Spectroscopic and microscopic characterization of dipicolinic acid and its salt photoproducts - A UVc effect study on DPA in solution and in bacterial spores.

Bacterial spores can cause significant problems such as food poisoning (like neurotoxin or emetic toxin) or serious illnesses (like anthrax or botulism). This dormant form of bacteria, made of several layers of barriers which provide extreme resistance to many abiotic stresses (radiation, temperature, pressure, etc.), are difficult to investigate in situ. To better understand the biological and chemical mechanisms involved and specific to spores resistance, the acquisition of environmental parameters is necessary. For that purpose, our research has been focused on the detection and analysis of a unique spore component, dipicolinic acid (DPA), used as the main in situ metabolite for sporulating bacteria detection. In its native form, DPA is only weakly fluorescent but after Ultraviolet irradiation at the wavelength of 254 nm (UVc), DPA photoproducts (DPAp) exhibit a remarkable fluorescence signal. These photoproducts are rarely identified and part of this study gives new insights offered by mass spectrometry (MS) in the determination of DPA photoproducts. Thanks to DPA assay techniques and fluorescence spectrometry, we highlighted the instability of photoproducts and introduced new assumptions on the effects of UVc on DPA. Studies in spectroscopy and microscopy allowed us to better understand these native probes in bacterial spores and will allow the implementation of a new method for studying the physico-chemical parameters of spore resistance.

Multispectral fluorescence sensitivity to acidic and polyphenolic changes in Chardonnay wines - The case study of malolactic fermentation.

In this study, stationary and time-resolvedfluorescence signatures, were statistically and chemometrically analyzed among three typologies of Chardonnay wines (A, B and C) with the objectives to evaluate their sensitivity to acidic and polyphenolic changes. For that purpose, a dataset was built using Excitation Emission Matrices of fluorescence (N = 103) decomposed by a Parallel Factor Analysis (PARAFAC), andfluorescence decays (N = 22), mathematically fitted, using the conventional exponential modeling and the phasor plot representation. Wine PARAFAC component C4 coupledwith its phasor plot g and s values enable the description of malolactic fermentation (MLF) occurrence in Chardonnay wines. Such proxies reflect wine concentration modifications in total acidity, malic/lactic and phenol acids.Lower g values among fresh MLF + wines compared to MLF- wines are explained by a quenching effect on wine fluorophores by both organic and phenolic acids.The combination of multispectral fluorescence parametersopens a novel routinely implementable methodology to diagnose fermentative processes.

The molecular dynamics of bacterial spore and the role of calcium dipicolinate in core properties at the sub-nanosecond time-scale.

Bacterial spores are among the most resistant forms of life on Earth. Their exceptional resistance properties rely on various strategies, among them the core singular structure, organization and hydration. By using elastic incoherent neutron scattering, we probed the dynamics of Bacillus subtilis spores to determine whether core macromolecular motions at the sub-nanosecond timescale could also contribute to their resistance to physical stresses. In addition, in order to better specify the role of the various spore components, we used different mutants lacking essential structure such as the coat (PS4150 mutant), or the calcium dipicolinic acid complex (CaDPA) located in the core (FB122 mutant). PS4150 allows to better probe the core's dynamics, as proteins of the coat represent an important part of spore proteins, and FB122 gives information about the role of the large CaDPA depot for the mobility of core's components. We show that core's macromolecular mobility is not particularly constrained at the sub-nanosecond timescale in spite of its low water content as some dynamical characteristics as force constants are very close to those of vegetative bacteria such as Escherichia coli or to those of fully hydrated proteins. Although the force constants of the coatless mutant are similar to the wild-type's ones, it has lower mean square displacements (MSDs) at high Q showing that core macromolecules are somewhat more constrained than the rest of spore components. However, no behavior reflecting the glassy state regularly evoked in the literature could be drawn from our data. As hydration and macromolecules' mobility are highly correlated, the previous assumption, that core low water content might explain spores' exceptional resistance properties seems unlikely. Thus, we confirm recent theories, suggesting that core water is mostly as free as bulk water and proteins/macromolecules are fully hydrated. The germination of spores leads to a much less stable system with a force constant of 0.1 N/m and MSDs ~2.5 times higher at low Q than in the dormant state. DPA has also an influence on core mobility with a slightly lower force constant for the DPA-less mutant than for the wild-type, and MSDs that are ~ 1.8 times higher on average than for the wild-type at low Q. At high Q, germinated and DPA-less spores were very similar to the wild-type ones, showing that DPA and core compact structure might influence large amplitude motions rather than local dynamics of macromolecules.

