Hélène Agogué

Emilio O Casamayor

Muriel Bourrain

Ingrid Obernosterer

Fabien Joux

Gerhard Herndl

Philippe Lebaron

A survey on bacteria inhabiting the sea surface microlayer of coastal ecosystems

Bacterial populations inhabiting the sea surface microlayer from two contrasted Mediterranean coastal stations (polluted vs. oligotrophic) were examined by culturig and genetic fingerprinting methods and were compared with those of underlying waters (50 cm depth), for a period of two years. More than 30 samples were examined and 487 strains were isolated and screened. Proteobacteria were consistently more abundant in the collection from the pristine environment whereas Gram-positive bacteria (i.e., Actinobacteria and Firmicutes) were more abundant in the polluted site. Cythophaga-Flavobacter-Bacteroides (CFB) ranged from 8% to 16% of total strains. Overall, 22.5% of the strains showed a 16S rRNA gene sequence similarity only at the genus level with previously reported bacterial species and around 10.5% of the strains showed similarities in 16S rRNA sequence below 93% with reported species. The CFB group contained the highest proportion of unknown species, but these also included Alpha-and Gammaproteobacteria. Such low similarity values showed that we were able to culture new marine genera and possibly new families, indicating that the sea-surface layer is a poorly understood microbial environment and may represent a natural source of new microorganisms. Genetic fingerprinting showed, however, no consistent differences between the predominant bacterial assemblages from surface microlayer and underlying waters, suggesting that the presence of a stable and abundant neustonic bacterial community is not a common trait of coastal marine environments.

Introduction

The marine air-water interface, located between the atmosphere and the hydrosphere, form a specific life environment in the ocean with peculiar and characteristic physicochemical processes [START_REF] Liss | The sea surface and global change[END_REF]. Through this interface a dynamic exchange of gases, water and organic and inorganic compounds occur. Dissolved substances, particles and microorganisms are deposited at this interface by simple diffusion, rising bubbles, convection and upwelling of underlying waters (UW). At the same time, the sea surface microlayer (SML) is a sink for atmospheric deposition [START_REF] Garrett | Stabilization of air bubbles at the air-sea interface by surface-active material[END_REF][START_REF] Norkrans | Surface microlayers in aquatic environments[END_REF].

Microorganisms in the surface microlayer are submitted to a combination of both favourable and detrimental factors. Favourable factors are high concentrations of inorganic and organic nutrients, while detrimental factors are intense solar radiation in the ultraviolet and visible spectra, high concentrations of heavy metals and organic pollutants, temperature fluctuations and salinity changes. Therefore, the surface microlayer has often been considered as an extreme environment for microorganisms that may contain unusual species and taxa [START_REF] Maki | Neuston microbiology: life at the air-water interface[END_REF]. This fact led to the thinking that a specific near-surface complex assemblage of bacteria, the bacterioneuston, would have a wide distribution in the world ocean [START_REF] Tsyban | Marine bacterioneuston[END_REF]). In addition, different reports suggest that microbial concentration exceeds that of underlying waters by orders of magnitude [START_REF] Macintyre | The top millimeter of the ocean[END_REF][START_REF] Hardy | The sea surface microlayer: biology, chemistry and anthropogenic enrichment[END_REF]), but despite their abundance and expected widespread distributions, bacterial communities thriving at the surface microlayer are poorly characterized [START_REF] Norkrans | Surface microlayers in aquatic environments[END_REF].

Isolation and cultivation techniques remain essential to understand the physiology and ecology of marine bacteria. Previous cultivation efforts on bacteria from the surface microlayer have collected strains belonging to Proteobacteria and Actinobacteria, including mostly misidentified genera such as Pseudomonas, Bacterium, Chromobacterium, Aeromonas and Micrococcus [START_REF] Sieburth | Distribution and activity of oceanic bacteria[END_REF][START_REF] Tsyban | Marine bacterioneuston[END_REF][START_REF] Carty | A microbial study of air and surface water microlayer in the open ocean[END_REF][START_REF] Fehon | Taxonomy and distribution of surface microlayer bacteria from two estuarine sites[END_REF], which have also been found in these early studies in various marine water samples [START_REF] Zobell | Marine microbiology. A monograph on hydrobacteriology[END_REF][START_REF] Sieburth | Observations on bacteria planktonic in Narragansett Bay, Rhode Island; a resume[END_REF][START_REF] Fehon | Taxonomy and distribution of surface microlayer bacteria from two estuarine sites[END_REF]. Evidence of unusual neustonic bacterial genera inhabiting the surface microlayer is therefore not available so far. This may be due to the well-known limitations for bacterial identification during the 1970s and 1980s and the fact that often less than 0.1% of total bacteria cells form colony on standard culture media [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF][START_REF] Pinhassi | Dominant marine bacterioplankton species found among colony-forming bacteria[END_REF]. The application of DNA-based techniques [START_REF] Woese | Bacterial evolution[END_REF] leading to the rapid identification of isolated strains makes it easier to survey the culturable bacteria inhabiting the surface microlayer. In addition, high-resolution biological and chemical techniques are now available to study the sea surface microlayer at the microscale, preserving its structure [START_REF] Agogué | Comparison of samplers for the biological characterization of the sea surface microlayer[END_REF][START_REF] Monzikoff | Field study of the chemical characterization of the top ocean surface using various samplers[END_REF]).

The present study represents an effort to obtain a collection of bacterial strains from coastal surface seawater, and was undertaken within the European Union project AIRWIN.

We focused on two coastal sites in the Mediterranean Sea, including an oligotrophic site (Bay of Banyuls-sur-Mer, France) and a heavily humanimpacted station (off Barcelona, Spain).

Overall, 487 bacterial strains isolated on solid media were screened by RFLP (restriction fragment length polymorphism) and identified by 16S rRNA gene sequencing. Surface microlayer bacteria are good candidates for specific adaptations such as resistance to high solar radiation (Agogué et al. in press) or pharmacological applications. Moreover, a PCR fingerprinting method SSCP (single-stranded-conformational polymorphism) was used to quickly compare the general structure of the predominant bacterial assemblages from surface microlayer and underlying waters. In a series of companion papers, a comparison of several surface microlayer-sampling devices has been described [START_REF] Agogué | Comparison of samplers for the biological characterization of the sea surface microlayer[END_REF][START_REF] Monzikoff | Field study of the chemical characterization of the top ocean surface using various samplers[END_REF], the surface microlayer microbial food webs are presented (Joux et al. submitted) and the microbial heterotrophic activity is analyzed (Obernosterer et al. in press).

Materials and methods

Field sites and sample collection

Samples were collected at two coastal stations in the NW Mediterranean Sea: the Bay of Banyuls-sur-Mer (France) and off the Olympic Harbour in Barcelona (Spain). The Bay of Banyuls-sur-Mer is oligotrophic [START_REF] Médernach | Temporal changes in the amino acid contents of particulate organic matter sedimenting in the Bay of Banyuls-sur-Mer (northwestern Mediterranean)[END_REF][START_REF] Grémare | The effects of a strong winter storm on physical and biological variables at a shelf site in the Mediterranean[END_REF]. The Barcelona site is moderately eutrophic and heavily impacted by the urban sewage sludge outfall [START_REF] Valls | Broad spectrum analysis of ionic and nonionic organic contaminants in urban wastewaters and coastal receiving aquatic systems[END_REF][START_REF] Bayona | Partitioning of urban wastewater organic microcontaminants among coastal compartments[END_REF][START_REF] Chalaux | Determination of nonylphenols as pentafluorobenzyl derivatives by capillary gas chromatography with electroncapture and mass spectrometric detection in environmental matrices[END_REF]. Samples were collected early in the morning around 4-5 h during four field campaigns (March 2001, September 2001, March 2002and June-July 2002).

The surface microlayer (SML) was collected with different types of devices as described in detail by [START_REF] Agogué | Comparison of samplers for the biological characterization of the sea surface microlayer[END_REF]). The different samplers differed in the thickness of the surface microlayer sampled and in the volume of SML water collected per unit of time (see details in [START_REF] Agogué | Comparison of samplers for the biological characterization of the sea surface microlayer[END_REF]). The bacterial strain collection was mostly obtained from both the metal screen and the glass plate samplers, but also from the nylon screen, the Harvey_ roller and after sampling with two types of membranes (Teflon and polycarbonate) (Hühnerfuss 1981a, b, Agogué et al. 2004). We did not detect differences in the bacterial species recovered from SML with the different samplers used. Samples from underlying waters (UW) were collected for direct comparison with each surface microlayer sample by submerging a polycarbonate bottle and opening it at a depth of 0.5 m.

