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

Abstract Background Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea (C. difficile-associated diarrhea, CDAD) which is life-threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 (Bio-K+) has demonstrated benefits in preventing CDI and has a strong inhibitory effect on the growth of several nosocomial C. difficile strains in vitro. Many Lactobacilli can inhibit CD growth though lactic acidification. Here, we have investigated novel acid-independent mechanisms by which these strains impair C. difficile virulence. Methods The hypervirulent strain C. difficile R20291 was co-cultured anaerobically with Bio-K+ probiotic strains in various media and glucose concentrations (5 g/L, 3 g/L, 0 g/L), for 24 hours at 37°C. Parameters such as Log CFU, pH, Toxin A and B, cell cytotoxicity were measured. Statistical comparisons using ANOVA one-way was performed in order to determine whether the groups were significantly different. Results At 5 g/L glucose, no C. difficile toxin was produced and co-culture with these lactobacilli resulted in potent acidification and growth inhibition. At 3 g/L glucose, C. difficile toxin production occurred and acidification by the lactobacilli resulted in growth inhibition as well as >99% reduced Toxin A and B production. In the absence of glucose and a starting pH of 7.0, TY broth, the lactobacilli did not acidify the medium and C. difficile growth was normal yet Toxin A and B production was partially reduced at, 20% and 41% lower. Toxin B from the supernatant of C. difficile grown in TY was cytotoxic to human fibroblast cells, but this was less cytotoxic when co-cultured with the Lactobacilli. Conclusion These results suggest that the combination of L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 interferes with C. difficile pathogenesis through: 1) inhibition of C. difficile growth (via lactic acid secretion), 2) reduced toxin A/B synthesis and (3) toxin neutralization. These results might explain the strain specificity of Bio-K+ probiotic bacteria in potentially preventing C. difficile-associated diarrhea in antibiotic treated patients. Disclosures All authors: No reported disclosures.

Download Full-text

Bacteroides fragilis prevents Salmonella Heidelberg translocation in co-culture model mimicking intestinal epithelium

Beneficial Microbes ◽

10.3920/bm2020.0004 ◽

2020 ◽

Vol 11 (4) ◽

pp. 391-401

Author(s):

T. Vernay ◽

I. Cannie ◽

F. Gaboriau ◽

S. David-Le Gall ◽

Z. Tamanai-Shacoori ◽

...

Keyword(s):

Bacterial Translocation ◽

In Vitro Model ◽

High Efficiency ◽

Bacteroides Fragilis ◽

Inhibitory Effect ◽

Foodborne Illnesses ◽

Culture Model ◽

Vitro Model ◽

Salmonella Heidelberg

Salmonella Heidelberg is one of the most common serovar causing foodborne illnesses. To limit the development of digestive bacterial infection, food supplements containing probiotic bacteria can be proposed. Commensal non-toxigenic Bacteroides fragilis has recently been suggested as a next-generation probiotic candidate. By using an original triple co-culture model including Caco-2 cells (representing human enterocytes), HT29-MTX (representing mucus-secreting goblet cells), and M cells differentiated from Caco-2 by addition of Raji B lymphocytes, bacterial translocation was evaluated. The data showed that S. Heidelberg could translocate in the triple co-culture model with high efficiency, whereas for B. fragilis a weak translocation was obtained. When cells were exposed to both bacteria, S. Heidelberg translocation was inhibited. The cell-free supernatant of B. fragilis also inhibited S. Heidelberg translocation without impacting epithelial barrier integrity. This supernatant did not affect the growth of S. Heidelberg. The non-toxigenic B. fragilis confers health benefits to the host by reducting bacterial translocation. These results suggested that the multicellular model provides an efficient in vitro model to evaluate the translocation of pathogens and to screen for probiotics that have a potential inhibitory effect on this translocation.

Download Full-text

1197. Inhibitory Effect of Ursodeoxycholic Acid on Clostridioides difficile Growth

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1382 ◽

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)

Download Full-text

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645978 ◽

1987 ◽

Vol 58 (02) ◽

pp. 744-748 ◽

Cited By ~ 11

Author(s):

A R Saniabadi ◽

G D O Lowe ◽

J C Barbenel ◽

C D Forbes

Keyword(s):

Platelet Aggregation ◽

Correlation Coefficient ◽

Whole Blood ◽

Blood Platelet ◽

Inhibitory Effect ◽

Time Interval ◽

Adp Receptor ◽

Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

Download Full-text

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646570 ◽

1989 ◽

Vol 61 (02) ◽

pp. 254-258 ◽

Cited By ~ 21

Author(s):

