2018 |
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Boudjemaa, R., Cabriel, C., Dubois-Brissonnet, F., Bourg, N., Dupuis, G., Gruss, A., Lévêque-Fort, S., Briandet, R., Fontaine-Aupart, M.-P., Steenkeste K. (2018). Failure of daptomycin to kill Staphylococcus aureus: impact of bacterial membrane fatty acid composition. Antimicrobial Agents and Chemotherapy, .
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Boudjemaa, R., Steenkeste, K., Jacqueline, C., Briandet, R., Caillon, J., Boutoille, D., Le Mabecque, V., Tattevin, P., Fontaine-Aupart, M.P., Revest, M. (2018). Live intramacrophagic Staphylococcus aureus as possible responsible for antibiotic therapy failure: observations in an in-vivo mouse model of prosthetic vascular material infections. Journal of Antimicrobial Chemotherapy, .
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Mathieu, M. - C., Toullec, A., Benoit, C., Berry, R., Validire, P., Beaumel, P., Vincent, Y., Maroun, P., Vielh, P., Alchab, L., Farcy, R., Moniz-Koum, H., Fontaine-Aupart, M. - P., Delaloge, S., & Balleyguier, C. (2018). Preclinical ex vivo evaluation of the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses. European radiology, , 1–9.
Résumé: OBJECTIVES: To assess the diagnostic performance of a new device for in situ label-free fluorescence spectral analysis of breast masses in freshly removed surgical specimens, in preparation for its clinical development. METHODS: Sixty-four breast masses from consenting patients who had undergone either a lumpectomy or a mastectomy were included. Label-free fluorescence spectral acquisitions were obtained with a 25G fibre-containing needle inserted into the mass. Data from benign and malignant masses were compared to establish the most discriminating thresholds and measurement algorithms. Accuracy was verified using the bootstrap method. RESULTS: The final histological examination revealed 44 invasive carcinomas and 20 benign lesions. The maximum intensity of fluorescence signal was discriminant between benign and malignant masses (p < .0001) whatever their sizes. Statistical analysis indicated that choosing five random measurements per mass was the best compromise to obtain high sensitivity and high negative predictive value with the fewest measurements. Thus, malignant tumours were identified with a mean sensitivity, specificity, negative and positive predictive value of 98.8%, 85.4%, 97.2% and 93.5%, respectively. CONCLUSION: This new in situ tissue autofluorescence evaluation device allows accurate discrimination between benign and malignant breast masses and deserves clinical development. KEY POINTS: A new device allows in situ label-free fluorescence analysis of ex vivo breast masses Maximum fluorescence intensity discriminates benign from malignant masses (p < .0001) Five random measurements allow a high negative predictive value (97.2%).
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Toullec, T., Mathieu, M. - C., Benoit, C., René Farcy, R., Tourasse, C., Boisserie-Lacroix, M., Fontaine-Aupart, M. - P., Delaloge, S., & Balleyguier, C. (2018). 25 gauge fibered-needle for label free fluorescence analysis of breast masses: a first in vivo study. In Proc.SPIE 10488, Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications, (Vol. XVIII).
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2017 |
Boudjemaa, R., Briandet, R., Fontaine-Aupart, M. P., & Steenkeste, K. (2017). How do fluorescence spectroscopy and multimodal fluorescence imaging help to dissect the enhanced efficiency of the vancomycin-rifampin combination against Staphylococcus aureus infections? Photochemical & Photobiological Sciences, 16(9), 1391–1399.
Résumé: Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections. Among current clinical antibiotics, very few enable long-term successful treatment. Thus, it becomes necessary to better understand antibiotic failures and successes in treating infections in order to master the use of proper antibiotic therapies. In this context, we took benefit from a set of fluorescence spectroscopy and imaging methods, with the support of conventional microbiological tools to better understand the vancomycin-rifampin combination (in) efficiency against S. aureus biofilms. It was shown that both antibiotics interacted by forming a complex. This latter allowed a faster penetration of the drugs before dissociating from each other to interact with their respective biological targets. However, sufficiently high concentrations of free vancomycin should be maintained, either by increasing the vancomycin concentration or by applying repetitive doses of the two drugs, in order to eradicate rifampin-resistant mutants.
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Boudjemaa, R., Briandet, R., Steenkeste, K., & Fontaine-Aupart, M. - P. (2017). Taking advantage of fluorescent-based tools to puzzle out successes and failures of antibiotics to inactivate infectious bacteria. Encyclopedia of Analytical Chemistry, , 1–18.
Résumé: In microbiology, fluorescence tools have undoubtedly proven their performance to analyze in real-time, non-invasively and quantitatively, the structure, composition, processes, and dynamics of microbial communities. Importantly, the available fluorescence-based methods enable to decipher multiscale events implicated in the failures and successes of antibiotics treatments. The goal of this article is to review the large range of fluorescence techniques and probes and their interest (i) to study bacterial cells viability in vitro and in vivo under antibiotics exposure, (ii) to elucidate the mechanisms of action of these drugs, and (iii) to dissect bacterial mechanisms to resist and tolerate antibiotics action.
