Charles Burdet
email: charles.burdet@inserm.fr

Olivier Pajot

Camille Couffignal

Laurence Armand-Lefèvre

Arnaud Foucrier

Cédric Laouénan

Michel Wolff

Laurent Massias

F Mentré

Population pharmacokinetics of single-dose amikacin in critically-ill patients with suspected ventilator-associated pneumonia

Keywords: Amikacin, Population pharmacokinetics, Intensive-care unit, Ventilator-associated pneumonia

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.

Amikacin clearance was decreased and its volume of distribution was increased as previously reported. A≥25 mg/kg single-dose is needed for empirical treatment of GNB-VAP.

Introduction

Ventilator-associated pneumonia (VAP), defined as pneumonia occurring more than 48 h after the initiation of mechanical ventilation, is the most common hospital-acquired infection in the intensive care unit (ICU) [START_REF] Bouadma | Ventilator-associated pneumonia and its prevention[END_REF]. Many previously published studies have shown that early and appropriate antibiotic therapy is associated with better outcome in critically-ill patients with severe infections [START_REF] Zahar | Outcomes in severe sepsis and patients with septic shock: pathogen species and infection sites are not associated with mortality[END_REF].The latest guidelines of the American Thoracic Society and Infectious Diseases Society of America recommend that the empirical antibiotic therapy for late-onset pneumonia or in patients with risk factors for infection by multidrug-resistant pathogens should include a combination of an anti-pseudomonal-lactam and an anti-pseudomonalfluoroquinolone or an aminoglycoside such asamikacin [3].The superiority of once-daily dosing of aminoglycosides over multiple-daily dosing has been established [START_REF] Barza | Single or multiple daily doses of aminoglycosides: a meta-analysis[END_REF]. However, no data supports a clinical benefit of a single-dose of aminoglycosides over multiple administrations. The rationale for an initial combination therapy is to broaden the antimicrobial spectrum of the empirical therapy; however some data suggest adverse effects linked to repeated administrations, such as adaptative resistance or nephrotoxicity. Amikacin, like other aminoglycosides, exhibits a concentration-dependent killing and producesa prolonged post-antibiotic effect. Previousclinical studies have shown that aratioof 10 or more between the concentration achieved 1 hour after the beginning of a 30-minute infusion (C 1h ) and the minimal inhibitory concentration (MIC) of the bacteria responsible of the infection was predictiveof therapeutic success [START_REF] Moore | Clinical response to aminoglycoside therapy: importance of the ratio of peak concentration to minimal inhibitory concentration[END_REF][START_REF] Kashuba | Optimizing aminoglycoside therapy for nosocomial pneumonia caused by gram-negative bacteria[END_REF]. Some authors recently suggested that a ratio between the area under the curve of concentration over time (AUC) and MIC (AUC/MIC) greater than 90 also has some predictive value for therapeutic success, with no difference in predictive capacities when compared to C 1h / MIC ratio [START_REF] Zelenitsky | Treatment and outcome of Pseudomonas aeruginosa bacteraemia: an antibiotic pharmacodynamic analysis[END_REF].Thosepharmacodynamic targets may be difficult to reach in criticallyill patients for several reasons. First, large interindividual variations of amikacin pharmacokinetic parameters have been reported [START_REF] Delattre | Population pharmacokinetic modeling and optimal sampling strategy for Bayesian estimation of amikacin exposure in critically ill septic patients[END_REF][START_REF] Bacopoulou | A study of once-daily amikacin with low peak target concentrations in intensive care unit patients: pharmacokinetics and associated outcomes[END_REF][START_REF] Romano | Influence of clinical diagnosis in the population pharmacokinetics of amikacin in intensive care unit patients[END_REF], with variations of C 1h and AUC as a consequence. This variability is partially explained by total body weight and creatinine clearance, which are the most frequently reported covariates of amikacin pharmacokinetic parameters [START_REF] Delattre | Population pharmacokinetic modeling and optimal sampling strategy for Bayesian estimation of amikacin exposure in critically ill septic patients[END_REF][START_REF] Bacopoulou | A study of once-daily amikacin with low peak target concentrations in intensive care unit patients: pharmacokinetics and associated outcomes[END_REF][START_REF] Romano | Influence of clinical diagnosis in the population pharmacokinetics of amikacin in intensive care unit patients[END_REF][START_REF] Lugo | Relationship between hemodynamic and vital support measures and pharmacokinetic variability of amikacin in critically ill patients with sepsis[END_REF]. Second, the current breakpoint of MICs defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for Enterobacteriaceaespecies and Pseudomonas aeruginosa is 8 mg/L [12].

