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Rapid Assay to Assess Bacterial Adhesion on Textiles.

ABSTRACT: Textiles are frequently colonized by microorganisms leading to undesired consequences like hygienic problems. Biocidal coatings often raise environmental and health concerns, thus sustainable, biocide-free coatings are of interest. To develop novel anti-adhesive textile coatings, a rapid, reliable, and quantitative high-throughput method to study microbial attachment to fabrics is required, however currently not available. Here, a fast and reliable 96-well plate-based screening method is developed. The quantification of bacterial adhesion is based on nucleic acid staining by SYTO9, with Pseudomonas aeruginosa and Staphylococcus aureus as the model microorganisms. Subsequently, 38 commercially available and novel coatings were evaluated for their anti-bacterial adhesion properties. A poly(l-lysine)-g-poly(ethylene glycol) coating on polyester textile substratum revealed an 80% reduction of bacterial adhesion. Both the coating itself and the anti-adhesive property were stable after 20 washing cycles, confirmed by X-ray analysis. The assay provides an efficient tool to rapidly screen for non-biocidal coatings reducing bacterial attachment.

SUBMITTER: Schmidt-Emrich S

PROVIDER: S-EPMC5502901 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Rapid Assay to Assess Bacterial Adhesion on Textiles.

Schmidt-Emrich Sabrina S Stiefel Philipp P Rupper Patrick P Katzenmeier Heinz H Amberg Caroline C Maniura-Weber Katharina K Ren Qun Q

Materials (Basel, Switzerland) 20160330 4

Textiles are frequently colonized by microorganisms leading to undesired consequences like hygienic problems. Biocidal coatings often raise environmental and health concerns, thus sustainable, biocide-free coatings are of interest. To develop novel anti-adhesive textile coatings, a rapid, reliable, and quantitative high-throughput method to study microbial attachment to fabrics is required, however currently not available. Here, a fast and reliable 96-well plate-based screening method is develop ...[more]

PMID: 28773373

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Project description:Background:Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. Results:In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. Conclusions:This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

Project description:Cigarette smoke (CS) has been reported to affect the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte-endothelial cell adhesion is promoted. Disease-relevant primary human coronary artery endothelial cells (HCAECs) were treated for 4 hours with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 hours (indirect treatment, I), (2) unconditioned media similarly prepared, however without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells-HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor alpha (TNF-alpha major inducer, was actually shed by unstable CS compound-activated TNFalpha-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. In conclusion, aqueous CS extract directly and indirectly promotes monocytic cell-endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms. Samples from E-MTAB-3644 dataset correspond to 3R4F sbPBS15 and PBS15 (vehicle control: 15% v/v). In the E-MTAB-3923 dataset, samples correspond to THS2.2 abPBS15, THS2.2 abPBS75 and PBS75 (75% v/v). All THS2.2 abPBS15 from E-MTAB-3923 must be compared with their respective control, PBS15 from E-MTAB-3644; while THS2.2 abPBS75 from E-MTAB-3923 must be compared with their respective control, PBS75 from E-MTAB-3923.

Dataset Information

Rapid Assay to Assess Bacterial Adhesion on Textiles.

Publications

Rapid Assay to Assess Bacterial Adhesion on Textiles.

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