Rym Boudjemaa
email: rym.boudjemaa@u-psud.fr

Romain Briandet

Matthieu Revest

Cédric Jacqueline

Jocelyne Caillon

Marie-Pierre Fontaine-Aupart

Karine Steenkeste

New insight into daptomycin bioavailability and localization in S. aureus biofilms by dynamic fluorescence imaging

Staphylococcus aureus (S. aureus) is one of the most frequent pathogens responsible for biofilmassociated 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 implantassociated S. aureus infections in clinical practice while its association with rifampicin 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 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 to eradicate biofilms while the matrix was not a shield to the antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampicin, daptomycin was still present in the vicinity of the bacterial cells allowing the prevention of the emergence of rifampicin-resistant mutants.

Conclusions derived from this study strongly suggest that S. aureus biofilms resistance/tolerance towards daptomycin may be more likely related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.

INTRODUCTION

Staphylococcus aureus (S. aureus) is a Gram-positive bacteria shown to be the most frequent cause of biofilm-associated infections (BAI) [START_REF] Sun | Biofilm-associated infections: antibiotic resistance and novel therapeutic strategies[END_REF] and one of the major cause of morbidity and mortality in hospitals and communities [START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF]. Unlike planktonic cells, biofilms exhibit specific phenotypic traits allowing them to resist host defenses and antibiotic treatments [START_REF] Lebeaux | Tolérance des biofilms aux antibiotiques : comprendre pour mieux traiter[END_REF], which frequently leads to chronic infections such as endocarditis, sinusitis, and osteomyelitis but also to implant-associated infections [START_REF] Costerton | The Role of Bacterial Surface Structures in Pathogenesis[END_REF].

Among the most recent clinically-used antibiotics, daptomycin is a cyclic lipopeptide approved for the treatment of serious staphylococcal infections such as bacteremia and implant-related infections [START_REF] Steenbergen | Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections[END_REF]. Daptomycin is a calcium-dependent antibiotic, acting by insertion into the Gram-positive cytoplasmic membranes where it forms oligomeric pores, causing potassium ion leakage and subsequent membrane depolarization, leading ultimately to cell death [START_REF] Straus | Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides[END_REF]. As is the case for many antibiotics, daptomycin has been shown to exhibit a significant bactericidal activity against planktonic cells [START_REF] Mascio | Bactericidal Action of Daptomycin against Stationary-Phase and Nondividing Staphylococcus aureus Cells[END_REF][START_REF] Cotroneo | Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus[END_REF][START_REF] Silverman | Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus[END_REF]. However, the eradication of adherent bacteria is rarely achieved despite the large number of in vitro and animal studies in which daptomycin activity was evaluated [START_REF] Khasawneh | In vitro testing of daptomycin plus rifampin against methicillin-resistant Staphylococcus aureus resistant to rifampin[END_REF][START_REF] Laplante | Activities of Daptomycin and Vancomycin Alone and in Combination with Rifampin and Gentamicin against Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Isolates in an Experimental Model of Endocarditis[END_REF][START_REF] Parra-Ruiz | Activities of High-Dose Daptomycin, Vancomycin, and Moxifloxacin Alone or in Combination with Clarithromycin or Rifampin in a Novel In Vitro Model of Staphylococcus aureus Biofilm[END_REF][START_REF] Olson | Rifampicin enhances activity of daptomycin and vancomycin against both a polysaccharide intercellular adhesin (PIA)-dependent andindependent Staphylococcus epidermidis biofilm[END_REF][START_REF] Nadrah | Antibiotic Combinations with Daptomycin for Treatment of Staphylococcus aureus Infections[END_REF][START_REF] Cirioni | Daptomycin and Rifampin Alone and in Combination Prevent Vascular Graft Biofilm Formation and Emergence of Antibiotic Resistance in a Subcutaneous Rat Pouch Model of Staphylococcal Infection[END_REF][START_REF] Salem | Pharmacodynamic assessment of vancomycinrifampicin combination against methicillin resistant Staphylococcus aureus biofilm: a parametric response surface analysis[END_REF]. Besides the results of the literature that appear controversial [START_REF] John | Reversible Daptomycin Tolerance of Adherent Staphylococci in an Implant Infection Model[END_REF], direct comparison between studies is not directly possible since biofilm growth and treatment protocols used greatly differ.

To get a realistic representation of daptomycin action against S. aureus biofilms, we developed an in vitro model built on the basis of our recently published in vivo study on S. aureus prosthetic vascular graft infections using the same strains [START_REF] Revest | New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection[END_REF]. The interest of this approach was the possibility to use fluorescence imaging techniques that cannot be employed in vivo (confocal laser scanning microscopy, time-lapse and fluorescence recovery after photobleaching) to examine the penetration, diffusion, bioavailability and localization of the fluorescently-labelled antibiotic inside the biofilms. To validate this approach, we monitored for 72 h the activity of daptomycin against biofilms formed by methicillin-susceptible and methicillin-resistant clinical and collection strains. In addition, we enriched the culture medium with proteins and calcium to mimic the in vivo physiological conditions of our mouse model [START_REF] Revest | New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection[END_REF]. The same experiments were performed in the presence of rifampicin, an antibiotic that can be combined with daptomycin for recalcitrant S. aureus BAI.

MATERIALS AND METHODS

Bacterial strains

Four S. aureus strains were tested in the present study: two of them were collection strains (methicillinsusceptible S. aureus (MSSA) ATCC 27217 and methicillin-resistant S. aureus (MRSA) ATCC 33591) and two others were isolated from patients with S. aureus bloodstream infections (MSSA 176 and MRSA BCB8). All the strains were kept at -80°C in Tryptic Soy Broth (TSB; Biomérieux, France) containing 20% (vol/vol) glycerol. The frozen cells were subcultured twice in TSB (one 8-hour culture followed by an overnight culture) to constitute the stock cultures from which aliquots were kept at -20°C. Bacterial growth and experiments were both conducted at 37°C.

