&lt;em&gt;In Vitro&lt;/em&gt; Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Letourneau, Jason; Levesque, Cynthia; Berthiaume, Frédéric; Jacques, Mario; Mourez, Michaël

doi:10.3791/2783-v

Cited by 7 publications

(6 citation statements)

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“…The same procedure was repeated once again in the following week. These mice were then sacrificed four weeks later [ 22 , 23 ], the dissected gastric tissues were subjected to the colony-forming unit (CFU) assay for examining the degree of bacterial colonization [ 37 , 38 ], i.e., to calculate the number of bacterial colonies per milligram of stomach tissue. H. pylori was detected in cryo-sections by using a specific monoclonal antibody.…”

Section: Resultsmentioning

confidence: 99%

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis

Ong,

Jan,

et al. 2024

J Biomed Sci

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Background Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6’-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. Methods Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori–AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. Results CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC–MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. Conclusions The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis

Ong,

Jan,

et al. 2024

J Biomed Sci

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“…E. coli showed a decrease in the total number of CFU over time in synovial fluid in SST but not in plain tubes. This could be the result of silica causing sequestration of bacteria within the clot, through cell agglutination, which may decrease bacteria proliferation under laboratory conditions (Letourneau et al., 2011 ). As bacterial proliferation is highly dependent on bacterial adhesion to the cell membrane, it is plausible to consider that the highest bacterial load within the synovial fluid sample may reside within the clotted part of the sample, rather than the fluid (Słotwińska et al., 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Effect of silica‐sprayed collection tubes on synovial fluid bacterial culture

Jimenez Rihuete,

Martin,

Villarino

et al. 2024

Veterinary Medicine & Sci

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IntroductionSilica‐sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture.Materials and methodsThe study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin‐resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no‐additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48 h. Outcome variables were negative culture, positive culture and total number of colony‐forming units (CFUs). Statistical analysis was performed using Mann–Whitney U test, and significance was set at p < 0.05.ResultsThe total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8 h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4 h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24 h.DiscussionSilica‐containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases.

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“…L. fermentum was used as a positive control as its ability to adhere to intestinal cell lines is well established (Archer et al, 2018). Cell concentrations for the late logarithmic/stationary phase for bacteria were determined prior to adhesion experiments to ensure the recommended multiplicity of infection (MOI, 1:10-1:100 mammalian cells: bacterial cells) (Letourneau et al, 2011), then 100 µl of bacterial suspension (containing 3.0-8.0 × 10 7 CFU/ ml) in DMEM were added to HT-29 and HT-29-MTX cells, mixed by a gentle swirl and incubated for 3.5 h in the CO 2 incubator at 37 • C. Control wells not containing mammalian cells were prepared and incubated in parallel in the same way. Upon incubation with bacterial strains HT-29 and HT-29-MTX cells were gently washed four times with 0.5 ml DPBS.…”

Section: Resistance Of B Coagulans Cgi314 Spores To Simulated Gastric...mentioning

confidence: 99%

In vitro safety and functional characterization of the novel Bacillus coagulans strain CGI314

Mazhar,

Simon,

Khokhlova

et al. 2024

Front. Microbiol.

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IntroductionBacillus coagulans species have garnered much interest in health-related functional food research owing to their desirable probiotic properties, including pathogen exclusion, antioxidant, antimicrobial, immunomodulatory and food fermentation capabilities coupled with their tolerance of extreme environments (pH, temperature, gastric and bile acid resistance) and stability due to their endosporulation ability.MethodsIn this study, the novel strain Bacillus coagulans CGI314 was assessed for safety, and functional probiotic attributes including resistance to heat, gastric acid and bile salts, the ability to adhere to intestinal cells, aggregation properties, the ability to suppress the growth of human pathogens, enzymatic profile, antioxidant capacity using biochemical and cell-based methods, cholesterol assimilation, anti-inflammatory activity, and attenuation of hydrogen peroxide (H2O2)-induced disruption of the intestinal-epithelial barrier.ResultsB. coagulans CGI314 spores display resistance to high temperatures (40°C, 70°C, and 90°C), and gastric and bile acids [pH 3.0 and bile salt (0.3%)], demonstrating its ability to survive and remain viable under gastrointestinal conditions. Spores and the vegetative form of this strain were able to adhere to a mucous-producing intestinal cell line, demonstrated moderate auto-aggregation properties, and could co-aggregate with potentially pathogenic bacteria. Vegetative cells attenuated LPS-induced pro-inflammatory cytokine gene expression in HT-29 intestinal cell lines and demonstrated broad antagonistic activity toward numerous urinary tract, intestinal, oral, and skin pathogens. Metabolomic profiling demonstrated its ability to synthesize several amino acids, vitamins and short-chain fatty acids from the breakdown of complex molecules or by de novo synthesis. Additionally, B. coagulans CGI314’s strong antioxidant capacity was demonstrated using enzyme-based methods and was further supported by its cytoprotective and antioxidant effects in HepG2 and HT-29 cell lines. Furthermore, B. coagulans CGI314 significantly increased the expression of tight junction proteins and partially ameliorated the detrimental effects of H2O2 induced intestinal-epithelial barrier integrity.DiscussionTaken together these beneficial functional properties provide strong evidence for B. coagulans CGI314 as a promising potential probiotic candidate in food products.

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<em>In Vitro</em> Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Cited by 7 publications

References 0 publications

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis

Effect of silica‐sprayed collection tubes on synovial fluid bacterial culture

In vitro safety and functional characterization of the novel Bacillus coagulans strain CGI314

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