Enumeration of total bacteria

Total heterotrophic bacteria were counted by flow cytométrie. Subsamples of 3 ml were fixed with formaldehyde (2% final concentration) in cryotubes and were left at room temperature for 10-15 min, frozen in liquid nitrogen and stored at -80°C until analysis. Bacterial cells were stained with a nucleic acid dye (SYBR-Green I; final concentration 0.01% [vol/vol] of the commercial solution; Molecular Probes) and stored for at least 15 min in the dark at room temperature before counting. The samples were run through a flow cytometer (FACScan, Becton Dickinson) equipped with a 488 nm, 15 mW Argon laser. Stained cells were discriminated and counted according to their right angle light scatter and green fluorescence measured at 530/30 nm [START_REF] Marie | Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I[END_REF][START_REF] Marie | Flow cytometry analysis of marine picoplankton[END_REF]. Other biological components were also evaluated and have been presented elsewhere (Agogué et al. 2004, Joux et al. submitted).

Enumeration and isolation of culturable heterotrophic bacteria

Samples for culturable heterotrophic bacteria were processed within 4 h after sampling. Serial dilutions (1:10 and 1:100 using filter-sterilized seawater) were prepared and 100 µl of each dilution was plated on 3 replicate plates on Marine Agar 2216 (Difco). Colony forming units were counted after incubation at 25°C in the dark after 2 weeks. Previous tests with low nutrient media did not offer higher culturability for the surface microalyer samples. Colonies were selected, picked and purified according to differences in colour and shape. We assigned a triple code for each strain, for example the code 14III/A01/020 correspond to a strain number 20 isolated on March 14 (14III), the year 2001 (A01).

Molecular characterization of the strains

For DNA extraction, bacterial cells were lysed either by thermal shock (the colony was resuspended in 500 µl of MilliQ sterile water and heated for 3 min at 94°C) or by chemical treatment. For chemical treatment, bacterial colonies were diluted in 500 µl lysis buffer (EDTA 40 mM, Tris 50 mM, pH 8, 750 mM saccharose) and incubated with lysozyme (final concentration, 1 mg ml _1 ) at 37°C for 45 min with gentle agitation. Sodium dodecyl sulfate (final concentration, 0.5% [w/v]) and proteinase K (final concentration 0.1 mg ml _1 ) were added, and the samples were incubated at 55°C for 1 h. DNA was extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1 [v/v/v]) and chloroformisoamyl alcohol (24:1 [v/v]). DNA was precipitated with 2 vol of isopropanol and recovered by centrifugation, washed with 70% cold ethanol (-20°C), air dried and diluted in 50 µl of sterile water. The 16S rRNA gene was amplified by PCR using the primer sets, SAdir (5'-AGAGTTTGATCATGGCTCAGA-3'; Escherichia coli 16S rRNA gene positions 8-27, forward) and S17 Rev (5'-GTTACCTTGTTACGACTT-3'; E. coli 16S rRNA gene positions 1491-1508, reverse) (numbering according to [START_REF] Brosius | Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli[END_REF]

Phylogenetic analysis

About 1300 bp of the 16S rRNA gene were used in a BLAST search [START_REF] Altschul | Basic Local Alignment Search Tool[END_REF], and the phylogenetic position of putative new ''species'' (i.e., strains with less than 97% similarity in the 16S rRNA gene as compared with already described species) was investigated. A multiple sequence file was produced, including the new sequences and their closest relatives given by the BLAST query (http://www.ncbi.nlm.nih.gov). Alignments were performed using the CLUSTAL W program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence aligment through sequence weighing, position-specific gap penalyites and weight matrix choice[END_REF]) and were then manually refined using SEAVIEW [START_REF] Galtier | SEAVIEW and PHYLO-WIN : two graphic tools for sequence alignement and molecular phylogeny[END_REF]. Phylogenetic trees were constructed by the neighbor-joining [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] and maximum-likelihood [START_REF] Felsentein | Evolutionary trees from DNA sequences: amaximum likelihood approach[END_REF]) methods using the PHYLO-WIN software [START_REF] Galtier | SEAVIEW and PHYLO-WIN : two graphic tools for sequence alignement and molecular phylogeny[END_REF], and graphic representation of the resulting trees was made using NJPLOT software [START_REF] Perrière | WWW-Query: an on-line retrieval system for biological sequence banks[END_REF]. Regions of ambiguous alignment were omitted from subsequent analyses. Evolutionary distance values were calculated using Kimura 2-state parameters and used in neighbor-joining analyses.

Bootstrap values were determined according to [START_REF] Felsentein | Confidence limits on phylogenies : an approach using the bootstrap[END_REF] SSCP fingerprinting Natural assemblages from SML and UW were quickly compared by SSCP fingerprinting.

High volume water samples (20-50 l) were collected and pooled together from the surface microlayer using the metal screen sampler previously washed with bleach, and were transferred to the laboratory within 1-3 h interval. There, a 1 l sample was filtered through 0.2 µm pore size polycarbonate filters using a vacuum pump and the filters were stored at -80°C.

Filters were thawed and resuspended in 900 µl of lysis buffer and digested as reported above [START_REF] Dumestre | Changes in bacterial and archaeal assemblages in an equatorial river induced by the water euthrophication of Petit Saut dam reservoir (French Guiana)[END_REF]. DNA was then extracted with an equal volume of phenolchloroform-isoamyl alcohol (25:24:1 [v/v/v]) and was precipitated by addition of one-tenth of the volume of 3 M sodium acetate and two volumes of cold isopropanol, followed by incubation for 3 h at -20°C [START_REF] Casamayor | Identification and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis[END_REF]. The DNA was recovered by centrifugation at 10,000g for 30 min and the pellet was washed with cold ethanol (70%), dried and resuspended in 20 µl of sterile MQ water. For PCR amplification, the bacterial primers W49 dir (E. coli position 331; 5'-ACG-GTC-CAGACT-CCT-ACG-GG-3') and W34 rev (E. coli position 533; 5'-TTA-CCG-CGG-CTG-CTG-GCA-C-3'; 5'-labelled with fluoresceine phosphoramidite TET, Applied Biosystems, Courtaboeuf, France) were used as reported [START_REF] Servais | Activity and diversity of bacterial cells with high and low nucleic acid content[END_REF]. For SSCP fingerprinting analysis, PCR products were heat-denatured at 94 °C for 5 min and immediately placed on ice for at least 10 min. A fluorescently labeled internal size marker (Genescan-400 Rox, Applied Biosystems) was added. The size standard contained a different fluorophore and permitted, after computing correction, a reliable comparison of patterns from many samples. SSCP electrophoresis was carried out as previously described [START_REF] Servais | Activity and diversity of bacterial cells with high and low nucleic acid content[END_REF], using the ABI 310 Genetic Analyzer equipped for capillary electrophoresis (Applied Biosystems). Computer files were analyzed with the 310 Genescan Analysis software (Applied Biosystems).

Results

Total counts and percentage of culturability

Total averaged bacterial counts for surface microlayer and underlying waters at the Barcelona sampling point were 1.43x10 6 and 1.30.10 6 cells ml _1 , respectively (n = 10) (Table 1). At the Banyuls-sur-Mer site, total counts were 0.97x10 6 and 0.93x10 6 cells ml _1 for SML and UW, respectively (n = 13). Thus, higher cell concentrations were found in Barcelona than in Banyuls-sur-Mer and slightly higher, statistically non-significant numbers in the surface microlayer samples compared to the underlying water samples. Viable counts on marine agar show higher percentage culturability vs. total counts in the surface microlayer than in underlying waters (Table 1). In Barcelona waters, culturable bacteria represented 4.16% and 2.51%, respectively. In Banyuls-sur-Mer waters, the percentages were 1.58% and 0.34%, respectively. Therefore, the percentage of culturable bacteria was higher in Barcelona and in the surface microlayer at both sites.