Antimicrobial agents and medium

Daptomycin and rifampicin were both purchased from Sigma (France). The fluorescently-labelled antibiotic BODIPY-FL®-daptomycin was a kind gift from Cubist Pharmaceuticals (MA, USA) and BODIPY-FL® was purchased from Invitrogen (France). According to the manufacturer's instructions, the stock solutions were prepared by diluting daptomycin and BODIPY®-FL-daptomycin in dimethylsulfoxide (1 mg/mL), and rifampicin in sterile water (2 mg/mL), further kept at -20°C. Before use, the solutions were diluted in TSB enriched with proteins (BSA, Bovine Serum Albumin, 36 g/L; Sigma, France) and calcium (CaCl 2 , 2H 2 O, 50 mg/L; Sigma, France) to mimic in vivo physiological levels. It has been checked that, in these conditions, the final concentration of dimethylsulfoxide was non-cytotoxic for the bacteria. Clinically-meaningful concentrations were used in this study: 20 µg/mL for both daptomycin and rifampicin. When combined, the antibiotics were mixed together before application above the biofilm surface.

Susceptibility testing

The MICs of daptomycin and rifampicin were determined by the broth microdilution method in cationadjusted Mueller-Hinton broth (CAMHB), according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Media were supplemented with 50 mg/L Ca 2+ for daptomycin.

Characterization of molecular interactions between daptomycin and rifampicin by absorption and fluorescence spectroscopies

Absorption spectra of antibiotics alone and in combination were measured with a Varian's Cary® 300 spectrophotometer (Agilent Technologies, France). The corresponding fluorescence emission spectra were recorded using a Fluorolog-3 (Jobin Yvon, Inc., France) fluorescence spectrophotometer mounted with front-face detection geometry by exciting daptomycin at 360 nm. The measurements were made in quintuplicate on each sample.

In vitro biofilm preparation and antibiotics activities

Biofilms were studied in a polystyrene microtiter plate-based assay since it has been shown that this material has physicochemical properties close to that of biomaterial surfaces such as polyethylene terephthalate that is used in vascular grafts [START_REF] Wang | Adhesion of Staphylococcus epidermidis to biomedical polymers: contributions of surface thermodynamics and hemodynamic shear conditions[END_REF][START_REF] Mafu | Adhesion of Pathogenic Bacteria to Food Contact Surfaces: Influence of pH of Culture[END_REF]. For the preparation of S. aureus biofilms, 250-µL portions of an overnight subculture adjusted to an optical density of 0.02 at 600 nm (corresponding to ~10 8 CFU/mL) were added to 96-well microplates (µClear; Greiner Bio-One, France). After a 1.5-h adhesion period at 37°C, the wells were rinsed with sterile physiological water (150 mM NaCl) in order to eliminate non-adherent cells, refilled with sterile TSB enriched with proteins and calcium (TSBpc) and then incubated for 24 h at 37°C to allow biofilm growth.

To assess antibiotics activities, the 24 h-biofilms were rinsed with a 150 mM NaCl aqueous solution before adding the antibiotics solutions diluted in TSBpc as described previously. Viable culturable bacteria were then counted at regular interval times: 0 h (when antibiotics are added), 24, 48 and 72 h after antibiotics injection. For each time, bacterial cultures were centrifuged 10 min at 7000g in order to eliminate the excess of antibiotic. The bacterial pellet was dispersed in NaCl 150 mM sterile saline solution, centrifuged again and dispersed in the same conditions. Successive decimal dilutions were then realized. For each dilution, six drops (10 µL) were deposited on Tryptic Soy Agar (TSA) plates (Biomérieux, France) and incubated at 37°C during 24 h. CFUs were counted and averaged for each dilution at each time. The detection limit of viable culturable cells was 100 CFU/mL.

Percentage of rifampicin-resistant mutants in biofilms

To measure percentages of rifampicin-resistant mutants, the antibiotics solutions (rifampicin combined or not with daptomycin) were added to 24 h-biofilms. CFUs were counted on TSA plates containing or not rifampicin (20 µg/mL) at 24 and 48 h after antibiotics injections. The percentages were obtained by calculating the ratio between the number of CFUs grown on rifampicin-containing TSA plates and the number of CFUs grown on rifampicin-free TSA plates.

Statistical analysis

The mean log 10 CFU/mL and biovolumes for each therapy were compared with each other using ANOVA variance analyses. They were performed using the Statgraphics software (Manugistics, Rockville, USA). Significance was defined as a P-value associated with a Fisher test value lower than 0.05.

Confocal Laser Scanning Microscopy (CLSM)

Visualization of antibiotics activities against biofilms using Live/Dead® staining 24 h-biofilms prepared as described previously were observed 24, 48 and 72 h after antibiotics addition using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, France) implemented at the Centre de Photonique Biomédicale (CPBM, Orsay, France). Prior to each observation, bacteria were stained with 2.5 µM Syto9® (Life Technologies, France), a green cell-permeant nucleic acid dye, and 30 µM propidium iodide (PI) (Life Technologies, France), a red nucleic acid dye that can penetrate cells with compromised membranes (dead cells) only. Syto9® and PI were sequentially excited at 488 nm and 543 nm respectively and their fluorescence emissions were collected between 500 and 600 nm for Syto9® and between 640 and 750 nm for PI. Images were acquired using a 63x oil immersion objective with a 1.4 numerical aperture. The size of the confocal images was 512 x 512 pixels (82 x 82 µm²), recorded with a z-step of 1 µm. For each biofilm, four typical regions were analyzed. Images were reconstructed in 3D using ICY® Software.

Quantification of biovolumes and maximum thickness

Maximum thickness (µm) was measured directly from xyz stacks. Biovolumes (µm 3 ) were calculated by binarizing images with a java script executed by ICY as described previously [START_REF] Sanchez-Vizuete | Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities[END_REF]. The biovolume was then defined as the overall volume of cells in the observation field. The percentage of dead cells corresponds to the ratio between biovolumes of PI-stained bacteria and Syto9®-stained bacteria.

