Emmanuel Rocca

Hadri Faiz

Philippe Dillmann

Delphine D Neff

François Mirambet

Electrochemical behavior of thick rust layers on steel artefact: Mechanism of corrosion inhibition

Keywords: corrosion layer, corrosion inhibition, FeOOH, impedance spectroscopy, carbon paste electrode

Introduction

Since the middle of the 20 th century, an increasing number of materials, objects of the industrial heritage are preserved in heritage institutions. Their preservation is of great importance considering their historical, technological, architectural and scientific values. This heritage consists of buildings and machinery, workshops, mills and factories, mines and sites for processing and refining… [START_REF] Alfrey | The Industrial heritage: Managing resources and uses[END_REF]. Among the objects conserved in museum collections, a significant part are covered by thick rust layers, especially items in cast iron or steel from the industrial revolution in the last half of the 18th century. These corrosion layers (CL) should be preserved because they also contain some traces of manufacturing and use of the objects.

Moreover, the important size of many industrial artefacts needs to expose or to store them outside or in sheltered conditions without control of the environmental parameters.

In cyclic atmospheric conditions, Evans and Taylor [START_REF] Evans | Mechanism of Atmospheric Rusting[END_REF] have first proposed a cyclic electrochemical mechanism to explain the formation of thick rust layers on iron substrate in atmospheric conditions. This cyclic mechanism can be described as follows. During a first wetting step, the iron(III) oxyhydroxides (FeOOH) are progressively dissolved into iron(III) cations which oxidize the iron metal into iron(II) compounds throughout the pores of the corrosion layer. Then, once the metal is covered by a thick water layer, the iron metal is mainly oxidized by dissolved oxygen into iron(II) compounds. Next, the drying step induces a progressive decrease of water thickness on surface and a rapid diffusion of oxygen gas across the thin water layer leading to both the oxidation of iron(II) species into iron(III) oxyhydroxide and the oxidation of iron into Fe(II). This mechanism, experimentally demonstrated by M. Stratman et al. [START_REF] Stratmann | An electrochemical study of phase transitions in rust layers[END_REF][START_REF] Stratmann | The atmospheric corrosion of iron -a discussion of the physico-chemical fundamentals of this omnipresent corrosion process[END_REF], is mainly controlled by the dissolution-precipitation process of different iron(II) and iron(III) solid compounds during wet-dry cycles. During this cycle, the oxygen access inside the CL has been underlined as an important parameter by several authors [START_REF] Stratmann | The mechanism of the oxygen reduction on rust-covered metal substrates[END_REF]. Moreover, the electronic conductivity of each iron oxide phase and their connectivity with the iron substrate are important parameters defining the localization of the oxygen reduction, but don't modify the overall Stratman's mechanism [START_REF] Monnier | Localisation of oxygen reduction sites in the case of iron long term atmospheric corrosion[END_REF][START_REF] Neff | Corrosion of iron archaeological artefacts in soil: characterisation of the corrosion system[END_REF][START_REF] Bouchar | New Insights in the Long-Term Atmospheric Corrosion Mechanisms of Low Alloy Steel Reinforcements of Cultural Heritage Buildings[END_REF][START_REF] Bernard | Understanding corrosion of ancient metals for the conservation of cultural heritage[END_REF]. In fact, ancient corrosion layer are often characterized by a complex mixing of several oxyhydroxide phases with different stratification, mainly depending on atmospheric conditions along their ageing [START_REF] Neff | Raman imaging of ancient rust scales on archaeological iron artefacts for long-term atmospheric corrosion mechanisms study[END_REF].

Only few studies propose a comprehensive electrochemical characterization of the phenomenon occurring inside a thick CL on steel [START_REF] Frateur | Free chlorine consumption induced by cast iron corrosion in drinking water distribution systems[END_REF][START_REF] Barcia | Application of the impedance model of de Levie for the characterization of porous electrodes[END_REF][13][START_REF] Sancy | Tribollet Mechanism of corrosion of cast iron covered by aged corrosion products: Application of electrochemical impedance spectrometry[END_REF]. From impedance measurements on corroded cast iron pipe, I. Frateur et al. have first proposed a description of the electrochemical phenomenon of cast iron in presence of CL [START_REF] Frateur | Free chlorine consumption induced by cast iron corrosion in drinking water distribution systems[END_REF][START_REF] Barcia | Application of the impedance model of de Levie for the characterization of porous electrodes[END_REF] based on de Levie's conductive porous electrode model [13], in immersed conditions in water electrolyte. This electrochemical model was confirmed and adapted in the case of corroded cast iron in water pipe [START_REF] Sancy | Tribollet Mechanism of corrosion of cast iron covered by aged corrosion products: Application of electrochemical impedance spectrometry[END_REF].

For cultural heritage, the cleaning of corroded surface can be required by curators to homogenize the surface aspect and to avoid the formation of red pits on surface during exposure in uncontrolled atmospheres [15,[START_REF] Gust | Sealing and Anti-Corrosive Action of Tannin Rust Converters[END_REF]. Nowadays, different treatments are implemented by conservators such as conversion treatments with polyphenol macromolecules based on flavonoids groups, named tannins [START_REF] Pardini | Formulation and testing of a waterborne primer containing chestnut tannin[END_REF][START_REF] Rahim | Mangrove (Rhizophora apiculata) tannins: an eco-friendly rust converter[END_REF], or with more aggressive phosphating solutions [START_REF] Rahim | Inhibitive action of mangrove tannins and phosphoric acid on pre-rusted steel via electrochemical methods[END_REF]. Unfortunately, these treatments can induce important modifications of the color aspect. The use of varnish or paints is also an interesting method of protection for objects exposed in outdoor in unsheltered conditions, but the change of aspect can be drastic in this case. Now, the conservation of the corrosion layers without modifications of aspect requires the development of specific treatments. Moreover, the important size of these objects requires that the treatment is realized on site with non-toxic and easy process.

In this framework, the present work aims to study the effect of non-toxic corrosion inhibitors as carboxylate and flavonoid compounds on the electrochemical behavior of aged corrosion layers on steel. In fact, sodium carboxylate or carboxylic acid have been already investigated as corrosion inhibitors on many metals such as zinc, copper, steel, magnesium alloys or lead [START_REF] Georges | Synergistic effect of tolutriazol and sodium carboxylates on zinc corrosion in atmospheric conditions[END_REF][START_REF] Rocca | Inhibition of copper aqueous corrosion by non-toxic linear sodium heptanoate : mechanism and ECAFM study[END_REF][START_REF] Rocca | Inhibition of lead corrosion with saturated linear aliphatic chain monocarboxylates of sodium[END_REF][START_REF] Rocca | Inhibition treatment of the corrosion of lead artefacts in atmospheric conditions and by acetic acid vapour : use of sodium decanoate[END_REF][START_REF] Mesbah | Inhibitors for magnesium corrosion: Metal organic frameworks[END_REF]. And catechin is the main flavonoid species, which constitutes the monomer of condensed tannin [START_REF] Rahim | Mangrove tannins and their flavanoid monomers as alternative steel corrosion inhibitors in acidic medium[END_REF].

The objective is to investigate the corrosion inhibition mechanism by studying, both the electrochemical behavior of the metal/CL system with or without corrosion inhibitor, and the influence of corrosion inhibitor on the electrochemical activity of different iron(III) oxyhydroyde phase of CL by the carbon paste electrode (CPE) technique.

Firstly, the metal/CL system was studied by electrochemical impedance spectroscopy (EIS) in corrosive water with or without inhibitor. Then, a corrosion inhibition treatment of the metal/CL system is proposed and was characterized by EIS in corrosive conditions. The corrosion layer and iron(III) oxyhydroxide treated and non-treated by corrosion inhibitors were characterized by Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX).

Experimental

The corroded steel samples, provided by the Musée Les Mineurs Wendel in the ancient site of a coal mine at Petite-Roselle in France, are supports of railroad tracks used in the pit head during around 50 to 60 years and then stored in outdoor unsheltered conditions, displayed in Figure 1a. These samples have a relative homogeneous thickness of corrosion layer of around 100 µm without traces of residual soil or coal. They were cut in small pieces of 1*1 cm 2 and carefully embedded in epoxy resin to mask the cut edges for electrochemical measurements, according to the scheme in Figure 1b. Then, the prepared samples were used as working electrode.

For metallographic observations and analysis, the metallographic cross-sections were polished with successive SiC emery papers up to 2400 under absolute ethanol, then with different diamond pastes without water. Samples were finally rinsed with ethanol and dried with hot air (around 40 °C).

The chemical analysis of the CL was realized by micro-Raman spectroscopy measurements performed on the metallographic cross section (Renishaw inVia reflex micro-Raman spectroscopy). Both the incident and the scattered lights are collected through a Leica microscope. Raman excitation was provided by a frequency-doubled Nd:YAG laser operating at 532 nm with a power of about 0.1 mW and with a probe diameter of about 1 µm. All spectra were calibrated using the 520.5 cm -1 line of a silicon wafer.

For X-ray diffraction analysis (XRD), the corrosion layers were sampled by scraping the surface and were crushed in an agate mortar to be analyzed by a Philips XPert Pro diffractometer equipped with copper anticathode (λkα = 1.5418 Å). Surface observations of the CL were carried out by Field Emission Scanning Electron Microscopy (FE-SEM JEOL J7600F), in backscattering electron (BSE) or secondary electron (SE) detection modes.

The reference corrosive medium in electrochemical experiments was the ASTM D1384-87 solution (noted ASTM [START_REF] Astm Standard | Standard test method for corrosion test engine coolants in glassware[END_REF]) with the following composition: 148 mg l -1 Na2SO4, 138 mg l -1 NaHCO3 and 165 mg l -1 NaCl, at pH=8.3. Sodium decanoate solution CH3(CH2)8COONa noted NaC10 was obtained through the neutralisation of decanoic acid (from Aldrich) by sodium hydroxide solution until pH=8. Then, sodium decanoate or catechin (from Aldrich) were mixing with ASTM water at a desired concentration at pH=8 for the electrochemical measurements. Decanoic acid solution, CH3(CH2)8COOH noted HC10, was just prepared by mixing decanoic acid and deionized water at natural pH = 4.5.