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Chan, A. J., Sarkar, P., Gaboriaud, F., Fontaine-Aupart, M. P., & Marliere, C. (2017). Control of interface interactions between natural rubber and solid surfaces through charge effects: an AFM study in force spectroscopic mode. Rsc Advances, 7(69), 43574–43589.
Résumé: This work presents a detailed investigation of interface interactions between natural rubber (NR) particles and solid surfaces in aqueous mediumat high ionic strength (0.1 M) using AFM in fast force spectroscopy mode. In this study, an original method for fixing the NR on the substrate was developed. This avoided the usual perturbations common in standard immobilization techniques. We proved that the adhesion process of the NR is monitored by slight changes in the surface charge state of the contacting solid surfaces made of silicon oxide or silicon nitride. The results were interpreted using Dynamic Force Spectroscopy theory, with the introduction of a supplementary term describing the electrostatic energy. Furthermore, these experiments revealed that adhesion between NR and tip was time dependent in a cumulative process. In addition, an increase of the adhesion between NR and AFM tip with the size of the rubber particles was measured. This was related to the higher concentration in lipids versus proteins for larger NR particles. These results are of great importance both for practical applications in solution-based industrial processes and to the fundamental knowledge of adhesion process involved for biopolymers or living cells.
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2016 |
Boudjemaa, R., Briandet, R., Revest, M., Jacqueline, C., Caillon, J., Fontaine-Aupart, M. P., & Steenkeste, K. (2016). New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging. Antimicrobial Agents And Chemotherapy, 60(8), 4983–4990.
Résumé: Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/ tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/ tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.
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Chagas, G. R., Xie, X., Darmanin, T., Steenkeste, K., Gaucher, A., Prim, D., Meallet-Renault, R., Godeau, G., Amigoni, S., & Guittard, F. (2016). Electrodeposition of Polypyrenes with Tunable Hydrophobicity, Water Adhesion, and Fluorescence Properties. Journal Of Physical Chemistry C, 120(13), 7077–7087.
Résumé: The control in surface hydrophobicity and water adhesion is extremely important for various applications in water harvesting, oil/water separation membrane, energy systems or biosensing, for example. Here, for the first time we show that the use of fluorescent monomers such as pyrene with various substituents differing by their hydrophobicity, size, or rigidity/flexibility can lead to surfaces with tunable hydrophobicity, water adhesion and fluorescence properties by a direct electropolymerization process. Seven original monomers with fluoroalkyl, alkyl, phenyl, adamantyl, and triethylene glycol substituents were synthesized and studied. The surface roughness is highly dependent on the substituent, and it seems that the fluorescence, although complex, correlates with the surface roughness. Superhydrophobic properties and highly oleophobic properties are obtained using fluoroalkyl chains due to the presence of nanostructured microparticles. In comparison to the structured absorption and emission bands of pyrene monomers, the pyrene polymers (oligomers) exhibit a broad structureless spectral shape both in absorption and in emission, which seems to be due to pyrene oligomerization and aggregation. This work is a first tentative attempt to combine superhydrophobic and fluorescent properties using an innovative strategy and opens new doors to explore in this domain.
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Chan, A., Clairand, M., Steenkeste, K., Eloy, M., Canette, A., Alousque, F., Ceccia, S., Gaboriaud, F., & Fontaine-Aupart, M. P. (2016). NR – Filler Interactions in aqueous condition by Multi-Modal Fluorescence and Electronic Microscopies. In Communications in Physics (Vol. ISSN 0868).
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Revest, M., Jacqueline, C., Boudjemaa, R., Caillon, J., Le Mabecque, V., Breteche, A., Steenkeste, K., Tattevin, P., Potel, G., Michelet, C., Fontaine-Aupart, M. P., & Boutoille, D. (2016). New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection. Journal Of Antimicrobial Chemotherapy, 71(5), 1291–1299.
Résumé: Prosthetic vascular graft infection (PVGI) is an emerging disease, mostly caused by staphylococci, with limited data regarding efficacy of current antistaphylococcal agents. We aimed to assess the efficacy of different antibiotic regimens. Six different strains of MSSA and MRSA were used. We compared results of minimal biofilm inhibitory and eradicating concentrations (MBICs and MBECs) obtained with a Calgary Biofilm Pin Lid Device (CBPD) with those yielded by an original Dacron (R)-related minimal inhibitory and eradicating concentration measure model. We then used a murine model of Staphylococcus aureus vascular prosthetic material infection to evaluate efficacy of different antibiotic regimens: vancomycin and daptomycin combined or not with rifampicin for MRSA and the same groups with cloxacillin and cloxacillin combined with rifampicin for MSSA. We demonstrated that classical measures of MBICs and MBECs obtained with a CPBD could overestimate the decrease in antibiotic susceptibility in material-related infections and that the nature of the support used might influence the measure of biofilm susceptibility, since results yielded by our Dacron (R)-related minimal eradicating assay were lower than those found with a plastic device. In our in vivo model, we showed that daptomycin was significantly more bactericidal than comparators for some strains of MRSA or MSSA but not for all. For the majority of strains, it was as efficient as comparators. The addition of rifampicin to daptomycin did not enhance daptomycin efficacy. Despite the heterogeneity of results according to bacterial strains, these innovative models represent an option to better evaluate the in vitro efficacy of antibiotics on Dacron (R)-related biofilm S. aureus infections, and to screen different antibiotic regimens in a mouse model of PVGIs.