Antibiotics penetration and localization inside biofilms

To study the penetration of BODIPY®-FL-daptomycin and its combination with rifampicin within 24 h-biofilms, we employed time-lapse microscopy, as described before [START_REF] Oubekka | Correlative timeresolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms[END_REF], using the same Leica TCS SP5 confocal microscope. Briefly, the fluorescence intensity evolution over time was observed in a defined focal plane (5 µm above the substratum surface). As soon as the TSBpc-diluted solutions of BODIPY-FL®-daptomycin combined or not with rifampicin were added above the biofilm, fluorescence intensity images were acquired every second during 15 min. Simultaneously, transmission images were acquired to ensure that no structural alteration of the biofilm occurred during this process.

The labelled antibiotic was excited with a continuous argon laser line at 488 nm through a 63x oil immersion objective and the emitted fluorescence was recorded within 500 and 600 nm.

The corresponding diffusive penetration coefficients (D p ) through the biofilms were determined according to the relationship previously described by Stewart (23):

D p = 1.03 x L²/t 9 0 (Eq. 1)
where L is the biofilm thickness and t 90 is the time required to attain 90% of the equilibrium staining intensity at the deeper layers of the biofilm.

To observe the localization of the fluorescently-labelled daptomycin within biofilms, bacteria were counterstained with the FM4-64 dye (Life Technologies, France): this dye was also excited at 488 nm but its fluorescence emission was collected within the range 640-750 nm. The images (512 x 512 pixels) of both fluorophores were simultaneously recorded with a z-step of 1 µm.

Antibiotic diffusion and bioavailability inside biofilms using FRAP experiments

Image-based Fluorescence Recovery after Photobleaching (FRAP) measurements was used to assess local diffusion and bioavailability of the fluorescently-labelled daptomycin. Briefly, FRAP is based on a brief excitation of fluorescent molecules by a very intense light source in a user-defined region to irreversibly photobleach their fluorescence. Fluorescence recovery is then probed over time at a low light power in the same photobleached region [START_REF] Oubekka | Correlative timeresolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms[END_REF][START_REF] Oubekka | Imagebased fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusionreaction processes in Staphylococcus aureus[END_REF]. All time-resolved measurements were obtained using the same confocal microscope. The time course of fluorescence intensity recovery was analyzed with mathematical models, giving access to the quantitative mobility of the fluorescent molecules and allowing to determine the diffusion coefficients. For all FRAP experiments, the fluorescence intensity image size was fixed to 512 x 128 pixels with an 80-nm pixel size and recorded using a 12-bit resolution. The line scan rate was fixed to 1400 Hz, corresponding to a total time between frames of ~ 265 ms. As determined previously, the full widths at half maximum in xy and z (along the optical axis)

of the bleached profile were 0.8 µm and 14 µm, respectively, allowing to neglect diffusion along the axial/vertical axis and thus to consider only two-dimensional diffusion. Each FRAP experiment started with the acquisition of 50 images at 7% of laser maximum intensity (7 µW) followed by a 200-ms single bleached spot at 100% laser intensity. A series of 450 single section images was then collected with the laser power attenuated to its initial value (7% of the bleach intensity). The first image was recorded 365 ms after the beginning of bleaching.

RESULTS

Susceptibility testing

The MICs obtained for daptomycin and rifampicin against the four S. aureus strains are presented in Table 1. All isolates in planktonic conditions were susceptible to daptomycin and rifampicin.

Breakpoint values of drugs according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2016 are: 2 mg/L for vancomycin; 1 mg/L for daptomycin; for rifampicin S ≤ 0.06 and R > 0.5 mg/L.

Spectroscopic characterization of the interaction between rifampicin and daptomycin

Neither the photonic absorption properties nor the fluorescence emission spectrum of daptomycin were significantly influenced by the addition of rifampicin (Fig. 1), revealing the absence of cross-reaction between the two antibiotics.

BODIPY-FL®-daptomycin penetration, diffusion, bioavailability and localization inside S. aureus biofilms

Time-lapse imaging: time-lapse experiments were performed to visualize in situ the penetration of the fluorescently-labelled daptomycin and its combination with rifampicin throughout the deepest layers of S. aureus biofilms. By setting the focal plane 5 µm above the substratum surface, we demonstrated that BODIPY-FL®-daptomycin penetrated the biofilms (~30 µm thickness) within few minutes:

fluorescence intensity was measured a few seconds after antibiotic addition and increased rapidly to reach 90% of the maximal intensity in 9 min (Fig. S1 and Movie S4). The penetration coefficients obtained from Eq. 1 ranged from 2.5 ± 0.7 µm²/s for S. aureus 27217, 176 and 33591 to 4.9 µm²/s ± 0.7 µm²/s for S. aureus BCB8. These values are both of the same order by comparison with BODIPY-FL® alone for which the penetration coefficient is superior to 140 µm²/s [START_REF] Oubekka | Correlative timeresolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms[END_REF]. The coefficients were not statistically different in comparison with those of daptomycin in the presence of rifampicin.

FRAP imaging: FRAP was used to measure the local diffusion of BODIPY-FL®-daptomycin and its interaction with bacteria within biofilms. According to the FRAP principle [START_REF] Oubekka | Correlative timeresolved fluorescence microscopy to assess antibiotic diffusion-reaction in biofilms[END_REF][START_REF] Oubekka | Imagebased fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusionreaction processes in Staphylococcus aureus[END_REF][START_REF] Bridier | Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis[END_REF], if the fluorescently-labelled daptomycin molecules are allowed to move freely in the sample, a total fluorescence recovery is observed, meaning that the fluorescence is redistributed in the defined region.

Conversely, if the fluorescence recovery is not total after the photobleaching, it means that a fraction of molecules is not diffusing freely and thus interacts with its local environment. The other fraction diffuses and is thus bioavailable.

First, we checked that no bacterial movement occurred during image acquisition by representing kymograms, two-dimensional graphs of fluorescence intensity measured along a line (here a straight line drawn on the full width of the images) for each image of the FRAP series (Fig. 2a). A typical FRAP curve of BODIPY-FL®-daptomycin in S. aureus biofilms is presented in Fig. 2b. Whatever the bacterial strains or the treatments used (daptomycin alone and in combination with rifampicin), we demonstrated here that the fluorescence recovery was not total after photobleaching: only 20% of BODIPY-FL-®-daptomycin molecules interacted with the environment, meaning that a large excess of molecules (80%) were diffusing freely in the defined-regions and thus bioavailable (mean local diffusion coefficient, 7.1 +/-0.6 µm²/s).

susceptible to daptomycin: ~ 60 % of cell death was quantified at 24 h, a value that decreased beyond (40% at 72 h, P < 0.05) due to cell regrowth (see below).