The electrochemical tests were performed, in aerated conditions, with a three-electrode electrochemical cell, connected to an EGG PAR 273A potentiostat and a frequency response analyzer (Solartron FRA 1255), driven by a computer. In this configuration, the working electrode surface is horizontal, facing the Pt-grid counter electrode. The reference electrode was a KCl-saturated calomel electrode (Hg2Cl2/Hg, E = +0.242 V/SHE) and all the working electrode potentials were given versus this reference. Electrochemical impedance spectra were recorded at open-circuit potential (or corrosion potential) between 10 6 and 10 -3 Hz with a 50 mV amplitude and 10 points per frequency decade, after a minimum of 30 min of immersion at open-circuit potential. The voltage amplitude was chosen in order to increase the signal/noise ratio, especially at low frequency, without changing the overall state of the system, by verifying the symmetry of the Lissajous plot (AC current versus AC potential).

Carbon pastes were prepared by mixing at 60 °C: 10 wt% of studied compound with 50 wt% of graphite powder and 40 wt% of paraffin wax as inert binder (in a liquid state). The liquid paste was inserted in a Plexiglas tube in contact with a Pt-wire. Then after cooling, the carbon paste surface was carefully polished with SiC emery paper. Voltammetric measurements with the carbon paste were carried out at 10 mV s -1 in Na2SO4 0.1 M support electrolyte (pH=7).

Goethite was provided from Alfa Aesar. Lepidocrocite and ferrihydrite were synthesised at 25 • C, following a procedure drawn from Cornell et al. [START_REF] Cornell | The iron oxides -Structure, Properties, Occurrences and Uses 2 nd[END_REF], and analysed by XRD.

Results and interpretations

Analysis of thick corrosion layer (CL)

After metallographic etching with Nital reagent (5%vol HNO3 in ethanol), the iron alloy of the railroad tracks reveals some zones of ferrite with ferrito-pearlitic structure with the presence of inclusions (Figure 1c). EDX analysis shows that carbon and phosphorous content is respectively inferior to 1 wt% and 0.1 wt%.

Concerning the composition of the corrosion layer (CL), X-ray diffraction pattern exhibit the presence of goethite, lepidocrocite and maghemite or magnetite, as often analyzed on this kind of aged CL (Figure 2a) [START_REF] Bouchar | New Insights in the Long-Term Atmospheric Corrosion Mechanisms of Low Alloy Steel Reinforcements of Cultural Heritage Buildings[END_REF]. More precisely, microRaman spectroscopy allows distinguishing two distinct layers at the microscopic scale, as displayed in Figure 3 [28]:

-An internal layer, close to the metal, is mainly formed with goethite, characterized by the Raman peak at 380 cm -1 with areas of ferrihydrite and maghemite, identified by the peak at 700 cm -1 which is a very low-crystallized phase [START_REF] Mazzetti | Raman spectra and thermal transformations of ferrihydrite and schwertmannite[END_REF][START_REF] De Faria | Raman microspectroscopy of some iron oxides and oxyhydroxides[END_REF]. This layer is relatively compact, particularly in the neighboring of the metal/oxide interface, and is sometimes called "black layer".

-An external layer is very cracked and porous, and mainly composed by lepidocrocite, identified by the Raman band at 250 cm -1 [START_REF] Neff | Raman imaging of ancient rust scales on archaeological iron artefacts for long-term atmospheric corrosion mechanisms study[END_REF][START_REF] De Faria | Raman microspectroscopy of some iron oxides and oxyhydroxides[END_REF].

EIS measurements

In the ASTM corrosive electrolyte without corrosion inhibitor (containing chloride, sulfate, hydrogenocarbonate at pH=8.3), the corrosion potential of the metal/CL system rapidly reaches a stable value by decreasing from -370 mV to -400 mV after 50 h of immersion according to Figure 4. During the immersion, the electrochemical impedance spectra exhibit qualitatively the same features with immersion time (Figure 5). So, the CL rapidly reaches a steady state in this electrolyte.

These impedance spectra can be described with three main relaxation phenomena:

-At high frequency (between 10 5 and 10 4 Hz), the first capacitive loop is assigned to a charge transfer phenomenon, characterized by a very low capacitance value. By considering a resistance value around 1000 Ω cm 2 and a characteristic frequency (fc) around 5 10 4 Hz, the capacitance value can be estimated between 10 -7 to 10 -8 F.cm -2 (fc = (2πRC) -1 ). This value is lower than a usual double layer capacitance of a metal and could be attributed to the capacitance of a dielectric oxide film in contact with metal [START_REF] Frateur | Free chlorine consumption induced by cast iron corrosion in drinking water distribution systems[END_REF].

-At medium frequency (around 10 3 to 10 2 Hz), a second relaxation time can be distinguished, characterized by a very depressed capacitive loop on the Nyquist diagram, and can be assigned to the impedance of conductive porous electrode which could be explained by the theory of de Levie [31].

-At low frequency (below 1 Hz), a third time constant is clearly assigned to diffusion phenomenon in the electrolyte inside the large pores of the external CL. In the Nyquist representation, the impedance data show an angle of about 30° with the real axis, which suggest a Warburg-like component or an impedance in a semi-infinite porous medium [START_REF] Frateur | Free chlorine consumption induced by cast iron corrosion in drinking water distribution systems[END_REF][START_REF] Barcia | Application of the impedance model of de Levie for the characterization of porous electrodes[END_REF].

Nevertheless, as simulated in the theoretical work of S.J. Cooper et al. on the diffusion impedance in porous media, a clear assignment of the phenomenon at medium and low frequency is difficult because different relaxation times can appear in function of conductivity, microstructure and geometry of pores in this kind of aged corrosion layer [START_REF] Cooper | Simulated impedance of diffusion in porous media[END_REF].

Despite the fact that the thickness of CL is important, the overall impedance modulus without inhibitor does not exceed 10000 Ω cm 2 at low frequency after 48 h of immersion in ASTM water. According microstructural analysis and EIS measurements, a comprehensive structural model of this metal/CL system can be proposed in Figure 6a.

With addition of 10 -2 M of sodium decanoate in ASTM water, the corrosion potential of the electrochemical system is sharply increased in the first hours of immersion, around 0 V, and stabilize around -100 mV after 20 h of immersion. This result reveals that decanoate ions have mainly an inhibiting action on the anodic reactions of the metal/CL system (Figure 4). In contrary, the addition of catechin has a very poor effect on the corrosion potential of the system. In fact, after some hours of immersion, the potential reach a value similar to the one measured without inhibitor.

After 24 hours of immersion, the measurements of impedance spectra with and without corrosion inhibitors are displayed and compared on Figure 7. Globally, the impedance spectra have the same features and can be interpreted with the same three relaxation phenomena detailed for the spectra without inhibitor in Figure 5.

At high frequency (between 10 4 and 10 5 Hz), the NaC10 addition slightly modifies the impedance modulus, from approximately 1000 to 2000 Ω cm 2 , which indicates that decanoate ion has a weak effect on the layer at the metal interface.

Nevertheless, the presence of decanoate ions significantly increases the impedance modulus of the relaxation phenomenon at medium and particularly at low frequency. Indeed the impedance modulus at low frequency (below 1 Hz) is approximatively multiplied by a 3 to 4 factor with decanoate ions, which means that its addition decreases the diffusion of ionic species such as iron cations and hydroxyl ions in the pores network.

X-ray diffraction analysis of the metal/CL system immersed in 10 -2 M NaC10 during 5 days shows the characteristic diffraction (001) and (002) peak at low angle of iron decanoate, which can be noted Fe(C10)3 in Figure 2c, according to previous study on iron carboxylate compounds by X-ray absorption spectroscopy at the iron K-edge and XRD [START_REF] Mirambet | A complementary set of electrochemical and X-ray synchrotron techniques to determine the passivation mechanism of iron treated in a new corrosion inhibitor solution specifically developed for the preservation of metallic artefacts[END_REF]. So, at neutral pH in ASTM corrosive water, decanoate anions are able to form insoluble iron decanoate in the CL and to block the diffusion of iron cations in the layer (figure 2b). However, the chemical conversion of FeOOH compounds into iron decanoate formation seems to be limited in this neutral pH in ASTM water.

Regarding the catechin addition, the presence of catechin compound in ASTM water has a weak influence on the overall impedance modulus of the metal/CL system. At high frequency, the impedance modulus increases from 1000 Ω cm 2 without inhibitor to 2000 Ω cm 2 with catechin (at 5 10 4 Hz), which indicates a slight increase of the resistance at the metal interface with catechin (Figure 7a). Thus, according to EIS analysis and surface analysis, decanoate species are able to increase the impedance modulus of the metal/CL system by forming iron decanoate inside the corrosion layer. In the following sections, a treatment in acid media (at pH=4.5) with a decanoic acid (HC10) solution will be used to increase the chemical conversion of FeOOH compounds into iron decanoate. Then, the corrosion behavior of HC10-treated metal/CL system was evaluated by EIS in ASTM corrosive water.

Electrochemical activity of iron oxyhydroxides in presence of corrosion inhibitor

In order to evaluate the influence of the HC10 conversion treatment on FeOOH compounds, the electrochemical activity of the main iron oxyhydroxide phases in the CL was studied in carbon paste electrode in neutral support electrolyte. Figure 8 gathers the voltammograms of lepidocrocite, goethite and ferrihydrite without inhibitor, which shows all a broad oxidation current peak around -0.3 to -0.2 V, and a reduction current peak around -1 V. However, the maximum oxidation and reduction current are very different in these two potential domains in function of FeOOH phases. According to most authors [START_REF] Vago | Electrocatalysis of oxygen reduction at iron oxide (Fe3O4) electrodes in alkaline solutions[END_REF][START_REF] Allen | The electrodissolution of magnetite: Part I. The electrochemistry of Fe3O4/C discs-potentiodynamic experiments[END_REF], these two peaks are due to an electronic exchange between Fe(III) and Fe(II) species, and the broadness of the peaks likely corresponds to several electrochemical processes, which are not yet well-known.