Few authors investigated the amikacin dosage needed in a single infusion setting to achieve the defined pharmacodynamic target in a homogenous groupof patients hospitalised in ICU with suspected VAP.

Using data from a prospective clinical trial, we aimed (i) to characterize amikacin pharmacokinetic parameters and their variability in ICU patients with suspected VAP, (ii) to study covariates of amikacin pharmacokinetic parameters, and (iii) toevaluate several amikacin dosing regimens using Monte Carlo simulations.

Material and methods

Patients and sampling

This study is part of the IMPACTtrial, a prospective multicenterclinical trial conducted between 2009 and 2011 inthreeICUs, at Bichat university hospital, Paris, France, and Victor Dupouy hospital, Argenteuil, France (ClinicalTrials #NCT00950222).

Patients were included in the trial if they presented the following criteria: mechanical ventilation for more than 48 hours, a clinical suspicion of Gram-negative bacilli VAP, risk factors for multidrug resistant bacteria, and if a microbiologic sample was obtained before initiation of antimicrobial therapy using blinded protected telescoping catheteror bronchoalveolar lavage. Clinical suspicion of VAP was defined by the onset of new lung infiltrates on chest radiography, fever greater than 38.3°C, purulent tracheal aspirates or a leukocytosis> 10000/mL. Risk factors for multidrug-resistant bacteria were antimicrobial therapy in the preceding 15 days or late-o set VAP ≥6 days [3]. Patients were not eligible if they were younger than 18-year-old, if they had renal failure requiring renal replacement therapy or received a treatment by imipenem or amikacin at the time of inclusion.

Written consent was obtained from patients or their legal representative. The study was approved by the Ethics Committee of the Hôtel-Dieu university hospital (APHP, Paris).

All patients were treated with a combination of imipenem and amikacin.Imipenem was administered every 8 hours over a 30-minute infusion and should not be changed for the first 48 hours. Doses ranged from 500 mg to 1000 mg, according to creatinine clearance (CrCL).Amikacintreatment consisted in onesingle 30-minute infusion of a suggested dose of 20 mg/kg administered the first day of antimicrobial therapy. This dose was recommended by guidelines at the time of this study was designed [3].

De-escalation therapy was encouraged after isolation of the Gram-negative pathogen and obtaining the results of susceptibility tests.

Usual clinical and demographic characteristics were recorded at ICU admission, as well as ventilator parameters, two scores for assessment of severity (SOFA [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF]and SAPS-II [START_REF] Gall | A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study[END_REF])and routine biologic markers.

Blood samples were collected 0.5, 1, 8, 16 and 24 hours after the onsetof infusion. Exact times of beginning and end of infusion were recorded, as well as exact sampling times. Amikacin concentrations were determined by fluorescence polarization immunoassay using the amikacinInnofluor® kit [START_REF] White | Evaluation of Innofluor fluorescence polarization immunoassay kits for the determination of serum concentrations of gentamicin, tobramycin, amikacin and vancomycin[END_REF]. The limit of quantification of the technique is 0.5 mg/L, and coefficients of variation for intra-and inter-assay were4.1% and 5.8%, respectively.

Population pharmacokinetic analysis

Basic model

Population pharmacokinetic analysis was performed using the Stochastic Approximation Expectation Minimization (SAEM) algorithm in Monolix v4.2 (Lixoft, Orsay, France, available at http://www.lixoft.com). This algorithm handles concentrations below the limit of quantification to improve parameters estimation [START_REF] Samson | Extension of the SAEM algorithm to left-censored data in non-linear mixed-effects model: application to HIV dynamics model[END_REF]. We used the total dose administered to each patient for model building.

