Romain Magny

Karima Kessal

Anne Regazzetti

Asma Ben Yedder

Christophe Baudouin

Stéphane Mélik Parsadaniantz

Françoise Brignole-Baudouin

Olivier Laprévote

Nicolas Auzeil
email: nicolas.auzeil@parisdescartes.fr

Lipidomic analysis of epithelial corneal cells following hyperosmolarity and benzalkonium chloride exposure: New insights in dry eye disease

Keywords: Dry eye, lipidomic analysis, benzalkonium chloride, hyperosmolarity stress, UPLC-HRMS Highlights

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

Introduction

Dry eye disease (DED) is a multifactorial chronic inflammatory disease of the ocular surface, affecting 20% of the population [START_REF] Stapleton | TFOS DEWS II Epidemiology Report[END_REF]. Its incidence is in constant growth, affecting millions of people worldwide. DED results in visual disorders and neurosensory abnormalities -discomfort sensations, burning, itching and pain -and alters occupational performances and quality of life. DED has been defined as "a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles" [START_REF] Craig | The Ocular Surface TFOS DEWS II De fi nition and Classi fi cation Report[END_REF].

DED is furthermore self-maintained by a vicious circle, enclosing alteration of tear film, hyperosmolarity, inflammation of the ocular surface leading to release of pro-inflammatory cytokines such as IFNγ, TNFα, IL-1β or IL-6 [START_REF] Massingale | Analysis of inflammatory cytokines in the tears of dry eye patients[END_REF][START_REF] Na | Correlations between tear cytokines, chemokines, and soluble receptors and clinical severity of dry eye disease[END_REF][START_REF] Baudouin | Role of hyperosmolarity in the pathogenesis and management of dry eye disease: Proceedings of the ocean group meeting[END_REF][START_REF] Pflugfelder | The Pathophysiology of Dry Eye Disease: What We Know and Future Directions for Research[END_REF][START_REF] Rhee | Inflammation in Dry Eye Disease[END_REF].

Hyperosmolarity (HO), known to be the core mechanism of DED, contributes to promoting and/or nurturing the pathology [START_REF] Craig | The Ocular Surface TFOS DEWS II De fi nition and Classi fi cation Report[END_REF][START_REF] Baudouin | Role of hyperosmolarity in the pathogenesis and management of dry eye disease: Proceedings of the ocean group meeting[END_REF]. DED etiology includes autoimmune origin such as in Sjögren syndrome and also exogenous causes such as exposure to environmental toxics or iatrogenic agents [START_REF] Gomes | TFOS DEWS II iatrogenic report[END_REF]. DED onset may also be related to the toxic effect of preservatives required by the pharmacopeia guidelines as excipients in multidose eyedrops, the most common of them being benzalkonium chloride (BAK) [START_REF] Gomes | TFOS DEWS II iatrogenic report[END_REF][START_REF] Baudouin | Preservatives in eyedrops: The good, the bad and the ugly[END_REF]. BAK is a quaternary ammonium salt with detergent and microbicidal properties and quaternary ammonium compounds are widely found in disinfecting sprays both at home and at work [START_REF] Ferk | Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations[END_REF]. Initially described in glaucomatous patients who are constrained to a chronic eyedrop administration [START_REF] Baudouin | Preservatives in eyedrops: The good, the bad and the ugly[END_REF][START_REF] Rasmussen | Benzalkonium Chloride and Glaucoma[END_REF], BAK toxicity impacts the different structures of the ocular surface, the conjunctiva, the cornea but also deeper structures such as the trabecular meshwork, the lens or even the retina [START_REF] Vaede | Les conservateurs des collyres: Vers une prise de conscience de leur toxicité[END_REF][START_REF] Brignole-Baudouin | A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits[END_REF]. In addition, because of its pro-inflammatory, pro-apoptotic and pro-oxidative effects, BAK may be responsible for DED or worsen it [START_REF] Stevens | Benzalkonium chloride induces anterior chamber inflammation in previously untreated patients with ocular hypertension as measured by flare meter: A randomized clinical trial[END_REF][START_REF] Bonniard | Expert Opinion on Drug Metabolism & Toxicology Ocular surface toxicity from glaucoma topical medications and associated preservatives such as benzalkonium chloride[END_REF][START_REF] Baudouin | ODISSEY European Consensus Group Members, Clinical impact of inflammation in dry eye disease: proceedings of the ODISSEY group meeting[END_REF].

The ocular surface epithelial cells represent a physiological barrier playing a key role in the protection of the eye. Indeed, corneal and conjunctival cells are the first impacted during the alteration of the tear film contributing to the pathophysiology of DED. Many molecular mediators involved in DED pathophysiology have been widely described in in vitro models [START_REF] Craig | The Ocular Surface TFOS DEWS II De fi nition and Classi fi cation Report[END_REF][START_REF] Pflugfelder | The Pathophysiology of Dry Eye Disease: What We Know and Future Directions for Research[END_REF][START_REF] Stevenson | Dry Eye Disease[END_REF]. Indeed, corneal and conjunctival cells exposed to BAK or HO undergo deleterious effects, especially apoptosis and oxidative stress [START_REF] Saint Jean | Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells[END_REF][START_REF] Li | Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells[END_REF][START_REF] Clouzeau | Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro[END_REF][START_REF] Deng | Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells[END_REF][START_REF] Warcoin | In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells[END_REF]. In addition, an increase in cytokines such as IL-1β, TNFα, IL-6, chemokines such as CCL2 or matrix metalloproteases (MMP) such as MMP9, which are all pro-inflammatory molecular features of the DED pathology, was reported in these models [START_REF] Deng | Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells[END_REF][START_REF] Cavet | Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells[END_REF][START_REF] Hua | Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells[END_REF].

Both inflammatory processes and cell death phenomena may involve second messengers derived from lipids. Indeed, lipids are not only the key components of biological cell membranes as well as sources of energy, they are also key mediators of intercellular and intracellular processes [START_REF] Magtanong | Emerging roles for lipids in non-apoptotic cell death[END_REF][26][START_REF] Pettus | Ceramide in apoptosis: an overview and current perspectives[END_REF]. During the last two decades, some of them have been described as "bioactive lipids" tightly associated with several chronic diseases including diabetes, inflammatory bowel disease, multiple sclerosis, atherosclerosis [START_REF] Kurz | Ceramides as Novel Disease Biomarkers[END_REF][START_REF] Hannun | Principles of bioactive lipid signalling: Lessons from sphingolipids[END_REF]. Several studies have been dedicated to the role of lipids in ocular pathologies. An increase in sphingolipid abundance was thus reported in the cornea of diabetic patients [START_REF] Priyadarsini | Description of the sphingolipid content and subspecies in the diabetic cornea[END_REF]. In addition, Robciuc et al. reported the role of lipids in ocular pathologies highlighting the importance of sphingolipid homeostasis [START_REF] Robciuc | Ceramides in the Pathophysiology of the Anterior Segment of the Eye[END_REF]. During DED, lipid composition of the tear film, which is under control of the meibomius gland secretion, is altered as it was previously shown by several studies focusing on its lipid characterization [START_REF] Ham | Identification, quantification and comparison of major non-polar lipids in normal and dry eye tear lipidomes by electrospray tandem mass spectrometry[END_REF][START_REF] Lam | Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome[END_REF][START_REF] Ambaw | Tear eicosanoids in healthy people and ocular surface disease[END_REF][START_REF] Chen | Untargeted lipidomic analysis of human tears: A new approach for quantification of O-acyl-omega hydroxy fatty acids[END_REF]. In contrast, to our knowledge, no study aimed at describing the modulation of epithelial cell lipids, involved in cell death process and promoting inflammatory cell recruitment, and its pathophysiological consequences in DED.

The purpose of this study was to characterize the changes of the lipid composition in a human epithelial corneal cell line following BAK or HO exposure, two well-known distinct stressors acting on cell membranes [START_REF] Warcoin | Hyperosmolarité : effets intracellulaires et implication dans la sécheresse oculaire[END_REF][START_REF] Chang | Benzalkonium Chloride and Gentamicin Cause a Leak in Corneal Epithelial Cell Membrane[END_REF]. The aim was also to understand their differential effects on cell-membrane lipids and in fine, to identify lipid species as possible key markers of DED. Based on cytotoxicity assay and on gene expression of proinflammatory cytokines, we first determined the osmolarity levels and BAK concentrations to be used for cell exposures. We then performed a comprehensive lipidomic analysis to characterize qualitatively and quantitatively the changes occurring in the cellular lipid profile resulting either from HO or BAK exposures. Finally, to support lipidomic analysis results, we 93 assessed the gene expression of enzymes involved in the modulated lipid biosynthesis. 94

Material and Methods

Cell line and culture conditions

Human corneal epithelial cell line (HCE) was obtained from the RIKEN biobank (Tsukuba, Japan) [START_REF] Araki-Sasaki | An SV40-immortalized human corneal epithelial cell line and its characterization[END_REF]. HCE cells were grown in culture flasks using DMEM/F12 (1/1), 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin all from Gibco (Paisley, UK). At confluence, every 3 days, cells were harvested with trypsin-EDTA 0.05% in dubelco phosphate-buffered saline (DPBS). Cells were used in this study from passages 3 to 12.