Nevertheless, the experimental studies and theoretical calculation of Charlet et al. show that the FeOOH/Fe 2+ redox couple have a very low equilibrium potential, around -0.5 to -0.6 V /SCE, compared to classical equilibrium potential of Fe(III)/Fe(II) redox couple [START_REF] Charlet | N-compound reduction and actinide immobilization in surficial fluids by Fe(II): the surface ≡FeIIIOFeIIOH° species, as major reductant[END_REF]. So, these peaks can correspond to the overall redox equation between FeOOH solid phases and aqueous or adsorbed Fe 2+ cations: FeOOH(s) + e -+ 3H + Fe 2+ (aq) + 2H2O. So, the maximum reduction peak current at -1 V is meaningful to evaluate the electrochemical reactivity of each phase. In this case, the most reactive phase, with the highest reduction peak, is ferrihydrite, then lepidocrocite, and the more inert phase is goethite, as already observed by Anthony et al. with another coulometric method [START_REF] Antony | Electrochemical study of indoor atmospheric corrosion layers formed on ancient iron artefacts[END_REF].

The most abundant phase in the external part of the CL, lepidocrocite (Figure 9), has been treated by simple immersion in corrosion inhibitor, decanoic acid solution and catechin solution at pH 4.5 during 5 days.

For the HC10 treatment, the presence of the diffraction peak at low angle in Figure 10 is characteristic of the formation of crystallized iron carboxylate Fe(C10)3, shown in Figure 10d. HC10 treated-lepidocrocite is partially transformed in iron carboxylate during this immersion treatment (Figure 10b,c) compared to non-treated lepidocrocite in Figure 10a. The voltammetric measurement in CPE of the HC10-treated lepidocrocite shows a sharp decrease of the reduction peak current, which reveals that the electrochemical activity of lepicocrocite is almost completely inhibited by the immersion treatment in HC10 solution (Figure 9a).

Concerning the catechin treatment, the immersion of lepidocrocite in 0.1 M catechin solution does not modify the X-ray diffractogram and Raman spectra of the FeOOH phase, although the lepidocrocite powder becomes blue-black color (data not shown). This observation indicates that the reaction of lepidocrocite with catechin to form blue black iron catecholate complex only occurs on the grains surface of FeOOH. The low chemical conversion of FeOOH in blue-black color catecholate complex is well-corroborated with the low reduction of current peak of the catechin-treated lepidocrocite in CPE. Consequently, the electrochemical activity of FeOOH phase is poorly affected by the catechin treatment in these conditions.

Consequently, the immersion in acidic solution leads to an increase of iron(III) dissolution, then precipitation into iron decanoate in bulk or iron catecholoate, only on the grain surfaces, according to the schematic reactions [START_REF] Mirambet | A complementary set of electrochemical and X-ray synchrotron techniques to determine the passivation mechanism of iron treated in a new corrosion inhibitor solution specifically developed for the preservation of metallic artefacts[END_REF][START_REF] Antony | Electrochemical study of indoor atmospheric corrosion layers formed on ancient iron artefacts[END_REF]: Fe(III) + 3HC10 Fe(C10)3 + 3H + Fe(III) + 3Cat Fe(Cat)3 (where cat represents catecholate organic function).

Inhibiting treatment of CL

The inhibiting treatment in HC10 decanoic acid solution was then performed in the overall metal/CL system. After treatment, SEM observations in BSE mode (Figure 11) display that the HC10-treated surface is partially covered by thick iron carboxylate deposit. The SEM zoom image in SE mode confirms the crystallization of iron carboxylate platelets. In fact, at a micrometer scale, the iron carboxylate coating is not homogeneous because the composition of the CL and its reactivity are not homogeneous. Nevertheless, the HC10-treated corrosion layers show macroscopically no change of colors and aspects after 5 days of treatment.

The corrosion potential and impedance spectra of treated system were regularly recorded during 30 days in ASTM corrosive water. As can be seen in Figure 12, the corrosion potential of HC10-treated metal/CL system remains approximately 400 mV higher than the one of nontreated metal/CL system in the ASTM corrosive water. It is noteworthy that the corrosion potential remains very stable even during long immersion time for the non-treated and treated system.

Throughout the immersion in the corrosive water, the EIS spectra of the non-treated and HC10-treated sample have the same features and can be also described with three main relaxation phenomena (Figure 13).

In the first hours of immersion (at 48 hours), the impedance spectra of HC10-treated sample, displayed on Figure 13, show an overall increase impedance modulus in all the frequency range. Particularly, the impedance modulus assigned to the diffusion phenomena at low frequency (below 1 Hz) in the external layer is multiplied by a ten factor (Figure 13a). Even after a long immersion time (at 480 h), the impedance modulus values of the HC10-treated CL remain high which underlines the good stability of the HC10-treated corrosion layer all over the immersion time in this corrosive media (Figure 13b).

Consequently, the treatment of metal/CL system in a 0.1 M HC10 solution at pH 4.5 clearly induces the formation of iron decanoate by chemical conversion, which covers the surface of FeOOH phase and seals some pores and some areas.

A schematic structural model of the HC10-treated metal/CL system is proposed in Figure 6b.

Even though the chemical conversion of corrosion layer is not homogeneous as shown by SEM observations, the impedance measurements show that this conversion layer increases the overall impedance of the system by inhibiting the dissolution phenomenon of FeOOH phases and reducing the diffusion phenomenon through the pores network.

Conclusion

Through the results of this study, the mechanism of corrosion inhibition in metal/CL system is mainly based on the blocking of the dissolution of FeOOH-type phase and hindering of the diffusion of ionic species inside the pore network of CL. Indeed, addition of decanoate anions in corrosive medium or a decanoic acid treatment of CL allow one to increase the impedance of CL layer and limit the diffusion inside the layer. The inhibition mechanism is a chemical conversion of the reactive iron oxyhydroxide, as lepidocrocite, into iron carboxylates (or iron soaps). This iron soap inhibits the electrochemical activity of FeOOH-type phases and blocks the dissolution of FeOOH, as demonstrated with CPE experiments.

Regarding the catechin, although many rust compounds are transformed in surface into blueblack colored compounds, which are appreciated by curators, the catechin has a slight influence on the electrochemical activity of FeOOH type phases .

From an electrochemical point of view, even if the different relaxation phenomenon are difficult to identify in details in the complex metal/CL systems on iron, electrochemical

Figure 1 :

 1 Figure 1: (a) Macroscopic photo of corroded railroad track (The black underlined area are sampling zones) , b) Schematic representation of prepared working electrode with the corroded railroad track (c) Optical micrograph of steel of corroded railroad tracks.

Figure 2 :Figure 3 :

 23 Figure 2: X-ray diffractograms of corrosion layer without inhibition treatments (a), after 5 days of immersion in NaC10 10 -2 M solution (b), after 5 days of immersion in 0.1 M HC10 solution (c)

Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 4567 Figure 4: Corrosion potential measurements of "metal/CL" system versus immersion time in ASTM corrosive water without or with corrosion inhibitors.

Figure 8 :Figure 9 :

 89 Figure 8: Voltammogram of different iron oxyhydroxide phase in CPE in Na2SO4 0.1 M electrolyte

Figure 11 :Figure 12 :Figure 13 :

 111213 Figure 11: SEM observation of surface of CL after HC10-based conversion treatment (5 days in HC10 0.1 M) in BSE mode and zoom of iron carboxylate deposit in SE mode


Camille Roux
email: roux.cam@hotmail.fr

Frédérique Gandjbakhch

Audrey Pierreisnard

Marion Couderc

Cédric Lukas

Racha Masri

Jean-Philippe Sommier

Isabelle Clerc-Urmès

Cédric Baumann

Isabelle Chary-Valckenaere

Isabelle Clerc-Urmes

Damien Loeuille

Ultrasonographic criteria for the diagnosis of erosive rheumatoid arthritis using osteoarthritic patients as controls compared to validated radiographic criteria

Keywords: rheumatoid arthritis, osteoarthritis, erosion, ultrasonography, radiography, diagnosis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Rheumatoid arthritis (RA) is the most prevalent chronic inflammatory joint disease [START_REF] Alamanos | Incidence and prevalence of rheumatoid arthritis, based on the 1987 American College of Rheumatology criteria: a systematic review[END_REF] responsible for structural damage. To limit these consequences, an international consensus recommended a therapeutic strategy based on early diagnosis and search for poor prognostic factors in order to optimize the tight control of disease activity. [START_REF] Van Aken | Radiological outcome after four years of early versus delayed treatment strategy in patients with recent onset rheumatoid arthritis[END_REF] Erosions on radiography (RX), high levels of biologic inflammation parameters, and the presence of anti-cyclic citrullinated protein antibodies (ACPA)

(3) are the main predictive factors of bone erosions on radiography, which is the gold standard for visualizing and quantifying bone lesions in RA (erosion and joint space).( 4) The modified Sharp/van der Heijde score (5) demonstrated good intraand inter-reader reliabilities, good sensitivity to change [START_REF] Van Der Heijde | Reliability and sensitivity to change of a simplification of the Sharp/van der Heijde radiological assessment in rheumatoid arthritis[END_REF] and is considered as the standard scoring method for assessing structural damage in RA.

Musculoskeletal ultrasound (US) is booming in clinical practice for the diagnosis and evaluation of inflammatory lesions from inflammatory disorders, and it has been proven to be effective in the evaluation of bone erosions in musculoskeletal disease osteoarthritis (OA) [START_REF] Usón | Symptomatic and asymptomatic interphalageal osteoarthritis: An ultrasonographic study[END_REF], gout [START_REF] Gutierrez | International Consensus for ultrasound lesions in gout: results of Delphi process and web-reliability exercise[END_REF], and in psoriatic arthritis (PsA) [START_REF] Weiner | Ultrasonography in the assessment of peripheral joint involvement in psoriatic arthritis : a comparison with radiography, MRI and scintigraphy[END_REF]. In RA, many studies have shown that US can detect more erosions than RX at the joint level, especially at an early stage of the disease, with higher sensitivity and specificity than RX when a CT scan is taken as the gold standard imaging method [START_REF] Wakefield | The value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: A comparison with conventional radiography[END_REF].