Both one-and two-compartment(s) models with first order elimination were tested [START_REF] Delattre | Population pharmacokinetic modeling and optimal sampling strategy for Bayesian estimation of amikacin exposure in critically ill septic patients[END_REF][START_REF] Romano | Influence of clinical diagnosis in the population pharmacokinetics of amikacin in intensive care unit patients[END_REF][START_REF] Lugo | Relationship between hemodynamic and vital support measures and pharmacokinetic variability of amikacin in critically ill patients with sepsis[END_REF][START_REF] Taccone | Revisiting the loading dose of amikacin for patients with severe sepsis and septic shock[END_REF].

We used an exponential random effects model for each pharmacokinetic parameter. We assumed the random effects to have a normal dist i utio ith a ea of a d a a ia e of ω 2 . Correlation () between individual random effects was kept in the model if the estimated correlation coefficient as ≥ . . The residual error model was supposed to be additive, proportional or combined, with a being the standard deviation of the additive component and b the standard deviation of the proportional component.

The best model was chosen using the Bayesian information criteria (BIC), derived for each model from the computation of likelihood by importance sampling [START_REF] Lavielle | Estimation of population pharmacokinetic parameters of saquinavir in HIV patients with the MONOLIX software[END_REF].

Covariate model building

The influence of the following covariates at initiation of treatment on amikacin pharmacokinetic parameters was tested: 3 demographic variables (age, gender and total body weight), 2 clinical variables (shock and edema score [START_REF] Gomez | Altered pharmacokinetics of ceftazidime in critically ill patients[END_REF]), 2 severity scores (SOFA [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF]

Final model determination

We graphically studied the influence of covariates on their related pharmacokinetic parameters.

Outliers were studied and excluded from analysis when there was a suspicion of incomplete data collection, in particular for creatinine clearance which was estimated using a 4-hour urine collection.

Patients for whom covariates had been imputed were also excluded. The covariate model was adjusted to the reduced population obtained after exclusion of these patients, and a backward selection method was used in order to obtain a final model in which all covariates had a p-value < 0.05 using the likelihood-ratio test.

The coefficient estimated for creatinine clearance was compared to 1 using the likelihood ratio test.

Model evaluation

Evaluation of the final model was conducted using graphical methods. Basic goodness-of-fit plots were used, as well as the individual weighted residuals (IWRES) and the normalized prediction distribution errors (NPDE) over time and the visual predictive check (VPC).NPDE and VPC were generated using 500 Monte Carlo simulations.

Evaluation of doses by simulation

Using the estimated distribution of amikacin pharmacokinetic parameters in the final model with covariates, we simulated amikacin concentration obtained 1 hour after the start of a 30-minute infusion and AUC for 1000 patients by Monte Carlo simulation for several dosing regimen. This timing for amikacin sampling is commonly used for studying amikacin efficacy [START_REF] Taccone | Revisiting the loading dose of amikacin for patients with severe sepsis and septic shock[END_REF].For pharmacokinetic simulations, we randomly re-sampled 1000 vectors of covariatesamong those observed in the patients included in the analysis and simulated individual pharmacokinetic parameters from their estimated distribution in the final model with covariates. Simulated doseregimens were the following: 20, 25, 30, 35 and 40 mg/kg. We computed the probability to achieve a C 1h /MIC ratio ≥ a d a AUC/MIC atio ≥ 9 for MICs ranging from 0.25mg/L to 64mg/L for each or these dosing regimens. These values are usually observed in clinical practice (http://www.eucast.org/mic_distributions/), with an 8 mg/l susceptibility breakpoint for Enterobacteriaceaespecies and Pseudomonas aeruginosa.