By comparison with the monotherapy treatment, the biofilms structures and thicknesses were not affected when they were treated with daptomycin in combination with rifampicin. However, in these conditions, a significantly higher proportion of cell death was observed, achieving 85% at 72 h (P < 0.01) (Fig. 4b). For the two MSSA strains (27217, 176) and the MRSA collection strain (33591), the antibiotics association activity gradually increased over time (Fig. 4) while the maximum activity against the MRSA clinical strain (BCB8) was reached within 24 h.

Another observation of interest (Fig. 4a) is that upon daptomycin exposure, dead cells were observed over the whole biofilm depth, including the basal layer of cells in contact with the substratum, providing further evidence of the antibiotic penetration throughout the deepest layers of the biofilms.

This result is even more observable for the BCB8 strain because of the greater proportion of dead cells involved by the action of daptomycin. This process was also observed when daptomycin was used in association with rifampicin.

These data from fluorescence imaging were supported by CFU counts of suspended biofilms (Fig. 5).

The results confirm that daptomycin alone was ineffective against biofilms formed by the two MSSA strains (27217, 176) and the MRSA collection strain (33591). For the MRSA clinical isolate (BCB8), only ~1-2-log reduction in bacterial counts was measured at 24 h before regrowth was observed to reach the same values as for the MSSA biofilms.

In accordance with fluorescence imaging data, the activity of the antibiotics combination was much greater than the monotherapy. The sensitivity of the microbiological method allowed determining that the cell population decreased by ~ 4 log after 72 h of treatment (P < 0.05). The emergence of rifampicin-resistant mutants was verified. As presented in Fig. 6, daptomycin dramatically prevented the emergence of rifampicin-resistant mutants when used in combination.

DISCUSSION

The choice of antibiotics to treat S. aureus BAI remains a challenge for the medical community.

In this context, the ambivalence of the published results on daptomycin activity is a relevant example.

Despite increasing data about daptomycin as an option to treat implant-associated S. aureus infections, as many failures [START_REF] Revest | New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection[END_REF][START_REF] Humphries | A Current Perspective on Daptomycin for the Clinical Microbiologist[END_REF] as successes [START_REF] Mascio | Bactericidal Action of Daptomycin against Stationary-Phase and Nondividing Staphylococcus aureus Cells[END_REF][START_REF] Cotroneo | Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus[END_REF][START_REF] Silverman | Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus[END_REF][START_REF] Parra-Ruiz | Activities of High-Dose Daptomycin, Vancomycin, and Moxifloxacin Alone or in Combination with Clarithromycin or Rifampin in a Novel In Vitro Model of Staphylococcus aureus Biofilm[END_REF] have been reported both in clinical practice and in laboratory models. This highlights that S. aureus BAI resistance/tolerance mechanisms to antimicrobials deserve more attention.

The biofilm-associated exopolymeric matrix may be considered to act as a shield to the antimicrobial diffusion-reaction [START_REF] Pibalpakdee | Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms[END_REF][START_REF] Singh | Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms[END_REF][START_REF] Jefferson | Use of Confocal Microscopy To Analyze the Rate of Vancomycin Penetration through Staphylococcus aureus Biofilms[END_REF][START_REF] Siala | Comparison of the Antibiotic Activities of Daptomycin, Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates[END_REF][START_REF] Stewart | Daptomycin Rapidly Penetrates a Staphylococcus epidermidis Biofilm[END_REF] by delaying its penetration and/or reducing its bioavailability.

To verify this hypothesis non-invasively, we took advantage of dynamic fluorescence imaging methods: confocal microscopy, time-lapse imaging and FRAP. For our biofilm model and whatever the bacterial strain, no failure of daptomycin penetrability or bioavailability was observed. The opposite finding described by Siala et al. [START_REF] Siala | Comparison of the Antibiotic Activities of Daptomycin, Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates[END_REF] may be related to the conditions of fluorescence acquisition that were not well-adapted to BODIPY-FL® fluorescence. In this study, time-lapse fluorescence imaging experiments demonstrated that daptomycin rapidly reached the biofilms deepest layers while section views of fluorescence intensity images presented in Fig. 3 ascertain the presence of the fluorescentlylabelled antibiotic through the whole biofilm structure. Furthermore, FRAP results ascertained that only ~20% of the antibiotic molecules were immobilized. Thus, the majority of the antibiotic molecules were in free movement and could be bioavailable through the biomass (~80% of non-immobilized molecules).

We further addressed the question of whether or not daptomycin reached its bacterial target.

Fluorescence intensity images provided interesting information, showing that the fluorescently-labelled antibiotic was distributed majorly in the extracellular matrix rather than in the bacterial cell membranes (Fig. 3). This is in agreement with the well-known property of daptomycin to have a very high degree of protein-binding, especially with serum albumin (90-93%) [START_REF] Benvenuto | Pharmacokinetics and Tolerability of Daptomycin at Doses up to 12 Milligrams per Kilogram of Body Weight Once Daily in Healthy Volunteers[END_REF][START_REF] Zeitlinger | Protein Binding: Do We Ever Learn?[END_REF] which is naturally present in physiological conditions. Nevertheless, the fluorescence recovery curves obtained by FRAP experiments in free medium and in the biofilms strongly suggested the reversibility of daptomycin protein-binding [START_REF] Zeitlinger | Protein Binding: Do We Ever Learn?[END_REF][START_REF] Schmidt | Effect of Protein Binding on the Pharmacological Activity of Highly Bound Antibiotics[END_REF]: the equilibrium between the bound and unbound states may conserve the apparent mobility of the antibiotic. Additional experiments were performed in a protein-free medium (a saline solution supplemented with calcium ions). In this case, bacterial cell membranes appeared as hot spots on fluorescence images, consistently with the described antibiotic interaction with its target [START_REF] Straus | Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides[END_REF].