It is now recognized that PsA [START_REF] Jadon | Psoriatic Arthritis Mutilans: Characteristics and Natural Radiographic History[END_REF], connective tissue diseases [START_REF] Alarcón-Segovia | Deforming arthropathy of the hands in systemic lupus erythematosus[END_REF], and metabolic diseases [START_REF] Dalbeth | Mechanisms of bone erosion in gout: a quantitative analysis using plain radiography and computed tomography[END_REF], are associated with bone erosions in hands and feet on RX, but these diagnosis, not only based on radiography, are finally established by specific clinical and biological features [START_REF] Rudwaleit | The development of Assessment of SpondyloArthritis international Society classification criteria for axial spondyloarthritis (part II): validation and final selection[END_REF].

In patients over 50 years of age, the diagnosis of RA is usually established based on clinical and biological features. However, the diagnosis of erosive RA disease based on RX or US is sometimes difficult since bone erosions related to degenerative changes (osteoarthritis) may coexist with erosions due to RA [START_REF] Guillemin | Prevalence of rheumatoid arthritis in France: 2001[END_REF][START_REF] Haugen | Prevalence, incidence and progression of hand osteoarthritis in the general population: the Framingham Osteoarthritis Study[END_REF].

The aims of this study are:

-to compare characteristics of RX and US erosive lesions in RA and OA patients (prevalence, topography and severity), -to determine thresholds for the diagnosis of erosive RA based on US, -to compare the performance of US and RX to establish a diagnosis of erosive RA differentiated from hand OA.

Methods

Patients

All consecutive patients hospitalized in the department of Rheumatology in Hospital of Nancy since 2005 for suspicion of RA were screened. Only patients fulfilling the following inclusion criteria were selected for this study: patients fulfilling ACR 1987 and/or ACR/EULAR 2010 criteria for RA, patients fulfilling only ACR OA criteria of hand OA served as control group [START_REF] Altman | The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hand[END_REF]. US exam and RX exam of hand and feet should be performed within 6 months. The only exclusion criterion was the presence of severe joint deformities that could prevent a complete appropriate US, RX and clinical evaluation. Finally, 122 RA patients and 46 OA patients were included. A complete assessment of their disease was performed (clinical, biological, radiological, and ultrasound evaluations). For RA patients, data collection included the Disease Activity Score 28-joint count (DAS 28) and treatments at the time of evaluation (bDMARD, sDMARD, corticosteroids, and NSAIDs). For OA patients, no treatment has been registered.

Biological assessment

The following measures were assessed: C-reactive protein level (CRP: normal value <5 mg/L), erythrocyte sedimentation rate at the first hour (ESR: normal value <5 mm), rheumatoid factor (RF) titres (normal value <20 UI), and ACPA titres (normal value <20 UI).

Radiography assessment

Postero-anterior views of hands and antero-posterior views of feet have been obtained according to the usual clinical practice recommendations for patients followed up for RA and for patients suffering from hands and/or feet OA. RX analyses have been performed blindly from clinical and US information's. Two independent readers (AP, MC) performed the modified Sharp/van der Heijde score (SHS) for erosions (SHSe) [START_REF] Van Der Heijde | EULAR definition of erosive disease in light of the 2010 ACR/EULAR rheumatoid arthritis classification criteria[END_REF][START_REF] Scheel | Prospective 7 year follow up imaging study comparing radiography, ultrasonography, and magnetic resonance imaging in rheumatoid arthritis finger joints[END_REF]. with sub-scores for hands and feet. Based on this RX evaluation, patients were classified as erosive RA according to EULAR 2013 Definition [START_REF] Van Der Heijde | EULAR definition of erosive disease in light of the 2010 ACR/EULAR rheumatoid arthritis classification criteria[END_REF]; an erosive disease is defined when an erosion (defined as a cortical break) is observed in at least three separate joints at any of the following sites: proximal interphalangeal joints (PIP), metacarpophalangeal joints (MCP), wrist (counted as one joint), and metatarsophalangeal joints (MTP) on radiographs of both hands and feet. In the case of discordance between the two readers for a number of eroded joints less than or equal to three (corresponding to the EULAR 2013 Definition of erosive RA), a third reader (ICV) served as adjudicator. For each patient, the SHSe corresponded to the mean score of the two or three readers.

Ultrasound assessment

Standardized US examinations were performed by senior US analysts (ICV, DL, JPS) after several sessions of harmonization for calibration of erosion. The equipment used throughout the study was the same: a Philips HD11 machine with a multi-frequency linear array transducer (5-12 MHz) with the focal length adjusted to the joint depth. US information's were acquired at optimal technical conditions at 12 MHz (spatial resolution 0.1 mm) blinded to clinical, biologic, and radiologic findings.

Twelve pre-selected targeted joints have been systematically examined on B mode: MCPs 2, 3, and 5 and MTPs 2, 3, and 5. MCP4 and MTP4 joints are less commonly affected by erosions in RA and were not included [START_REF] Wakefield | The value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: A comparison with conventional radiography[END_REF][START_REF] Scheel | Prospective 7 year follow up imaging study comparing radiography, ultrasonography, and magnetic resonance imaging in rheumatoid arthritis finger joints[END_REF][START_REF] Szkudlarek | Ultrasonography of the metacarpophalangeal and proximal interphalangeal joints in rheumatoid arthritis: a comparison with magnetic resonance imaging, conventional radiography and clinical examination[END_REF]; MCP1, PIP and MTP1 joints, which are frequently damaged by degenerative changes or metabolic diseases, have also been excluded [START_REF] Iagnocco | Usefulness of high resolution US in the evaluation of effusion in osteoarthritic first carpometacarpal joint[END_REF]. Wrists were also excluded because of the lack of precise anatomic localization of erosions when both axial and longitudinal plans were required. Moreover numerous cortical defects and joint ligament attachments were present embarrassing the US exam.

Localization and grading of erosions

Erosions were searched on the dorsal (D) and palmar or plantar (P) facets of each joint and on the lateral (L) facet when accessible (MCP2, MCP5 and MTP5). On each facet, erosion was defined as a cortical defect with an irregular bone surface, observed in two perpendicular planes (axial and longitudinal). Erosions were scored semi-quantitatively according to 4 grades: grade 0 = no erosion; grade 1 = single erosion <2 mm in its largest dimension; grade 2 = single erosion ≥2 mm and < 3 mm in its largest dimension or no more than two erosions < 2 mm; and grade 3 = single erosion ≥ to 3 mm in its largest dimension or multiple erosions (>n=2). The total US score for erosions (USSe) was the sum of erosion grades for all eroded joints facets (n=30) and ranged from 0 to 90.

Intra-and inter-examiner US reproducibility

Intra-examiner reproducibility was assessed on 11 RA patients according to two complete examinations per patient within 24 hours. Inter-examiner reproducibility was assessed on 11 RA patients examined independently on the same day by each US operator. For intra-examiner reproducibility, the median value of the total US erosion score was 21 (range: for the first exercise and 21 (range: 3-34) for the second.

The intra-class correlation coefficient (ICC) values of the erosion US score for intraand inter-examiner studies were 0.96 (CI95: 0.93-0.98) and 0.97 (CI95: 0.92-0.99), respectively. The inter reader reliability for the diagnosis of erosion was excellent (Gwet's AC1: 0.80)

Statistical analysis

Characteristics of patients were described by number and percentage for categorical variables and mean and standard deviation for continuous variables. For comparison, parametric statistics (Chi-square test and ANOVA F-statistic) or non-parametric statistics (Fisher exact, Wilcoxon tests) were used when appropriate. Inter-reader reliability for radiography and ultrasonography were tested using Gwet's AC1 statistic for binary variables and intra-class correlation coefficient (ICC) for continuous variable. The diagnostic performances (sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV)) have been analysed. Sensitivity analyses of the threshold for dichotomizing the diagnosis of erosive RA based on RX and US were performed. Alpha risk was set at 5% for all analyses. These statistical analyses were performed using SAS 9.4 software (SAS Institute, Inc, Cary, N.C.).

The ethical committee of Nancy approved this study in June 2017 (Number of recording: R2017-16).

Results

Demographic characteristics of the population

During the study period, 168 patients were included. Among them, 122 patients (72.6%) belonged to the RA group and were separated into early RA (disease duration of less than 2 years, n=32) and late RA (disease duration of 2 years or more, n=90), and 46 patients (27,4%) belonged to the OA group. Gender and age did not differ between all groups. The demographic characteristics are detailed in table1.

Radiographic evaluation

Reproducibility

The ICC between the two readers was 0.81 (IC95: 0.75, 0.86). Among the 168 exams, 99 (58.9%) matched between the two readers for a number of eroded joints less than or equal to three (data not shown). A third reading was necessary for 69 patients (41.1%) who were younger (58.8 ±13.6 vs 53.1±11.2; p=0.0035).

Localization and severity of erosions

For the 168 patients, 2,856 joints were analysed (2, With a threshold set at three eroded joints, the positive and negative likehood ratios were 3.16 and 0.73 respectively. Considering the severity of erosion at the joint level, whatever its localization, an erosion of a grade 2 had a sensitivity of 18.8% and a specificity of 86.9%; see table 3. Analyses for each threshold and their PPV and NPV are detailed in table 3.

Ultrasound evaluation

Localization and severity of erosions

For the 168 patients, 5,040 joint facets were analysed [START_REF] Aletaha | rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative[END_REF]660 There was a total of 549 eroded joint facets (10.9%), 537 (10.6%) in the RA group and 12 (0.2%) in the OA group (p<.001). In the OA group, 12 (26.1%) patients presented with one erosion, and five patients had an erosion of grade 2 (table 4). In RA groups, the distribution of erosions prevailed on lateral facets, independent of disease duration. MTP5 joints were the most frequently eroded joints (46.5%), followed by MCP2 joints (27.3%) and MCP5 joints (14.7%); details are presented in table 4. The severity of erosions prevailed on MTP5 followed by MCP2 joints (table 4). The mean (±SD) USSe was 0.4 (±0.7) in the OA group and 10.0 (±12.0) in the RA group (p<.0001). The difference between mean USSe between early and late RA patients reached statistical significance (4.3±7.5 in the early RA group and 12.1±12.7 in the late RA group (p<.0001)) and the difference was also statistically significant between OA and early RA patients (p<.0001) (table 4).

Definition of erosive RA on ultrasound

For the diagnosis of erosive RA, the presence of at least two eroded joint facets presented the better compromise in terms of sensitivity (68.0%) and specificity (100.0%) in comparison with other combinations (table 3) The positive likehood ratio were not computable (specificity =100%) and the negative likelihood ratio was 0.31.