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2015 |
Chan, A. J., Steenkeste, K., Canette, A., Eloy, M., Brosson, D., Gaboriaud, F., & Fontaine-Aupart, M. - P. (2015). Natural Rubber-Filler Interactions: What Are the Parameters? Langmuir, 31(45), 12437–12446.
Résumé: Reinforcement of a polymer matrix through the incorporation of nanoparticles (fillers) is a common industrial practice that greatly enhances the mechanical properties of the composite material. The origin of such mechanical reinforcement has been linked to the interaction between the polymer and filler as well as the homogeneous dispersion of the filler within the polymer matrix. In natural rubber (NR) technology, knowledge of the conditions necessary to achieve more efficient NR-filler interactions is improving continuously. This study explores the important physicochemical parameters required to achieve NR-filler interactions under dilute aqueous conditions by varying both the properties of the filler (size, composition, surface activity, concentration) and the aqueous solution (ionic strength, ion valency). By combining fluorescence and electron microscopy methods, we show that NR and silica interact only in the presence of ions and that heteroaggregation is favored more than homoaggregation of silica-silica or NR-NR. The interaction kinetics increases with the ion valence, whereas the morphology of the heteroaggregates depends on the size of silica and the volume percent ratio (dry silica/dry NR). We observe dendritic structures using silica with a diameter (d) of 100 nm at a similar to 20-50 vol % ratio, whereas we obtain raspberry-like structures using silica with d = 30 nm particles. We observe that in liquid the interaction is controlled by the hydrophilic bioshell, in contrast to dried conditions, where hydrophobic polymer dominates the interaction of NR with the fillers. A good correlation between the nanoscopic aggregation behavior and the macroscopic aggregation dynamics of the particles was observed. These results provide insight into improving the reinforcement of a polymer matrix using NR-filler films.
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Chan, A. J., Steenkeste, K., Ely, M., Brssn, D., Gaboriaud, F., & Fontaine-Aupart, M. - P. (2015). Lipid Content In Small And Large Natural Rubber Particles. Rubber Chemistry And Technology, 88(2), 248–257.
Résumé: Knowledge of the surface composition of natural rubber (NR) latex is essential to manufacturers of latex goods. Films made from only small rubber particles (SRPs) and a mix of SRPs and large rubber particles (LRPs) differ in mechanical properties. The reason for this difference, which is still under debate, is hypothesized to be linked with biomolecules (proteins and lipids) present in the NR particle surface. In this study, we characterize the surface chemistry, particularly lipid content of the SRP and LRP, by performing investigations directly on these particles in aqueous conditions. Fluorescent probes were used to display protein and lipid affinity and analyze them in situ with steady-state fluorescence spectroscopy, fluorescence correlation spectroscopy, and fluorescence lifetime measurements. Results are atypical in showing that lipids are more abundant in LRPs than in SRPs, suggesting thicker and/or denser membranes in LRPs. The degree of membrane compacity affects rigidity, influences biomolecular interactions, and might impact natural rubber coagulation. These results provide additional insights into colloidal behavior of NR for more efficient industrial applications.
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Farcy, R., Benoit, C., Moustie, R., Eyrignoux, T., Beaumel, P., Toullec, A., & Fontaine-Aupart, M. P. (2015). Aiguille fibrée pour l’aide au diagnostic du cancer du sein par fluorescence endogène. Photoniques, 79, 22–25.
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2014 |
Monsel, A., Lecart, S., Roquilly, A., Broquet, A., Jacqueline, C., Mirault, T., Troude, T., Fontaine-Aupart, M. - P., & Asehnoune, K. (2014). Analysis of Autofluorescence in Polymorphonuclear Neutrophils: A New Tool for Early Infection Diagnosis. PLOS ONE, 9(3), 1790–1800.