Exposure solutions

A stock solution of 0.1% (w/v) BAK (Sigma, Saint Quentin Fallavier, France) corresponding to 2.65 mM was used to prepare the 10 -4 % BAK exposure solution, one hundred times lesser than the average concentration used in standard eye drop formulations. The 500 mOsM solutions were obtained after dilution of a 1000 mOsM stock solution prepared by overloading the culture medium with sodium chloride (Sigma-Aldrich, Saint Quentin Fallavier, France). Medium osmolarity was controlled using an osmometer Roebling 13DR (Roebling, Berlin, Germany).

Cell viability assay

Neutral Red (NR) uptake assay is based on the lysosome staining of viable cells after uptake of the dye through an active transport. To assess viability of HCE cells after exposure to HO or BAK, a solution of NR at 50 µg/mL was added to the cells grown at subconfluence in a 96-wells plate (20.000 cells/well), we performed NR assay, in accordance with previously published data [START_REF] Repetto | Neutral red uptake assay for the estimation of cell viability/cytotoxicity[END_REF]. The solution of NR was left to incubate for 3h at 37°C. Cells were washed with DPBS, then lysed using a mixture containing water, ethanol, acetic acid (49.5/49.5/1, v/v/v) and finally homogenized at room temperature for 15 minutes on a stirring plate. Fluorescence intensity was measured using a spectrofluorometer Infinite® 1000 (TECAN, Neuville-Sur-Oise, France) at 540 nm excitation and 600 nm emission wavelengths.

Reactive oxygen species production

assay. The H2DCFDA 0.1 M stock solution in DMSO (Thermo Fisher Scientific, Saint-Quentin-Fallavier, France) was used to prepare a 20 μM solution in DPBS. Cells were incubated for 24 hours with BAK or under HO condition on a 96 well plate, then washed with DPBS and a volume of 200 μL of the H2DCFDA solution was distributed in each well. Following a 30-minute incubation at 37°C in the dark under a 5% CO2 atmosphere, cells were washed with DPBS and fluorescence intensity was measured at 485 nm excitation and 535 nm emission wavelengths using a spectrofluorometer Infinite 1000 ® (TECAN, Neuville-Sur-Oise, France).

Lipid droplet staining

Cells were seeded on glass slide in 12-well plate at 50,000 cells/well. After a 24-hour exposure to BAK or HO, cells were washed with DPBS and fixed with 4% paraformaldehyde solution in water.

Oil Red O (OR) staining was performed using a 0.5% OR stock solution (Sigma-Aldrich, Saint-Quentin Fallavier, France) diluted to 3/2 (v/v) in distillated water. This solution was distributed in each well. Following a 15-minute incubation, the cells were washed three times in DPBS.

Nile Red (NiR) staining was performed using a stock solution (Thermo Fisher Scientific, Saint-Quentin-Fallavier, France) diluted to 1/1000 (v/v) in a Mowiol ® mounting medium. Nuclei were counterstained with DAPI. Glass slides were subsequently mounted under a cover glass in a Mowiol ® mounting medium and were observed by epifluorescence microscopy. Quantification of area and size of lipid droplet was performed using imageJ ® software (National Institutes of Health).

Lipidomic analysis

Chemicals and reagents

Chloroform (Carlo Erba Reactifs SDS, Val-de-Reuil, France), acetonitrile, methanol, isopropanol of LC-MS grade (J.T. Baker, Phillipsburg, NJ, USA), 3,5-di-tert-4-butylhydroxytoluene (Sigma Aldrich, Saint-Quentin Fallavier, France) and methyl-tertbutyl-ether (Acros Organics Thermo Fisher Scientific, Villebon-sur-Yvette, France) were used to prepare cell lipid extracts and mobile phase for reverse phase liquid chromatography. LC-MS grade water (Sigma Aldrich, Saint-Quentin Fallavier, France) was used in sample preparation and analysis. All standard lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and are listed in the Table S1 of Supplementary Information.

Sample preparation for lipidomic analysis

Lipidomic analysis was performed as previously described [START_REF] Petit | Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP[END_REF][START_REF] Lanzini | Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin[END_REF]. Briefly, cells were harvested using trypsin-EDTA 0.05%, washed with DPBS, centrifuged at 2000 rpm for 10 min. Dry cell pellets were adjusted to 3 million cells and stored at -80°C until analytical process. After thawing, the cell pellets were resuspended in ultra-pure water (1mL) containing a mixture of lipid internal standards (Suppl. Inform. S2) at a final concentration of 1 µM and were sonicated for 5 minutes.

Lipids were extracted using a chloroform/methanol/water (5:5:2, v/v/v) mixture containing 3,5-di-tert-4-butylhydroxytoluene 0.01% (w/v) as antioxidant agent. Samples were subsequently centrifuged at 3000 rpm for 10 min, supernatants were collected, and solvents evaporated under reduced pressure at 45 °C. Dry residues were resuspended in an acetonitrile/isopropanol/chloroform/water (35:35:20:10 v/v/v/v) mixture before injection into the UPLC-MS system. Liquid chromatography-electrospray ionization mass spectrometry analysis of lipid extracts was performed on a Synapt ® G2 High Definition MS™ (Q-TOF) mass spectrometer (Waters ® ) combined with a UPLC system (Waters ® ).

Chromatographic separation was performed on an Acquity ® CSH C18 column (100× 2.1 mm; 1.7 µm) set at 50°C. Lipids were eluted using a binary gradient system consisting in 10 mM ammonium acetate in an acetonitrile/water mixture (40:60, v/v) as solvent A and 10 mM ammonium acetate in an acetonitrile/isopropanol mixture (10:90, v/v) as solvent B. The eluent increased from 40% B to 100% B in 10 min and was held at 100% B for 2 min before a return to 40% B followed by an equilibration period of 2.5 min. The flow rate was kept at 0.4 mL/min for 15 min. Data were collected in the full scan mode at m/z 50-1200 in both positive (ESI +) and negative (ESI-) ion modes. The source parameters were as follows: capillary voltage 3,000 V (ESI +) and 2,400 V (ESI-), cone voltage 30 V (ESI +) and 45 V (ESI-), source temperature 120°C, desolvation temperature 550°C, cone gas flow 20 L/h, and desolvation gas flow 1,000 L/h. Leucine enkephalin (2 ng/mL) was used as the external reference compound (Lock-Spray™) for mass correction. Data were acquired in the so-called resolution mode (20,000 FWHM a m/z 500) with a scan time of 0.1 s. Data acquisition was managed using Waters MassLynx™ software (version 4.1; Waters MS Technologies).

Data pre-processing

Raw data files (.raw format) acquired on UPLC-ESI-MS were processed using XCMS set up with parameters suitable for high resolution LC-MS, to generate in both ESI+ and ESI-, a matrix listing peak areas associated to a unique m/z and retention time. These matrixes were normalized and filtered as previously described [START_REF] Lanzini | Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin[END_REF].

Lipid structure assignment

The structure assignment of lipids was based on the following criteria. An annotation of lipid species was first performed through the use of the online databases LIPID MAPS and METLIN using the mass accuracy with a tolerance window of 5 ppm. The annotation was confirmed using retention time. Indeed, by using the UPLC-ESI-MS analysis of the standard lipid mixture, each lipid class can be determined by the linear relationship between retention time and equivalent carbon number [START_REF] Lanzini | Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin[END_REF].

Finally, MS/MS fragmentation data was used to provide structural information on the annotated lipid structure.

Lipid amount estimation

The lipid quantities, expressed as mol%, were estimated using internal standard lipid mixture spiked in cell suspension samples before extraction, according to procedure in agreement to current guideline [START_REF] Burla | MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines[END_REF]. Indeed, lipid species intensities were individually normalized to the one of the corresponding internal standards chosen in the same subclass. The standard lipid mixture includes one lipid species representative of each investigated subclass (Suppl. Inform. S2). Lipid species used as internal standard contain fatty acid side chain with total odd carbon number or are deuteriated derivatives and thus cannot overlap with endogenous lipid species.

Gene expression analysis by RT-qPCR

Dry cell pellets were stored at -80°C until analysis. Total RNAs were extracted from cells using Nucleospin ® RNA kit (Macherey Nagel, Neumann-Neander, Germany). RNA content was measured using a Nanodrop Detector (ND-1000 spectrophotometer, ThermoFischer Scientific).