Considering the severity of the erosion at the joint facet level, whatever its localization, the presence of one erosion of grade 2 presented the best sensitivity and specificity ratios (72.1% and 89.1%, respectively) compared to other combinations. The positive and negative likelihood ratios were 6.64 and 0.31 respectively. The analysis for each threshold is detailed in table 3. According to ultrasound assessment, 83 RA (14 early RA and 69 late RA) and zero OA patient satisfied with definition of erosive RA based on US with at least two eroded joint facets. Considering at least a grade 2 for erosion severity on at least one joint facet as the diagnosis threshold, 81 RA patients (11 early RA and 70 late RA) and five OA patients were classified with erosive disease.

Comparison of performance of radiography and ultrasound

The agreement between RX (threshold of 3 eroded joints) and US (threshold of two joint facets eroded) for erosive RA was 90.4%: 38 US patients with erosive RA were identified among the 42 erosive RA patients on radiography (three out of six in early RA and 35 out of 36 in late RA). In the four discordant cases (positive RX and negative US), three patients presented one US erosion (two patients with grade 2 and one with grade 1), and the other patient presented no erosion. Interestingly, the eroded sites based on radiography for three of the patients were wrists and MCP1, sites that were not explored using US. For the 45 patients with negative RX and positive US, the mean (±SD) number of eroded joint facets was 4.38 (±2.52) with a mean USSe of 8.91 (±6.78), mainly localized on MTP5 followed by MCP2 then MCP5 joints.

The agreement between RX (threshold of 3 eroded joints) and US (threshold of at least a grade 2 at the joint facet level) for erosive RA was 92.8%: 39 US patients with erosive RA were identified among the 42 erosive RA patients based on radiography (four out of six in early RA and 35 out of 36 in late RA). In the three discordant cases (positive RX and negative US), one patient presented one erosion of grade 1, the second patient presented two erosions of grade 1 and the last patient had no erosion according to the ultrasound evaluations. According to the radiography evaluations, the three patients presented erosions on wrists (n=3), MCP1 (n=2), and MCP4 joint (n=1). For the 42 patients with negative RX and positive US, the mean (±SD) number of eroded joint facets was 4.3 (±2.7) with a mean USSe of 9.3 (±6.8).

Discussion

The aim of this study was to compare radiography and ultrasound erosion characteristics (prevalence, localization and severity) in RA and OA patients in order to establish thresholds for the diagnosis of erosive RA on US, according to two different approaches: 1) a minimal number of eroded joint facets 2) a minimal grade of severity for at least one joint facet eroded. We demonstrated through the use of these two approaches that ultrasound has a better sensitivity and similar specificity in comparison to radiography to detect patients with erosive disease. This technique also offers many advantages (accessibility, cost, lack of irradiation) with good intra-and inter-reliabilities for assessing structural damage and especially erosion. Like other authors, we demonstrated excellent intra-reader reliability [START_REF] Zayat | The specificity of ultrasound-detected bone erosions for rheumatoid arthritis[END_REF][START_REF] Wakefield | The value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: a comparison with conventional radiography[END_REF][START_REF] Bajaj | Ultrasound detects rapid progression of erosive disease in early rheumatoid arthritis: a prospective longitudinal study[END_REF][START_REF] Scheel | Interobserver reliability of rheumatologists performing musculoskeletal ultrasonography: results from a EULAR "Train the trainers" course[END_REF][START_REF] Funck-Brentano | Benefits of ultrasonography in the management of early arthritis: a cross-sectional study of baseline data from the ESPOIR cohort[END_REF][START_REF] Gutierrez | Interobserver reliability of high-resolution ultrasonography in the assessment of bone erosions in patients with rheumatoid arthritis: experience of an intensive dedicated training programme[END_REF], at the joint facet level with same ranged of values calculated by Wakefield and Zayat.

In this study, US joint examination was limited at some selected joints because not all the joints evaluated using the modified SHS can be explored in clinical practice. We decided, as other authors have done, to exclude the wrist because of its anatomic characteristics that make it difficult to localize erosions with precision in axial and longitudinal planes, except for the ulnar styloid process, where detection of these erosions seem easier [START_REF] Zayat | The specificity of ultrasound-detected bone erosions for rheumatoid arthritis[END_REF]. We also excluded: MCP1, MTP1 and all PIP joints because these joints may be eroded by other musculoskeletal diseases osteoarthritis (the most prevalent disease in patients over 50 years old) [START_REF] Iagnocco | Usefulness of high resolution US in the evaluation of effusion in osteoarthritic first carpometacarpal joint[END_REF] and patients with severe joint deformities because this exam performs poorly in this situation and is inappropriate (joints not well visualized and erosion not clearly identified on two perpendicular planes) [START_REF] Szkudlarek | Summary Findings of a Systematic Literature Review of the Ultrasound Assessment of Bone Erosions in Rheumatoid Arthritis[END_REF].

Our results confirmed that the MTP5 joint and the MCP2 joint are the most prevalent eroded joints especially in the lateral facets in RA, as shown by other studies. [START_REF] Zayat | The specificity of ultrasound-detected bone erosions for rheumatoid arthritis[END_REF][START_REF] Szkudlarek | Summary Findings of a Systematic Literature Review of the Ultrasound Assessment of Bone Erosions in Rheumatoid Arthritis[END_REF][START_REF] Tămaş | Bone erosions in rheumatoid arthritis: ultrasound findings in the early stage of the disease[END_REF] In OA, MCP2 followed by MTP5 joints are the most frequently eroded joints with a prevalence varying from 5 to 13% according to the joint examined, but neither of these joints are severely eroded [START_REF] Zayat | The specificity of ultrasound-detected bone erosions for rheumatoid arthritis[END_REF]. In our study, five OA patients presented with erosion of grade 2 (10.3%), whereas Zayat, et al. [START_REF] Zayat | The specificity of ultrasound-detected bone erosions for rheumatoid arthritis[END_REF] did not find severe erosion in OA patients. With a threshold of two eroded joint facets, whatever the grade of severity observed, we calculated a sensitivity of 68.0% with a specificity of 100.0%. Our results showed that RA, in comparison to hand OA, affects more joints with more severe bone damages in early and late stages of RA disease.

A recent meta-analysis and other studies also showed that erosions in RA are larger and bigger than in other rheumatisms, with a "target size" varying between 2 and 2.5 mm [START_REF] Bajaj | Ultrasound detects rapid progression of erosive disease in early rheumatoid arthritis: a prospective longitudinal study[END_REF][START_REF] Szkudlarek | Summary Findings of a Systematic Literature Review of the Ultrasound Assessment of Bone Erosions in Rheumatoid Arthritis[END_REF][START_REF] Finzel | A detailed comparative study of high-resolution ultrasound and micro-computed tomography for detection of arthritic bone erosions[END_REF]. Our study confirmed that erosion with the same size (grade 2: single erosion ≥2 mm and < 3 mm in its largest dimension or no more than two erosions < 2 mm) presented the best compromise in terms of sensitivity and specificity in comparison to OA patients. It is true that an erosion on ultrasound is defined by a cortical defect observed on two planes (axial and longitudinal) and is not always related to musculoskeletal damage but may be related to physiological "vascular channels" that are mainly observed on the plantar facet [START_REF] Finzel | A detailed comparative study of high-resolution ultrasound and micro-computed tomography for detection of arthritic bone erosions[END_REF][START_REF] Etchepare | Ultrasonography and rheumatoid arthritis[END_REF]. These vascular channels are probably graded in the same manner (frequency and severity) in our population, independent of the disease. Osteophytes may also mimic erosion but are rarely observed in the two planes of the examination. In our study, no significant difference with respect to age or gender was noted between the two groups (OA versus RA patients), limiting the biases related to age structural damages during the ultrasonography examination.

In the literature, four scores of US erosions were compared to radiographic assessment [START_REF] Funck-Brentano | Benefits of ultrasonography in the management of early arthritis: a cross-sectional study of baseline data from the ESPOIR cohort[END_REF][START_REF] Luz | A new musculoskeletal ultrasound scoring system (US10) of the hands and wrist joints for evaluation of early rheumatoid arthritis patients[END_REF][START_REF] Ohrndorf | Evaluation of a new erosion score by musculoskeletal ultrasound in patients with rheumatoid arthritis: is US ready for a new erosion score?[END_REF][START_REF] Leng | Ultrasound7 versus ultrasound12 in monitoring the response to infliximab in patients with rheumatoid arthritis[END_REF]. The authors showed the superiority of ultrasound to detect erosions, but they were unable to define a threshold from which an erosive RA disease on US can be established due to the lack of a control group.

On radiography, the diagnosis of erosive RA in hands and feet is routinely performed in clinical practice. The diagnosis of eroded disease in RA is crucial since structural damages are associated to poor functional outcome and are considered as poor prognostic factors leading to a more aggressive therapy. RX serves as the gold standard for erosive disease if at least three selected joints are eroded [START_REF] Van Der Heijde | EULAR definition of erosive disease in light of the 2010 ACR/EULAR rheumatoid arthritis classification criteria[END_REF].

Radiography presents many advantages: the views and the technical parameters are well defined and the diagnosis of erosive RA is easily established after a careful reading performed for few seconds. The modified SHS is considered one of the standard methods for assessing structural effects of RA DMARDs (synthetic or biologic) in clinical trials because it presents excellent intra-or inter-reader reliabilities and a good sensitivity to change (6). To limit reading biases, two senior readers of the ESPOIR's cohort blindly assessed the SHS, and an adjudication was made by the third senior reader, On the 44 joints assessed, MTP5 and wrists joints were most frequently eroded as described in the literature [START_REF] Brook | Radiographic changes in early rheumatoid disease[END_REF]. When OA patients served as a control group, the diagnosis of erosive RA disease based on radiography was calculated with a sensitivity of 34.4% and a specificity of 89.1%. As a result, the positive and negative like ratios were 3.16 and 0.73 respectively. These results are close to those validated by the EULAR task force in two cohorts of early arthritis (a sensitivity of 15-29% and a specificity of >80%) [START_REF] Funck-Brentano | Benefits of ultrasonography in the management of early arthritis: a cross-sectional study of baseline data from the ESPOIR cohort[END_REF][START_REF] De Rooy | Predicting arthritis outcomes--what can be learned from the Leiden Early Arthritis Clinic?[END_REF].