Résumé: Diagnosing bacterial infection (BI) remains a challenge for the attending physician. An ex vivo infection model based on human fixed polymorphonuclear neutrophils (PMNs) gives an autofluorescence signal that differs significantly between stimulated and unstimulated cells. We took advantage of this property for use in an in vivo pneumonia mouse model and in patients hospitalized with bacterial pneumonia. A 2-fold decrease was observed in autofluorescence intensity for cytospined PMNs from broncho-alveolar lavage (BAL) in the pneumonia mouse model and a 2.7-fold decrease was observed in patients with pneumonia when compared with control mice or patients without pneumonia, respectively. This optical method provided an autofluorescence mean intensity cut-off, allowing for easy diagnosis of BI. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope. Assessing the autofluorescence of PMNs provides a fast, simple, cheap and reliable method optimizing the efficiency and the time needed for early diagnosis of severe infections. Rationalized therapeutic decisions supported by the results from this method can improve the outcome of patients suspected of having an infection.
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Nodin, L., Méallet-Renault, R., & Piard, J. (2014). Séparation et étude des pigments des épinards par spectrofluorimétrie. Bulletin de l’Union des Physiciens, 961, 293–310.
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Quoilin, C., Mouithys-Mickalad, A., Lecart, S., Fontaine-Aupart, M. - P., & Hoebeke, M. (2014). Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1837(10), 1790–1800.
Résumé: To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacteria] endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects. LPS treatment resulted in an increase in the expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX-4), suggesting the cytosolic overexpression of nitric oxide and superoxide anion, the primary reactive nitrogen species (RNS) and reactive oxygen species (ROS). This oxidant state seemed to interrupt mitochondrial oxidative phosphorylation by reducing cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the mitochondrial inner membrane occurred, leading to a decrease of the mitochondrial membrane potential, a release of apoptotic-inducing factors and a depletion of adenosine triphosphate. Interestingly, after being targeted by RNS and ROS, mitochondria became in turn producer of ROS, thus contributing to increase the mitochondrial dysfunction. The role of oxidants in mitochondrial dysfunction was further confirmed by the use of iNOS inhibitors or antioxidants that preserve cytochrome c oxidase activity and prevent mitochondria] membrane potential dissipation. These results suggest that sepsis-induced AKI should not only be regarded as failure of energy status but also as an integrated response, including transcriptional events, ROS signaling, mitochondrial activity and metabolic orientation such as apoptosis. (C) 2014 Elsevier B.V. All rights reserved.
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2013 |
Aldeek, F., Schneider, R., Fontaine-Aupart, M. - P., Mustin, C., Lecart, S., Merlin, C., & Block, J. - C. (2013). Patterned Hydrophobic Domains in the Exopolymer Matrix of Shewanella oneidensis MR-1 Biofilms. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 79(4), 1400–1402.
Résumé: Water-dispersible amphiphilic surface-engineered quantum dots (QDs) were found to be strongly accumulated within discrete zones of the exopolymer network of Shewanella oneidensis MR-1 biofilms, but not on the cell surfaces. These microdomains showed a patterned distribution in the exopolymer matrix, which led to a restricted diffusion of the amphiphilic nanoparticles.
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Quoilin, C. C., Mouithys-Mickalad, A., Lecart, S., Gallez, B., Fontaine-Aupart, M. - P., & Hoebeke, M. (2013). Disruption in energy metabolism and mitochondrial function in a cellular model of inflammation-induced acute kidney injury. In Free Radical Biology And Medicine (Vol. 65, pp. S44–S45).
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Videcoq, P., Steenkeste, K., Bonnin, E., & Garnier C. (2013). A multi-scale study of enzyme di ff usion in macromolecular solutions and physical gels of pectin polysaccharides. Soft Matter, 9, 5110–5118.
Résumé: Pectin accounts for one of the major polysaccharides of the plant cell wall. Pectin methylesterases (PMEs)are enzymes that are able to alter the pectin structure and affect its gelling properties by de-esterifying galacturonic acid. In this work, the diffusion of two PMEs with different origins and modes of action
was characterised with a multi-scale approach in different media consisting of pectin macromolecular solutions and physical gels. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescent Correlation Spectroscopy (FCS) investigations showed similar diffusion behaviour for both enzymes at a microscopic scale. In contrast, their behaviours tended to differ when increasing the observation scale up to mm. Indeed, fungal PMEs diffused faster and in a larger range than plant PMEs. These results are discussed with regard to the respective modes of action, processivity and in vivo roles of the two enzymes.
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2012 |
Bulard, E., Fontaine-Aupart, M. - P., Dubost, H., Zheng, W., Bellon-Fontaine, M. - N., Herry, J. - M., & Bourguignon, B. (2012). Competition of Bovine Serum Albumin Adsorption and Bacterial Adhesion onto Surface-Grafted ODT: In Situ Study by Vibrational SFG and Fluorescence Confocal Microscopy. LANGMUIR, 28(49), 17001–17010.