Margaret L Rand ◽

Peter L Gross ◽

Donna M Jakowec ◽

Marian A Packham ◽

J Fraser Mustard

Keyword(s):

Cyclic Amp ◽

Inhibitory Effect ◽

Rabbit Platelet ◽

Inhibitory Effects ◽

Low Concentrations ◽

Rabbit Platelets ◽

High Concentrations ◽

In Vitro Effects ◽

Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Download Full-text

The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649080 ◽

1973 ◽

Vol 30 (02) ◽

pp. 315-326

Author(s):

J. Heinz Joist ◽

Jean-Pierre Cazenave ◽

J. Fraser Mustard

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Barbituric Acid ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Primary Response ◽

Sodium Pentobarbital ◽

Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

Download Full-text

Inhibition of Human and Animal Platelet Adhesiveness to Glass Bead Columns by Adenosine, Dipyridamole, Chlorpromazine and Acetylsalicylic Acid

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648055 ◽

1976 ◽

Vol 36 (02) ◽

pp. 401-410 ◽

Cited By ~ 6

Author(s):

Buichi Fujttani ◽

Toshimichi Tsuboi ◽

Kazuko Takeno ◽

Kouichi Yoshida ◽

Masanao Shimizu

Keyword(s):

Guinea Pig ◽

Acetylsalicylic Acid ◽

Guinea Pigs ◽

Glass Bead ◽

Animal Species ◽

Inhibitory Effect ◽

Rabbit Platelet ◽

Platelet Adhesiveness

SummaryThe differences among human, rabbit and guinea-pig platelet adhesiveness as for inhibitions by adenosine, dipyridamole, chlorpromazine and acetylsalicylic acid are described, and the influence of measurement conditions on platelet adhesiveness is also reported. Platelet adhesiveness of human and animal species decreased with an increase of heparin concentrations and an increase of flow rate of blood passing through a glass bead column. Human and rabbit platelet adhesiveness was inhibited in vitro by adenosine, dipyridamole and chlorpromazine, but not by acetylsalicylic acid. On the other hand, guinea-pig platelet adhesiveness was inhibited by the four drugs including acetylsalicylic acid. In in vivo study, adenosine, dipyridamole and chlorpromazine inhibited platelet adhesiveness in rabbits and guinea-pigs. Acetylsalicylic acid showed the inhibitory effect in guinea-pigs, but not in rabbits.

Download Full-text

Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657151 ◽

1982 ◽

Vol 47 (02) ◽

pp. 150-153 ◽

Cited By ~ 13

Author(s):

P Han ◽

C Boatwright ◽

N G Ardlie

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Antiarrhythmic Drugs ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Second Phase ◽

Ionophore A23187 ◽

High Concentrations ◽

Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

Download Full-text

Effects of Ticlopidine of Platelet Function in Men with Stable Angina Pectoris

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660138 ◽

1985 ◽

Vol 54 (04) ◽

pp. 808-812 ◽

Cited By ~ 7

Author(s):

Ulf Berglund ◽

Henning von Schenck ◽

Lars Wallentin

Keyword(s):

Platelet Aggregation ◽

Angina Pectoris ◽

Platelet Function ◽

Plasma Levels ◽

Platelet Reactivity ◽

Inhibitory Effect ◽

Controlled Study ◽

Double Blind ◽

Platelet Factor

SummaryThe effects of ticlopidine (T) (500 mg daily) on platelet function were investigated in a double-blind placebo-controlled study in 38 middle-aged men with stable incapacitating angina pectoris. The in vitro platelet reactivity to aggregating agents, the platelet sensitivity to prostacyclin and the plasma levels of platelet specific proteins and fibrinogen were determined before and after 4 and 8 weeks of treatment. T exerted a potent inhibitory effect on ADP- and collagen-induced platelet aggregation. The effect of T was proportional to the pretreatment reactivity to ADP and collagen. The inhibitory effect of T on the epinephrine response was less pronounced. The plasma levels of beta-thromboglobulin, platelet factor 4 and fibrinogen were not influenced by T. The platelet inhibition of prostacyclin was potentiated by T, and it was demonstrated that T and prostacyclin had synergistic inhibitory effects on platelet aggregation.

Download Full-text

Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657338 ◽

1983 ◽

Vol 49 (02) ◽

pp. 132-137 ◽

Cited By ~ 16

Author(s):

A Eldor ◽

G Polliack ◽

I Vlodavsky ◽

M Levy

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Competitive Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

Ionophore A23187 ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

Download Full-text