mint (Mentha piperita × ‘Chocolate’), pineapple mint (Mentha suaveolens ‘Variegata’), apple mint (Mentha × rotundifolia), spearmint (Mentha spicata), orange mint (Mentha × piperita ‘Granada’) and strawberry mint (Mentha × villosa ‘Strawberry’) – were investigated for antimicrobial effects against plant pathogenic bacteria: Agrobacterium tumefaciens, Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. corylina. The screening was carried out in vitro on agar plates filled with the target organism. All essential oils screened exhibited a higher level of antibacterial activity against A. tumefaciens and X. arboricola pv. corylina than streptomycin used as a standard in all tests. The antimicrobial effect of streptomycin and five mint oils was at the same level for P. syringae pv. syringae. There were no significant differences in the influence of the chocolate mint oil on the growth inhibition of all bacteria tested. Plant essential oils from pineapple mint, apple mint, spearmint and strawberry mint showed the weakest antimicrobial activity against P. syringae pv. syringae and the strongest towards A. tumefaciens and X. arboricola pv. corylina. The essential oils from strawberry mint, pineapple mint, spearmint and apple mint had the strongest effect on A. tumefaciens, and the lowest inhibitory activity was exhibited by the chocolate mint and orange mint essential oils. X. arboricola pv. corylina was the most sensitive to the strawberry mint, pineapple mint and spearmint oils. The chocolate mint oil showed the greatest activity against P. syringae pv. syringae.

scholarly journals Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gautier Follain ◽  
Naël Osmani ◽  
Valentin Gensbittel ◽  
Nandini Asokan ◽  
Annabel Larnicol ◽  
...  
Keyword(s):  
Blood Flow ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Metastatic Progression ◽  
Metastatic Dissemination ◽  
Secondary Tumors ◽  
Flow Profiles ◽  
Endothelial Response ◽  
Life Threatening

AbstractTumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

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