Results

Patients' characteristics

Of the 61 patients included in the IMPACTtrial, one had a kinetic profile which was not compatible with a unique injection and was withdrawn from analysis. Thus, data from 60 patients were available for modelling. Patie ts' characteristics are presented in Table 1. Median (minmax) age and total body weight were respectively 61.5 years (28 -84) and 78 kg (45 -126), andmost patients were males (n=47, 78%). At inclusion, 26 patients hadseptic shock (43%). Median SAPS-II and SOFA scores were 42 (19 -90) and 7 (2 -17), respectively,andmedian 4-hour creatinine clearance was 82 mL/min (4 -412).

Pharmacokinetic data and modelling

A total of 291 values of amikacin concentrations were available, with a median of 5 per patient (3 -5). Median amikacin dose administered was 20 mg/kg (11 -28). Median serum concentration observed 1 hour after the beginning of infusion was 45 mg/L (22 -87). Individual observed concentrations are presented in Figure 1. Fourteen values (4.8%) of amikacin concentration were below the limit of quantification. Serum concentrations were best described by a 2-compartment model with a combined residual error model (Online Resource 2). Correlations between estimates in the basic model were ≥ 0.2 between all individual random effects, and thus kept in the analysis. In this model, amikacin clearance (CL) was estimated to 4.0L/h, central volume of distribution (V1) to 15.3L, peripheral volume of distribution (V2) to 22.1L and inter-compartmental clearance (Q) to 12.2 L/h (Table 2).

Their relative standard errors were satisfactory, all being <10%. The inter-individual variability of pharmacokinetic parameters ranged from 30% for Q, to 60% for CL. Estimates of inter-individual variability of pharmacokinetic parameters were also satisfactory (Table 2). The goodness of fit plots of this model without covariate did not show any model deficiency (data not shown).

Among the 12studiedcovariates, 8 were significantly associated with the individual Bayes estimates of pharmacokinetic parameters (Online Resource 1).The best model included 4 covariates (Online resource 1): creatinine clearance for CL, total body weight and 2 /FIO 2 ratio for V1, and creatinine clearance for V2. Two patients presented a low creatinine clearance but a high amikacin clearance.

Their individual fits were satisfactory, and we hypothesised that their urine collection was incomplete. They were excluded from the analysis. In another patient, amikacin clearance was estimated to 11 mL/min, but creatinine clearance was missing and had been imputed to the median value. The 5 patients in whom creatinine clearance was not available were excluded. The 4-covariate model was adjusted to the 53 remaining patients.

Creatinine clearance was no longer a significant covariate for V2 (p=0.4), and the final model included 3 covariates: creatinine clearance (p<0.001)for CL, total body weight(p<0.001) and PaO 2 /FIO 2 ratio (p<0.001) for V1 (Table 2).The coefficient for creatinine clearance on CL was significantly different from 1 (p<0.001).The variations of pharmacokinetic parameters according to covariates are presented in Figure 2. All three of them were positively correlated with the pharmacokinetic parameters. Amikacin clearance was estimated in the final model at4.3 L/h (72 mL/min), V1 at 15.9L and V2 at 21.4 L (Table 2).Variancecovariance matrix between the individual random effects is presented in Online Resource 3.

Goodness-of-fit plots did not show any model misspecification. The IWRES and NPDE were centered to zero and did not show any trend over time (Online Resource 4). The VPC did not show any model deficiency (Figure 3).

Pharmacokinetic simulations

Probabilities to achieve a C 1h /MIC atio ≥ a o di g to the single-dose of amikacinsimulated and MICs are presented in Figure 4. A20 mg/kg dosewas sufficient to achieve the pharmacokinetic target in 100%of patients for a MIC of 2 mg/L or less. With this dose only 80% and 4% of patients achieved the target for a MIC of 4 and 8 mg/L, respectively. With a25mg/kg simulated infusion, the probability to reach the target was 96% for a MIC of 4 mg/L and 20% for a MIC of 8 mg/L. With the 40 mg/kg regimen, the probability to achieve a C 1h /MIC atio ≥ was 80% for a MIC of 8 mg/L (Figure 5).Online Resource 5 presents the boxplots of the 1000 simulated C 1h /MIC ratio for a MIC of 8 mg/L, for doses from 20 to 40 mg/kg.