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing.

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).

High pressure sensitization of heat-resistant and pathogenic foodborne spores to nisin.

Today, there is no effective non-thermal method to inactivate unwanted bacterial spores in foods. High-Pressure (HP) process has been shown to act synergistically with moderate heating and the bacteriocin nisin to inactivate spores but the mechanisms have not been elucidated. The purpose of the present work was to investigate in depth the synergy of HP and nisin on various foodborne spore species and to bring new elements of understandings. For this purpose, spores of Bacillus pumilus, B. sporothermodurans, B. licheniformis, B. weihenstephanensis, and Clostridium sp. were suspended in MES buffer, in skim milk or in a liquid medium simulating cooked ham brine and treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Nisin (20 or 50 IU/mL) was added at three different points during treatment: during HP, during and or in the plating medium of enumeration. In the latter two cases, a high synergy was observed with the inhibition of the spores of Bacillus spp. The evaluation of the germinated fraction of Bacillus spp. spores after HP revealed that this synergy was likely due to the action of nisin on HP-sensitized spores, rather than on HP-germinated spores. Thus, the combination of nisin and HP can lead to Bacillus spp. spore inhibition at 20 °C. And Nisin can act on HP-treated spores, even if they are not germinated. This paper provides new information about the inhibition of spores by the combination of HP and nisin. The high synergy observed at low temperature has not been reported yet and could allow food preservation without the use of any thermal process.

Analysis of multivariate images in fluorescence microscopy.

A multivariate image is an image stack in which each pixel contains several variables. Such images are common in many fields (medicine, imaging microscopy, satellite imaging...) and their analysis requires adapted multivariate statistical methods. In fluorescence imaging microscopy, different probes or different measurements such as intensity, fluorescence lifetime or spectral information can be observed from one view. However, this is not yet analysed as multivariate images. Here, we are presenting a full approach of multivariate analysis of fluorescence microscopy images and we are proposing a free R package (multifluo) to conduct it.

Inactivation of dried spores of Bacillus subtilis 168 by a treatment combining high temperature and pressure.

Specific treatments combining high temperatures of up to 150 °C and moderate pressure of up to 0.6 MPa have been applied to Bacillus subtilis 168 spores conditioned at different aw levels (between 0.10 and 0.70) corresponding to different residual water contents within the spore core. The spores were treated as a dry powder in a pressurized nitrogen environment or in water/glycerol solutions. These thermodynamic conditions were intended to prevent any water evaporation from the spore core during time/temperature treatments. Our results clearly show that retaining liquid water in the core by applying pressure during the treatment resulted in greater spore destruction (between 2.4 and 4.9 log at 150 °C, 120 s and aw 0.5 in powder) than the destruction observed after the treatment at atmospheric pressure (0.7 log), during which the water rapidly evaporated because its boiling point was reached. Moreover, we found that the water activity level of the spore had a significant impact on spore destruction: the higher the aw level, the greater the spore inactivation. We obtained similar results from spores heat-treated in powder and in water/glycerol solution at the same aw, confirming the strong influence of this parameter. We hypothesized that the increased spore inactivation was related to the well-known thermal sensitivity of vital organic molecules such as proteins, enzymes, and ribosomes in the presence of water.

Understanding the Effects of High Pressure on Bacterial Spores Using Synchrotron Infrared Spectroscopy.