Résumé: The interaction of hydrophilic and hydrophobic ovococcoid bacteria and bovine serum albumin (BSA) proteins with a well ordered surface of octadecanethiol (ODT) self assembled monolayer (SAM) has been studied in different situations where proteins were either preadsorbed on ODT or adsorbed simultaneously with bacterial adhesion as in life conditions. The two situations lead to very different antimicrobial behavior. Bacterial adhesion on preadsorbed BSA is very limited, while the simultaneous exposure of ODT SAM to proteins and bacteria lead to a markedly weaker antimicrobial effect. The combination of sum frequency generation spectroscopy and fluorescence confocal microscopy experiments allow one to draw conclusions on the factors that govern the ODT SAM or BSA film interaction with bacteria at the molecular level. On the hydrophobic ODT surface, interaction with hydrophobic or hydrophilic biomolecules results in opposite effects on the SAM, namely, a flattening or a raise of the terminal methyl groups of ODT. On an amphiphilic BSA layer, the bacterial adhesion strength is weakened by the negative charges carried by both BSA and bacteria. Surprisingly, preadsorbed BSA that cover part of the bacteria cell walls increase the adhesion strength to the BSA film and reduce hydrophobic interactions with the ODT SAM. Finally, bacterial adhesion on a BSA film is shown to modify the BSA proteins in some way that change their interaction with the ODT SAM. The antimicrobial effect is much stronger in the case of a preadsorbed BSA layer than when BSA and bacteria are in competition to colonize the ODT SAM surface.
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Bulard, E., Fontaine-Aupart, M. - P., Dubost, H., Zheng, W., Herry, J. - M., Bellon-Fontaine, M. - N., Briandet, R., & Bourguignon, B. (2012). The Effect of Bacterial Adhesion on Grafted Chains Revealed by the Non-Invasive Sum Frequency Generation Spectroscopy. SPECTROSCOPY-AN INTERNATIONAL JOURNAL, 27(5-6), 571–579.
Résumé: In biomedical and food industry, surface colonization by bacteria is harmful: it leads to biofilm formation, a microbial consortia more resistant to antibiotics than planktonic bacteria. In order to design materials able to limit the biofilm formation, the effect of bacteria on materials has to be well characterized. In this work, a well-defined surface composed of a self-assembled monolayer (SAM) of octadecanethiol (ODT) onto a gold surface is probed in situ. The SAM conformation is obtained using the femtosecond vibrational sum frequency generation (SFG) spectroscopy. This technique provides selectively the molecular vibrational signature of the interface. The behaviour of the ODT SAM is studied in different environments: in air, in water, and upon exposure to hydrophilic or hydrophobic Lactococcus lactis bacteria. Modelling the experimental SFG spectra reveals a measurable change of the SAM conformation which depends on the environment, especially on the hydrophilic-hydrophobic character.
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Daddi Oubekka, S., Briandet, R., Fontaine-Aupart, M. - P., & Steenkeste, K. (2012). Correlative time-resolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms. Antimicrobial agents and chemotherapy, 56(6), 3349–58.
Résumé: The failure of antibiotics to inactivate in vivo pathogens organized in biofilms has been shown to trigger chronic infections. In addition to mechanisms involving specific genetic or physiological cell properties, antibiotic sorption and/or reaction with biofilm components may lessen the antibiotic bioavailability and consequently decrease their efficiency. To assess locally and accurately the antibiotic diffusion-reaction, we used for the first time a set of advanced fluorescence microscopic tools (fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence lifetime imaging) that offer a spatiotemporal resolution not available with the commonly used time-lapse confocal imaging method. This set of techniques was used to characterize the dynamics of fluorescently labeled vancomycin in biofilms formed by two Staphylococcus aureus human isolates. We demonstrate that, at therapeutic concentrations of vancomycin, the biofilm matrix was not an obstacle to the diffusion-reaction of the antibiotic that can reach all cells through the biostructure.
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Deniset-Besseau, A., Miannay, F. - A., Laplace-Builhe, C., Vielh, P., Lecart, S., Lwaleed, B. A., Eschwege, P., & Fontaine-Aupart, M. - P. (2012). A fluorescence-based assay for monitoring clinical drug resistance. JOURNAL OF CLINICAL PATHOLOGY, 65(11), 1003–1007.
Résumé: Background and aims Multidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor patients' responses to chemotherapy. Methods The assay is based on the fluorescent properties of doxorubicin alone as well as in combination with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). The slide-based cell imaging technique was first optimised using a panel of breast and urothelial cancer cell lines and then extended to fine needle breast aspiration biopsy and urine cytology. Results The drug fluorescence behaviour observed on smears of clinical specimens is identical to that obtained using fixed cultured cells. The fluorescence of sensitive cells to chemotherapy is mainly localised in the nucleus, whereas resistant cells show a weak fluorescence signal localised in the cytoplasm. The difference in terms of fluorescence intensity is also highlighted through fluorescence spectra. Conclusions The results suggest that the assay provides clinically valuable information in predicting responses to doxorubicin and/or MVAC therapy. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope; as such it is technically simple, reliable and inexpensive.
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Grammenos, A., Fillet, M., Collodoro, M., Lecart, S., Quoilin, C., Fontaine-Aupart, M. - P., & Hoebeke, M. (2012). An ESR, Mass Spectrometry and Fluorescence Microscopy Approach to Study the Stearic Acid Derivatives Anchoring in Cells. APPLIED MAGNETIC RESONANCE, 43(3), 311–320.
