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1. Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions

Author

Claire Saulou, Muriel Mercier-Bonin, Isabelle Fourquaux, Paul Dumas, Claude Maranges, Frédéric Jamme, Muriel Cocaign-Bousquet, Laurence Girbal, Génie et Microbiologie des Procédés Alimentaires (GMPA), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), The authors gratefully acknowledge the 'BioPleasure' project of the Research National Agency (ANR-07-BLAN-0196-01) for funding., The research described in this paper was performed at the French national synchrotron facility SOLEIL (Gif-sur- Yvette, France), using the SMIS beamline (proposal no 20090556)., ANR-07-BLAN-0196,BIOPLEASURE,Plasma - surface engineering for biofilm prevention(2007), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées, Institut National des Sciences Appliquées (INSA), Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Hôpital de Rangueil, and CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]

Subjects
In situ, Analytical chemistry, medicine.disease_cause, Biochemistry, Analytical Chemistry, 03 medical and health sciences, Stress, Physiological, Spectroscopy, Fourier Transform Infrared, medicine, Escherichia coli, Fourier transform infrared spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein secondary structure, 030304 developmental biology, 0303 health sciences, biology, Ionic silver, 030306 microbiology, Chemistry, Synchrotron FTIR-ATR microspectroscopy, Single-cell scale, [CHIM.MATE]Chemical Sciences/Material chemistry, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Antibacterial effect, Transmission electron microscopy, Attenuated total reflection, Ultrastructure, Biophysics, Silver Nitrate, Single-Cell Analysis, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, Synchrotrons, Bacteria
Abstract

International audience; The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO3 concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag+-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag+ concentrations (

Published
2013

2. Synchrotron FTIR microspectroscopy of the yeast Saccharomyces cerevisiae after exposure to plasma-deposited nanosilver-containing coating

Author

Claire Saulou, Isabelle Fourquaux, Muriel Mercier-Bonin, P. Dumas, C. Maranges, Patrice Raynaud, Bernard Despax, Frédéric Jamme, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées, LAboratoire PLasma et Conversion d'Energie (LAPLACE), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA), Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Matériaux et Procédés Plasmas (LAPLACE-MPP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), The research described in this paper was performed at the French national synchrotron facility SOLEIL (Gif-sur-Yvette, France), using the SMIS beamline (proposal no 20060176)., ANR-07-BLAN-0196,BIOPLEASURE,Plasma - surface engineering for biofilm prevention(2007), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3)

Subjects
Antifungal effect, Antifungal Agents, Silver, Cell Survival, Surface Properties, Scanning electron microscope, Thin films, Analytical chemistry, Metal Nanoparticles, Nanoparticle, Infrared spectroscopy, 02 engineering and technology, Saccharomyces cerevisiae, Biochemistry, Silver nanoparticle, Analytical Chemistry, Biomaterials, Plasma, 03 medical and health sciences, Nanosilvercontaining coating, Biological samples, Microscopy, Electron, Transmission, X-ray photoelectron spectroscopy, Synchrotron FTIR microspectroscopy, Spectroscopy, Fourier Transform Infrared, IR spectroscopy/Raman spectroscopy, Fourier transform infrared spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, 030304 developmental biology, 0303 health sciences, Nanocomposite, Chemistry, 021001 nanoscience & nanotechnology, Nanoparticles/ Nanotechnology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Transmission electron microscopy, Cold plasma, Interface/Surface analysis, 0210 nano-technology, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, Synchrotrons, Nuclear chemistry
Abstract

International audience; The present work was focused on elucidating changes in the model yeast Saccharomyces cerevisiae (cell composition, ultrastructure) after exposure to antimicrobial plasma-mediated nanocomposite films. In order to achieve this, a nanosilver-containing coating was deposited onto stainless steel using radiofrequency HMDSO plasma deposition, combined with simultaneous silver sputtering. X-ray photoelectron spectroscopy (XPS) confirmed the presence of silver nanoparticles embedded in an organo-silicon matrix. In addition, scanning electron microscopy (SEM) demonstrated the nanoparticle-based morphology of the deposited layer. The antifungal properties towards S. cerevisiae were established, since a 1.4 log reduction in viable counts was observed after a 24-h adhesion compared to control conditions with the matrix alone. Differences in cell composition after exposure to the nanosilver was assessed for the protein region using, for the first time, synchrotron Fourier-transform infrared (FTIR) microspectroscopy of single S. cerevisiae cells, through in situ mapping with sub-cellular spatial resolution. IR spectrum of yeast cells recovered after a 24-h adhesion to the nanosilver-containing coating revealed a significant downshift (20 cm −1) of the amide I peak at 1655 cm −1 , compared to freshly harvested cells. This lower band position, corresponding to a loss in α-helix structures, is indicative of the disordered secondary structures of proteins, due to the transition between active and inactive conformations under nanosilver-induced stress conditions. No significant effect on the nucleic acid region was detected. The inhibitory action of silver was targeted against both cell wall and intracellular proteins such as enzymes. Transmission electron microscopy (TEM) observations of the yeastultrastructure confirmed serious morphological and structural damages. A homogeneous protein-binding distribution of nanosilver all over the cell was assumed, since the presence of electron-dense silver clusters was detected not only on the cell surface but also within the cell. For control experiments with the organosilicon matrix alone, no antimicrobial effect was observed, which was consistent with synchrotron FTIR results and TEM observations.

Published
2010