Reverse transcription was performed with 600 ng RNA using Multiscribe reverse transcriptase (TaqMan Reverse Transcription Reagents, Applied Biosystems, Life Technologies). Concentrations of each sample were adjusted to 5 ng/μL of cDNA. The reaction mixture was preheated at 50°C for 2 min, followed by 40 cycles (95°C for 15 s and 60°C for 1 min). Target cDNA was amplified using the 7300 Real-Time PCR system (Applied Biosystems, Life Technologies) with Taqman ® probes for IL1B (Hs015554136), IL6 (Hs00174131), CCL2 (Hs00234140), CerS2 (Hs00371958), SMPD2 (Hs00906924), ASAH2 (HsO1O15655), DGAT1 (Hs01020362), PLA1 (Hs01056915) (Thermo Fisher Scientific, Saint-Quentin-Fallavier, France). Each assay was normalized by amplifying the housekeeping cDNA HPRT (Hs02800695). Changes in mRNA expression were calculated according to the 2 -ΔΔCT method (CT, cycle threshold), with ΔCT = CTtarget gene -CTHPRT and ΔΔCT = ΔCTstimulated -ΔCTcontrol.

PLA2 activity assay

PLA2 activity was determined using Red/Green BODIPY based EnzCheck Phospholipase A2 assay kit according to manufacturer procedure. Briefly, cells were incubated for 24 hours with BAK or HO in 6-well plates. Culture media was removed, and cells were washed with DPBS. Harverest cells were centrifugated, DPBS removed and cell pellet was suspended in 100 μL of PLA2 reaction buffer with protease inhibitor and finally sonicated for 10 sec. A volume of 50 μL of cell lysate were then transferred in a 96 well plate and mixed with liposomes prepared with the EnzChek Phospholipase A2 substrate at a ratio of 1:1. Following a 30-minute incubation at 37°C in the dark, PLA2 activity was determined as a FRET ratio (λex = 460 nm λem = 515/575 nm). Fluorescence were measured using a spectrofluorometer Spark ® (TECAN, Neuville-Sur-Oise, France).

Statistical analysis

Unsupervised and supervised multivariate analyses were performed using SIMCA-P+ software version 13.0.3 (Umetrics, Umeå, Sweden) as previously described [START_REF] Petit | Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP[END_REF][START_REF] Lanzini | Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin[END_REF][START_REF] Ayciriex | Neuronal Cholesterol Accumulation Induced by Cyp46a1 Down-Regulation in Mouse Hippocampus Disrupts Brain Lipid Homeostasis[END_REF]. Briefly, a Pareto scaling was applied to the variables prior to unsupervised principal component analyses (PCA) and supervised partial least squares-discriminant analyses (PLS-DA). Permutation tests on the class labels were conducted to assess over-fitting of models. Orthogonal partial least squares discriminant analyses (OPLS-DA) model was subsequently built based on the corresponding selected PLS-DA models. S-plot was generated from each OPLS-DA model to investigate the lipids involved in the statistically significant differences between control cells and exposed cells. A cross-validated analysis of variance (CV-ANOVA) was carried out on each supervised model to assess the statistical significance of group separation. Finally, a misclassification test was performed to validate the models. Univariate data analysis (Wilcoxon) with a false discovery rate [(FDR)-adjusted p<0.01] controlling the false-positive rate associated with multiple comparisons, was performed to assess the whole lipids identified and the statistical significance of the difference in BAK-or HO-treated cells vs control cells.

Each experiment was performed independently at least three times. Results are expressed in percentage compared to control and are presented as means ± standard deviation (SD). The statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA).

Verification of the normality assumption with an Agostino-Pearson test was first performed. The comparison of the means of more than two groups was then performed using ANOVA test followed by a Dunnet multiple comparison test with a risk set at 0.05. Comparisons of two averages were done using a Student t-test and after normality assessment. The significance thresholds compared to control were: *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Cell viability, reactive oxygen species and inflammatory cytokine production following exposure of HCE cells to BAK or HO

Human corneal epithelial cells were exposed for 24 h to BAK concentrations ranging from 10 - 6 % to 10 -3 % or to HO values ranging from 350 to 600 mOsM respectively. Both BAK and HO led to a significant decrease in viability (data not shown). For subsequent experiments, cells were exposed to 10 -4 % BAK concentrations, and 500 mOsM HO corresponding to a decrease in viability of 40% (p < 0.001), and 35% (p < 0.001) (Figure 1A) respectively. A 24-hour exposure of HCE to BAK 10 -4 % led to a ROS production increase of 190% (Figure 1B). In contrast, HO 500 mOsM did not induce any change in ROS production (Figure 1B). While BAK led to an increase in gene expression of IL1B (p < 0.05) and IL6 (p < 0.01), HO induced a significant enhancement of the CCL2 (p < 0.01) and IL6 (p < 0.001) gene expressions (Figure 1C).

Lipid distribution

HO led to the formation of lipid droplets in the cytoplasm of HCE cells (Figure 2A). Indeed, size area (Figure 2B) and number of lipid droplets (Figure 2C) were significantly increased after HO exposure. In contrast, BAK did not induce any change in neutral lipids. It is noteworthy that following HO exposure, the number of LD per HCE cell is lower when staining is performed using Red Oil O than in Red Nil. This discrepancy is related to the fact that droplet fusion specifically occurs in Red Oil O staining protocol [START_REF] Fukumoto | Deformation of lipid droplets in fixed samples[END_REF].

Changes in lipid composition of HCE cells exposed to BAK or HO

To investigate the impact of BAK or HO exposure on the lipid composition of HCE cells, we performed an untargeted lipidomic analysis using liquid chromatography coupled to mass Validation of the lipidomic analysis was performed as previously described [START_REF] Petit | Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP[END_REF][START_REF] Lanzini | Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin[END_REF]. Briefly, an unsupervised principal component analysis (PCA) was performed based on three quality controls (QCs) dilutions (QC 1/1, 1/3 and 1/6). The built PCA model for the ESI + and ESI-analysis showed compact clusters of replicates for each QC levels ((Suppl. Inform. S1) thus confirming that differences between biological samples were not related to analytical variations.

Unsupervised analysis (PCA) comparing BAK-exposed and control cells was first performed.

Both in ESI+ and ESI-, the score plots corresponding to the PCA model which had been created

clearly exhibited two clusters ascribed to BAK-exposed and control cells (Figure 3B). The percentage of explained and predicted variances generated exhibited a moderated value for data acquired in ESI+ (R² = 0.42, Q² = 0.32) and a quite high one for data acquired in ESI-(R² = 0.62, Q² = 0.52).

Unsupervised analysis was also performed to compare the lipidomes of HO-exposed and control cells.

Separation between the HO-exposed and control groups was clearly displayed in the score plots (Figure 3B). The percentage of explained and predicted variances generated showed a moderated value for data acquired in ESI+ (R² = 0.47, Q² = 0.43) and a good one for data acquired in ESI-(R² = 0.77, Q² = 0.67).

The analysis of the data set corresponding to BAK exposure led to the selection of 1200 variables exhibiting a p(corr) value > 0.7 from the S-Plot (Figure 3C). Among these discriminant variables, 168 were identified as lipids, 120 species were increased and 48 were decreased.

Accordingly, exposing HCE cells to BAK led to an increase of sphingolipids including 13 Cer and 10 SM species while 4 hexosylceramides were decreased (Figure 4A). Moreover, the cell level of 17 PC and 14 PE species was significantly decreased while three LPC and two LPE species were enhanced.

Finally, seven DG species were decreased, among which three compounds contained saturated or mono-unsaturated fatty acid (Figure 4A).

Exposure of HCE cells to 500 mOsM HO made it possible to select 1102 variables with a p(corr) > 0.7 on the corresponding S-Plots (Figure 3C). A total of 132 lipids were identified, 87 lipids being increased and 45 decreased. Results show an increase in sphingolipid species, including 7 Cer and 5 SM (Figure 4). Among glycerophospholipids, only one LPC species decreased while 2 increased and 6 PC and 9 PE increased. Finally, 56 TG species exhibited an increased cell level (Figure 4A).

The Venn diagram displayed the lipid species modulated in a common and specific manner following exposure of the HCE cells to BAK or HO (Figure 4B). At the lipid class levels, both BAK and HO respectively increased and decreased the total sphingolipid and phospholipid cell level (Figure 4C). In contrast, following exposure of HCE cells to HO, the level of glycerolipids was strikingly increased while fatty acid content was decreased.

Gene expression and activity of enzymes involved in lipid metabolism

To investigate the origins of the lipidome changes of HCE cells exposed to BAK or HO, we assessed the gene expression of several key enzymes involved in lipid metabolism. Regarding metabolism of sphingolipids (Figure 5A), the total cell level of Cer and SM was increased following exposure to BAK or HO (Figure 5B). The gene expression study showed that ASAH2, CerS2 and SMPD2 were significantly up regulated after exposure to HO (p < 0.01) while incubation with BAK induced no change in expression of these genes (Figure 5C). As total TG cell level was increased following exposure to HO (Figure 6A), enzymes involved in TG biosynthesis (Figure 6B) were also investigated. Results showed an increase in DGAT1 gene expression (p < 0.01) following HO exposure. In contrast, no DGAT1 modulation was observed after BAK exposure (Figure 6C).