Résumé: Lateral phase separations in biological membranes are of great interest, making electron spin resonance (ESR) spectroscopy combined with spin labeling a nondestructive and sensitive technique for the study of lipid rafts. It is currently accepted that the spin probe is localized on the plasma membrane. However, no study confirms this hypothesis. Herein, we report, for the first time, an accurate multispectral method for the quantification of the lipid spin label presence in every subcellular fraction. Cells were incubated with the 5-DOXYL stearic acid derivative and then subfractionated. Results of our multimodal spectroscopy approach ubiquitously demonstrate that the ESR spin label presence only sets in the plasma membranes.
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2011 |
Aldeek, F., Mustin, C., Balan, L., Roques-Carmes, T., Fontaine-Aupart, M. - P., & Schneider, R. (2011). Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms. BIOMATERIALS, 32(23), 5459–5470.
Résumé: Quantum dots (QDs) nanoprobes are emerging as alternatives to small-molecule fluorescent probes in biomedical technology. This paper reports an efficient and rapid method of producing highly dispersed and stable CdSe-core QDs with a hydrophobic gradient. Amphiphilic core/shell CdSe/ZnS QDs were prepared by ligand exchange at the surface of lipophilic CdSe/ZnS QDs using the dihydrolipoic acid (DHLA) dithiol ligand linked to Leucine or Phenylalanine aminoacids. Contact angle relaxations on a hydrophobic surface and surface tension measurements indicated that aqueous dispersions of CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs exhibit increased hydrophobicity compared to CdSe-core QDs capped by the hydrophilic 3-mercaptopropionic acid (MPA) ligand. We found that the surface functional groups and the ligand density at the periphery of these QDs significantly dictated their interactions with a complex biological matrix called biofilm. Using fluorescence confocal microscopy and an autocorrelation function (semi-variogram), we demonstrated that MPA-capped QDs were homogeneously associated to the biopolymers, while amphiphilic CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs were specifically confined allowing identification of hydrophobic microdomains of the biofilms. Results obtained clearly point out that the final destination of QDs in biofilms can properly be controlled by an appropriate design of surface ligands. (C) 2011 Elsevier Ltd. All rights reserved.
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Bridier, A., Tischenko, E., Dubois-Brissonnet, F., Herry, J. - M., Thomas, V., Daddi-Oubekka, S., Waharte, F., Steenkeste, K., Fontaine-Aupart, M. - P., & Briandet, R. (2011). Deciphering Biofilm Structure and Reactivity by Multiscale Time-Resolved Fluorescence Analysis. In BACTERIAL ADHESION: CHEMISTRY (Vol. 715, pp. 333–349).
Résumé: In natural, industrial and medical environments, microorganisms mainly live as structured and organised matrix-encased communities known as biofilms. In these communities, microorganisms demonstrate coordinated behaviour and are able to perform specific functions such as dramatic resistance to antimicrobials, which potentially lead to major public health and industrial problems. It is now recognised that the appearance of such specific biofilm functions is intimately related to the three-dimensional organisation of the biological edifice, and results from multifactorial processes. During the last decade, the emergence of innovative optical microscopy techniques such as confocal laser scanning microscopy in combination with fluorescent labelling has radically transformed imaging in biofilm research, giving the possibility to investigate non-invasively the dynamic mechanisms of formation and reactivity of these biostructures. In this chapter, we discuss the contribution of fluorescence analysis and imaging to the study at different timescales of various processes: biofilm development (hours to days), antimicrobial reactivity within the three-dimensional structure (minutes to hours) or molecular diffusion/reaction phenomena (pico- to milliseconds).
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Bulard, E., Dubost, H., Fontaine-Aupart, M. - P., Zheng, W., Herry, J. - M., Bellon-Fontaine, M. - N., Briandet, R., & Bourguignon, B. (2011). Non invasive SFG spectroscopy: a tool to reveal the conformational change of grafted chains due to bacterial adhesion. In CLINICAL AND BIOMEDICAL SPECTROSCOPY AND IMAGING II (Vol. 8087).
Résumé: In many fields such as biomedical or food industry, surface colonization by micro-organisms leads to biofilms formation that are tridimentional biostructures highly resistant to the action of antimicrobials, by mechanisms still unclear. In order to deepen our understanding of the initial interaction of bacteria cells with a solid surface, we analyze by in situ vibrational Sum Frequency Generation (SFG) spectroscopy the effect of the adhesion of hydrophilic Lactoccocus lactis bacteria and its hydrophobic mutants in distilled water on a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film. When a homogeneous bacterial monolayer is deposited on this ordered surface, SFG spectrum of the ODT SAM shows significant intensity changes from that in air or in water. Its modelling as a function of conformation allows to distinguish optical effects due to the water solution surrounding bacteria from conformational changes of the ODT SAM due to the presence of the bacteria cells. Futhermore, bacterial adhesion induces different measurable effects on the ODT SAM conformation, depending on the hydrophobic / hydrophilic character of the bacterial surface. Such a result deserves to be taken into account for the design of new materials with improved properties or to control biofilm formation.