Surprisingly, whether the medium was protein-free or not, daptomycin exhibited the same lack of effectiveness, as revealed by time-kill studies performed by fluorescent live-dead staining and conventional plating on agar (data not shown in the absence of proteins). Thus, the interaction with the matrix components cannot explain biofilm tolerance to the antibiotic.

The particular physiology of embedded bacteria should be thus considered and more specifically cells with a low metabolic activity. Previous studies using a BrdU immunofluorescent labelling technique demonstrated that the large majority of staphylococci cells in a biofilm were actually in a low-metabolic state [START_REF] Oubekka | Dynamique réactionnelle d'antibiotiques au sein des biofilms de Staphylococcus aureus : apport de la microscopie de fluorescence multimodale[END_REF][START_REF] Rani | Spatial Patterns of DNA Replication, Protein Synthesis, and Oxygen Concentration within Bacterial Biofilms Reveal Diverse Physiological States[END_REF]. Additionally, in the present study, the comparison of cell viability results obtained by CFU counts and fluorescence imaging highlighted a significant proportion of viable cells detected by Live-Dead® staining but not by CFU measurements. This subpopulation may be considered as viable but non-culturable (VBNC), a subpopulation known to have a slow metabolism [START_REF] Ayrapetyan | Bridging the gap between viable but nonculturable and antibiotic persistent bacteria[END_REF][START_REF] Li | The importance of the viable but nonculturable state in human bacterial pathogens[END_REF][START_REF] Pasquaroli | Role of Daptomycin in the Induction and Persistence of the Viable but Non-Culturable State of Staphylococcus Aureus Biofilms[END_REF]. Moreover, it has been demonstrated that daptomycin is poorly effective against bacteria in stationary stage [START_REF] Mascio | Bactericidal Action of Daptomycin against Stationary-Phase and Nondividing Staphylococcus aureus Cells[END_REF][START_REF] Humphries | A Current Perspective on Daptomycin for the Clinical Microbiologist[END_REF]. One can thus reasonably suggest that for bacteria with low-metabolic activity, the cell membrane dysregulation induced by daptomycin may be slower and/or more difficult to attain due to structural modifications of the cell membrane. This assumption is supported by the reported data revealing that daptomycin displays a concentration-dependent bactericidal activity against dormant cells [START_REF] Mascio | Bactericidal Action of Daptomycin against Stationary-Phase and Nondividing Staphylococcus aureus Cells[END_REF][START_REF] Silverman | Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus[END_REF][START_REF] Parra-Ruiz | Activities of High-Dose Daptomycin, Vancomycin, and Moxifloxacin Alone or in Combination with Clarithromycin or Rifampin in a Novel In Vitro Model of Staphylococcus aureus Biofilm[END_REF]. In the present study, we tested a double concentration of daptomycin (40 µg/mL) on the different S. aureus biofilms, almost leading to bactericidal effects after 24 h of drug exposure (Fig. S3) and showing no cell-regrowth over time.

However, biofilm clearance was not reached. This achievement was reported to occur in a daptomycin concentration equal to or greater than 100 µg/mL but may not be relevant in clinical practice [START_REF] Mascio | Bactericidal Action of Daptomycin against Stationary-Phase and Nondividing Staphylococcus aureus Cells[END_REF][START_REF] Silverman | Correlation of Daptomycin Bactericidal Activity and Membrane Depolarization in Staphylococcus aureus[END_REF][START_REF] Parra-Ruiz | Activities of High-Dose Daptomycin, Vancomycin, and Moxifloxacin Alone or in Combination with Clarithromycin or Rifampin in a Novel In Vitro Model of Staphylococcus aureus Biofilm[END_REF].

One particularity in this study concerns the BCB8 clinical isolate, which discriminated itself by a twice higher penetration coefficient by comparison with the other strains tested and a greater susceptibility as revealed by the observation of a larger proportion of dead cells over the whole biofilm thickness, including the basal layer in contact with the substratum. These results are in line with those obtained in vivo [START_REF] Revest | New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection[END_REF], which demonstrated a strain-dependent activity of daptomycin against S. aureus biofilms. In view of the antibiotic mechanism of action which is supposed to target the plasma membrane, the observed variable response depending on the bacterial strain may be due to a change in membrane composition or conformation from a strain to another.

Facing the lack of daptomycin efficiency in treating recalcitrant S. aureus BAI, the addition of rifampicin has raised great interest (10-13, 15, 40-42). Through this study, we demonstrated that the combined therapy was indeed highly efficient against S. aureus biofilms, but did not allow total bacterial clearance. Both antibiotics have been shown not to cross-react with each other, as evidenced by steady-state fluorescence spectroscopy. Moreover, the penetration, diffusion and localization of the fluorescently-labelled daptomycin were not affected by the presence of rifampicin. We also proved here that rifampicin-resistant mutants emerged when biofilms were treated with rifampicin alone, but not when treated with the antibiotic combination. Altogether, presented data confirm that daptomycin prevents the emergence of rifampicin resistant mutants, allowing the bactericidal activity of rifampicin to quickly occur in time, regardless of the cell physiological state.

In conclusion, consistently with the previous in vivo study aiming at evaluating the antibiotic efficacy in S. aureus prosthetic vascular graft infections [START_REF] Revest | New in vitro and in vivo models to evaluate antibiotic efficacy in Staphylococcus aureus prosthetic vascular graft infection[END_REF], we demonstrated in the present in vitro model a strain-dependent lack of daptomycin activity toward biofilms. Dynamic fluorescence microscopy allowed discarding a lack of antibiotic availability and interaction with bacteria. Given the mode of action of daptomycin, these observations suggest a membrane-dependent factor of tolerance in such biofilms. Therefore, to provide a better understanding of daptomycin reduced activity against biofilms, a further step should concern the analysis of the membranes composition. Dead cells were stained red with propidium iodide and all bacteria were stained green with Syto9®.

The acquisition was performed on the whole biofilm thickness with an axial displacement of 1 µm.

Images dimension is 82x82µm².

Fig. 1 :Fig. 2 :Fig. 3 :

 123 Fig. 1: Absorption (straight lines) and fluorescence spectra (dashed lines) of daptomycin (20 µg/mL) alone and combined to rifampicin (20 µg/mL). Excitation wavelength: 360 nm. Daptomycin alone is represented in black and its combination with rifampicin in grey.