Phospholipid metabolism (Figure 7B) was also as BAK and HO induced an increase and a decrease of LPC species, respectively (Figure 7A). Indeed, HO exposure led to an increase in gene expression of PLA1 (p < 0.01) while BAK did not induce such change (Figure 7C). Exposure of HCE cells to BAK

Discussion

Dry eye disease is a chronic inflammatory pathology of the ocular surface with significant impact on everyday life for patients. Despite its high prevalence, only few treatments are available. It is thus important to better understand molecular mechanisms of the pathology in order to develop new targeted treatments and to find new markers to improve patient monitoring. Lipids are now recognized as mediators of the signal transduction with molecular impact on cell homeostasis and with an important role in inflammatory process. Our purpose was therefore to investigate the lipid changes in an in vitro model of human epithelial corneal cell line, exposed to BAK or hyperosmolarity (HO).

These both cell stress, HO, key feature in the pathophysiology of DED and BAK, common eye drop excipient, are known to be contributing factor of this eye disease [START_REF] Baudouin | Preservatives in eyedrops: The good, the bad and the ugly[END_REF].

BAK concentration and HO value were chosen according to eye drop concentration for BAK and HO value determinated in the context of DED and also using HCE cell viability assays and previously published reports. Indeed, a 24-hour exposure to BAK 10 -4 % or HO 500 mOsM leads to a significant decrease in cell viability associated to an increase in gene expression of pro-inflammatory cytokines as previously described in DED patients [START_REF] Liang | Correlation of clinical symptoms and signs with conjunctival gene expression in primary Sjögren syndrome dry eye patients[END_REF][START_REF] Kessal | Conjunctival Inflammatory Gene Expression Profiling in Dry Eye Disease: Correlations With HLA-DRA and HLA-DRB1[END_REF]. Our results are in accordance with previous studies investigating BAK and HO [START_REF] Clouzeau | Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro[END_REF][START_REF] Brasnu | Comparative study on the cytotoxic effects of benzalkonium chloride on the Wong-Kilbourne derivative of Chang conjunctival and IOBA-NHC cell lines[END_REF][START_REF] Warcoin | Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro[END_REF]. It must be emphasized, that the aforementioned BAK concentration is 1/50 to 1/200 times that used in commercially available eye-drops (0.005% -0.02%).

Regarding HO, in DED patients, mean HO levels range from 310 to 330 mOsM [START_REF] Baudouin | Role of hyperosmolarity in the pathogenesis and management of dry eye disease: Proceedings of the ocean group meeting[END_REF] and values up to 800 mOsM have been previously reported in tears of DED patients [START_REF] Liu | A Link between Tear Instability and Hyperosmolarity in Dry Eye[END_REF]. In addition, a HO level of 500 mOsM has been widely used in in vitro models of DED [START_REF] Deng | Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells[END_REF][START_REF] Cavet | Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells[END_REF][START_REF] Hua | Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells[END_REF][START_REF] Ren | Hyperosmolarityinduced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells[END_REF].

In order to exhaustively list the lipid species which level is modified following BAK or HO exposure in HCE, we performed an untargeted lipidomic analysis involving UPLC-ESI-HRMS as well as supervised and unsupervised multivariate analyses. Indeed, this analytical approach is recognized to be able to extensively characterize qualitative and quantitative changes in lipid composition without a priori, especially in cells [START_REF] Petit | Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP[END_REF][START_REF] Saunier | Resveratrol reverses the Warburg effect by targeting the pyruvate dehydrogenase complex in colon cancer cells[END_REF]. In HCE incubated either to BAK 10 -4 % or to HO 500 mOsM, three main lipid classes display marked alterations: sphingolipids, glycerolipids and glycerophospholipids.

In HCE exposed to BAK or HO, a major change in sphingolipid level was observed. Changes involving this lipid subclass was previously reported in a 3D-reconstructed human-cornea-like epithelium exposed to 0.1% BAK and in a human corneal cell line exposed to HO [START_REF] Robciuc | Hyperosmolarity-induced lipid droplet formation depends on ceramide production by neutral sphingomyelinase 2[END_REF][START_REF] Lee | Nervonoylceramide (C24:1Cer), a lipid biomarker for ocular irritants released from the 3D reconstructed human cornea-like epithelium, MCTT HCE TM[END_REF]. In our study, both BAK and HO also increased ceramide species levels, especially Cer (44:2), Cer (42:2), Cer (36:2) and Cer (34:1). HO mainly increased long chain ceramides. This result is consistent with an increase in gene expression of CerS2 also only confirmed in HCE cells exposed to HO. Indeed, CerS2 catalyzes acetylation of sphinganine or sphingosine by C20 to C26 fatty acid to form dihydroceramides and ceramides, respectively [26]. In contrast, BAK mainly increasing short chain ceramides is therefore not expected to change in CerS2 gene expression. It would be of interest to investigate gene expression of CerS6, an enzyme specifically catalyzing short chain ceramide biosynthesis. Beside de novo synthesis, ceramides are produced through the cleavage of sphingomyelins by sphingomyelinase enzymes. nSMase2 gene expression was increased following HO exposure, suggesting that ceramide accumulation could be due to sphingomyelin hydrolysis, as previously reported after a 2 hour-HO exposure in corneal cells [START_REF] Robciuc | Hyperosmolarity-induced lipid droplet formation depends on ceramide production by neutral sphingomyelinase 2[END_REF]. Metabolism of ceramides also includes degradation into sphingosines via a ceramidase, especially ASAH2, an enzyme whose gene exhibited an increased expression in HCE exposed to HO. Mechanisms of Cer accumulation involve different metabolic pathways related to specific cell locations. On one hand, de novo synthesis of ceramides take place in the endoplasmic reticulum and involves enzymes such as serine palmitotoyl transferase and ceramide synthase. On the other hand, the hydrolysis of SM into Cer can be achieved at the plasma membrane by neutral SMase but also in lysosomes by acid SMase. In dry eye disease models, a global profiling of the enzymes involved in the synthesis of ceramides could be of particular interest to target one or more enzymes to be investigated in subsequent pharmacological studies.

Ceramides promote inflammation through IL-1β release, induce apoptosis and alter cell survival through PKCζ and PP2A activation [START_REF] Pettus | Ceramide in apoptosis: an overview and current perspectives[END_REF][START_REF] Maceyka | Sphingolipid metabolites in inflammatory disease[END_REF][START_REF] Chaurasia | Ceramides -Lipotoxic Inducers of Metabolic Disorders[END_REF]. Ceramides thus play key roles in various cell processes, and the dysregulation of ceramide metabolism is involved in many inflammatory diseases such as atherosclerosis, inflammatory bowel disease or multiple sclerosis [START_REF] Kurz | Ceramides as Novel Disease Biomarkers[END_REF][START_REF] Stith | Advances in determining signaling mechanisms of ceramide and role in disease[END_REF][START_REF] Park | Ceramide synthases as potential targets for therapeutic intervention in human diseases[END_REF]. Our results show that HO leads to an increase in both CerS2 and CCL2 gene expression. On one hand, de novo synthesis of ceramide was shown to induce a CCL2 release in a macrophage cell line [START_REF] Hamada | Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte-macrophage interaction[END_REF] and on the other hand, HO is a well-known inducer of CCL2, stimulating gene expression and protein release [START_REF] Warcoin | In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells[END_REF][START_REF] Hua | Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells[END_REF][START_REF] Warcoin | Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro[END_REF]. De novo synthesis of ceramide may thus stimulate CCL2 release leading to the recruitment of macrophages which, in turn, triggers inflammatory pathways. In contrast to HO, incubation with BAK did not modify CerS2 and CCL2 gene expression in HCE.

The increase in ceramide levels, described in our study, following a 24-hour incubation of HCE with BAK or HO, previously reported by Robciuc et al. but following a shorter exposure of 2 hours, indicates effect during at least 24 hours. In a short time exposure, Cer is produced through the action of SMase, a well-known mechanism occurring in the acute phase of apoptosis, while our study indicates that following a 24-hours exposure, de novo synthesis pathway is also involved. Regarding apoptosis and inflammatory process induced by this lipid subclass [START_REF] Claus | Inhibition of sphingomyelin hydrolysis: targeting the lipid mediator ceramide as a key regulator of cellular fate[END_REF], ceramides may be regarded as important mediators of the deleterious effects due to HO or BAK.