In 2000, Wakefield, et al. [START_REF] Wakefield | The value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: A comparison with conventional radiography[END_REF] demonstrated that ultrasonography permits to detect, at the joint level, more erosions in early and late RA than radiography. The superiority of ultrasound was confirmed in other studies [START_REF] Szkudlarek | Ultrasonography of the metacarpophalangeal and proximal interphalangeal joints in rheumatoid arthritis: a comparison with magnetic resonance imaging, conventional radiography and clinical examination[END_REF][START_REF] Szkudlarek | Ultrasonography of the metatarsophalangeal joints in rheumatoid arthritis: comparison with magnetic resonance imaging, conventional radiography, and clinical examination[END_REF] but also when CT scans served as the gold standard to evaluate both imaging techniques [START_REF] Døhn | Are bone erosions detected by magnetic resonance imaging and ultrasonography true erosions? A comparison with computed tomography in rheumatoid arthritis metacarpophalangeal joints[END_REF]. Our study demonstrated that ultrasound detected 2.0 times more eroded RA patients than radiography with a threshold of at least two eroded joint facets and 1.9 times more patients with a threshold of at least one erosion of grade 2 at a joint facet level with excellent specificities (100.0% and 89.1%, respectively) when compared to OA patients. Moreover, we showed that ultrasonograhic assessment was able to detect two times more patients with early RA erosive disease than radiograph (14 or 11 on US versus 6 on RX). Erosion diagnosis on US is relevant since a longitudinal follow-up in the ESPOIR Cohort demonstrated that US erosion at baseline was predictive of eroded joint on radiography (OR= 1.44 (IC95%: 1.01-1.98)) at one year [START_REF] Funck-Brentano | Prediction of radiographic damage in early arthritis by sonographic erosions and power Doppler signal: a longitudinal observational study[END_REF]. This pilot study performed by trained sonographers required the development of the USSe score according to an external validation process such as the OMERACT filter.

The sensitivity to change should also be tested, and the relationship between bone erosion and joint inflammation at the joint level should also be investigated. To reduce the time of the US examination, varying between 10 and 20 minutes, the number of joint facets could probably be optimized. Of course, radiographic and ultrasonographic patterns of erosions in other musculoskeletal diseases were not evaluated in this study and could be detemined in the future. Finally, joint space assessment, not performed in this study and recently validated by Mandl et al. (40), should also be investigated according to a complementary manner.

Finally, we demonstrated that bone erosion assessed using US was reliable and may be observed in both OA and RA diseases, with a higher prevalence and severity in RA. According to two different criteria (number of facets eroded and severity of erosion at the joint facet level) the USSe detected approximately 2.0 times more eroded RA patients than radiography, especially in early RA.

.3 Number of patients (RA and OA) fulfilling to the definition of erosive RA regarding to EULAR 2013 definition

  074 joints in the RA group and 782 in the OA group): 72 patients (12 early RA and 60 late RA) and 12 patients were eroded in RA and OA groups respectively. There were 301 (10.5%) eroded joints, 272 (9.5%) in the RA group and 29 (1.0%) in the OA group (p<.0001) (table 2). Erosions were preferentially observed in wrists and PIPs in the OA group, and in MTP5 joints and wrists in the RA group (table 2). The mean SHSe was 1.09±2.58 and 5.98±11.09 in the OA and RA groups, respectively (p<.0001) (1.62±3.09 in early RA, 7.53±12.44 in late RA groups, (p<.0001)) (table 2). In the OA group, 12 patients (26.1%) presented at least one erosion, for whom five (10.8%) satisfied with EULAR 2013 definition of erosive disease (table 2). In the RA group, 72 patients (59.0%) presented at least one erosion for whom 42 (34.4%) satisfied with EULAR 2013 definition of erosive disease (six (4.9%) in early RA and 36 (29.5%) in late RA) (table 2). When OA patients served as controls, the EULAR 2013 definition of erosive disease with a threshold of three eroded joints was confirmed as the best compromise in terms of sensitivity and specificity (34.4% and 89.1%, respectively) in comparison with other combinations; see results in table 3.

	3.2

Table 1 Characteristics of patients

 1

		Rheumatoid Arthritis	Rheumatoid Arthritis <2 years	Rheumatoid Arthritis ≥2 years	Osteoarthritis
			N=122		N=32		N=90		N=46
			(72.6%)		(19.0%)		(53.6%)		(27.4%)
		N(%)	Mean(SD)	N(%)	Mean(SD)	N(%)	Mean(SD)	N(%)	Mean(SD)
	Age	122	54.9(13.5)	32	54.3(15.7)	90	55.1(12.7)	46	56.8(9.6)
	Gender
	Male	34(27.9)		10(31.3)		24(26.7)		10(21.7)	21.7
	Female	88(72.1)		22(68.8)		66(73.3)		36(78.3)	78.3
	Disease duration (years)	122	7.1(7.2)	32	0.9(0.4)	90	9.3(7.1)	46	6.2(8.1)
	Delay between RX and US (days)	122	0.6(6.9)	32	0.8(7.5)	90	0.5(6.7)	46	39.7(269.2)
	NSAIDs	16(13.1)		1(3.1)		15(16.7)		0	0.0
	Corticosteroids	68(55.7)		18(56.3)		50(55.6)		0	0.0
	sDMARDS	94(77)		23(71.9)		71(78.9)		0	0.0
	bDMARD	37(30.3)		2(6.3)		35(38.9)		0	0.0
	ESR	121	22.5(20.2)	32	32.2(26.5)	89	19.1(16.3)	0
	CRP (mg/l)	122	10.8(22.6)	32	19.5(31.6)	90	7.7(17.6)	0
	ACPA	84(68.9)		21(65.6)		63(70.0)		0	0.0
	ACPA (titers)	106	357.7(696.1)	27	526.1(893.5)	79	300.1(610.8)	0
	RF	70(57.4)		17(53.1)		53(58.9)		0	0.0
	RF (titers)	98	88.4(161.8)	27	69.5(139.2)	71	95.5(170.0)	0
	DAS 28	117	3.6(1.4)	29	4.3(1.8)	88	3.4(1.2)	0
	N: number; SD: Standard deviation; NSAIDS: non-steroidal anti-inflammatory drugs, sDMARDs: synthetic DMARDS, bDMARDs: biologic DMARDS, ESR: erythrocyte sedimentation rate, CRP: C-
	reactive protein, ACPA: anti-cyclic citrullinated peptide antibodies, RF: rheumatoid factor, DAS 28: Disease Activity Score 28-joint count.

Table 2 Distribution and severity of erosions on radiography according to modified Sharp/van der Heijde scoring

 2

		WRIST MCP1 MCP2 MCP3 MCP4 MCP5 IPP1 IPP2 IPP3 IPP4 IPP5	Total Hands	MTP1 MTP2 MTP3 MTP4 MTP5 IP1	Total Feet	Total Hands +Feet	Mean of eroded joints±SD	Mean SHSe ±SD
	Rheumatoid arthritis
	Erosion (N)	50	17	23	12	4	5	0	2	6	2	1	122	16	20	24	17	63	10	150	272
	SHSe mean	0,27	0,14	0,25	0,11	0,04	0,07	0	0,01	0,08	0,01	0,02		0,18	0,28	0,35	0,21	0,9	0,08			2,23	5,98
	SD	0,74	0,56	0,87	0,51	0,31	0,5	0	0,14	0,54	0,15	0,26		0,76	1,08	1,27	0,96	1,79	0,43			3,1	11,09
	Early rheumatoid arthritis
	Erosion (N)	5	2	1	1	1	1	0	2	1	0	0	14	1	0	3	2	8	3	17	31
	SHSe mean	0,04	0,04	0,02	0,02	0,03	0,02	0	0,05	0,02	0	0		0,02	0	0,07	0,1	0,33	0,05			0,97	1,62
	SD	0,16	0,21	0,17	0,17	0,25	0,13	0	0,26	0,17	0	0		0,17	0	0,35	0,6	1,04	0,25			1,58	3,09
	Late rheumatoid arthritis
	Erosion (N)	45	15	22	11	3	4	0	0	5	2	1	108	15	20	21	15	55	7	133	241
	SHSe mean	0,35	0,18	0,33	0,14	0,04	0,08	0	0	0,1	0,02	0,02		0,23	0,38	0,45	0,25	1,1	0,09			2,68	7,53
	SD	0,85	0,64	0,99	0,58	0,33	0,58	0	0	0,62	0,18	0,3		0,88	1,25	1,45	1,06	1,96	0,48			3,38	12,44
	Osteoarthritis
	Erosion (N)	10	0	1	0	0	0	0	4	5	5	0	25	2	1	0	0	0	1	4	29
	SHSe mean	0,06	0	0,03	0	0	0	0	0,09	0,14	0,17	0		0,03	0,01	0	0	0	0,01			0,63	1,09
	SD	0,21	0	0,31	0	0	0	0	0,44	0,6	0,7	0		0,19	0,14	0	0	0	0,1			1,24	2,58

N: Number, SHSe: Modified Sharp/van der Heijde Score for erosion, SD: Standard deviation, MCP: metacarpophalangeal joints, IPP/IP: proximal Interphalangeal joints, MTP: metarsophalangeal joints.