Fig. 4 :

 4 Fig. 4: (a) Visualization of MSSA 27217 and MRSA BCB8 biofilms using 3D reconstruction to observe biofilm thickness. Images were collected without any drug exposure (control) and after 72 h exposure to unlabeled daptomycin (20 µg/mL) alone and in association with rifampicin (20 µg/mL).

Fig. 5 :

 5 Fig. 5: Time-kill curves of daptomycin (20 µg/mL) activities combined or not with rifampicin (20 µg/mL) against MSSA 27217 and MRSA BCB8 biofilms. Filled circles: controls; filled squares: daptomycin; filled triangles: daptomycin + rifampicin. Error bars represent the standard deviation.

Fig. 6 :

 6 Fig. 6: Number of rifampicin-resistant mutants determined in MSSA 27217 biofilms counted on rifampicin-containing TSA plates. Above each bar is shown the percentage of rifampicin-resistant mutants in S. aureus biofilms among the total bacterial population. Error bars represent the standard deviation. White bars, rifampicin alone (20 µg/mL); black bars, daptomycin-rifampicin combination (20 µg/mL for both antibiotics).

ACKNOWLEDGEMENTS

The authors want to thank the Centre de Photonique Biomédicale (CPBM) of the Centre Laser de l'Université Paris-Sud (CLUPS/LUMAT FR2764, Orsay, France) for the confocal microscope and L2 microbiology facilities, Rachel Méallet-Renault for the spectrofluorimeter facilities at the Ecole Normale Supérieure (ENS Cachan) and Jared Silverman (Cubist Pharmaceuticals) for providing BODIPY-FL®-labelled daptomycin.

Localization of the fluorescently-labelled daptomycin combined or not with rifampicin depending on the surrounding environment:

As daptomycin is known to be highly bound to serum proteins (90-93%) [START_REF] Benvenuto | Pharmacokinetics and Tolerability of Daptomycin at Doses up to 12 Milligrams per Kilogram of Body Weight Once Daily in Healthy Volunteers[END_REF], we addressed the question of whether there could be a different localization of the fluorescently-labelled daptomycin in a protein-enriched medium. As shown in Fig. 3, fluorescence intensity images were significantly different depending on the surrounding medium: regardless of the strain tested, the antibiotic appeared to be colocalized with the FM4-64 dye at the bacterial site when the surrounding medium was a NaCl aqueous solution supplemented in calcium while the antibiotic localization appeared to be mainly extracellular when the medium was enriched with proteins (TSBpc).

The addition of rifampicin did not affect daptomycin localization in the biofilm.

Combining 3D-fluorescence imaging and time-kill studies to assess S. aureus biofilms inactivation in the presence of daptomycin alone and in association with rifampicin

We used confocal microscopy to describe the three-dimensional structures of biofilms and the temporal distribution of both live and dead cells throughout the biofilm thickness.

Fluorescence intensity images showed that the biofilms formed by the four strains yielded similar compact structures (Fig. 4a and Fig. S2 controls). Their thicknesses were neither significantly variable from one strain to another (25-29 µm, P > 0.05), nor between 24 h and 72 h. This is a reasonable result given that the culture medium (TSBpc) was renewed before the first observation but not over time: thus biofilm development mainly occurred during the first 24 h. When treated with daptomycin, biofilms exhibited more free-of-cells areas compared with controls (Fig. 4a and Fig. S2). This can be related to significant decreases in biofilms thicknesses (19-21 µm in the presence of daptomycin, P < 0.05) by comparison with the control biofilms. Moreover, in the presence of daptomycin, no statistically significant change in the proportion of cell death over time was quantified (10-30 %, P > 0.05) (Fig. 4b). However, the MRSA clinical isolate (BCB8) was more

FUNDING INFORMATION

This work was supported by a grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, Université Paris-Sud, for Rym Boudjemaa's PhD thesis (grant n° 2014-172).


Michel Sablier
email: michel.sablier@mnhn.fr

Philippe Garrigues
email: p.garrigues@ism.u-bordeaux1.fr

Cultural Heritage and its environment: an issue of interest for Environmental Science and Pollution Research

whether they are published or not. The documents may come

ventilation and air-conditioning systems may act as discernable sources of gaseous pollutants and particulate matter. Moreover, due to the growing popularity of works of art and monuments, the increasing impact of the flow of visitors to monuments, caves or museums may be also influential.

The difficulties faced by curators and persons involved in art conservation under polluting conditions are then relevant to techniques able to provide an answer to the large set of questions raised by the natural and non-natural weathering of Cultural Heritage. As a first approach, this observation confirms how the choice of a journal involved in both environmental and pollution research is suited for the publication of articles reporting studies in a domain relevant to Conservation Sciences.

On the basis of a brief overview of articles published in ESPR within the five last years, this editorial proposes to re-examine the problems of pollution in the domain of Cultural Heritage. This outing amongst the recently published articles in ESPR confirms that atmospheric pollution and air contaminants, airborne microorganisms and heavy metals are the common denominators for recent investigations dealing with Cultural Heritage published in ESPR.

The reviewed articles were mostly referenced under the principal discipline heading "environmental sciences" and under several sub-discipline headings: "pollution and remediation", "environmental chemistry", "environmental health-public health" and "environmental toxicology". Diversity of these sub-headings demonstrates objectively how Cultural Heritage and its surrounding environment inscribes itself in the different scopes of studies of ESPR.

Most of these articles treat the deterioration of monuments, in particular, with the adverse effects of inorganics. Indeed, atmospheric pollution acts as a noticeable accelerating factor in the material deterioration of buildings.

Briefly, gaseous NOx, SO 2 , O 3 , HNO 3 , particulate matter and acid rainfall are the chemicals or the chemical processes responsible for these adverse effects [START_REF] Varotsos | The enhanced deterioration of the cultural heritage monuments due to air pollution[END_REF]. The hazardous pollutants with strong oxidizing power (NO 2 , SO 2 , O 3 ) are of concern for their potential to initiate chain reactions giving rise to acidic substances that can directly act on the various substrates, while volatile organic compounds (VOCs) are monitored for their potential to generate O 3 under lightexposition conditions.