Bacterial spores are extremely resistant life-forms that play an important role in food spoilage and foodborne disease. The return of spores to a vegetative cell state is a three-step process, these being activation, germination, and emergence. High-pressure (HP) processing is known to induce germination in part of the spore population and even to inactivate a high number of Bacillus spores when combined with other mild treatments such as the addition of nisin. The aim of the present work was to investigate the mechanisms involved in the sensitization of spores to nisin following HP treatment at ambient temperature or with moderate heating leading to a heterogeneous spore response. Bacillus subtilis spores were subjected to HP treatment at 500 MPa at 20 and 50°C. The physiological state of different subpopulations was characterized. Then Fourier transform infrared (FTIR) microspectroscopy coupled to a synchrotron infrared source was used to explore the heterogeneity of the biochemical signatures of the spores after the same HP treatments. Our results confirm that HP at 50°C induces the germination of a large proportion of the spore population. HP treatment at 20°C generated a subpopulation of ungerminated spores reversibly sensitized to the presence of nisin in their growth medium. Regarding infrared spectra of individual spores, spores treated by HP at 50°C and germinated spores had similar spectral signatures involving the same structural properties. However, after HP was performed at 20°C, two groups of spores were distinguished; one of these groups was clearly identified as germinated spores. The second group displayed a unique spectral signature, with shifts in the spectral bands corresponding to changes in membrane fluidity. Besides, spores spectra in the amide region could be divided into several groups close to spectral properties of dormant, germinated, or inactivated spores. The part of the spectra corresponding to α-helix and ß-sheet-structures contribute mainly to the spectral variation between spores treated by HP at 20°C and other populations. These changes in the lipid and amide regions could be the signature of reversible changes linked to spore activation.

Cell Death Mechanisms Induced by Photo-Oxidation Studied at the Cell Scale in the Yeast Saccharomyces cerevisiae.

Blue light (400-430 nm) is known to induce lethal effects in some species of fungi by photo-oxidation caused by the excitation of porphyrins but the mechanisms involved remain poorly understood. In this work, we exposed the yeast Saccharomyces cerevisiae to a high density light flux with two-photon excitation (830 nm equivalent to a one-photon excitation around 415 nm) and used quasi real-time visualization with confocal microscopy to study the initiation and dynamics of photo-oxidation in subcellular structures. Our results show that the oxidation generated by light treatments led to the permeabilization of the plasma membrane accompanied by the sudden expulsion of the cellular content, corresponding to cell death by necrosis. Moreover, excitation in the plasma membrane led to very fast oxidation and membrane permeabilization (<60 s) while excitation at the center of the cell did not induce permeabilization even after a period exceeding 600 s. Finally, our study shows that the relationship between the laser power used for two-photon excitation and the time required to permeabilize the plasma membrane was not linear. Thus, the higher the power used, the lower the energy required to permeabilize the plasma membrane. We conclude that fungal destruction can be generated very quickly using a high density light flux. Better knowledge of the intracellular processes and the conditions necessary to induce necrosis should make it possible in the future to improve the efficiency of antimicrobial strategies photo-oxidation-based.

Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder.

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20-40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70-80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time-temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product.

Cellular imaging using BODIPY-, pyrene- and phthalocyanine-based conjugates.

Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in the green/red have been synthesized (as the form of dyads-pentads), studied by spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanine-pyrene 1 was achieved by cyclotetramerization of pyrenyldicyanobenzene, whereas phthalocyanine-BODIPY 2c was synthesized by Sonogashira coupling between tetraiodophthalocyanine and meso-alkynylBODIPY. The standard four-steps BODIPY synthesis was applied to the BODIPY-pyrene dyad 3 starting from pyrenecarbaldehyde and dimethylpyrrole. 1H, 13C, 19F, 11BNMR, ICP, MS, and UV/Vis spectroscopic analyses demonstrated that 2c is a mixture of BODIPY-Pc conjugates corresponding to an average ratio of 2.5 BODIPY per Pc unit, where its bis, tris, tetrakis components could not be separated. Fluorescence emission studies (µM concentration in THF) showed that the design of the probes allowed excitation of their antenna (pyrene, BODIPY) in the blue/green region of the spectrum, and subsequent transfer to the acceptor platform (BODIPY, phthalocyanine) followed by its emission in the green/red (with up to 140-350 nm overall Stokes shifts). The fluorescent probes were used for cellular imaging of B16F10 melanoma cells upon solubilization in 1% DMSO containing RPMI or upon encapsulation in liposomes (injection method). Probes were used at 1-10 µM concentrations, cells were fixed with methanol and imaged by biphoton and/or confocal microscopy, showing that probes could achieve the staining of cells membranes and not the nucleus.