Phylogenetic diversity of the bacterial strains

Overall, 487 isolates were isolated from both sites and both layers, yielding 124 different ribotypes after analysis of RFLP patterns. We did not detect differences in the bacterial species recovered from SML with the different samplers used. All the strains could be clustered in 5 phylogenetic bacterial groups (Table 2): Alpha-and gamma subdivisions of the Proteobacteria, CFB (Cytophaga-Flavobacter-Bacteroides), Actinobacteria (high G+C Gram-positive) and Firmicutes (low G+C Gram-positive). Proteobacteria were more abundant in the Banyuls-sur-Mer collection, whereas Gram-positive (i.e., Actinobacteria and Firmicutes) were more abundant in the Barcelona sampling point. The group of Gammaproteobacteria was the predominant cultured group in all the samples, except for the SML of Barcelona, where Actinobacteria were more abundant (Table 2).

Similarity values for the 16S rRNA gene sequences from the culture collection with previously reported species or clones in data bases are presented in Table 3. Strains with sequence similarities P97% were considered to be representatives of the same species, similarities between 93% and 97% were considered to be representatives of the same genus and sequence similarities <93% would represent differences above genus [START_REF] Hagstrom | Biogeographical diversity among marine bacterioplankton[END_REF]. Overall, 22.5% of our isolates showed a 16S rRNA gene sequence similarity at the genus level with previously reported bacterial species and around 10.5% showed similarities below 93% (Table 3, considering both surface microlayer and underlying water strains). Therefore, we found consistent novelty at the genus level within our strain collection. The CFB group appeared to contain the highest proportion of unknown species (i.e., 7 out of 15 strains showed a sequence similarity less than 97% with cultured species), followed by Alphaand Gammaproteobacteria.

We also compared the cultured strains in our collection with the closest environmental clone known from databases (Table 3). Obviously, the percentage of strains with sequence similarities >97% to already reported 16S rRNA gene sequences, was higher. Nevertheless, nine strains showed similarities below 97% and three other strains (two surface microlayer Gammaproteobacteria isolated in Barcelona and one underlying water Gammaproteobacteria isolated in Banyuls-sur-Mer) had similarities lower than 93% with known environmental clones.

Phylogenetic trees were constructed with those surface microlayer-and underlying water strains that were not closely related to already known species (<97% similarity in the 16S rRNA gene). Only two Actinobacteria strains were putative new species (the Nocardioidaceae 14III/A01/020 and the Microbacteriaceae 18III/A01/077) (Fig. 1).

Conversely, seven strains related to CFB showed low similarities with previously described species (Fig. 2): five strains (14III/A01/006, 14III/A01/012, 18III/A01/061, 18III/A01/068 and 12IX/A01/169) belonged to Flavobacteriaceae and two (5IX/A01/134 and 13IX/A01/164) to Flexibacteraceae. Among these, four strains were isolated from the surface microlayer and three from underlying waters.

Finally, the Alpha-and Gammaproteobacteria were grouped on the same tree (Fig. 3).

For Gammaproteobacteria (Table 3), we included 10 sequences out of 12 (two strains were very similar to the others). Most of Gammaproteobacteria (i.e., strains 14III/A01/015, 18III/A01/067, 26III/A02/220, 3IV/A02/222, 3X/A02/235, 17X/A02/237 and 17X/A02/240) clustered into the genera Vibrio, Pseudoalteromonas, Pseudomonas and Alteromonas. For Alphaproteobacteria, seven putative new species were found and most of them (except 3X/A02/232) belonged to Rhodobacteriales, mainly the genera Roseobacter and Erythrobacter.

Putative neustonic isolates

To explore whether some strains were specific to the air-seawater interface, we included in a table the 41 isolates that were exclusively isolated from the surface microlayer either of Barcelona or Banyuls-sur-Mer stations (Table 4). Initially, we considered these strains as potential neustonic bacteria. Most of the isolates showed ≥97 % similarity in the 16S rRNA gene, with either already known bacterial species (71% of the isolated strains) or environmental clones (95% of the strains). Environmental clones had diverse marine origins ranging from deep-sea sediments or hydrothermal vents to Arctic sea ice. However, we also found two potentially ''neustonic'' Gammaproteobacteria strains isolated from Barcelona (5IX/A01/131 and 14III/A01/031), which were poorly related to any previously described sequence (less than 93%). Most of the Gram-positive strains were closely related to environmental clones found in diverse ecosystems, mainly from soils and polluted areas.

Therefore, only a few unusual bacterial taxa were collected from the surface microlayer isolates and most of the strains were closely related to bacteria of widespread distribution.

General structure of bacterial communities

To explore whether bacterial communities inhabiting both the surface microlayer and underlying waters were consistently different or not, we targeted the whole bacterial assemblage by SSCP genetic fingerprinting. We examined several vertical profiles from Banyuls-sur-Mer (n = 18) and Barcelona (n = 12) over two years. Overall, the number of SSCP peaks recovered from both surface microlayer and underlying waters was within the same range (18-24 predominant peaks). For all the samples collected in the Banyuls-sur-Mer station, we found very similar SSCP profiles for the surface microlayer and underlying waters (e.g., March 27, 2002 in Fig. 4). In contrast, differences in SSCP fingerprints between surface microlayer-and underlying water samples were often found at the Barcelona station (7 samples out of 12, Table 5). In those cases, only a few additional (2 or 3) peaks were found in the SML, but these peaks were highly significant, representing more than 30% of the total surface of the SSCP profile (e.g., September 7, 2001 in Fig. 4). Unfortunately, SSCP peaks cannot be directly recovered and used for 16S rRNA gene sequencing. Interestingly, such differences between surface microlayer and underlying waters profiles were not reproducible from one day to another. For instance, the Barcelona station was visited along three 5). As an example, Fig. 4 shows the bacterial community structure found for two consecutive days (6 and 7 September 2001): on the first day, only one minor peak was specific for the surface microlayer, whereas in the following days, several major peaks appeared.

Discussion

Range of culturability

Bacterial culturability in oligotrophic to mesotrophic marine habitats generally range from 0.001% to 1%, whereas this percentage can increase in nutrient-rich environments (e.g., activated sludge) [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF]. The percentages reported in this study greatly varied (from 0.34% up to 14.5% of total cells counts in Barcelona), but the mean value was consistently lower at the oligotrophic site (Banyuls-sur-mer), in agreement with this general trend. In addition, at both stations culturability was higher for the surface microlayer. This may be easily explained by the accumulation of organic matter at this interface (POC enrichment: 1.8 [n = 5] and 3.2 [n = 7] for Banyuls-sur-Mer and Barcelona sites, respectively) (Joux et al. submitted). We tested different media at the beginning of our survey, including low-nutrient media such as R2A and using several samples from the surface microlayer. We observed that the largest diversity (in terms of conspicuous morphotypes and ribotypes after RFLP profiles) was consistently recovered with the Zobell medium. Other reports have shown biochemical and bacterial activities to increase in the surface microlayer when compared to underlying waters [START_REF] Sieburth | Distribution and activity of oceanic bacteria[END_REF][START_REF] Carlucci | Surface-film microheterotrophs: amino acid metabolism and solar radiation effects on their activities[END_REF][START_REF] Mudryk | Bacteriological investigation of the surface microlayer of the Gulf of Gdansk[END_REF]. This would explain why bacterial species isolated from the surface microlayer represent a higher fraction of total bacteria than in underlying waters.