The present untargeted lipidomic analysis indicates a striking increase in TG species induced by HO exposure. This result is supported by lipid droplet (LD) formation and by DGAT1 gene overexpression in the HCE cells. HO is known to induce LD formation in renal as well as corneal epithelial cells [START_REF] Robciuc | Hyperosmolarity-induced lipid droplet formation depends on ceramide production by neutral sphingomyelinase 2[END_REF][START_REF] Weber | TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells[END_REF] and DGAT1 is one of the main enzymes involved in TG biosynthesis. LD are an active field of research because their role has been described as either protective or deleterious [START_REF] Welte | Lipid droplet functions beyond energy storage[END_REF][START_REF] Thiam | The biophysics and cell biology of lipid droplets[END_REF]. Indeed, LD are involved in free fatty acids storage playing a protective role against cell lipotoxicity [START_REF] Thiam | The biophysics and cell biology of lipid droplets[END_REF][START_REF] Welte | Expanding Roles for Lipid Droplets[END_REF]. However, LD are also known to be deleterious to cells as they are key effectors of inflammation [START_REF] Xu | Lipid droplet proteins and metabolic diseases[END_REF], especially through the COX2 activity, an enzyme located in LD catalyzing synthesis of pro-inflammatory eicosanoids [START_REF] Xu | Lipid droplet proteins and metabolic diseases[END_REF]. We may thus hypothesize that HO leads to eicosanoid formation. A target lipidomic analysis would therefore be valuable to assess eicosanoid level changes after BAK and HO exposure. In consistency with our results, an increase in COX2 protein was reported in primary epithelial corneal cells and in a conjunctival cell line after HO exposure [START_REF] Deng | Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells[END_REF][START_REF] Chen | Global Metabonomic and Proteomic Analysis of Human Conjunctival Epithelial Cells (IOBA-NHC) in Response to Hyperosmotic Stress[END_REF].

LD could therefore be explored as a cell marker of hyperosmolarity of the ocular surface.

20

In vitro BAK exposure led to both a striking decrease in PC and PE associated to an increase in LPC and LPE. These results are in complete agreement with the increase of PLA2 activity, the enzyme responsible for the sn2 acyl chain hydrolysis of phospholipids. Furthermore, an important decrease in PC-O (36:4) level which contain arachidonic acid in sn2 position was especially observed.

This result may be expected since PC containing arachidonic acid in sn2 position is a key substrate of PLA2 enzyme. As PUFA are mainly targeted by PLA2, the decrease in PC (O-18:0/22:6) level may also be related to the increase of DHA rate which could constitute a cell response against BAK exposure. As an increase of PLA2 has been previously reported in tears of DED patients [START_REF] Chen | sPLA2-IIa is an inflammatory mediator when the ocular surface is compromised[END_REF], this enzyme could thus promote BAK toxicity in human corneal epithelial cells. Indeed, it was demonstrated that in a macrophage cell line, LPC promote inflammation through IL-6 and TNFα release [START_REF] Sevastou | Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes[END_REF][START_REF] Chen | Resveratrol alleviates lysophosphatidylcholine-induced damage and inflammation in vascular endothelial cells[END_REF]. In addition, in vitro, LPC triggers signaling pathways of TLR-4, a major lipopolysaccharide pattern recognition receptor widely described in DED pathophysiology [START_REF] Craig | The Ocular Surface TFOS DEWS II De fi nition and Classi fi cation Report[END_REF][START_REF] Redfern | Dry eye modulates the expression of toll-like receptors on the ocular surface[END_REF][START_REF] Lee | Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease[END_REF].

LPC may thus contribute to inflammation mediated by BAK and hence, play a role in its toxicity.

In contrast, HO exposure induced a marked increase in PE levels. A similar change has been previously described in renal cells exposed to HO [START_REF] Weber | TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells[END_REF]. A slight decrease in LPC levels associated to an increase in PLA1 gene expression was also exhibited. PLA1 catalyzes LPC conversion to glycerylphosphorylcholines, known to be osmoprotective compounds. Our results are compatible with the previously reported increase in glycerylphosphorylcholine level in the IOBA-NHC conjunctival cell line exposed to HO [START_REF] Chen | Global Metabonomic and Proteomic Analysis of Human Conjunctival Epithelial Cells (IOBA-NHC) in Response to Hyperosmotic Stress[END_REF]. Furthermore, a metabolomic study on DED patient serum exhibited glycerylphosphorylcholine and lysolipid changes [START_REF] Vehof | A Metabolome-Wide Study of Dry Eye Disease Reveals Serum Androgens as Biomarkers[END_REF] indicating an alteration of this lipid subclass, in consistency with our results. Increase in catabolism of PC may thus be regarded as a self-protective mechanism of cells to osmotic stress via osmoprotective glycerylphosphorylcholine [START_REF] Gallazzini | What's New About Osmotic Regulation of Glycerophosphocholine[END_REF].

In addition, numerous PC, PE and PI plasmalogen species are strikingly decreased in HCE cells under BAK or HO exposure. This result may be explained by the fact that vinyl ether located in sn1 position is very sensitive to oxidative stress [START_REF] Braverman | Functions of plasmalogen lipids in health and disease[END_REF][START_REF] Wallner | Plasmalogens the neglected regulatory and scavenging lipid species[END_REF]. Oxidative stress is known to be part of the mechanisms explaining deleterious effect observed under BAK and HO exposure [START_REF] Clouzeau | Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro[END_REF][START_REF] Pauloin | In vitro modulation of preservative toxicity: High molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride[END_REF][START_REF] Vitoux | Benzalkonium chloride-induced direct and indirect toxicity on corneal epithelial and trigeminal neuronal cells: proinflammatory and apoptotic responses in vitro[END_REF]]. An increase in ROS production observed in HCE cells following a 24-hour exposure of BAK reinforced the above observations. In contrast, HO exposure did not lead to any ROS production after 24 hours.

Nevertheless, it has been shown that HO lead to oxidative stress but only following 1 to 3 hours exposure [START_REF] Deng | Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells[END_REF]. Regarding oxidative stress in DED pathophysioplogy, plasmalogen species may finally be regarded as cell marker.

In summary, exposure of HCE to BAK or HO highlights a large set of lipid mediator modulation. Interestingly, these two in vitro models showed common and specific alterations in the cell lipidome. An overview of major lipid alterations reported in this study is displayed in the Figure 8.

While ceramides were increased both after BAK or HO exposure, changes in glycerolipids and phospholipids depend on the type of the stressor involved. Indeed, TG accumulation and LD formation were specifically induced by HO while a slight TG decrease arouse following BAK exposure.

Moreover, LPE and LPC drastically decreased following HO exposure, while BAK induces an increase in these two lipid subclasses. Using epithelial corneal cells exposed to BAK, a microbicidal, detergent and pro-oxidative agent, or HO, a pathophysiological feature of DED, this untargeted lipidomic investigation showed common and contrasted cell lipid alterations. It also underlines the weight of lipid metabolism in cell death and inflammation processes. Finally, all the impacted lipid species could be tightly intricated in a metabolic network underlying specific regulation pathways in addition to other biological processes.

Conclusion

  spectrometry (UPLC-MS). Typical UPLC-ESI-MS in positive (ESI+) and negative (ESI-) ion mode chromatograms of cell lipid extracts from HCE cells are displayed in Figure3. In positive ion mode, lysophospholipids (LP) and monoacylglycerols (MG) appear between 1.8 and 3 min, phospholipids phosphatidylserine PS) and sphingolipids (Sphingomyelin SM and Ceramide Cer) between 5 and 8 min, Diacylgycerol (DG) between 6 and 9 min and Triacylglycerol (TG) between 8 and 11 min. In negative ion mode, fatty acid (FA) are first eluted before 6 min, followed by Cers and phospholipids (PE, PI, PA, PG, and PS) between 6 and 9 min.

From a clinical pointFiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Lipid staining following BAK or HO exposure. HO induces lipid droplet formation (A) in the cytoplasm of HCE cells with an increase of their size and area (B). Results are expressed as mean ± SD (ANOVA with Tukey's multiple comparisons test. n=4. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3 :

 3 Figure 3: Liquid chromatography time-of-flight mass spectrometry-based lipidomics analysis following BAK or HO exposure. Chromatograms (A) and PCA built models (B) of control and exposed HCE cells. Variable selections for lipid annotation were based on S-Plot (C). Score plots of BAK-exposed and control cells are presented in both ESI+ (B1) and ESI-(B3). Metabolites with significant changes in cellular level between BAK-exposed and control cells are presented in ESI+ (C1) and ESI-(C3) S-plots (orange boxes delimited the variables of interest). Score plots of HOexposed and control cells are presented in both ESI+ (B2) and ESI-(B4) ion mode. Metabolites with significant changes in cellular level between HO-exposed and control cells are presented in ESI+ (C2) and ESI-(C4) S-plots (orange boxes delimited the variables of interest).

Figure 4 :

 4 Figure 4: Comparative cell lipid levels in HCE exposed to BAK or HO. (A) Change in discriminant lipid species in HCE cells exposed to BAK or HO. Results are expressed as fold changes compared to control (FDR-adjusted p < 0.01). Blue color corresponds to a decrease in the cell lipid level compared to control while red color indicates an increase. (B) Venn diagram displays common and specific modulated lipid species to BAK or HO HCE exposure. (C) Distribution of the total lipid classes between control, BAK and HO exposed HCE cells.