Caco-2 Invasion by Cronobacter sakazakii and Salmonella enterica Exposed to Drying and Heat Treatments in Dried State in Milk Powder.

Due to the ability of foodborne pathogens to survive in low moisture food, the decontamination of milk powder is an important issue in food protection. The safety of food products is, however, not always insured and the different steps in the processing of food involve physiological and metabolic changes in bacteria. Among these changes, virulence properties may also be affected. In this study, the effect of drying and successive thermal treatments on the invasion capacity of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii was assessed. Bacteria were dried on milk powder at three different water activity levels (0.25, 0.58, and 0.80) and heated at two different temperatures (90°C and 100°C) for 30 and 120 s. After recovery, stressed bacterial populations were placed in contact with Caco-2 cells to estimate their invasion capacity. Our results show that drying increases the invasion capacity of foodborne pathogens, but that heat treatment in the dried state did not exert a selective pressure on bacterial cells depending on their invasion capacity after drying. Taken together, our findings add to the sum of knowledge on food safety in dried food products and provide insight into the effects of food processing.

Modeling the heat inactivation of foodborne pathogens in milk powder: High relevance of the substrate water activity.

Due to the ability of foodborne pathogens to survive in low moisture foods, the decontamination of these products is an important issue in food hygiene. Up to now, such decontamination has mostly been achieved through empirical methods. The intention of this work is to establish a more rational use of heat treatment cycles. The effects of thermal treatment cycles on the inactivation of dried Salmonella Typhimurium, Salmonella Senftenberg, Cronobacter sakazakii and Escherichia coli were assessed. Bacteria were mixed with whole milk powder and dried down to different water activity levels (0.11, 0.25, 0.44 and 0.58). The rate of inactivated bacteria was determined after thermal treatment at 85°C, 90°C, 95°C and 100°C, from 0s to 180s in closed vessels, in order to maintain aw during treatment. In a first step, logarithmic bacterial inactivation was fitted by means of a classical loglinear model in which temperature and aw have a significant effect (p<0.05). DT,aw values were estimated for each T, aw condition and the results clearly showed that aw is a major parameter in the thermal decontamination of dried foods, a lower aw involving greater thermal resistance. In a second step, Bigelow's law was used to determine zT, a classical parameter relative to temperature, and yaw values, a new parameter relative to aw resistance. The values obtained for zT and yaw showed that the bacterium most resistant to temperature variations is Salmonella Typhimurium, while the one most resistant to aw variations is Escherichia coli. These data will help design decontamination protocols or processes in closed batches for low moisture foods.

Drying parameters greatly affect the destruction of Cronobacter sakazakii and Salmonella Typhimurium in standard buffer and milk.

Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal infections from milk powder and infant formula. Their ability to survive in low-moisture food and during processing from the decontamination to the dried state is a major issue in food protection. In this work, we studied the effects of the drying process on Salmonella Typhimurium and Cronobacter sakazakii, with the aim of identifying the drying parameters that could promote greater inactivation of these two foodborne pathogens. These two bacteria were dried under different atmospheric relative humidities in milk and phosphate-buffered saline, and the delays in growth recovery and cultivability were followed. We found that water activity was related to microorganism resistance. C. sakazakii was more resistant to drying than was S. Typhimurium, and milk increased the cultivability and recovery of these two species. High drying rates and low final water activity levels (0.11-0.58) had a strong negative effect on the growth recovery and cultivability of these species. In conclusion, we suggest that effective use of drying processes may provide a complementary tool for food decontamination and food safety during the production of low-moisture foods.

Recovery Estimation of Dried Foodborne Pathogens Is Directly Related to Rehydration Kinetics.