Table 3 Diagnostic values for erosive RA of radiography and ultrasonography considering the number of eroded joints or the grade of erosion at the joint level. Sensitivity Specificity PPV NPV Number of eroded joints or joint facets

 3

	RX	2	43.4	80.4	85.4	34.9
		3*	34.4	89.1	89.4	33.9
		4	26.2	95.6	94.1	32.8
	US	1	77.9	73.9	88.8	55.7
		2	68.0	100.0	100.0	54.1
		3	58.2	100.0	100.0	47.4
		4	45.9	100.0	100.0	41.1
		5	36.9	100.0	100.0	37.4
	Erosion score at the joint or facet level
	RX	2	18.8	86.9	79.3	28.8
		3	9.8	89.1	70.6	27.1
	US	1	77.9	73.9	88.8	55.7
		2	72.1	89.1	94.6	54.7
		3	67.2	100.0	100.0	53.5

RX: radiography; US: ultrasounds; PPV: positive predictive value; NPV: negative predictive value; * EULAR 2013 Definition of erosive rheumatoid arthritis

Table 4 Distribution and severity of US erosions in joint assessed in USSe

 4 : Number, SD: standard deviation; Grade 1= single erosion <2 mm in its largest dimension; Grade 2 = single erosion ≥2 mm and < 3 mm in its largest dimension, or no more than two erosions < 2 mm; Grade 3 = single erosion ≥ 3 mm in its largest dimension, or multiple erosions, USSe: Ultrasound score for erosions, MCP: metacarpophalangeal joints, MTP: Metarsophalangeal joints, D: dorsal, P: plantar/palmar, L: lateral facets

														Total	Eroded	USSe
						Total							Total	Hands+	joints	mean
		MCP2	MCP3	MCP5	Hands MTP2	MTP3	MTP5		Feet	Feet	mean ±SD	±SD
		D P L	D	P	D P L		D	P	D	P	D P	L
	Rheumatoid Arthritis
	Erosion (N)	33 26 88	25	3 17 7 55	254	6	10	6	11	33 87 130 283	537	4.4 ± 4.7
	Grade1 Grade2	33 43	9 7		31 24	73 74	4 5		4 7		41 57		49 69	122 143		10.0 ± 12.0
	Grade3	71	12		24	107	7		6		152		165	272
	US erosion grade/joint (mean±SD) 2.72±4.29 0.48±1.38 1.24±2.77		0.29±0.86 0.29±1.05	5.01±5.28
	Early Rheumatoid Arthritis
	Erosion (N)	2 4 17	2	0	0 1 7	33	1	1	2	1	6 10 19	40	73	2.3 ± 3.4
	Grade1 Grade2	13 5	2 0		4 4	19 9	0 1		1 1		12 6		13 8	32 17		4.3 ± 7.5
	Grade3	5	0		0	5	1		1		17		19	24
	US erosion grade/joint (mean±SD) 1.19±2.04 0.06±0.24 0.38±0.98		0.16±0.88 0.19±1.06	2.34±4.06
	Late rheumatoid Arthritis
	Erosion (N)	31 22 71	23	3 17 6 48	221	5	9	4	10	27 77 111 243	464	5.2 ± 4.9
	Grade1 Grade2	20 38	7 7		27 20	54 65	4 4		3 6		29 51		36 61	90 126		12.1 ± 12.7
	Grade3	66	12		24	102	6		5		135		146	248
	US erosion grade/joint (mean±SD) 3.27±4.74 0.63±1.58	1.5 ±3.12		0.33±0.85 0.33±1.06	5.96±5.35
	Osteoarthritis
	Erosion (N)	0 0 4	0	0	0 0 1	5	1	0	0	0	1 1	4	7	12	0.3 ± 0.4
	Grade1 Grade2	3 1	0 0		0 1	3 2	0 1		0 0		4 2		4 3	7 5		0.4 ± 0.7
	Grade3	0	0		0	0	0		0		0		0	0
	US erosion grade/joint (mean±SD) 0.11±0.37	0		0.04±0.29		0.04±0.29	0		0.17±0.49

N

Disclosure of interest

The authors declare that they have no competing interest.


Ghiles Grine

Romain Lotte

David Chirio

Alicia Chevalier

Didier Raoult

Michel Drancourt

Raymond Ruimy
email: ruimy.r@chu-nice.fr.

Prof Raymond Ruimy

Co-culture of Methanobrevibacter smithii with enterobacteria during urinary infection

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Forty-eight-hour culturing of a large volume of urine samples collected from women by transurethral catheter in parallel with suprapubic aspiration has shown that urine hosts a resident microbiota, which includes bacteria missed by routine procedures [START_REF] Wolfe | Evidence of uncultivated bacteria in the adult female bladder[END_REF] . This resident urinary microbiota has been further explored using an expanded quantitative urine culture (EQUC) method 2, [START_REF] Xu | Mini-review: perspective of the microbiome in the pathogenesis of urothelial carcinoma[END_REF] . Metagenomic analyses have confirmed that urine is not sterile in healthy individuals 4-8 .

An interesting concept currently emerging is that cases of urinary tract infection (UTI) may result from disequilibrium in the repertoire of the urinary microbiota rather than from the introduction of an exogenous pathogenic organism 8, [START_REF] Whiteside | The microbiome of the urinary tract -A role beyond infection[END_REF] . Therefore, efforts to characterise the repertoire of organisms residing in the urinary microbiota are important for clinical microbiology and medicine.

Current culture-dependant and culture-independent studies of the urinary microbiota rely on methods that are unable to detect archaea. These micro-organisms form a living domain distinct from that of bacteria and have been detected and cultured from the oral and gut microbiota [START_REF] Nguyen-Hieu | Methanogenic archaea in subgingival sites: A review[END_REF][START_REF] Grine | Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach[END_REF][START_REF] Demonfort | Archaea : Essential inhabitants of the human digestive microbiota[END_REF] . Moreover, the specific archaea group of methanogens, characterised by the ability to produce methane from hydrogen, are recognised as emerging pathogens in a few clinical situations, including life-threatening brain abscess 12-15. In this study, we aimed to characterise the repertoire of human urine microbiota using laboratory protocols specifically designed for the detection and culture of methanogens in urines. Surprisingly, we found that among the six previously-described methanogens in nonurine human microbiota, only Methanobrevibacter smithii can be detected and cultured from urines. Routine investigations. Urine leukocyte and erythrocyte counts were obtained with a UF-1000i ® analyser (Sysmex, bioMérieux, Craponne, France) in laboratory 1 and with an IQ 200 IRIS analyser (Beckman Coulter, Villepinte, France) in laboratory 2. Gram staining was carried out with the automated PREVI Color Gram (bioMérieux) at both laboratories in order to stain bacteria. Indeed, methanogens are not usually coloured by using Gram staining. A 200-µL urine volume was cultured on a COLUMBIA COS medium (bioMérieux) (laboratory 1) or on Uriselect medium (Bio-Rad, Marnes-la-Coquette, France) (laboratory 2) and incubated at 37 °C for 24 h (both laboratories), while visible colonies were identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (Brucker, Wissembourg, France), as previously described [START_REF] Seng | Ongoing Revolution in Bacteriology : Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry[END_REF] .

Patients and methods

Sample

Methanogen DNA extraction, PCR and sequencing. The manual protocol was carried out using the NucleoSpin® Tissue Mini Kit (Macherey Nagel, Hoerdt, France) as previously described 11, [START_REF] Grine | Tobaccosmoking-related prevalence of methanogens in the oral fluid microbiota[END_REF] . Extraction of 250 µL of sterile PBS was included in each DNA extraction series as a negative control.

Methanogen PCR and sequencing was performed as described in previous articles 11, [START_REF] Grine | Tobaccosmoking-related prevalence of methanogens in the oral fluid microbiota[END_REF] using the 16S rRNA broad range archaeal forward primer 5'-CCGGGTATCTAATCCGGTTC-3' and reverse primer 5'-CTCCCAGGGTAGAGGTGAAA-3' 18 , and the mcrA gene forward primer 5'-GCTCTACGACCAGATMTGGCTTGG-3' and reverse primer 5'-CCGTAGTACGTGAAGTCATCCAGCA-3' [START_REF] Vianna | T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype[END_REF] . All PCR products were sequenced and the various fragments were assembled using the ChromasPro1.34 software (Technelysium Pty.

Ltd., Tewantin, Australia) and compared with sequences available in the GenBank database using the online NCBI BLAST program (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi).

Extracted DNA was also analysed by real-time PCR targeting M. smithii using the following primer pair: M. smithii-F (5'-ACCATAACyATCAGCAGCATTAT-3') and M. smithii-R (5'-AGTATTGGTGAAGGATTTaCTGT-3') (Eurogentec, Seraing, Belgium) and the M. smithii probe (6-carboxyfluorescein [FAM]-5'ACCyTTATCAGCTTTACCA TTAATyAAAG-3') (Applied Biosystems, Courtaboeuf, France) as described in a previous study [START_REF] Drancourt | Evidence of Archaeal Methanogens in Brain Abscess[END_REF] .

Methanogen isolation and culture. A 250 µL volume of urine sample was seeded in ambient air in a sterile Hungate tube 20 (Dominique Dutscher, Brumath, France). The Hungate culture tube was developed specifically for growing and storing strictly anaerobic bacteria and archaea. It features an autoclavable screw cap with a 9-mm opening, a non-toxic, gasimpermeable butyl rubber stopper and a disposable screw cap. We placed 5 mL of SAB broth [START_REF] Khelaifia | A Versatile Medium for Cultivating Methanogenic Archaea[END_REF] into each Hungate tube, seeded it with urine then inoculated it with Bacteroides thetaiotaomicron (10 5 cells/mL) for hydrogen production [START_REF] Scola | Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report[END_REF] . The mixture in the Hungate tube was then incubated at 37 °C with agitation for seven days. Methanogen growth was inferred from the production of CH 4 detected by gas chromatography, as previously described [START_REF] Grine | Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach[END_REF] . The subculture was seeded on a Petri dish containing SAB medium supplemented with 15 g/L agar and placed in the upper chamber of a double-chamber box. Tubes inoculated with sterile PBS were used as negative controls in the archaea isolation experiments (one negative control every five tubes); culture dishes containing the SAB medium inoculated with sterile PBS were used as negative controls in the sub-culture experiments (one negative control every five dishes).

Characterisation of isolates.

Isolates were further genotyped using the Multi Spacer Typing method, as previously described [START_REF] Nkamga | Diversity of Human-Associated Methanobrevibacter smithii Isolates Revealed by Multispacer Sequence Typing[END_REF] . In addition, we determined the antibiotic susceptibility profile of the isolates, as previously described, by incorporating the following antibiotics: fosfomycin (100 mg/L), sulfamethoxazole-trimethoprim (50 mg/L), amoxicillin-clavulanate (100 mg/L) and ofloxacin (1 mg/L) (BIOGARAN, Colombes, France) [START_REF] Khelaifia | A Versatile Medium for Cultivating Methanogenic Archaea[END_REF] . M. smithii culture with metronidazole (1 mg/L) was used as the positive control and M. smithii culture without metronidazole as the negative control.