Taxonomic groups of culturable bacteria from both sites

The dominant bacterial groups found in the surface microlayer-and underlying water isolates collected at Banyuls-sur-Mer site were similar to those commonlyfound in seawater samples [START_REF] Benlloch | Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing[END_REF][START_REF] Bowman | Diversity and association of psychrophilic bacteria in Antartic sea ice[END_REF][START_REF] Pinhassi | Dominant marine bacterioplankton species found among colony-forming bacteria[END_REF][START_REF] Eilers | Culturability an in situ abundance of pelagic bacteria from the north sea[END_REF], 2001). Around one half of the bacteria isolated from the Banyuls-sur-Mer site belonged to Gammaproteobacteria, and a significant proportion of the strains belonged to Alphaproteobacteria and CFB. Gammaproteobacteria represent a large phylogenetic group of cosmopolitan species generally well-represented in culture collections [START_REF] Bowman | Diversity and association of psychrophilic bacteria in Antartic sea ice[END_REF][START_REF] Pinhassi | Dominant marine bacterioplankton species found among colony-forming bacteria[END_REF][START_REF] Suzuki | Bacterial diversity among small-subunit rRNA genes clones and cellular isolates from the same seawater sample[END_REF][START_REF] Eilers | Culturability an in situ abundance of pelagic bacteria from the north sea[END_REF]. The high proportion of Gammaproteobacteria in collections can be partly biased, since the isolation procedure may favour bacterial strains able to rapidly grow on nutrient-rich media. Similarly, the Cytophaga-Flavobacteria cluster is characterized by mainly aerobic species, especially effective at degrading complex polymers (Kirchman, 2002) and able to develop conspicuous colonies on solid media. The same types of Gammaproteobacteria and CFB were found in both surface microlayer and underlying waters, suggesting that culturable surface microlayer species are not different from underlying water species with the growth medium used, and were in agreement with SSCP fingerprinting profiles. Overall, the molecular fingerprints indicated that in most cases the same bacterial populations dominated surface microlayer and underlying waters. The fact that similar SSCP profiles were found suggests that, at least for predominant bacterial populations (above 1% of total bacteria), there was no evidence for unusual neustonic species that do not occur in underlying waters. This is congruent with the hypothesis that most bacteria in the surface microlayer are planktonic cells that accumulate from a passive flotation process, although these cells may potentially grow in the surface microlayer. We cannot discard, however, differences for minor populations that could escape detection limits of the SSCP fingerprinting. The Barcelona station seemed to behave quite different and the dominant cultured groups included species commonly found in wastewater and polluted coastal areas [START_REF] Hugenholtz | Impact of culture-independant studies on the emerging phylogenetic view of bacterial diversity[END_REF]) in both surface microlayer and underlying waters. The Gram-positive species isolated at Barcelona are abundant in activated sludge [START_REF] Kampfer | Characterization of bacterial communities from activated sludge: culture-dependant numerical identification versus in situ identification using group-and genus-specific r RNA-targeted oligonucleotide probes[END_REF][START_REF] Snaidr | Phylogenetic analysis and in situ identification of bacteria in activated sludge[END_REF]) and were probably released from the sewage outfall.

Interestingly, we found Actinobacteria, (i.e., the high G+C Gram-positive group) [START_REF] Stackebrandt | Proposal for a new hierarchic classification system, Actinobacteria classis nov[END_REF]) to be more abundant in the surface microlayer than in underlying waters. Actinobacteria are known to degrade toxic compounds such as hydrocarbons, pesticides and heavy metals in soil and aquatic environments [START_REF] Kashner | Enumeration and characterization of the soil microflora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic-hydrocarbons (PAH)[END_REF][START_REF] De Schrijver | Degradation of pesticides by actinomyctes[END_REF][START_REF] Gremion | Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil[END_REF][START_REF] Zhuang | Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments[END_REF]. Such compounds have been already found to accumulate in the surface microlayer at the Barcelona station [START_REF] Valls | Broad spectrum analysis of ionic and nonionic organic contaminants in urban wastewaters and coastal receiving aquatic systems[END_REF][START_REF] Bayona | Partitioning of urban wastewater organic microcontaminants among coastal compartments[END_REF] and, therefore, these strains would probably be well-adapted to handle high concentrations of these toxic substances in the surface microlayer. Duration of the surface microlayer and mixing with water from the deeper layer seems to be similar for both stations. [START_REF] Romano | Photographic records of sea-surface microlayers as a survey of pollution daily rhythm in coastal waters[END_REF] showed, after an extensive study carried out in the north-western Mediterranean coast, that when the wind speed is below 4-5 m/s slicks are large and frequent, whereas when the wind increases slicks are smaller and rare. The slicks were maintained under natural conditions as far as the wind speed was low. Most of the days selected for sampling in our study had wind speed below this threshold. In Banyuls, most of the days wind speed was below 4 m/s, but we did not detect significant differences between surface microlayer-and underlying water bacterial assemblages. In Barcelona, we did detect differences, although wind data were in the same range than in Banyuls (see Table 5).

Phylogenetic richness of the culture collection

The phylogenetic analyses revealed high genetic diversity among the isolated bacteria, with 16S rRNA gene sequence similarities ranging from 88% to 100% to already known species (Table 3). Particularly, around 25% of the isolated strains showed sequence similarities lower than 97% to previously reported species, 10% of them having sequence similarities lower than 93%. Such low similarity values indicate that we were able to bring into culture new marine genera or even new families [START_REF] Devereux | Diversity and origin of Desulfovibrio species: phylogenetic definition of a family[END_REF]). Most of these strains were CFB (i.e., 47%), whereas Proteobacteria generally showed higher similarity values to already known species. These observations agreed with others reports on the prevalence of novel marine bacteria among the culturable bacterial fraction [START_REF] Bowman | Diversity and association of psychrophilic bacteria in Antartic sea ice[END_REF][START_REF] Pinhassi | Dominant marine bacterioplankton species found among colony-forming bacteria[END_REF][START_REF] Suzuki | Bacterial diversity among small-subunit rRNA genes clones and cellular isolates from the same seawater sample[END_REF][START_REF] Hagstrom | Biogeographical diversity among marine bacterioplankton[END_REF] that may have a great interest for future research.

Although, we initially expected that these new culturable bacterial taxa might be the dominant bacterial components in the unexplored surface microlayer habitat, these potential ''new'' bacterial genera were not specific for the surface microlayer. This reinforces the idea that the sea-surface layer from 0 to 100 cm is still a poorly known microbial environment that represents a natural source of new microorganisms.

Bacterioneuston specificity

Although some strains were only found in the surface microlayer, the 16S rRNA gene sequence analysis indicated that they were closely related to sequences retrieved from different marine environments and different water depths. In addition, the apparent higher occurrence of Actinobacteria in the surface microlayer at both stations should be interpreted with caution because of (i) the low number of strains obtained (especially in the case of Banyuls-sur-Mer, with only three isolates) and (ii) the influence of the treated-wastewater outfall in the Barcelona sampling station. [START_REF] Norkrans | Surface microlayers in aquatic environments[END_REF] explained the dominance of Bacillus species in the surface microlayer by the fact that spore-forming species coming from the air are more resistant to desiccation and to solar radiation than the bubble-transported marine species. For [START_REF] Carty | A microbial study of air and surface water microlayer in the open ocean[END_REF], bacteria belonging to Bacillus and Micrococcus genera were also found in abundance in air and may have a terrestrial origin.

Although terrestrial origin cannot be excluded in coastal areas, these species cannot be considered as neustonic bacteria. From this field study and considering these two coastal sites, we hypothesize that most of the bacteria living in the sea surface microlayer are planktonic and may accumulate via a flotation process when they attach to particles and/or bubbles coming from the water column. A fraction of these bacteria may also accumulate via atmospheric deposition. As a consequence, viable cells collected from the surface microlayer may be those that have developed mechanisms to resist intense solar radiations and/or high concentrations of toxic compounds. We cannot disregard, however, the existence of resistance-forms in the surface microlayer, such as spores, that could grow under laboratory conditions. From this field experiment we can suggests that specific neustonic bacterial species are scarce rather than abundant. However, the existence of specific neustonic populations at the marine surface microlayer cannot be excluded, as some adapted species have already been described in freshwater environments [START_REF] Glöckner | Phylogeny and identification in situ of Nevskia ramosa[END_REF][START_REF] Paddies | Distribution of Nevskia ramosa and other rosette-forming neustonic bacteria[END_REF]). If this is the case for marine environments, they do not seem to be strongly enriched in the marine neuston of coastal areas.

CFB SML 4 1 2 1 4 0 0 UW 6 5 (83%) 0 1 (17%) 5 (83%) 1 (17%) 0 Gram + Actinobacteria SML 2 2 0 0 2 0 0 UW 1 0 1 0 0 1 0 FLGC SML 3 3 0 0 3 0 0 UW 4 4 0 0 4 0 0 Barcelona site Gram - Gamma SML 9 5 (56%) 0 4 (44%) 7 (78%) 0 2 (22%) UW 6 6 0 0 6 0 0 Alpha SML 7 5 (71%) 2 (29%) 0 7 0 0 UW 3 1 2 2 1 0 CFB SML 3 2 1 0 3 0 0 UW 2 0 1 1 0 2 0 Gram + Actinobacteria SML 10 9 (90%) 1 (10%) 0 10 0 0 UW 1 1 0 0 1 0 0 FLGC SML

  consecutive days duringMarch 2001, September 2001, March 2002 and June 2002, and well- developed specific surface microlayer bacterial assemblages were only found in a few occasions(14 March 2001, 7 September 2001, 20 and 21 March 2002; Table

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

 123 Fig.1. Phylogenetic tree constructed using almost complete 16S rDNA gene sequences of isolated strains and related species of Gram-positive bacteria. The tree was built with the neighbor-joining method by using the Kimura distance. Values at nodes indicate bootstrap percentages for 500 replicates. Values less than 50 % are not reported. The scale bar indicate Kimura distance of 0.01. Asterisk (*) indicates branches that were also found using the Maximum Likelihood method.

