In vitro and in situ abrogation of biofilm formation in E. coli by vitamin C through ROS generation, disruption of quorum sensing and exopolysaccharide production

2021 ◽  
Vol 341 ◽  
pp. 128171
Author(s):  
DP Shivaprasad ◽  
Neetu Kumra Taneja ◽  
Anupama Lakra ◽  
Divya Sachdev
Keyword(s):  
Quorum Sensing ◽  
Vitamin C ◽  
Biofilm Formation ◽  
Ros Generation ◽  
Exopolysaccharide Production ◽  
E Coli

scholarly journals Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

2021 ◽  
Author(s):  
Zachary Morrison ◽  
Alexander Eddenden ◽  
Adithya S Subramanian ◽  
P. Lynne Howell ◽  
mark nitz
Keyword(s):  
Biofilm Formation ◽  
Chain Termination ◽  
Salvage Pathway ◽  
Biofilm Inhibition ◽  
E Coli ◽  
Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated in Vitro

2020 ◽  
Author(s):  
Francisca Espinoza ◽  
Rocio Magdalena ◽  
Natalia Saldivia ◽  
Nery Jara ◽  
Fernando Martínez ◽  
...  
Keyword(s):  
Vitamin C ◽  
Bystander Effect ◽  
Dehydroascorbic Acid ◽  
Neurite Growth ◽  
Protein Carbonylation ◽  
Reduced Form ◽  
Ros Generation ◽  
Lysine Production ◽  
Neuritic Growth

Abstract Background: The reduced form of vitamin C, ascorbic acid (AA), has been related to antioxidant defense as well as gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown.Methods: To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells (astrocytes, HL60 and U87). Reactive oxygen species (ROS) generation was also analyzed by flow cytometry and protein carbonylation / carboximetil-lysine production.Results: AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells (HL60, U87, or astrocytes) that efficiently capture DHA (Bystander effect). In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. Finally, our data indicate that DHA induces ROS generation, a condition that results in protein carbonylation and carboximetil-lysine production. Conclusions: We have demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

scholarly journals In-situ monitoring AHL-mediated quorum-sensing regulation of the initial phase of wastewater biofilm formation

2020 ◽  
Vol 135 ◽  
pp. 105326 ◽  
Author(s):  
Jinfeng Wang ◽  
Qiuju Liu ◽  
Xianhui Li ◽  
Sijia Ma ◽  
Haidong Hu ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Initial Phase ◽  
In Situ Monitoring

scholarly journals Synthesis, In Silico, and In Vitro Evaluation of Long Chain Alkyl Amides from 2-Amino-4-Quinolone Derivatives as Biofilm Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (2) ◽  
pp. 327 ◽  
Author(s):  
Mariana Espinosa-Valdés ◽  
Sara Borbolla-Alvarez ◽  
Ana Delgado-Espinosa ◽  
Juan Sánchez-Tejeda ◽  
Anabelle Cerón-Nava ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Active Sites ◽  
Multidrug Resistant ◽  
Health Concern ◽  
Resistant Bacteria ◽  
Biofilm Inhibition ◽  
Amide Derivatives ◽  
Long Chain

Infection from multidrug resistant bacteria has become a growing health concern worldwide, increasing the need for developing new antibacterial agents. Among the strategies that have been studied, biofilm inhibitors have acquired relevance as a potential source of drugs that could act as a complement for current and new antibacterial therapies. Based on the structure of 2-alkyl-3-hydroxy-4-quinolone and N-acylhomoserine lactone, molecules that act as mediators of quorum sensing and biofilm formation in Pseudomonas aeruginosa, we designed, prepared, and evaluated the biofilm inhibition properties of long chain amide derivatives of 2-amino-4-quinolone in Staphylococcus aureus and P. aeruginosa. All compounds had higher biofilm inhibition activity in P. aeruginosa than in S. aureus. Particularly, compounds with an alkyl chain of 12 carbons exhibited the highest inhibition of biofilm formation. Docking scores and molecular dynamics simulations of the complexes of the tested compounds within the active sites of proteins related to quorum sensing had good correlation with the experimental results, suggesting the diminution of biofilm formation induced by these compounds could be related to the inhibition of these proteins.

scholarly journals Peptide-Based Formulation from Lactic Acid Bacteria Impairs the Pathogen Growth in Ananas Comosus (Pineapple)

Coatings ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 457 ◽  
Author(s):  
Gabriela N. Tenea ◽  
Daniela Olmedo ◽  
Clara Ortega
Keyword(s):  
Significant Proportion ◽  
High Capacity ◽  
Total Cell ◽  
Ananas Comosus ◽  
Street Vending ◽  
E Coli ◽  
Cell Counts ◽  
Total Cell Density