An original approach towards establishing the relation between monuments and pollution [START_REF] Rampazzi | Monuments as sampling surfaces of recent traffic pollution[END_REF] was proposed for the quantitative sampling on the cement mortar surface of a modern sculpture, the Camerlata Fountain in Como, Italy. Based on the consideration that monuments may act as passive samplers, the authors used voltammetry to measure platinum and rhodium concentrations, each of them tracers of automotive emission, and thereby probe for environmental pollution coming from the vehicular pollution.

Chemical analysis of the stained-glass windows and characterization of the patinas developed at their surface are an unavoidable first step in the definition of the best restoration practices to be followed.

For the preservation of stained-glass window in the Pedralbes Monastery, Barcelona, Spain, the combination of X-ray diffraction techniques and Fourier transform infrared spectroscopy techniques has demonstrated its potential for the determination of the degree of weathering via the characterization of their patinas [START_REF] Aulinas | Weathering patinas on the medieval (S. XIV) stained glass windows of the Pedralbes Monastery (Barcelona, Spain)[END_REF]. The preliminary evaluation of the degree of weathering deduced from these analyses was correlated with the composition of the observed patinas, the composition of the original glass as well as the environmental conditions of the deposition in the city of Barcelona.

However, a clear quantification of the impact of the different extrinsic environmental factors of deterioration is difficult. The study of medieval-like model glasses, exposed to simulated atmospheric conditions, permitted the evaluation of the role of both the composition and the surrounding environment on potash-lime glass weathering [START_REF] Gentaz | Early stage of weathering of medievallike potash-lime model glass: evaluation of key factors[END_REF]. This study also highlighted the differentiation to make between wet and dry deposition conditions in the weathering process.

In concert with restoration campaigns, studies can be initiated for a better understanding of factors affecting the degradation of buildings. The restoration project of historical limestone buildings in Bordeaux (France) using laser cleaning among other cleaning methods gave the opportunity to investigate, in the meantime, the composition of the black crust deposited on the surface of the monuments [START_REF] Chapoulie | Laser Cleaning of Historical Limestone Buildings in Bordeaux. Appraisal Using Cathodoluminescence and Electron Paramagnetic Resonance[END_REF]. This study gave an insight into how porous carbon microparticles, atmospheric dusts and aluminosilicate particles can be cemented by gypsum.

Owing to the varying extent of hydration of magnesium sulfate which contributes to the formation of the salt efflorescences, the identification of sulfur sources deserves a particular attention to be able to trace its natural or anthropogenic origin. The isotope ratio measurement of  34 S and  18 O values by isotope ratio mass spectrometry (IRMS) have proven to be an efficient way to clarify the origin of salt efflorescence on buildings in the Old Town of Salamanca, Spain [START_REF] Schleicher | Source identification of sulphate forming salts on sandstones from monuments in Salamanca, Spain-a stable isotope approach[END_REF]. The combination of  34 S and  18 O value measurements has clarified the potential contribution of vehicle exhausts and heating fuel combustion during wintertime whilst the role of mortars was proposed to explain the span of  34 S value distribution among the examined samples. These results demonstrated the need to investigate the isotopic composition of atmospheric aerosols for a definitive attribution of the main sulfur sources in the processes of salt efflorescences that contribute to the alteration and the deterioration of building materials.

Each of these papers highlighted the primary role played by the environment and pollution in the weathering processes. Nevertheless, due to the multiple factors susceptible to explain the origin of weathering, the scope of investigation must be enlarged, and combined studies are often necessary to explain the origin of degradation mechanisms.

The effect of pollution due to air contaminants as well as soluble salts inside churches and monuments is not easily predicted because these contaminants may behave differently on the different materials: for example, wood and brick react differently than stone and mortar to the damaging action of salts and pollutants [START_REF] Morabito | Deposition in St. Mark's Basilica of Venice[END_REF]. Internal or external sources may be deduced from the distribution of elements and pollutants depending on the place of sample collection. However, due to the different behavior of different contaminants on different materials, the effect of pollution inside monuments is not easy to predict. Moreover, synergistic effects are often expected, which call for innovative approaches.

In this context, laser ablation inductively-coupled plasma mass spectrometry associated with traditional micro-morphological and microscopy analysis, and infrared spectroscopy techniques constitute an original methodological approach for the chemical characterization of black crust deposits on stone monuments [START_REF] Barca | Application of laser ablation ICP-MS and traditional techniques to the study of black crusts on building stones: a new methodological approach[END_REF] have demonstrated how the concentration of heavy metals and the composition of black crusts observed on stone surfaces could be a fingerprint of fuel usage over time [START_REF] Belfiore | Application of spectrometric analysis to the identification of pollution sources causing cultural heritage damage[END_REF]). These results corroborated how investigations can reveal in which ways crust formation was greatly influenced by the varying degrees of anthropogenic pollution over time.

Passive sampling was performed to study the coverage rate of airborne particulate matter in the indoor environment of the National Tile Museum, Lisbon (Portugal) in order to evaluate the effectiveness of adding glass panes in the building to restrain airborne pollution inside the museum [START_REF] Anaf | Effects of a constructional intervention on airborne and deposited particulate matter in the Portuguese National Tile Museum, Lisbon[END_REF]). These investigations revealed the utility of particulate matter sampling to control risks of blackening, abrasion, catalysis and biodeterioration favored by particulate matter.

Besides the influence of air pollution, organics and microorganisms are also known for their adverse effects in the preservation of historical works of art. The physical parameters of influence (temperature, relative humidity) in the likely degradation of artworks in confined environments like museums must be controlled, but airborne microorganisms should also be monitored [START_REF] Lee | Cultural heritage: a potential pollution source in museum[END_REF].

Estimation of the influence of microorganisms under exhibition conditions in caves or grottos focuses mainly on the determination of the ratio of the entry and the dispersion of microorganisms and their nutrients into the interior of the cave. This ratio commonly depends on the exchange rates between the inner atmosphere and exterior through the main entrance, galleries or other ventilation outlets.