Statistical analyses. Data were analysed with Prism 7.0 (GraphPad Software) by unpaired Student's t-test and chi-square test (** p < 0.01, * p < 0.05, ns: non-significant). We used the former to compare the quantitative variables from the two centres (pH, urine salinity, white and red blood cells), and the latter to compare the categorical variables (sex ratio, % of standard cultures positive, % of urine samples positive for methanogens by molecular or culture methods).

Results

Population and routine investigations. A total of 383 urine specimens from 383 patients were collected and analysed prospectively, 183 specimens in laboratory 1 and 200 specimens in laboratory 2. Mean patient age was 56 years (0-95 years) and 61% of the patients were women. Leukocyturia was significant in 205 of the 383 specimens ( > 10 4 leukocytes/mL) and 142 specimens yielded bacterial growth. Comparison of the data collected from laboratory 1 and from laboratory 2 is shown in Table 1. samples in laboratory 2. Fifteen of the 18 cultured samples in laboratory 1 and all 16 cultured samples in laboratory 2 yielded colonies, which were obtained after 20 days of incubation. All the colonies were identified as M. smithii on the basis of PCR sequencing of the 16S rRNA and mcrA genes, which revealed 100% sequence similarity with the reference homologous genes in M. smithii strain NVD (accession NCBI: LT223565) (Table 1). MST genotyping indicated that the M. smithii isolates belonged to genotype 2 (n=13), genotype 3 (n=10) and genotype 1 (n=8) (Supplementary Table 1). Antibiotic susceptibility testing indicated that fosfomycin (100 mg/L), sulfamethoxazole-trimethoprim (50 mg/L), amoxicillin-clavulanate (100 mg/L) and ofloxacin (1 mg/L) were inactive against M. smithii, whereas metronidazoleused as a positive control -inhibited the growth of M. smithii.

Methanogen quantitative PCR

Microbiological and clinical analyses. M. smithii was detected in 34 (9%) of the 383 urine samples analysed in the study. Escherichia coli, Klebsiella pneumoniae, Enterobacter sp. and Enterococcus faecium were detected along with M. smithii in 18, 6, 3 and 1 urine samples, respectively. The other six samples in which M. smithii was detected yielded mixed cultures including Enterobacteriaceae. Of the 34 patients with M. smithii in their urine samples, 63.4% were women, and mean patient age was 63 years (20-95). Ten patients (30%) had an underlying urinary tract disease, 14 (40%) were immunosuppressed and 19 (44%) had chronic renal failure (GFR < 60L/min). It is worth noting that M. smithii was associated with UTI in 19 (56%) patients and with colonisation in 15 (44%). Of the 19 patients with UTIs, 17 (88%) were community acquired, 13 presented with cystitis, while four were diagnosed with pyelonephritis and two with prostatitis. All 19 patients received antibiotic treatments targeting the isolated bacteria and commonly used to treat UTIs, including beta-lactams, fluoroquinolones and fosfomycin-trometamol, with favourable outcomes (Table 2).

Discussion

M. smithii was the only methanogen prospectively detected in the urines using a polyphasic approach, including molecular detection by PCR sequencing of two unrelated genes, and isolation and culture methods specifically developed for methanogenic archaea.

Given that the negative controls included in every experimental procedure remained negative, that M. smithii was detected by different unrelated technical approaches, and that similar data were obtained in two unrelated laboratories, we can exclude the possibility of in-laboratory contamination and centre bias in the study. These pieces of experimental evidence, therefore, validate the data reported here.

M. smithii was first detected as a normal organism in the gut microbiota of the vast majority of individuals 11,24 , although it is depleted in children diagnosed with severe malnutrition and kwashiorkor [START_REF] Million | Increased Gut Redox and Depletion of Anaerobic and Methanogenic Prokaryotes in Severe Acute Malnutrition[END_REF] . It was then shown to be a component of the oral cavity microbiota in the saliva [START_REF] Grine | Tobaccosmoking-related prevalence of methanogens in the oral fluid microbiota[END_REF] and the dental plaque, including periodontitis 26,27 and periimplantitis lesions 10, [START_REF] Faveri | Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis[END_REF] . More recently, the pathogenic potential of M. smithii was illustrated by its isolation and culture from a muscular abscess [START_REF] Nkamga | Methanobrevibacter smithii and Bacteroides thetaiotaomicron cultivated from a chronic paravertebral muscle abscess[END_REF] and by its detection in one case of brain abscess [START_REF] Drancourt | Evidence of Archaeal Methanogens in Brain Abscess[END_REF] . Our study is of interest because it reports for the first time M. smithii, methanogens and archaea at large in urines, as all previous culture-based and culture-independent studies have failed to detect archaea in this type of fluid. Our success in detecting M. smithii was down to the use of laboratory tools that we developed specifically for detecting methanogens 22, 29-31 . These tools, including the technique we set up for easy isolation and culture of methanogens, could be easily implemented in other clinical microbiology laboratories enabling them to investigate the presence of methanogens in urines [START_REF] Khelaifia | Aerobic culture of methanogenic archaea without an external source of hydrogen[END_REF] .

We detected M. smithii in 9% of urine samples, and, furthermore, in the same proportions of patients in two unrelated laboratories (10% in laboratory1 and 8% in laboratory 2). This supports the idea that M. smithii may be part of "the urinary microbiota", a new concept that has emerged since the expansion of urine metagenomic analyses and EQUC approaches. It is worth noting that all the urine samples which cultured M. smithii also cultured enterobacteria, such as E. coli, K. pneumoniae, Enterobacter sp.,which are known to produce hydrogen as a substrate for methanogenesis [START_REF] Mckay | Methane and hydrogen production by human intestinal anaerobic bacteria[END_REF] . The fact that M. smithii was isolated along with enterobacteria and never detected in a urine sample without bacterial growth suggests that it could play a role in or even induce dysbiosis, which facilitates the growth of enterobacteria, acknowledged agents of UTI. In our study, 19 (54%) of the 34 patients with M. smithii in their urine samples presented with UTIs, were most of them community acquired. These preliminary results need to be confirmed by further studies, including epidemiological, clinical and fundamental approaches, in order to determine the role of M. smithii in UTIs. Finally, our study indicates that the antibiotics commonly used to treat bacterial urinary tract infections are inactive against M. smithii, consistent with a previous report of its narrow spectrum of in vitro susceptibility [START_REF] Dridi | The Antimicrobial Resistance Pattern of Cultured Human Methanogens Reflects the Unique Phylogenetic Position of Archaea[END_REF] . Interestingly, the 19 patients in whose urine samples we detected M. smithii and who presented with UTI symptoms showed clinical improvement after receiving antimicrobial treatment active against enterobacteria but inactive against methanogens. We hypothesise that the inhibition of enterobacterial growth by appropriate antibiotic treatment induces a decrease ²in hydrogen production, a major substrate for methanogenesis, and therefore destroys the metabolic cooperation between these two microorganisms.

In conclusion, our data show that M. smithii is part of the urinary microbiota in some patients and suggest that it plays a role in community-acquired UTIs in association with enterobacteria. Therefore, we encourage colleagues to use the above-described methods to investigate methanogens in urines, including M. smithii, in order to determine its potential contribution to the pathogenesis of UTIs.

  collection. The retrospective study was approved by the Ethics Committee of the IHU Méditerranée Infection under n° 2016-01. A total of 383 urine specimens taken for microbiological diagnosis of UTI in 383 patients without urinary catheter were prospectively collected at the IHU Méditerranée Infection Laboratory, Marseille, France (henceforth referred to as laboratory 1) (183 urines) and at the Laboratory of Bacteriology at the University Centre of Nice (henceforth referred to as laboratory 2) (200 urines) using a BD Vacutainer (Becton Dickinson, Le Pont de Claix, France). In this study, clean catch midstream urine specimen were collected by from September 2017 to November 2017 at laboratory 1 and from January 2018 to March 2018 at laboratory 2. A total of 50 BD vacutainer tubes filled with sterile phosphate buffered saline (PBS) (Thermo Fisher Scientific, Villebon-sur-Yvette, France) were intercalated in every 8 urine specimens to serve as negative controls in all further laboratory steps. The following data on each patient included in the study were anonymously collected: age, sex, underlying disease of the urinary tract, immune status, symptoms of urinary tract infection, antibiotic treatment (molecule and duration) and clinical outcome.

  and sequencing. Of the 383 urine samples analysed by standard PCR sequencing, 18/183 (10%) from laboratory 1 and 16/200 (8%) from laboratory 2 were positive for both archaeal 16S rRNA PCR and the mcrA PCR, while 165/183 (90%) and 184/200 (92%), respectively, were negative in both PCR assays in the presence of 50 negative controls, all of which remained negative. Sequencing the 16S rRNA PCR products revealed 99% sequence similarity with the homologous fragment of the reference 16S rRNA gene of M. smithii strain NVD (accession NCBI: LT223565). Further sequencing of the PCRamplified mcrA gene revealed 99% sequence similarity with the homologous fragment of the reference mcrA gene of M. smithii strain NVD (accession NCBI: LT223565) and M. smithii ATCC 35061 (accession NCBI: NR_074235). Quantitative PCR analyses targeting the M. smithii 16S rRNA gene yielded a median Ct of 33.7 ± 2.47, indicative of an M. smithii load of 1.24*10 3 ±1.56*10 3 / mL. Detection of M. smithii in urine samples was significantly associated with leukocyte counts greater than 10 4 /mL, P-value = 0.0015 (Chi-square test). Isolation and culture of M. smithii. To test the viability of the M. smithii organisms detected by the PCR-based methods, methanogen was cultured on 18 samples in laboratory 1 and 16

Acknowledgements/funding

GG benefits a PhD grant from the Fondation Méditerranée Infection, Marseille, France. This work was supported by the French Government under the «Investissements d'avenir» (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (reference: Méditerranée Infection 10-IAHU-03). S

Funding This study was supported by IHU Méditerranée Infection, Marseille, France.

Author contributions

GG and RL wrote the manuscript. GG, RL cultivated the methanogens and carried out the PCR. DC, RL and AC performed the clinical analyses. MD, DR and RR supervised the study.

Conflicts of interests

GG, RL, DC, AC, DR, MD, RR declare no potential conflict of interest.