Figure 5 :

 5 Figure 5: Alteration of sphingolipid metabolism. (A) Total ceramide and sphingomyelin level change following BAK or HO exposure (B) Metabolic pathways of sphingolipids. (C) Gene expression of enzyme involved in sphingolipid metabolism. Results are expressed as mean ± SD (ANOVA with Tukey's multiple comparisons test. n=5. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6 :

 6 Figure 6: Alteration of glycerolipid metabolism. (A) Change in total DG and TG levels following BAK or HO exposure. (B) Metabolic pathways of glycerolipids. (C) Gene expression of DGAT1, enzyme involved in TG biosynthesis. Results are expressed as mean ± SD (ANOVA with Tukey's multiple comparisons test. n=5. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 7 :

 7 Figure 7: Alteration of glycerophospholipid metabolism. (A) Change in total PC and LPC levels following BAK or HO exposure. (B) Metabolic pathways of lipids containing choline head group. (C) Gene expression of PLA1 which is implicated in LPC degradation to glycerylphoshorylcholine. (D) PLA2 activity change following BAK or HO exposure. Results are expressed as mean ± SD (ANOVA with Tukey's multiple comparisons test. n=5. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 8 :

 8 Figure 8: Synopsis highlighting the main changes in lipid subclasses related to BAK and HO exposure. BAK and HO shared alterations are displayed in the white box at the center. Those specific to BAK and HO are respectively shown on the left and right side of the diagram.

  Figure 1 737

(Figure7D).

Aknowledgment

This study was funded by Sorbonne Université and the Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique. The authors thank the core facilities of the Institut de la vision, the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Région Ile-de-France and Ville de Paris.

Conflict of interest

The authors declare that they have no conflicts of interest.


Tania Nawfal Dagher

Charbel Al-Bayssari

Selma Chabou

Sophie Baron

Linda Hadjadj

Seydina M Diene

Eid Azar

Jean-Marc Rolain
email: jean-marc.rolain@univ-amu.fr

Intestinal Carriage of Colistin Resistant Enterobacteriaceae at Saint Georges Hospital in Lebanon

Keywords: Colistin resistance, Gram-negative bacteria, pmrAB, phoPQ, mgrB, Lebanon Eclo-14B V38I, W47S, ins 48V No mutation No mutation D46V, I47F, I49F, E140del, F141del, I143D, N144A, del148-

Objectives

The increase in resistance to antibiotics has led to the revival of colistin, as the last option for treatment, which automatically led to an increase of colistin-resistant Gram-negative bacteria. In this study, we report the presence of clinical colistin-resistant Enterobacteriaceae isolated from a Lebanese hospital.

Method

From twenty-three rectal swabs, eight colistin-resistant clinical strains (5 Escherichia coli, 2

Enterobacter cloacae, and 1 Klebsiella pneumoniae) were isolated. Antibiotic susceptibility testing was performed using the disk diffusion method and E-test. Broth microdilution method was performed to determine colistin susceptibility. RT-PCR, standard PCR, and sequencing were used to investigate genes encoding for extended-spectrum β-lactamases, carbapenemases, and colistin resistance. Genotyping of these isolates was conducted by MLST.

Results

Results of antibiotic susceptibility testing revealed that all isolates were resistant to colistin. They have MICs for colistin that ranged from 8 mg/L to 32 mg/L. Real-time PCR results showed that 5 strains harbored blaTEM-1 and one strain harbored blaTEM-163. Moreover, 4 strains were positive for blaCTX-M-15, blaCTX-M-103, and blaCTX-M-189, and K. pneumoniae harbored blaSHV-1. Observed colistin resistance was linked to amino acid substitutions into protein sequences of pmrA/B, phoP/Q, and mgrB. Interestingly, we report here mutation in the mgrB regulator and pmrA/B, phoP/Q in colistin resistant E. cloacae and E. coli clinical isolates for the first time in Lebanon.

This study highlights the presence of colistin-resistant Gram-negative bacteria in a Lebanese hospital, which is worrisome. An urgent strategy needs to be adopted in order to avoid the spread of such bacteria.

Introduction

Resistance to antibiotics in Gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae has become a field of interest over the last decade. Those bacteria develop high resistance to most available antibiotics such as beta-lactams, aminoglycosides, and fluoroquinolones [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. This growing infection caused by multidrug-resistant (MDR) Gram-negative bacteria has led to the renewal of colistin as a last-resort treatment option especially for patients in intensive care units (ICUs) [START_REF] Olaitan | Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study[END_REF]. Unfortunately, the resistance to polymyxins has been increasingly reported worldwide [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. Polymyxin E (colistin) and polymyxin B have been used in humans and are bactericidal toward Gram-negative bacteria except for the bacteria that are intrinsically resistant to colistin such as Burkholderia, Edwardsiella, Proteus, Providencia, Morganella and Serratia,. From 1959, colistin was available to treat infections caused by Gram-negative bacteria. Conversely, its utilization is limited due to the daily dose that cannot be increased because it will lead to nephrotoxicity [START_REF] Falagas | Colistin: The Revival of Polymyxins for the Management of Multidrug-Resistant Gram-Negative Bacterial Infections[END_REF]. Between the late 1990s and early 2000s, colistin was re-introduced into clinical medicine due to the emergence of resistance to beta-lactams and carbapenems. This led to the emergence of colistin-resistant bacteria among patients treated with this compound [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. The bacterial cell membrane is the main target for the antimicrobial activity of colistin: colistin binds to the anionic lipid A moiety of lipopolysaccharide (LPS) and disrupts the cell membrane [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF][START_REF] Falagas | Colistin: The Revival of Polymyxins for the Management of Multidrug-Resistant Gram-Negative Bacterial Infections[END_REF]. Several strategies are used by Gram-negative bacteria to escape from polymyxin. The major mechanism of resistance occurs by the alteration of the negatively charged lipopolysaccharide (LPS), by the addition of phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (L-Ara4N), to the lipid A moiety of the LPS. This can be accomplished by specific mutations of the two-component systems (TCSs) (pmrA/pmrB, phoP/phoQ), or its negative regulator mgrB. The modified LPS with this positive charge reduces its binding to polymyxins and produces this resistance [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. Moreover, it has been demonstrated that resistance to colistin can also be due to the loss of LPS production due to mutations in lpxACD genes [START_REF] Olaitan | Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria[END_REF], in addition to the use of efflux pumps, the capsules formation, and overexpression of OprH, which are all efficiently regulated at the molecular level [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. Finally, colistin resistance can also be due to the presence of a plasmid-mediated mcr gene that encodes for the phosphoethanolamine transferase enzyme which is capable of modifying the lipid A moiety of LPS with the addition of phosphoethanolamine [START_REF] Okdah | First report of colistin-resistant Klebsiella pneumoniae clinical isolates in Lebanon[END_REF]. Finally, the increased use of colistin as a last-resort therapeutic medication to treat patients infected with multidrugresistant (MDR) Gram-negative bacteria, has been followed by an increase in the number of Gram-negative bacteria resistant to colistin [START_REF] Baron | Molecular mechanisms of polymyxin resistance: knowns and unknowns[END_REF]. The present study aims to investigate the prevalence of colistin-resistant Enterobacteriaceae isolated from rectal swabs of patients remaining at ICU of Saint-George Hospital in Beirut, Lebanon for more than one week and who were treated with carbapenem and colistin combination therapy.

Materials and methods

Microbiology procedure:

Study Design

Between October 2016 and February 2017, a rectal swab was collected for each patient hospitalized in the ICU of Saint-George Hospital in Beirut who received colistin and carbapenem during their stay in ICU for more than one week. These patients were treated with combination therapy because it was given as a precaution for patients to prevent their infections by carbapenem-resistant bacteria that were spreading in the hospital. Only patients of the ICU treated with combination therapy were selected because we believe that patients receiving colistin have a bigger chance to have an intestinal carriage of colistin-resistant isolates. The rectal swab was collected after 7 days of therapy for the analysis of colistin and carbapenemresistant bacteria. They were kept at -80°C before being transported to the laboratory in Marseille, France.

Microbiological procedures

Screening of colistin-resistant enterobacteria

Rectal swabs were transferred to an enrichment Tryptic Soy Broth (TSB) medium and incubated overnight at 37°C. After incubation, 100 μL of the enrichment medium were cultivated for 24 hours at 37°C on the selective medium LBJMR for the screening of colistin-resistant organisms, and on MacConkey agar plates supplemented with Ertapenem (2 µg/ml) for the screening of carbapenem-resistant organisms. Species intrinsically resistant to colistin were excluded from this study. Bacterial identification at the species level was performed using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) method (Microflex; Bruker Daltonics).