Fig. 4 .

 4 Fig. 4. Comparison between surface microlayer (SML) and underlying water (UW) bacterial community structure using SSCP fingerprints of 16S rRNA genes from Barcelona (6 and 7 September, 2002) and Banyuls-sur-Mer (27 March 2002) sampling stations

Table 1 .

 1 Cell counts and bacterial culturability (in percentage vs. total cells count) in the sea surface microlayer (SML) sampled by the metal screen and underlying waters (UW) from Barcelona and Banyuls-sur-Mer sites, respectively

		Barcelona	Banyuls sur Mer
		SML	UW	SML	UW
	n	10	10	13	13
	Total counts a (10 6 cells ml -1 ) 1,43 ± 0,43 1,30 ± 0,38 0,97 ± 0,16 0,93 ± 0,14
	CFU / total counts (%)	4,16	2,51	1,58	0,34
	% CFU range	0,3 -14,5	0,001 -7,6	0,1 -7,3	0,05 -1,42
	a mean ± standard deviation
	n number of samples

Table 2 .

 2 Relative contribution (%) of taxonomic groups of culturable fraction of the sea surface microlayer (SML) and underlying waters (UW) bacterial communities from Banyulssur-Mer and Barcelona.

		Barcelona	Banyuls-sur-Mer
		SML	UW	SML	UW
	Alphaproteobacteria	19	18	19	24
	Gammaproteobacteria	24	35	53	47
	CFB group	8	12	13	16
	Total Gram-negative	51	65	85	87
	Actinobacteria	27	6	6	3
	Firmicutes	22	29	9	10
	Total Gram-positive	49	35	15	13

Table 3 .

 3 Sequence similarity in the 16S rRNA gene of isolated strains to the closest matching relative in the data bank (the closest species on the left and the closest clone on the right).

					Most closest sp. a			Most closest clone b
					Known sp. New putative sp.	New putative gen.	Known sp.	New putative sp. New putative gen.
		Taxonomic group	n c	≥ 97 %	93 % -97 %	< 93 %	≥ 97 %	93 % -97 %	< 93 %
	Bayuls-sur-Mer site
		Gamma	SML UW		17 14 (82%) 17 12 (71%)	2 (12%) 2 (12%)	1 (6%) 3 (18%)	17 16 (94%)	0 0	0 1 (6%)
	Gram -	Alpha	SML UW		6 4 (67%) 10 9 (90%)	1 (17%) 0	1 (17%) 1 (10%)	6 9 (90%)	0 1 (10%)	0 0

Table 4 .

 4 List of strains exclusively isolated from the SML in Banyuls-sur-Mer and Barcelona stations

			Closest species	Closest clone acc. N°
	Code	Acc. N°	(% homology)	(% homology)	Origin of closest clone
	Gamma proteobacteria
	Banyuls -sur -Mer isolates
	3X/A02/235	AY576769 Pseudomonas pseudoalcaligenes (90%)	AF468388 (98%)	Arctic sea ice
	17X/A02/240	AY576773 Microbulbifer maritimus (96%)	AF500211 (99%)	?
	18III/A01/067	AY576718 Pseudomonas anguilliseptica (96%)	AJ293824 (98%)	Arctic seawater
	18III/A01/066	AY576717 Pseudomonas putida (98%)	AJ297355 (98%)	freshwater fishfarm
	27III/A02/219	AY576760 Marinobacter litoralis (99%)	AF479689 (99%)	East Sea in Korea
	18III/A01/058	AY576713 Pseudoalteromonas agarivorans (99%)	AY028200 (99%)	Diatom detritus
	17X/A02/242a	AY576775 Alcanivorax venustensis (100%)	AF328762 (100%) Mediterranean Sea
	Barcelona isolates
	5IX/A01/131	AY576729 Marinobacter stanieri (90%)	AF228694 (91%)	marine environment
	14III/A01/031	AY576709 Oleispira antarctica (91%)	AF353237 (91%)	Arctic Ocean
	14III/A01/015	AY612750 Pseudoalteromonas agarivorans (92%)	AY028198 (98%)	diatom detritus
	7IX/A01/156	AY576745 Alteromonas infernus (98%)	AB015135 (99%)	deep-sea hydrothermal vent
	6IX/A01/151	AY576743 Enterobacter sakazakii (99%)	AB004746 (99%)	clinical specimens
	7IX/A01/137	AY576733 Pseudoalteromonas mariniglutinosa (99%)	AJ551143 (99%)	deep sea sediments
	14III/A01/025	AY576705 Pseudoxanthomonas broegbernensis (99%)	AF273082 (99%)	diverse environments
	Alpha proteobacteria
	Banyuls -sur -Mer isolates
	3X/A02/236	AY576770 Ruegeria atlantica (95%)	AJ391187 (97%)	Adriatic Sea
	18III/A01/069	AY576720 Paracoccus aminophilus (98%)	D32239 (98%)	soil
	18III/A01/070	AY576721 Paracoccus marcusii (99%)	AJ532681 (99%)	uranium mining waste piles
	Barcelona isolates
	5IX/A01/143	AY576739 Jannaschia helgolandensis (95%)	AB018689 (99%)	marine environments
	5IX/A01/132	AY615725 Salipiger muscesens (97%)	AY162092 (99%)	Sargasso Sea
	14III/A01/023	AY576704 Agrobacterium tumefaciens (99%)	AY221181 (99%)	soil
	5IX/A01/139	AY576735 Sulfitobacter pontiacus (99%)	AJ298355 (99%)	Mediterranean sea
	Cytophaga/Flavobacterium/Bacteroides group
	Banyuls -sur -Mer isolates
	12IX/A01/169	AY576752 Psychroflexus tropicus (91%)	U85882 (97%)	Antarctic sea ice
	18III/A01/068	AY576719 Salegentibacter salegens (95%)	AY319330 (99%)	Southern Ocean
	18III/A01/061	AY576714 Algibacter lectus (96%)	AF359540 (97%)	dinoflagellates
	Barcelona isolates
	14III/A01/010	AY576693 Maribacter sedimenticola (97%)	AF367849 (99%)	sea water
	7IX/A01/155	AY576744 Muricauda ruestringensis (97%)	AY445076 (98%)	East Sea in Korea
	Actinobacteria
	Banyuls -sur -Mer isolate
	17X/A02/245	AY576777 Arthrobacter agilis (99%)	X80748 (99%)	water, soil and human skin
	Barcelona isolates
	14III/A01/020	AY576701 Nocardioides jensenii (96%)	AJ316318 (99%)	mural painting environments
	14III/A01/014	AY576697 Corynebacterium ammoniagenes (97%)	X84440 (97%)	piggery waste
	14III/A01/017	AY576699 Brachybacterium arcticum (99%)	AF468445 (100%) Arctic sea ice
	14III/A01/011	AY576694 Microbacterium kitamiense (99%)	AB042082 (99%)	plant-nematode
	6IX/A01/148	AY576742 Microbacterium esteraromaticum (99%)	AB099658 (99%)	activated sludge
	14III/A01/022	AY576703 Micrococcus luteus (99%)	AJ441006 (99%)	mats from Antarctic lakes
	14III/A01/028	AY576707 Arthrobacter nitroguajacolicus (100%)	AJ512504 (100%) forest soil
	14III/A01/021	AY576702 Dietzia maris (100%)	Y18883 (100%)	soil, skin and intestine of carp
	Firmicutes Low GC content
	Banyuls -sur -Mer isolate
	5IX/A01/142b	AY576738 Paenibacillus glucanolyticus (99%)	AB073189 (99%)	soil
	13IX/A01/167	AY576750 Planococcus rifietoensis (99%)	AJ493659 (99%)	mat from a sulfurous spring
	Barcelona isolates

Table 5 .