Results were similar when considering the target of AUC/MIC ≥ 90 (Online Resource 6). With a dose of 20 mg/kg, 90% of patients achieved the target for a MIC of 2 mg/L or less, but this proportion was below 20%for a MIC value of 8 mg/L. Simulations for a dose of 25 mg/kg showed that 69% of patients achieved the pharmacodynamic target for a MIC of 4 mg/L, but only 22% did for a MIC of 8 mg/L. An increase to 40 mg/kg led to an increase of the probability to reach the pharmacodynamic target to 90% and 52% for a MIC of 4 mg/L or 8 mg/L, respectively.

Discussion

The present study confirms that the 20 mg/kg single-dose regimen traditionally used is not adequatebased on the analysed pharmacokinetic and pharmacodynamic indices and thatamikacin dosing regimen should be increased to at least 25 mg/kg for critically-ill patients when initiating empirical amikacin therapy for VAP caused by a Gram-negative rod. This increase has been recently suggested by Taccone et al [START_REF] Taccone | Revisiting the loading dose of amikacin for patients with severe sepsis and septic shock[END_REF]. To our knowledge, few data supported this recommendation [21,[START_REF] Mahmoudi | Influence of sepsis on higher daily dose of amikacin pharmacokinetics in critically ill patients[END_REF]. Those studies did not use a population approach and were performed in patients with severe sepsis or septic shock, thereby gathering patients with different varieties of infection. Although the influence of the nature of infection has not been thoroughly examined, some authors suggested that the clinical diagnosis might influence the pharmacokinetic parameters of aminoglycosides [START_REF] Romano | Influence of clinical diagnosis in the population pharmacokinetics of amikacin in intensive care unit patients[END_REF]. We focused our analysis on an homogenous group of patients. Our simulation-based analysis, using data from a prospective multicenter trial and a solid methodology, confirms theresults of Tacconeet al [START_REF] Taccone | Revisiting the loading dose of amikacin for patients with severe sepsis and septic shock[END_REF], and suggest that a higher initial dose is necessary, at least in patients requiring mechanical ventilation.

A comparative study of different initial amikacin dosing regimen would confirm our conclusions. It would also allow to study the impact of higher doses on renal function. Nephrotoxicity is a wellknown adverse effect of aminoglycoside therapy. However, this effect has beenassociated with duration of therapy [23].Available studies suggest that short-course treatments would allow to minimize toxicity while keeping maximal efficacy [24]. In the majority of severe infections, aminoglycosides are administered as a single infusion in combined antimicrobial therapy. Higher single doses would therefore have potentially limited or no effect on renal function, as suggested by recent studies [21,[START_REF] Mahmoudi | Influence of sepsis on higher daily dose of amikacin pharmacokinetics in critically ill patients[END_REF]. It should also be noticed that neuromuscular blockade might have an increased frequency when using higher doses of aminoglycosides [25]. Its main manifestation is respiratory weakness, and this side effect is reversible. It would have a limited impact on patients with mechanical ventilation.

Another result is thatamikacin clearance and volume of distribution were estimated to approximately 70 mL/min and 37 L, respectively. These values were in accordance with published data on ICU patients. This confirms the issues observed in antimicrobial pharmacokinetic parameters in critically-ill patients [26,[START_REF] Pea | Antimicrobial therapy in critically ill patients: a review of pathophysiological conditions responsible for altered disposition and pharmacokinetic variability[END_REF], i.e.adecreaseof drug clearance and an increaseof volume of distribution [START_REF] Lode | Pharmacokinetic and clinical studies with amikacin, a new aminoglycoside antibiotic[END_REF][29][30][START_REF] Bressolle | Population pharmacokinetics of amikacin in critically ill patients[END_REF][START_REF] Marik | The pharmacokinetic of amikacin in critically ill adult and paediatric patients: comparison of once-versus twice-daily dosing regimens[END_REF]. Significant covariates were 4-hour creatinine clearance for amikacin clearance, and total body weight and PaO 2 /FIO 2 ratio for central volume.