Antibiotic susceptibility testing

The standard disk diffusion method on Mueller-Hinton agar was performed to determine the antibiotic susceptibility testing of the strains as recommended by the European Committee of Antimicrobial Susceptibility Testing (EUCAST) 2017 (http://www.sfmmicrobiologie.org/UserFiles/files/casfm/CASFM%20V2_0_Mai2017.pdf ). The minimal inhibitory concentration (MIC) of colistin was determined using the broth microdilution method according to EUCAST 2017. Each strain with a MIC > 2 mg/L for colistin was considered as resistant to this antibiotic.

DNA extraction

The automatic robot EZ1 (Qiagen BioRobot EZ1-, Tokyo, Japan) was performed to extract the DNA of the bacteria, with the extraction kit (EZ1 DNA, Qiagen, Hilden, Germany), according to the manufacturer's guidelines and eluted in 200 μL of elution buffer and stored at -20°C.

Multilocus sequence typing (MLST)

To determine the genetic relationship between the clinical isolates, genotyping analysis was done using seven housekeeping genes for E. coli, K. pneumoniae and E. cloacae, as described on Institute Pasteur's MLST Web site (www.pasteur.fr/mlst).

Screening of isolates by real-time PCR, standard PCR, and sequencing of the antibiotic resistance genes

To detect the presence of carbapenemase encoding genes, a real-time PCR assay was done, using specific primers for blaOXA-48, blaOXA-58, blaKPC, blaNDM, and blaVIM genes. All colistin-resistant isolates were also screened for the presence of extended-spectrum β-lactamase (blaTEM, blaSHV, blaCTX-M,) genes and for the mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes.

Probes and primers used were described previously [START_REF] Rebelo | Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes[END_REF][START_REF] Christophy | Prevalence, antibiotic susceptibility and characterization of antibiotic resistant genes among carbapenem-resistant Gram-negative bacilli and yeast in intestinal flora of cancer patients in North Lebanon[END_REF]. Positive PCR amplification targeting antibiotic resistance genes were sequenced using BigDye terminator chemistry on an automated ABI 3130 sequencer (PE Applied Biosystems, Foster City, CA). ARG-ANNOT database was used to analyze the sequenced genes and was compared to other genes using the BlastN and BlastP analysis. The described colistin-resistant genes including pmrA, pmrB, phoP, phoQ, and mgrB were amplified and sequenced [START_REF] Olaitan | Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study[END_REF][START_REF] Olaitan | Clonal transmission of a colistin-resistant Escherichia coli from a domesticated pig to a human in Laos[END_REF][START_REF] Quesada | Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine[END_REF][START_REF] Jayol | National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014[END_REF]. Sequenced genes were compared with those of the reference strains including E. coli K-12 MG 1655 (NCBI GenBank accession no.CP000647), K. pneumoniae MGH 78578 (NCBI GenBank accession no.CP000647) and E. cloacae ATCC 13047 (NCBI GenBank accession no.CP000647) using the nps alignment software (https://prabi.ibcp.fr/htm/site/web/home). We used PROVEAN (Protein Variation Effect Analyser) software (http://provean.jcvi.org/seq_submit.php), to predict whether the identified amino acids substitutions that result from missense mutations would affect the function of the proteins. If the protein variant has a score below or equal to a predefined threshold (-2.5), it is predicted to have a "deleterious" effect. If it is above this threshold, it is predicted to have a "neutral" effect. Enterobacter cloacae, and 1 strain as Klebsiella pneumoniae). In addition, 4 isolates that were intrinsically resistant to colistin were also isolated (1Proteus mirabilis and 3 Morganella morganii) but were excluded from this study. The antibiotic resistance profile of all the isolates is presented in Table 1. Results showed that all isolates were resistant to amoxicillin, amoxicillin/clavulanic acid, cefalotin, doxycycline, and colistin. Colistin MICs ranged from 8 to 32 mg/L. Inversely, all isolates were susceptible to ertapenem, imipenem, amikacin, gentamicin, and nitrofurantoin. In addition, six strains were resistant to the third generation cephalosporins.

Results

rectal swabs

Real-time PCR and standard PCR results showed that E. coli (EC-5, EC-10, and EC-23), E. cloacae (Eclo-14A and Eclo-14B), and K. pneumoniae (KP-16), were positive for blaTEM gene.

Sequence analysis of detected penicillinase identifies blaTEM-1 gene in positive strains except for E. coli EC-23 that was identified as blaTEM-163. Moreover, 4 out of the 5 E. coli strains harbored blaCTX-M-15, blaCTX-M-103, and blaCTX-M-189 (Table 1). Single K. pneumoniae KP-16 harbored blaSHV-1 gene. None of the strains harbored mobile colistin resistance genes (from mcr-1 to mcr-5). MLST analysis reveals that five E. coli isolates belonged to five different sequence types (STs) including ST131, ST6174, ST405, ST162 and ST1451. K. pneumoniae has ST45, and the two E. cloacae have new sequence types ST924 and ST925 (Table 1). Due to the absence of mcr genes, the associated colistin resistance genes including mgrB, pmrA, pmrB, phoP, and phoQ were amplified and sequenced. As shown in Table 2, sequence analysis of these latter revealed that the two E. cloacae isolates exhibited non-synonymous mutations in mgrB, pmrA, pmrB, and phoP genes leading to amino acid changes. These mutations in mgrB were detected for the first time in lebanon and were considered as deleterious by the PROVEAN software. Moreover, sequence analysis of E. coli strains revealed that colistin resistance of E. coli EC-5 was due to different missense mutations in pmrAB operon resulting in amino acids substitutions. For E. coli EC-10, the missense mutations occurred in phoPQ proteins. E. coli EC-12 exhibited missense and deletion mutations in pmrB and phoPQ. E. coli EC-21 showed pmrA and phoP genes affected by missense mutations, and for E. coli EC-23 colistin resistance was due to missense and deletions mutations in pmrA and phoPQ. Analysis of K. pneumoniae KP-16 revealed that there were two genetic changes linked to colistin resistance: firstly, missense mutations leading to a premature stop codon of the mgrB and secondly, a missense in the pmrB gene that results in amino acid substitutions (Table 2). All these mutations were predicted as deleterious by the PROVEAN software.

Discussion

The increase of multidrug-resistant Gram-negative bacteria, especially carbapenemresistant bacteria, is a worldwide clinical problem. Indeed, this has led to the re-introduction of colistin into clinical treatment against infections caused by MDR bacteria [START_REF] Okdah | First report of colistin-resistant Klebsiella pneumoniae clinical isolates in Lebanon[END_REF]. This study describes the presence of intestinal carriage of colistin-resistant enterobacteria in patients who received colistin and carbapenem for treatment. We found that these patients carried only colistin-resistant bacteria but no carbapenem-resistant bacteria, suggesting that the use of these molecules may select colistin resistance but not carbapenem resistance. One limitation of our study is that no sample was collected before the instauration of the treatment. It is possible that these patients already carried these colistin-resistant bacteria, before the use of colistin. Different studies, in different countries such as France, Columbia, Spain, Laos, Thailand, USA, Nigeria, and Saudi Arabia have described the presence of colistin-resistant enterobacteria in humans without prior colistin exposure [START_REF] Olaitan | Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance[END_REF]. Moreover, a study done by Nakayama T et al. showed that even a short-term trip to some countries may result in the spread of strains that harbored mcr-1 and that are carried by international travelers where their numbers continue to increase, thereby increasing the risk of spreading of colistin-resistant to developed countries [START_REF] Nakayama | Carriage of colistinresistant, extended-spectrum β-lactamase-producing Escherichia coli harboring the mcr-1 resistance gene after short-term international travel to Vietnam[END_REF].

Genotyping results showed that our strains belonged to different sequence types (ST) suggesting, therefore, no link between strains isolated in the same hospital. It has been shown that the clone ST131 is a globally important pathogen among multidrug resistance E. coli and is linked to nosocomial and acquired infections [START_REF] Weissman | Epidemiology and Antimicrobial Resistance Characteristics of the Sequence Type 131-H30 Subclone Among Extraintestinal Escherichia coli Collected From US Children[END_REF]. Moreover, the clones of other E. coli ST405, ST162, ST1451, and K. pneumoniae ST45 have been reported in hospitalized patients in different counties, such as Brazil, Spain, Sweden and Uruguay [START_REF] Campos | Comprehensive Molecular Characterization of Escherichia coli Isolates from Urine Samples of Hospitalized Patients in Rio de Janeiro, Brazil[END_REF][START_REF] Coelho | Spread of Escherichia coli O25b:H4-B2-ST131 producing CTX-M-15 and SHV-12 with high virulence gene content in Barcelona (Spain)[END_REF][START_REF] Sundqvist | Withinpopulation distribution of trimethoprim resistance in Escherichia coli before and after a community-wide intervention on trimethoprim use[END_REF][START_REF] Garcia-Fulgueiras | Allodemic distribution of plasmids co-harbouring blaCTX-M-15/aac(6')-Ib-cr/qnrB in Klebsiella pneumoniae is the main source of extended-spectrum β-lactamases in Uruguay's paediatric hospital[END_REF]. In addition, E. coli ST405 has also been reported in animals in Algeria [START_REF] Bachiri | First Report of the Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli ST405 Isolated from Wildlife in Bejaia, Algeria[END_REF]. acid, and cephalosporins including cefalotin, cefepime, and ceftriaxone except the E. cloacae isolates, which are sensitive to ceftriaxone and cefepime. All isolates harbored β-lactamase genes such as TEM, SHV, and CTX-M. blaTEM-163, blaCTX-M-103, blaCTX-M-189, and blaSHV-1 detected in this present study have never been previously reported in Lebanon.