 5 Similarity values (Jaccard coefficient) for the absence/presence of data from the genetic fingerprintings carried out in the surface microlayer (SML) and underlying waters (UW) of the Barcelona coastal station

	Site	Date	SML vs. UW	Wind speed (m/s)
	Barcelona	March 13, 2001	1.00	5-8*
		March 14, 2001	0.58	2-4
		March 15, 2001	1.00	2-3
		September 5, 2001	1.00	4-6*
		September 6, 2001	0.90	2-4
		September 7, 2001	0.60	2-3
		March 19, 2002	1.00	<2
		March 20, 2002	0.77	<2
		March 21, 2002	0.60	<2
		June 25, 2002	1.00	<2
		June 26, 2002	0.90	<2
		June 27, 2002	0.90	<2
	No significant differences were detected for Banyuls-sur-Mer coastal station (see Results).
	* Sampling was carried out inside the sheltered harbour.

Acknowledgements

This work was supported by the European Commission (Research Directorate General-Environment Program-Marine Ecosystems) through the AIRWIN project "Structure and role of biological communities involved in the transport and transformation of persistent pollutants at the marine AIR-Water Interface" (contract EVK3-CT2000-00030). The AIRWIN project is part of the EC IMPACTS cluster. During the final stage of this work EOC was supported by the Programa Ramo´n y Cajal from the Spanish MCYT. We thank Nicole Batailler, Philippe Catala and Laurent Intertaglia from the OOB and Harry Witte from the NIOZ for technical assistance. We also thank Nyree West for input.


MD Nadjib Hammoudi
email: nadjib.hammoudi@psl.aphp.fr.

MD Malek Ihaddaden

PhD Sylvie Lang

MD Florent Laveau

MD Stephane Ederhy

MD Pierre-Louis Michel

MD Sonia Alamowitch

PhD Ariel Cohen

Three-dimensional transesophageal echocardiography for descending aortic atheroma: a preliminary study

Keywords: 3D TEE, Aortic plaque, Atheroma, Imaging, Transesophageal echocardiography

BACKGROUND

Atherosclerotic disease is a leading cause of cardiovascular death [START_REF] Go | Executive summary: heart disease and stroke statistics--2014 update: a report from the American Heart Association[END_REF] and constitutes a major public health issue. This disease, in its different localizations, is therefore generating increasing interest and remains a diagnostic and therapeutic challenge.

In recent years, significant progress has been made in the examination of atherosclerotic plaque due to improvements in imaging methods. Several techniques for the morphological and functional analysis of atherosclerotic plaques were developed for coronary artery disease,[2] [START_REF] Waksman | Intravascular ultrasound versus optical coherence tomography guidance[END_REF]and magnetic resonance imaging (MRI) provides new data on carotid plaques. [START_REF] Takaya | Association between carotid plaque characteristics and subsequent ischemic cerebrovascular events: a prospective assessment with MRI--initial results[END_REF] Atherosclerosis in the descending thoracic aorta is a marker of diffuse atherosclerotic cardiovascular disease; atherosclerotic plaques localized in this segment of aorta are associated with coronary artery disease and cardiovascular events.[5] [START_REF] Gu | Relation between the incidence, location, and extent of thoracic aortic atherosclerosis detected by transesophageal echocardiography and the extent of coronary artery disease by angiography[END_REF][7] [START_REF] Roos | Association of atherosclerosis in the descending thoracic aorta with coronary artery disease on multi detector row computed tomography coronary angiography in patients with suspected coronary artery disease[END_REF]Transesophageal echocardiography (TEE) is an established technique to study aortic atherosclerotic plaques (AAP) [START_REF] Cohen | Aortic plaque morphology and vascular events: a follow-up study in patients with ischemic stroke. FAPS Investigators. French Study of Aortic Plaques in Stroke[END_REF] [START_REF] Hahn | Guidelines for Performing a Comprehensive Transesophageal Echocardiographic Examination: Recommendations from the American Society of Echocardiography and the Society of Cardiovascular Anesthesiologists[END_REF]; moreover descending aortic atheroma is the most common extracardiac finding during routine TEE regardless of the indication. [START_REF] Alkhouli | Extracardiac Findings on Routine Echocardiographic Examinations[END_REF] Three-dimensional (3D) TEE was proposed and validated as a complementary ultrasound exploration to the two-dimensional (2D) approach, its utility in various circumstances including native and prosthetic valve analysis and for monitoring of percutaneous catheter-based interventions has been established. [START_REF] Lang | EAE/ASE Recommendations for Image Acquisition and Display Using Three-Dimensional Echocardiography[END_REF]However, the usefulness of 3D TEE in the examination of AAPs has not been yet well explored. [START_REF] Aggeli | Role of real time-3D transesophageal echocardiography in evaluating the atheromatous burden of thoracic aorta in patients with heterozygous familial hypercholesterolemia: comparison with 2D transesophageal study[END_REF] The objective of this study was to evaluate the feasibility and the contribution of 3D TEE in the assessment of AAPs of the descending thoracic aorta.

METHODS

Study Population

From January 1st to June 30th 2010, all patients referred to the ultrasound laboratory for TEE and examined by the same experienced operator (N.H.), who was familiar with the 3D mode, were considered for the study. The examination was carried out according to a standardized protocol.

Exclusion criteria were the classic contraindications for TEE, [10][14] the absence of aortic atheroma identified in 2D TEE, and an incomplete examination of the aorta due to poor tolerance of the examination (Figure 1). Ninety three patients were considered for the study. Of these, 11 did not meet all inclusion and exclusion criteria. The remaining 82 patients constituted the study population. Data on diabetes, hypertension, dyslipidemia, smoking, and serum creatinine level were collected from medical records.

The consent of all patients was obtained and the study was approved by the institutional committee on human research.

Acquisition of Images

All examinations were performed on an iE33 system (Phillips Medical Systems, Andover, MA, USA) equipped with a 3D TEE probe. After a complete transthoracic echocardiogram, local anesthesia of the pharynx by lidocaine, and mild sedation with midazolam (mean dose 1.3 ± 0.4 mg; 5 patients were not sedated), patients were placed in the left lateral decubitus position. Following a conventional 2D TEE examination, [10][14] in particular of the valves, the left atrium, and the interatrial septum, the operator focused on the aorta. To optimize image quality, the size of the aorta on the display was increased by reducing the depth, the focus was moved to the near field, and the transducer frequency was increased. [START_REF] Hahn | Guidelines for Performing a Comprehensive Transesophageal Echocardiographic Examination: Recommendations from the American Society of Echocardiography and the Society of Cardiovascular Anesthesiologists[END_REF] The probe was withdrawn very gradually until the aortic arch in its various segments was reached. [START_REF] Cohen | Aortic plaque morphology and vascular events: a follow-up study in patients with ischemic stroke. FAPS Investigators. French Study of Aortic Plaques in Stroke[END_REF]The descending thoracic aorta was visualized in its short axis (TEE at 0°), and whenever an AAP (defined as a hyperechoic thickening of the intima, regardless of the thickness) was identified in 2D images, a rotation of 90° was carried out for viewing on its long axis and a 3D zoom mode acquisition was performed. Image acquisition was standardized, alternating a 2D loop of an AAP followed systematically by a 3D loop of the same AAP. Thus, 2D and 3D loops of the same AAP followed each other during the recording of the images.

Image Analysis

The images were transferred to a computer equipped with Phillips QLAB software (version 8.1) for post-processing. The analyses were performed more than 6 months after the examinations by two investigators (N.H., M.I.). The total duration of the TEE (defined as the time between the first and the last image of the examination) and the specific duration of the examination of the aorta (defined as the time between the first and the last image of the thoracic aorta) were collected.

2D Data

2D data were analyzed blinded to 3D TEE images and to clinical data. The number of AAPs was quantified and each plaque was measured on the incidence where it appeared the thickest (mean of 3 measurements). The AAPs were then classified into three categories according to their thickness: < 1 mm, 1-3.9 mm, and ≥ 4 mm, [START_REF] Cohen | Aortic plaque morphology and vascular events: a follow-up study in patients with ischemic stroke. FAPS Investigators. French Study of Aortic Plaques in Stroke[END_REF]and the degree of calcification of each AAP was evaluated: not calcified (0), slightly calcified (+), or very calcified (++).The presence of mobile "debris" was noted; a disruption or marked irregularities of the AAPs' surface defined ulceration (≥2 mm in depth and width). [START_REF] Cohen | Aortic plaque morphology and vascular events: a follow-up study in patients with ischemic stroke. FAPS Investigators. French Study of Aortic Plaques in Stroke[END_REF] Particular attention was given to the precise location of the AAP analyzed in the segment of descending aorta studied. Indeed, it was decided to divide the descending thoracic aorta in TEE 0° into 4 segments, segments 1 and 2 corresponding to the portions closest to the probe, and segments 3 and 4 corresponding to the other half of the aorta.