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Bulard, E., Guo, Z., Zheng, W., Dubost, H., Fontaine-Aupart, M. - P., Bellon-Fontaine, M. - N., Herry, J. - M., Briandet, R., & Bourguignon, B. (2011). Non-Invasive Vibrational SFG Spectroscopy Reveals That Bacterial Adhesion Can Alter the Conformation of Grafted “Brush” Chains on SAM. LANGMUIR, 27(8), 4928–4935.
Résumé: Understanding bacterial adhesion on a surface is a crucial step to design new materials with improved properties or to control biofilm formation and eradication. Sum Frequency Generation (SFG) vibrational spectroscopy has been employed to study in situ the conformational response of a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film to the adhesion of hydrophilic and hydrophobic ovococcoid model bacteria. The present work highlights vibrational SFG spectroscopy as a powerful and unique non-invasive biophysical technique to probe and control bacteria interaction with ordered surfaces. Indeed, the SFG vibrational spectral changes reveal different ODT SAM conformations in air and upon exposure to aqueous solution or bacterial adhesion. Furthermore, this effect depends on the bacterial cell surface properties. The SFG spectral modeling demonstrates that hydrophobic bacteria flatten the ODT SAM alkyl chain terminal part, whereas the hydrophilic ones raise this ODT SAM terminal part. Microorganism-induced alteration of grafted chains can thus affect the desired interfacial functionality, a result that should be considered for the design of new reactive materials.
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Habimana, O., Steenkeste, K., Fontaine-Aupart, M. - P., Bellon-Fontaine, M. - N., Kulakauskas, S., & Briandet, R. (2011). Diffusion of Nanoparticles in Biofilms Is Altered by Bacterial Cell Wall Hydrophobicity. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(1), 367–368.
Résumé: Diffusion of entities inside biofilm triggers most mechanisms involved in biofilm-specific phenotypes. Using genetically engineered hydrophilic and hydrophobic cells of Lactococcus lactis yielding similar biofilm architectures, we demonstrated by fluorescence correlation spectroscopy that bacterial surface properties affect diffusion of nanoparticles through the biofilm matrix.
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Oubekka, S. D., Briandet, R., Waharte, F., Fontaine-Aupart, M. - P., & Steenkeste, K. (2011). Image-based Fluorescence Recovery After Photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms. In CLINICAL AND BIOMEDICAL SPECTROSCOPY AND IMAGING II (Vol. 8087).
Résumé: The diffusion capabilities of free fluorophores inside the heterogeneous three dimensional structure of Staphylococcus aureus biofilm were studied by an original image-based Fluorescence Recovery After Photobleaching method. The study was extended to BODIPY-vancomycin in order to better understand the mechanisms involved in the high tolerance of the bacteria embedded in a biofilm to the antibiotic.
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2010 |
Alchab, L., Dupuis, G., Balleyguier, C., Mathieu, M. - C., Fontaine-Aupart, M. - P., & Farcy, R. (2010). Towards an optical biopsy for the diagnosis of breast cancer in vivo by endogenous fluorescence spectroscopy. JOURNAL OF BIOPHOTONICS, 3(5-6), 373–384.
Résumé: The techniques of medical imaging allow the detection of suspect lesions in the breast, but they do not always evidence the malignant nature of these lesions. Breast biopsies and histological analyses are therefore implemented to establish a diagnosis. In order to reduce the number of these invasive procedures, a portable clinical system was designed based upon the excitation of Endogenous Fluorescence in vivo at 405 nm via a fiber-optics probe included in a disposable needle of small diameter (<1 mm). From the fluorescence signal, the authors are able to discriminate between diseased and healthy areas of human breast biopsies. Stronger fluorescence intensity and systematic spectral red shift of the tumor areas were observed. These results are confirmed by confocal microscopy. This new instrument is promising for the minimally invasive diagnosis of breast tumors in vivo with an appreciable limitation of patient trauma and of operational and financial cost. [GRAPHICS] Measurement of the endogenous fluorescence spectrum of a tissue sample via a fiber-optics probe included in a disposable needle of small diameter (<1 mm).
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Guelluy, P. - H., Fontaine-Aupart, M. - P., Grammenos, A., Lecart, S., Piette, J., & Hoebeke, M. (2010). Optimizing photodynamic therapy by liposomal formulation of the photosensitizer pyropheophorbide-a methyl ester: In vitro and ex vivo comparative biophysical investigations in a colon carcinoma cell line. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 9(9), 1252–1260.