Worldwide, street vending commerce has grown exponentially, representing in some countries, including Ecuador, a significant proportion of food consumed by the urban population. Pineapple is one of the common fruits sold as ready-to-eat slices by ambulant vendors in the street or on public transport at risk of contamination by various microorganisms. Previously, we selected Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis Gt28 strains producing peptides with high capacity to inhibit pathogen growth in vitro. In this study, the effect of different edited formulations containing a mixture of Cys5-4/Gt28 peptides was evaluated in vitro and ex vitro against a pathogenic cocktail containing E. coli (2), Salmonella (2) and Shigella (1). The growth of bacterial cocktail co-inoculated with cell-free supernatant containing peptides (formulation T1) and precipitated peptides (formulation T6), in a ratio of Cys5-4/Gt28:1:1 (v/v), results in a decrease of total cell viability with 1.85 and 1.2 log CFU/mL orders of magnitude at 6 h of incubation. About the same decrease (1.9 log CFU/g) was observed when pineapple slices artificially inoculated with the pathogenic cocktail were coated with T1 formulation, indicating the capacity to diminish simultaneous pathogens in situ, thus demonstrating its great biological control and protection. However, the E. coli cell counts reduced by 2.08 log CFU/g while Salmonella and Shigella cell counts reduced by 1.43 and 1.91 log CFU/g, respectively, at 5 days of refrigeration. In the untreated pineapple slices, the total cell density was maintained during storage, suggesting the adaptation of the pathogens to the fruit matrix. The peptide-based formulation exerted a bacteriolytic mode of action inducing pathogenic cell death. The results indicate that coating pineapple slices with peptide-based formulation is a promising approach to protect them from further contamination by microbial spoilage as well as an alternative to increase the food safety.

Materials Sealing Preventing Biofilm Formation in Implant/Abutment Joints: Which Is the Most Effective? A Systematic Review and Meta-Analysis

2020 ◽  
Vol 46 (2) ◽  
pp. 163-171
Author(s):  
Cecília Alves de Sousa ◽  
Maria Beatriz Bello Taborda ◽  
Gustavo Antônio Correa Momesso ◽  
Eduardo Passos Rocha ◽  
Paulo Henrique dos Santos ◽  
...  
Keyword(s):  
Systematic Review ◽  
Biofilm Formation ◽  
Meta Analysis ◽  
Significance Level ◽  
Gutta Percha ◽  
Significant Difference ◽  
Physical Barriers ◽  
External Connection

The purpose of this systematic review was to evaluate the literature available for materials exhibiting the best efficacy in preventing biofilm formation in the interior of implants. We searched PubMed/MEDLINE, Scopus, and Cochrane databases. This review is registered with the PROSPERO database and followed the suitability of the PRISMA protocol. The initial search resulted in 326 articles from the databases. After they were read, 8 articles remained, and the inclusion and exclusion criteria were applied. Six of these 8 articles were classified as in vitro and 2 were classified as in situ. The regions of the implants evaluated ranged from the interface of the pieces to the occlusal upper access of the abutment. The implant connections evaluated the Morse taper, external connection, and internal connection. Meta-analysis of the quantitative data was performed at a significance level of .05. Cotton exhibited poor control of infiltration, even in combination with other materials. Isolated gutta-percha (GP) and polytetrafluoroethylene (PTFE) tape with composite resin (CR) or GP performed better as physical barriers. The best results for chemical barriers were observed by the application of 1% chlorhexidine gluconate (CG) gel, thymol varnish, and the deposition of Ag films onto the surface. The applied meta-analysis did not show a significant difference in comparison between the different types of implant connections (P > .05). The application of CG and thymol varnish antimicrobials was effective in preventing biofilm formation and easy clinical execution; these could be used in combination with CR, GP, and PTFE.

Rosemary and Tea Tree Essential Oils Exert Antibiofilm Activities In Vitro against Staphylococcus aureus and Escherichia coli

2020 ◽  
Vol 83 (7) ◽  
pp. 1261-1267
Author(s):  
TING LIU ◽  
JINGFAN WANG ◽  
XIAOMAN GONG ◽  
XIAOXIA WU ◽  
LIU LIU ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Essential Oil ◽  
Biofilm Formation ◽  
Gas Chromatography Mass Spectrometry ◽  
Tea Tree ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT The purpose of the present study was to determine the bioactive compounds in rosemary essential oil (REO) and tea tree essential oil (TEO) and to investigate their antibacterial and antibiofilm activities against Staphylococcus aureus and Escherichia coli in vitro. The MIC and MBC assays were performed to assess the antibacterial activity of these two EOs against S. aureus and E. coli with the broth microdilution method. A crystal violet assay was used to ascertain the effects of EOs on the biofilm formation of the test strains, and a tetrazolium bromide (MTT) assay was used to measure the level of inactivation of mature biofilms by EOs. Gas chromatography–mass spectrometry revealed 15 compounds in REO and 27 compounds in TEO, representing 97.78 and 98.13% of the total EO, respectively. Eucalyptol and α-pinene were found in high concentrations in REO, and the two major compounds in TEO were 4-terpineol and terpinolene. The MICs of REO for the two S. aureus and E. coli test strains were both 0.5 mg/mL, and the MICs of TEO for the two strains were both 0.25 mg/mL. Therefore, these EOs can significantly inhibit the formation of biofilms and induced morphological biofilm changes, as verified by scanning electron microscopy. Both EOs had destructive effects on the mature biofilm of the two test strains. TEO was more inhibitory than REO for biofilm formation by the two test strains. HIGHLIGHTS

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

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