Drying is a common process which is used to preserve food products and technological microorganisms, but which is deleterious for the cells. The aim of this study is to differentiate the effects of drying alone from the effects of the successive and necessary rehydration. Rehydration of dried bacteria is a critical step already studied in starter culture but not for different kinetics and not for pathogens. In the present study, the influence of rehydration kinetics was investigated for three foodborne pathogens involved in neonatal diseases caused by the consumption of rehydrated milk powder: Salmonella enterica subsp. enterica serovar Typhimurium, Salmonella enterica subsp. enterica serovar Senftenberg and Cronobacter sakazakii. Bacteria were dried in controlled relative humidity atmospheres and then rehydrated using different methods. Our results showed that the survival of the three pathogens was strongly related to rehydration kinetics. Consequently, rehydration is an important step to consider during food safety assessment or during studies of dried foodborne pathogens. Also, it has to be considered with more attention in consumers' homes during the preparation of food, like powdered infant formula, to avoid pathogens recovery.

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores.

In this work, we investigated how a combination of ethanol and high temperature (70°C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C12 to study the microscopic viscosity in the inner membrane of B. subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000cP to 860cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of B. subtilis spores, as well as demonstrates the ability of FLIM - Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation.

Innovative High Gas Pressure Microscopy Chamber Designed for Biological Cell Observation.

An original high-pressure microscopy chamber has been designed for real-time visualization of biological cell growth during high isostatic (gas or liquid) pressure treatments up to 200 MPa. This new system is highly flexible allowing cell visualization under a wide range of pressure levels as the thickness and the material of the observation window can be easily adapted. Moreover, the design of the observation area allows different microscope objectives to be used as close as possible to the observation window. This chamber can also be temperature controlled. In this study, the resistance and optical properties of this new high-pressure chamber have been tested and characterized. The use of this new chamber was illustrated by a real-time study of the growth of two different yeast strains - Saccharomyces cerevisiae and Candida viswanathii - under high isostatic gas pressure (30 or 20 MPa, respectively). Using image analysis software, we determined the evolution of the area of colonies as a function of time, and thus calculated colony expansion rates.

Effects of hydrostatic pressure on yeasts isolated from deep-sea hydrothermal vents.

Hydrostatic pressure plays a significant role in the distribution of life in the biosphere. Knowledge of deep-sea piezotolerant and (hyper)piezophilic bacteria and archaea diversity has been well documented, along with their specific adaptations to cope with high hydrostatic pressure (HHP). Recent investigations of deep-sea microbial community compositions have shown unexpected micro-eukaryotic communities, mainly dominated by fungi. Molecular methods such as next-generation sequencing have been used for SSU rRNA gene sequencing to reveal fungal taxa. Currently, a difficult but fascinating challenge for marine mycologists is to create deep-sea marine fungus culture collections and assess their ability to cope with pressure. Indeed, although there is no universal genetic marker for piezoresistance, physiological analyses provide concrete relevant data for estimating their adaptations and understanding the role of fungal communities in the abyss. The present study investigated morphological and physiological responses of fungi to HHP using a collection of deep-sea yeasts as a model. The aim was to determine whether deep-sea yeasts were able to tolerate different HHP and if they were metabolically active. Here we report an unexpected taxonomic-based dichotomic response to pressure with piezosensitve ascomycetes and piezotolerant basidiomycetes, and distinct morphological switches triggered by pressure for certain strains.

Visualization of RNA-Quadruplexes in Live Cells.

Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

Harnessing medically relevant metals onto water-soluble subphthalocyanines: towards bimodal imaging and theranostics.

Subphthalocyanine (SubPc), a putative fluorophore for optical imaging (OI), was conjugated to chelating ligands (DOTA, DTPA) affording water-soluble conjugates complexed with (non-radioactive) metals relevant to the following medical imaging techniques/therapies: MRI (Gd), PET (Cu, Ga), SPECT (In, Ga, Lu), RIT (Cu, Lu, Y), and NCT (Gd). Magneto-optical properties of ditopic gadolinium species (and optical properties of other metal containing species) were examined (brightness (ε × ΦF) and relaxivity R1) and fluorescence confocal/biphoton microscopy studies were conducted.