Résumé: Photodynamic therapy (PDT), induced by a photosensitizer (PS) encapsulated in a nanostructure, has emerged as an appropriate treatment to cure a multitude of oncological and non-oncological diseases. Pyropheophorbide-a methyl ester (PPME) is a second-generation PS tested in PDT, and is a potential candidate for future clinical applications. The present study, carried out in a human colon carcinoma cell line (HCT-116), evaluates the improvement resulting from a liposomal formulation of PPME versus free-PPME. Absorption and fluorescence spectroscopies, fluorescence lifetime measurements, subcellular imaging and co-localization analysis have been performed in order to analyze the properties of PPME for each delivery mode. The benefit of drug encapsulation in DMPC-liposomes is clear from our experiments, with a 5-fold higher intracellular drug delivery than that observed with free-PPME at similar concentrations. The reactive oxygen species (ROSs) produced after PPME-mediated photosensitization have been identified and quantified by using electron spin resonance spectroscopy. Our results demonstrate that PPME-PDT-mediated ROSs are composed of singlet oxygen and a hydroxyl radical. The small amounts of PPME inside mitochondria, as revealed by fluorescence co-localization analysis, could maybe explain the very low apoptotic cell death measured in HCT-116 cells.
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Steenkeste, K., Tfibel, F., Perree-Fauvet, M., Briandet, R., & Fontaine-Aupart, M. - P. (2010). Tracking the Photosensitizing Antibacterial Activity of Mono(acridyl)bis(arginyl)porphyrin (MABAP) by Time-Resolved Spectroscopy. JOURNAL OF PHYSICAL CHEMISTRY A, 114(9), 3334–3339.
Résumé: Photodynamic inactivation (PDI) is currently receiving interest for its potential as an antimicrobial treatment. Although photosensitizing agents and light have been used for medical purposes for a very long time, only a little information is available about the mechanism of PDI for bacteria. Pseudomonas aeruginosa is a Gram negative bacteria involved in chronic infections in cystic fibrosis patients and also one of the commonest agents of hospital acquired infections. In the present study the sensitivity of Pseudomonas aeruginosa to the phototoxic effects of the mono(acridyl)bis(arginyl)porphyrin (MABAP) has been investigated as well as the photophysical and photochemical properties of this cationic porphyrin complexed to [poly(dG-dC)](2) to investigate the mechanisms that lead to bacteria inactivation. Both picosecond time-resolved fluorescence and femtosecond to nanosecond transient absorption measurements give evidence that while MABAP can react through its triplet state and/or an ultrafast electron transfer with guanine, its intercalation between GC base pairs is not the main target of MABAP photoactivity. The analysis of both fluorescence emission and excitation spectra reveals the occurrence of an energy transfer through the DNA double helix between the acridine and porphyrin chromophores of MABAP, as previously observed for the stacked free molecule in solution. This efficient process may lead to the excitation of twice more porphyrin chromophores in MABAP by comparison to other cationic porphyrins.
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Waharte, F., Steenkeste, K., Briandet, R., & Fontaine-Aupart, M. - P. (2010). Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 76(17), 5860–5869.
Résumé: Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside “live” biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactoccocus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.
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2008 |
Bojarska, E., Kazimierczuk, Z., Mouchard, C., Tfibel, F., & Fontaine-Aupart, M. - P. (2008). UVC induced oxidation of chloropurines: excited singlet and triplet pathways for the photoreaction. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 7(9), 1054–1062.
Résumé: The phototransformation of 2-chloro. 6-chloro and 2,6-dichloropurines under UVC excitation (254 nm) has been studied and the major photoproducts have been identified using absorption spectroscopy, HPLC and mass spectrometry It was shown that hydropurines were formed as the main products for all three investigated compounds both in the presence and absence of oxygen. In the case of 6-chloro- and 2,6-dichloropurine, a photodimer is also formed as a minor photoproduct in the absence of oxygen but is efficiently quenched in the presence of oxygen. Nanosecond photolysis experiments also revealed significant intersystem crossing to the triplet state of the chloropurines which has been characterized (transient absorption spectra, triplet formation quantum yields and rate constants of quenching by oxygen, Mn2+ ions and ground state). Experimental evidence allows to Conclude that the triplet state is involved in photodimer formation whereas the hydroxypurine is formed from the reaction of the excited singlet state of chloropurines with the solvent (water addition) through heterolytic C-Cl bond rupture. Mass spectrometry and H-1 NMR results allowed to propose a chemical pathway for dimer formation in the case of 2,6-dichloropurine in a two-step process: first a homolytic rupture of C-Cl bond in the triplet state of the molecule with the formation of purinyl radicals, which subsequently react with an excess of ground state molecules and/or hydroxypurine primarily formed.
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Briandet, R., Lacroix-Gueu, P., Renault, M., Lecart, S., Meylheuc, T., Bidnenko, E., Steenkeste, K., Bellon-Fontaine, M. - N., & Fontaine-Aupart, M. - P. (2008). Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74(7), 2135–2143.
Résumé: In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as “active” phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.
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