3D Data

The 3D analysis was performed blinded to the 2D TEE data to clinical data. The quality of the 3D images was graded as insufficient, good, or optimal. To make sure that the plaque analyzed in 3D was the same which was analyzed in 2D, in cases where several plaques were identified on 3D acquisition, the elements of localizations defined above and collected during 2D analysis were used to define the plaque to be analyzed in 3D.

Morphological Analysis in 3D

Initially, 20 3D acquisitions of randomly selected AAPs were analyzed simultaneously by three confirmed echocardiographers (M.I., N.H., A.C. respectively 5, 10 and 25years experience with TEE).

By consensus, AAP in 3D was defined as a persistent intimal thickening despite the reduction in gain to zero. In addition, three different morphological types of plaques were identified (Figure 2). Type I atheroma plaque was defined as an intimal thickening with regular contours and a smooth surface.

Type II atheroma plaque was defined as an intimal thickening, with irregular contours and a bumpy surface. Type III atheroma plaque was defined as an intimal thickening with strongly marked irregular contours, a tortuous surface and an empty band under the plaque separating it from the aortic wall.

In a second step, the morphological study of all 3D acquisitions was carried out by two investigators (N.H., M.I.) and the AAPs identified were classified according to the three types of plaques defined above.

2D reconstructions from 3D Data

After the 3D morphological analysis, a study in multiplanar reconstruction (MPR) mode, allowing for the extraction of 2D data from the 3D volume, was systematically carried out. From reconstructed 2D images, a measurement of the maximum thickness (mean of 3 measurements) was done, and the degree of calcification of each AAP was evaluated (Figure 3). In addition, to morphological analysis from 3D image, the presence of mobile "debris" and/or ulceration was also searched for carefully in whole AAP 3D volume with MPR mode.

Statistical Analysis

Continuous variables are presented as medians with interquartile ranges (IQRs). Qualitative variables are presented as counts and percentages. The data were compared using the Fisher and Kruskal-Wallis exact tests. Correlations between the different measures were evaluated using the Pearson correlation test. The agreement between 2D data and 2D data extracted from 3D acquisitions by MPR was evaluated by calculating the Kappa coefficient. Using the Bland-Altman approach, the mean difference and the 95 % limit of agreement was also calculated.

To evaluate intra-and inter-observer agreement in the analysis of plaque morphology in 3D, 40 randomly selected plaques were reviewed blinded to the initial evaluation and the Kappa coefficient was calculated. A P value < 0.05 was considered statistically significant. STATA software version 11 was used to perform all analyses.

RESULTS

Population Characteristics

The characteristics of the population are summarized in Table 1. Patients' median age was 62.3 (55.2-75.1) years and 33 patients (40.2%) out of a total of 82 were women.

Transesophageal Echocardiography

The tolerance to the examination was satisfactory in 98.8% of cases and no complication was observed. The median duration of examination was 9.2 (6.2-12.2) minutes, the median time to examine the aorta was 2.2 (1.3-3.7) minutes.

2D Analysis

A total of 192 AAPs were identified by 2D TEE, with a median of 2 (1-3) AAPs per patient. Ninety-five (49.4%) AAPs were not calcified, 65 (33.9%) were slightly calcified, and 32 (16.7%) were very calcified.

The median thickness of the AAPs was 2.0 mm (1.1-2.7 mm); 38 (19.8%) were< 1 mm, 132 (68.8%) were 1-3.9 mm, and 22 (11.4%) were≥ 4 mm. Ulcerations and mobile debris were identified in 13 and 4 AAPs respectively.

3D Analysis

Of the 192 AAPs viewed by 2D TEE, 189 (98.4%) were identified by 3D TEE. The missed plaques were thin (thickness between 0.5 and 1.2mm) and not calcified in 2D. The mean 3D TEE off-line review time was 2.5 minutes per plaque and the quality of the 3D images was rated as good or optimal in 95% of the cases.

Of the 189 AAPs identified in 3D, 77 (40.7%) were a type I, 63 (33.4%) were a type II, and 49 (25.9%) were a type III. The 2D TEE characteristics for the three morphological types of AAP in 3D TEE were different (Table 2). Type I AAPs are thin and rarely calcified, type III AAPs are thicker and often calcified, and type II AAPs have intermediate 2D TEE characteristics. The 3D morphology could not be formally predicted based on 2D TEE characteristics, due to significant overlap between the measurements of plaque thickness for types II and III (Figure 4).

There was similar intra-and inter-individual agreement for the 3D morphological analysis of AAPs, with 93% (kappa=0.88) of AAPs classified in agreement with the first reading.

MPR Analysis of 3D Acquisitions

A good correlation (r = 0.92, P < 0.001) was observed between 2D measurements extracted from 3D acquisitions and those carried out on the 2D TEE data. The mean difference between the both methods was -0.1mm, the 95% limits of agreement were -1.4 to 1.2mm (Figure 5). Table 3 presents the conventional classification of AAPs according to their 2D TEE characteristics and those extracted from 3D by MPR. The agreement between the two methods was good, at 83.6% (kappa = 0.69) for the measurements of thickness, and 82.5% (kappa = 0.72) for the degree of calcification.

All AAP ulcerations (n=13) and mobile debris (n=3) seen in 2D have been identified from 3D images (Figure 6) (Video 1).

DISCUSSION

In this study evaluating 3D TEE applied to the study of AAPs in the descending thoracic aorta, we have shown the feasibility of the method and an excellent agreement for AAP characterization between the 2D data extracted from 3D TEE and those measured from conventional 2D. In addition, 3D TEE provides an original morphological approach for the study of AAP.

Feasibility

We showed that 3D TEE is an appropriate tool for evaluating AAP. Indeed, 98.4% of plaques viewed in 2D were visualized in 3D. The median 3D TEE examination time seems to be acceptable for routine use, being relatively similar to that for a conventional 2D TEE examination. [START_REF] Daniel | Safety of transesophageal echocardiography. A multicenter survey of 10,419 examinations[END_REF][16]

2D TEE Aortic Atherosclerotic Plaques

With 2D TEE a total of 192 AAPs were identified on the descending thoracic aorta. Echocardiographic studies do not usually report the number of plaques studied by aortic segment.[5][6] [START_REF] Khoury | Relation of coronary artery disease to atherosclerotic disease in the aorta, carotid, and femoral arteries evaluated by ultrasound[END_REF]In 120 consecutive necropsy studies, Khatibzadeh et al. [START_REF] Khatibzadeh | Aortic atherosclerotic plaques as a source of systemic embolism[END_REF]reported a mean of 2.2 plaques per patient, including fatty streaks on the descending thoracic aorta. This result is very similar to our own.

3D TEE Aortic Atherosclerotic Plaques

We found good agreement between the 2D measurements and those extracted from 3D by MPR, which has been validated and routinely used for 3D visualization in computed tomography (CT). [START_REF] Rothman | Multiplanar reconstruction as an aid in CT diagnosis[END_REF] However, two cases of clear disagreement between the two methods were observed (figure 5).

Because these cases concerned very protrusive aortic plaques (> 6 mm), it is possible that the measurements extracted from 3D were not made at exactly the same level of cut as those carried out on the corresponding 2D images; the MPR mode makes it possible to "navigate" in the entire acquired 3D volume and to measure the thickest part of the plaque, as well as, to analyze the plaque morphology looking in particular for ulcerations or mobile debris (Figure 6). The optimized crosssectional planes of the heart that provides the 3D is one of the important advantage of this technology over the 2D, it allows accurate measurements of chamber dimensions as well as improved evaluation of the morphology of different structures. [START_REF] Lang | EAE/ASE Recommendations for Image Acquisition and Display Using Three-Dimensional Echocardiography[END_REF]. The analysis of the degree of calcification of the plaques also shows a good agreement between the 2D and 3D analyzed in MPR.

All AAP ulcerations and mobile debris seen in 2D were identified from 3D images. Similarly, a study reported the ability of 3D TEE for the identification of these morphological AAP characteristics. [START_REF] Haruki | Prevalence and clinical implication of complex atherosclerotic plaque in the descending thoracic aorta of Japanese patients assessed by transesophageal echocardiography[END_REF]In that study, the 3D TEE was dedicated to morphological assessment of large plaques without any quantitative evaluation from 3D. In another work, a good agreement between quantitative 2D and 3D evaluation of AAP has been shown. However, the evaluation of AAP calcification as well as the morphological analysis of AAP from 3D were not investigated. [START_REF] Aggeli | Role of real time-3D transesophageal echocardiography in evaluating the atheromatous burden of thoracic aorta in patients with heterozygous familial hypercholesterolemia: comparison with 2D transesophageal study[END_REF] A 3D acquisition offers a much greater volume of data than that acquired with 2D acquisition.