Among cultural properties, caves represent a special case because they usually show a stable and fragile confined environment that makes them very susceptible to disturbances resulting from human activities. As a consequence, caves are directly impacted by tourism. One effect resides in the erosion caused by mechanical removal of sediment particles. Other evident impacts interrupting the fragile geochemical / environmental balance are the increase of CO 2 concentration, the increase of water vapor and temperature variations. Moreover, these effects may provoke uncontrolled chain reactions at the origin of corrosion, which contribute to rock surface weathering independently from the presence of historical paintings and engravings. The direct impact of visitors, in terms of microbial dispersion, the replacement of the natural microbial communities by alien human-induced microbial populations or the pool of pathogenic microorganisms thriving inside the caves [START_REF] Fernandez-Cortes | Detection of human-induced environmental disturbances in a show cave[END_REF]) is less well known. Application of stable isotope determination experiments ( 13 C) in trace gases has demonstrated its usefulness in the characterization of gas exchange processes to explain the origin of dispersion of the microorganisms in the Altamira cave, Spain where the deterioration of rock art paintings by microbial action has been observed (Garcia-Anton et al. 2013).

The maintenance of very stable microclimates due to limited air exchange with the external atmosphere, a prerequisite for a proper state of conservation until now, bring into question the role of tourism in the preservation of World Heritage through the influence of massive visits and the hitherto abundance of available nutrients provided. Two recent studies illustrate this point: investigations on the Emperor Qin's Terra-Cotta Museum in Xi'an, China (Chen et al. 2010) and the show cave of Ardales, Spain [START_REF] Fernandez-Cortes | Detection of human-induced environmental disturbances in a show cave[END_REF]). In the case of Emperor Qin's Terra-Cotta Museum in Xi'an [START_REF] Cui | Variation of airborne bacteria and fungi at Emperor Qin's Terra-Cotta Museum, Xi'an, China, during the "Oct. 1" Gold Week Period of 2006[END_REF], passive sampling during the congested yearly rush of the goldenweek visits did not reveal microbial concentration in the outer range of regular indoor guidelines. The study focused rather on the side effect of particle sedimentation on surface of displays with the likely release of enzymes and organic acids. As a result, it was stated that investigation to assess the effects of fungal colonization of works of art with regard to any influence of fungal metabolism products is a necessity to determine the risks associated with the presence of fungi on their surfaces as a result of particle sedimentation. These studies shed light on the inner atmosphere dynamics, and contributed to the management and conservation of geological and cultural heritage sites.

Heavy metal poisoning, especially arsenic, is a matter of concern in Cultural Heritage since a large part of museum collections (herbarium, natural history specimens) have been exposed to such metal species mainly for conservation purposes. But the introduction of heavy metals in the environment through the mining and refining of metals is also relevant for environmental studies since the ancient modes of industrial production, agriculture, livestock management and the activities of everyday life may have increased the exposure of ancient populations towards toxic substances in their environment [START_REF] Cattani | Metallurgy, environmental pollution and the decline of Etruscan civilisation[END_REF].

In conclusion, the interface between disciplines currently applied in Conservation Sciences, and Environmental Sciences is particularly well-suited for what can be defined as a fertile field where methodologies, analytical techniques, and ideas issued from each specific scientific area may be optimized and ultimately applied with inventiveness. Cultural heritage materials due to their diversity are extremely challenging in that regard. There is no doubt that the aforementioned articles published during these five last years in ESPR corroborate it.

The entirety of the works reported in the field of environmental pollution applied to Cultural Heritage clearly shows how, today, chemical research associated with these investigations is a very active and productive field. As previously stated elsewhere [START_REF] Mazzeo | Analytical chemistry for cultural heritage: a key discipline in conservation research[END_REF], a new trend in the scientific community involved in conservation consists in focusing its interest into problem-solving approaches that favor studies aimed at providing feasible and durable solutions to specific conservation problems, beside studies focusing on a complete knowledge of the constitution of heritage materials and ancient production techniques.

From the above reviewed articles, won't it be possible to extract guidelines for future works? Noticeably, the standards for the accepted levels of microorganisms are usually set for the health of visitors to museums rather than for the preservation of cultural properties. One must consider that, as the management standards have not been established on the basis of the preservation of cultural properties, it may be necessary to modify these standards for airborne bacteria and fungi for the protection for the cultural properties. This necessitates further investigation.

Notwithstanding that viable airborne organisms are not necessarily air pollutants, they should at least be considered as factors affecting air quality. It is commonly accepted that exposure to large concentrations of airborne microbes is often associated with asthma and rhinitis, hypersensitivity pneumonitis, and to the so-called "sick building" syndrome. Examination of the relation between worker exposure to airborne microbes and health effects would be of great concern in a precautionary approach in considering the health of the personnel working daily in presence of these airborne organisms.

At present, considerable efforts are also under way to develop portable instrumentation for noninvasive analyses to be carried out on-site, where the object is located or exhibited. The act of bringing the laboratory near to the works of art reduces the risk and costs associated with the transportation of precious objects. Equally, it opens the way to the scientific examination of a greater number of works of art. One can expect that the development of new portable instruments (Portable X-Ray Fluorescence, unilateral portable NMR) will be at the origin of in situ experimentations in the scope of the journal.

  . Demonstration of the reliability of the laser ablation technique confirmed how this technique can help in accurate determination of the concentration of various trace elements in the black crusts, providing information on the state of conservation, the degree of deterioration of stones, and extent of pollution. Similarly, combined spectrometric analyses for the identification of pollution sources in three different churches belonging to the European built Heritage: the Corner Palace in Venice (Italy), the Cathedral of St. Rombouts in Mechelen (Belgium) and the Church of St. Eustache in Paris (France),

Concerning outdoor pollution, a future challenge for the influence of environment on the deterioration of built Cultural Heritage consists in the assessment of combined effects in multipollutant situations (for example, the effect of sulfur pollutants in combination with NOx, considering climatic parameters and the concerned materials). Moreover, the use of passive sampling devices that allow the characterization of the unknown deposited mass on control particle collectors besides water-soluble fractions may constitute an open field for further investigation.

All new contributions in the field of Cultural Heritage and its environment in ESPR are thus welcome and will be greatly appreciated.