Interestingly, many different gene mutations including missenses and deletions have been identified in genes associated with colistin resistance in our clinical isolates. However, some of these mutations have been reported earlier such as R81S found in PmrA of a colistin-resistant strain isolated from animal samples under the Spanish Surveillance Network of Antimicrobial Resistance in Bacteria [START_REF] Quesada | Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine[END_REF][START_REF] Poirel | Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes[END_REF]. R81S is also similar to the missense mutation R81H which was found in PmrA of S. Typhimurium [START_REF] Roland | Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence[END_REF]. The D150W mutation in PhoQ of our E. coli EC-10 isolate was previously reported in Taiwan in a K. pneumoniae isolate collected from patient at the Taipei Veterans General Hospital [START_REF] Cheng | Colistin Resistance Mechanisms in Klebsiella pneumoniae Strains from Taiwan[END_REF]. Mutation T92H found in pmrB of E. coli EC-12 isolate, were similar to T92A described in PmrB of S. Typhimurium [START_REF] Olaitan | Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria[END_REF][START_REF] Roland | Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence[END_REF]. D191Y mutation in PhoP of E. coli EC-21 was reported in South Africa in clinical K. pneumoniae isolate [START_REF] Jayol | Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system[END_REF].

Olaitan et al. have also identified mutation L96P in K. pneumoniae isolated from patients and healthy individuals in Nigeria, Thailand, Lao PDR, and France [START_REF] Olaitan | Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study[END_REF] which is similar to L96S found in PhoQ of our study. Regarding colistin-resistant K. pneumoniae KP-16 isolate, different missense mutations have been found, such as G37M, C39P and N42Y that were similar to the mutations identified in the mgrB of K. pneumoniae described by Poirel et al. In Lebanon, there is no report describing mechanism of colistin resistance of Enterobacteriaceae clinical isolates except a study reported recently by one of our group that describe three colistin-resistant K. pneumoniae isolated from Lebanese Hospital in Beirut in 2015. The resistance of those strains was due to mutations of mgrB, pmrA, pmrB, and phoQ genes [START_REF] Okdah | First report of colistin-resistant Klebsiella pneumoniae clinical isolates in Lebanon[END_REF]. Those strains were isolated from patients without previous use of colistin. On the other hand, two studies were done by Dandachi et al., which described the mechanism of colistin resistance in animals. In these studies, colistin resistance was due to the presence of a plasmid-mediated mcr-1 gene isolated from E. coli strain from poultry in Lebanon [START_REF] Dandachi | Prevalence of extended-spectrum beta-lactamase-producing gram-negative bacilli and emergence of mcr-1 colistin resistance gene in Lebanese swine farms[END_REF] and from 23 E. coli strains isolated from Lebanese swine farms [START_REF] Dandachi | Prevalence of extended-spectrum beta-lactamase-producing gram-negative bacilli and emergence of mcr-1 colistin resistance gene in Lebanese swine farms[END_REF]. In fact, carriage of mcr-1 in farms could constitute a potential key for the introduction of this gene into the community as well as to clinical settings in Lebanon through horizontal transfer from animals to humans. Due to the spread of carbapenemase producers in hospitals in Lebanon, it is expected that once mcr-1 would be introduced, this latter will be selected by the frequent use of colistin.

Few studies have described the mechanism of colistin resistance in E. cloacae. It seems that the PhoP/PhoQ two-component system may play a role in this colistin resistance. The mgrB gene acts as a repressor of this two-component system and inactivation of this gene leads to colistin resistance in this species. However, a previous study on colistin-resistant E. cloacae has detected no alteration of the mgrB gene, leading the mechanism still unknown [START_REF] Jayol | National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014[END_REF].On the other hand, a recent study had detected a missense mutation in the mgrB gene (I10V), in addition to many missense mutations in the pmrA/B genes which may contribute to the colistin resistance [START_REF] Chavda | The co-identification of mcr-4.6 and bla NDM-1 in a clinical Enterobacter cloacae isolate from China 2 3 Downloaded from[END_REF]. In our study, we have detected for the first time in Lebanon mutations in the two components system pmrA/B, phoP/Q and mgrB genes in E cloacae. Moreover, different missense mutations that have been found, such as C39G, N42S, I45Y, W47V, W47S, *48K and *48Y were similar to the mutation identified in the mgrB of K. pneumoniae described by Poirel et al [START_REF] Poirel | Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes[END_REF].

In conclusion, this study reports the emergence of colistin resistance in Gram-negative bacteria in Lebanon. The use of polymyxins to treat patients has probably contributed to the emergence of colistin-resistant strains in this hospital since no relationship between our strains has been observed. An urgent strategy must be implemented to prevent the spread of these resistant microorganisms among hospitalized patients.

E. coli EC-10 R R S R R R S S S S R S S R S R 16 - TEM-1 CTX-M-15 E. coli EC-12 R R S R R R S S S S S S S R S R 16 - - CTX-M-189 6174
E. coli EC-21 R R R R R R S S S S R S S R S R 16

  were collected from 23 different patients treated with colistin and carbapenem combination therapy. No carbapenem-resistant Enterobacteriaceae have been isolated from MacConkey agar medium supplemented with Ertapenem. Of the 23 patients, 12 patients have no colistin-resistant Enterobacteriaceae (ColiRE) whereas 11 patients carried at least one ColiRE. The mean carriage of ColiRE was 1 /sample except one patient carries 2ColiREs/sample. In summary, twelve ColiRE were collected with the LBJMR medium. Among the isolates, 8 colistin-resistant strains were identified as: (5 Escherichia coli, 2 strains as

  ), amoxicillin-clavulanic acid (AMC), piperacillin-tazobactam (TZP), Cefalotin (CF), ceftriaxone (CRO), cefepime (FEP), 359 ertapenem (ERT), imipenem (IPM), amikacin (AK), gentamicin (GEN), ciprofloxacin (CIP), fosfomycin (FF), nitrofurantoin (F), doxycycline 360 (DO), trimethoprim-sulfamethoxazole (SXT), colistin (CS). R, resistant; S, sensitive, sequence type (ST) 361 21

Table 1 :

 1 Phenotypic and genotypic features of the eight colistin-resistant strains isolated at the Saint Georges hospital in Lebanon

	358
	Strain name	AMX	AMC TZP CF	CRO	FEP	ERT	IPM	AK GEN	CIP	FF F DO SXT CS	Colistin MIC (μg/ml)	blaSHV	blaTEM	blaCTX-M-A	ST
	E. coli EC-5	R	R	S	R	R	R	S	S	S	S	S	S	S	R	S	R	16	-	TEM-1	-	1451

Table 2 :

 2 Mutations of the associated colistin-resistance proteins

	362
	Strain names	MgrB	PmrA	PmrB	PhoP	PhoQ
			L11Q
	E. coli EC-5	Not tested		A65del	No mutation	No mutation
			R81S
						I88T, L95P, P111T, W113R,
	E. coli EC-10	Not tested	No mutation	No mutation	R163P, N165del	L114S, S116W, R145W,
						D150W, H157P, L218F
				V77A, L81P, T92H
				R93P, L95A, E97G
				L98A, Q99A, L102S	Q113P, K171R, H198del
	E. coli EC-12	Not tested	No mutation			L231R, N235I, E246del
				E103W
	E. coli EC-21	Not tested	G15E, R81S D82del, D86R, K87del	No mutation	K171R, R185P, S187I, D191Y, K200R, I210S, V213G, Y218H	L96S, Q99A, I109H, W113M, F119S
	E. coli EC-23	Not tested	R81S	No mutation	N119I, E154del	K46I, del56-70
	K. pneumoniae KP-16	S36G, G37D, I38A, C39A, I41T, N42Y	No mutation	D150N, S205I, G207del	No mutation	No mutation
		G37_V38insG
	E. cloacae Eclo-14A	V38S, C39G, A40K	S64C, L216W, E217I L38S	No mutation	No mutation
		I41M, N42S, K43G,
		I45Y, P46G

Acknowledgments

The authors thank CookieTrad for English language corrections.

Funding source:

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).

Competing interests:

No conflicts of interest or financial disclosure for all authors

Ethical approval:

Not required.

Authors' affiliations

TND and ED collect samples and isolate strains. TND, CAB, SC, LH, SB, ED, and SMD performed experiments and analyzed the data. TND, SMD, ED, and JMR draft and proofreading authors read and approved the final version of the manuscript.

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