In this work, we have systematically acquired 2D and 3D images separately. Given the good agreement between the information extracted from the two modes, our findings support that a 3D TEE exploration protocol of descending aortic atheroma appear to be as efficient as the standard protocol.

In the new approach, the 2D is used for guiding 3D volume acquisitions; however, the technically difficult and time consuming on-line 2D multiplane imaging is avoided.

From a morphological perspective, most of the AAPs were classified as type I. While the three types of plaques in 3D acquisitions had different 2D characteristics, there was significant "overlap" of the 2D appearance between the plaque categories. It is not therefore possible to predict reliably the 3D morphology from the 2D characteristics. The clinical meaning of this new morphological AAP approach remains to be evaluated.

Type III AAP were associated with an empty band under the plaque giving the impression of a "floating" plaque. In 2D acquisitions, this type of plaque is most often calcified. Thus, the space below the plaque could correspond to a posterior acoustic shadowing.

Potential applications of 3D TEE

The management of the increased workload of the echocardiography laboratories is a current important issue. [START_REF] Hammoudi | Pretest probability of a normal echocardiography: validation of a simple and practical algorithm for routine use[END_REF]As descending aortic atheroma is the most common extracardiac finding during TEE, [START_REF] Alkhouli | Extracardiac Findings on Routine Echocardiographic Examinations[END_REF] the implementation of the new 3D protocol in routine appear to be a promising interest of 3D AAP approach. It might be helpful to reduce the examination duration. In addition to the optimization of the laboratory efficiency, this could also improve the patient comfort.

3D TEE might also be used to check carefully off-line by MPR mode the entire plaque volume and make sure that the measure of thickness is done at the right place (maximal thickness) with the right method (perpendicularly to the aortic wall). That could be very helpful to verify the appropriate classification of the AAP with a 2D thickness close to the 4mm validated cut-off value. [START_REF] Cohen | Aortic plaque morphology and vascular events: a follow-up study in patients with ischemic stroke. FAPS Investigators. French Study of Aortic Plaques in Stroke[END_REF] The standardization of on-line acquisition protocol and MPR post-processing that allow 3D TEE make this technology a promising tool for clinical research. MRI and CT could also be used for the assessment of AAP. However, MRI is expensive and cannot be performed at the bedside, while CT exposes patients to radiation and contrast injection.

Limitations

The review time for 3D acquisitions is an obstacle to the use of this technique. However, improved post-processing software should reduce the analysis time in the future.

In this study, the quantitative approach to AAP volume was not possible. Improved postprocessing software should soon allow for this to be evaluated. Recently, a study reported a quantitative approach for AAP volume, which correlated well with 2D measurements. However, in this work the volume was not measured; rather, it was estimated by multiplying the surface area by plaque thickness, which no doubt explains the correlation with 2D acquisitions. [START_REF] Aggeli | Role of real time-3D transesophageal echocardiography in evaluating the atheromatous burden of thoracic aorta in patients with heterozygous familial hypercholesterolemia: comparison with 2D transesophageal study[END_REF] Using a pre-commercial new software another group have reported very recently the feasibility of semiautomated volume quantification of AAP from 3D TEE images. [START_REF] Piazzese | Semiautomated detection and quantification of aortic plaques from three-dimensional transesophageal echocardiography[END_REF]In this latter work the correlation between maximum AAP thickness and volume was only moderate. Further studies are warranted to investigate the clinical impact of this very promising new quantitative approach of AAP.

The study of AAP in aortic arch involved in neurovascular disease was not carried out in this study. In fact, we decided as a first step to evaluate 3D TEE approach of AAP in descending thoracic aorta. Our very encouraging results need to be confirmed in aortic arch which is technically more complex to study by ultrasound.

Finally, in this feasibility study we excluded the subjects without aortic atheroma in 2D. It would be interesting in next studies to assess if 3D TEE could detect AAPs that are not detected in 2D.

Conclusion

3D TEE is a feasible method for the analysis of AAP of the descending thoracic aorta. In addition to conventional characterization, 3D TEE offers a promising approach to the disease as much morphologically as quantitatively. The clinical impact of 3D TEE approach of AAP should be investigated in larger studies.

FIGURE LEGENDS Figure 1 .Figure 2 .

 12 FIGURE LEGENDS Figure 1. Study flow chart. AAP, aortic atherosclerotic plaque; MPR, multiplanar reconstruction mode; TEE, Transesophageal echocardiography; TTE, transthoracic echocardiography

Figure 3 .

 3 Figure 3. MPR allowing the analysis of degree of calcification and the thickness measurement of an aortic atherosclerotic plaque (arrows) from a 3D image. (A) Long-and (B) short-axis reconstructions; note the hyperechogenic image with a posterior cone shadow (calcified plaque); (C) short-axis reconstruction with thickness measurement; and (D) original 3D morphology.

Figure 4 .

 4 Figure 4. 2D Thickness values in the three morphological 3D types of plaques. There was an overlap among the groups and the 3D morphology could not be predicted from 2D data. The box defines the interquartile range, the median value is indicated by the bar, and the whiskers indicate the minimal and maximal thickness.

Figure 5 .

 5 Figure 5. Bland-Altman analysis demonstrated a good agreement between plaque thickness derived from 2D TEE and 3D MPR. MPR, multiplanar reconstruction mode; SD, standard deviation; TEE, Transesophageal echocardiography

Figure 6 .

 6 Figure 6. Two short axis reconstruction plans of the aorta extracted with MPR mode from a 3D acquisition of a voluminous aortic plaque. (A) The red line cutting plane shows the maximum thickness of the plaque and an ulceration (arrow); (B) at the green line cutting plane the plaque appear to be thinner and the ulceration is not displayed.

Table 1

 1 TABLES Baseline characteristics of the study population (n = 82)

	Characteristic	n (%) or median (interquartile range)
	Age, years	62.3 (55.2-75.1)
	Gender, women	33 (40.2)
	Body mass index, kg/m²	25.4 (22.4-28.0)
	Hypertension	38/77 (49.4)
	Smoking (current or past)	34/77 (44.2)
	Diabetes	20/77 (26.0)
	Hypercholesterolemia	32/77 (41.6)
	Atrial fibrillation	25 (30.5)
	Serum creatinine*, µmol/L	77 (66-98)
	Prior myocardial infarction	10/77 (13.0)
	Prior stroke	9/77 (11.7)
	Left ventricular ejection fraction**, %	65 (60-70)
	Left atrial area**, cm²	21 (19-28)
	Transesophageal echocardiography indication
	Stroke	53 (64.6)
	Atrial fibrillation	8 (9.8)
	Valvulopathy	7 (8.5)
	Endocarditis	6 (7.3)
	Others	8 (9.8)
	*n=74; **from transthoracic echocardiography

Table 2

 2 Two-dimensional (2D) characteristics of the three-dimensional (3D) morphological types of atherosclerotic plaques in the descending aorta (n = 189)

	Variable

Table 3 .

 3 Agreement analysis between 2D and 2D data extracted from 3D images by multiplanar

	reconstruction mode (MPR)
				From 3D
		Thickness	< 1 mm	1-3.9 mm	≥ 4 mm	n
		< 1 mm	36 (97.3)	1 (2.7)	0 (0)	37
	From 2D	1-3.9 mm	23 (17.6)	104 (79.4)	4 (3.0)	131
		≥ 4 mm	0 (0)	3 (14.3)	18 (85.7)	21
				From 3D
		Calcification	Not calcified	Slightly calcified	Very calcified
		Not calcified	81 (88.0)	11 (12.0)	0 (0)	92
	From 2D	Slightly calcified	3 (4.6)	47 (72.3)	15 (23.0)	65
		Very calcified	0 (0)	4 (12.5)	28 (87.5)	32
	Results are expressed as n (%)

ACKNOWLEDGMENT

Sophie Rushton-Smith, PhD, provided editorial assistance including editing, checking content and language, formatting, and referencing, and was funded by the authors.

DISCLOSURES

The authors have no disclosures

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