The association between total body weight and volume of distribution has already been reported [START_REF] Bacopoulou | A study of once-daily amikacin with low peak target concentrations in intensive care unit patients: pharmacokinetics and associated outcomes[END_REF][START_REF] Romano | Influence of clinical diagnosis in the population pharmacokinetics of amikacin in intensive care unit patients[END_REF]33], and administered amikacin doses are commonly adjusted to total body weight. It is still not clear which measure of body size best describes pharmacokinetic parameters.A recent meta-analysis explored the relationship between drug clearance and body size metrics [34]. The authors found that lean body weight to the exponent 2/3 was more suitable for describing the relationship between drug clearance and body size. However, they did not question the link between the volume of distribution and body size, and this conclusion may not apply for all pharmacokinetic parameters. As body size was not collected in the IMPACT trial, we could not investigate the effect of this weight metrics or of body mass index on amikacin pharmacokinetic parameters. For hydrophilic drugs such as aminoglycosides, data on the influence of lean body weight and drugs volume of distribution are lacking and would be of interest.

The BIC was not improved by adding total body weight as a covariate for V2. This is quite unexpected, and V2 might represent a weight-independent compartment in which amikacin accumulates. It has previously been shown that total urine recovery of amikacin isnot complete 24 hours after its administration, and that aminoglycosides including amikacin accumulates in the kidney [START_REF] Edwards | Concentrations of gentamicin and amikacin in human kidneys[END_REF][START_REF] French | Amikacin and gentamicin accumulation pharmacokinetics and nephrotoxicity in critically ill patients[END_REF].

We found a high interindividual variability in amikacin pharmacokinetic parameters as previously reported in studies performed among critically-ill patients [START_REF] Delattre | Population pharmacokinetic modeling and optimal sampling strategy for Bayesian estimation of amikacin exposure in critically ill septic patients[END_REF][START_REF] Joubert | A population approach to the forecasting of amikacin plasma and urinary levels using a prescribed dosage regimen[END_REF]. Significant covariates reducedthis variability, with a 50% maximal decrease for amikacinCL in our study. To our knowledge, no published covariate model could fully explain the observed interindividual variability. It is therefore highly probable that inside diagnosis-homogenous groups of patients, subpopulations exist that are not individualized by usual covariates. Such variability makes the choice of the optimal dosing strategy extremely challenging from an individual perspective. Considering the absence of severe adverse effect expected in a single-dose setting,mechanically-ventilated patients in intensive care units should receive a high amikacine dose in order to maximize the probability of pharmacodynamic target attainment. Further studies should furthermore focus on identification of covariates allowing for reducing the high unexplained interindividual variability.

The main limitation of this study is that it was restricted to a limited number of patientswho had a suspicion on VAP. Therefore the results may not apply to patients who do not require mechanical ventilation. Nevertheless, the French surveillance network of nosocomial infections recently reported that about 60% of patients hospitalized in ICU would require mechanical ventilation during their stay [38]. Another limitation is the use of the MIC breakpoint for Monte Carlo simulations. To our knowledge, no published data report the distribution of MICs for Gram-negative bacilli responsible for infection in intensive care units. As bacteria involved in infection of critically-ill patients frequently have higher MICs than those isolated from patients hospitalised in other wards, we did not use published MICs distributions that gather strains obtained from all wards. A recent study of MIC breakpoints based on simulations suggested that pharmacokineticpharmacodynamic breakpoints are similar to those defined by the EUCAST or the CLSI [START_REF] Frei | Antimicrobial breakpoints for gram-negative aerobic bacteria based on pharmacokinetic-pharmacodynamic models with Monte Carlo simulation[END_REF]. In the empirical setting, the worst-case assumption should be preferred when initiating an antimicrobial therapy.This approach is currently used for pharmacokinetic-pharmacodynamic simulations [START_REF] Taccone | Revisiting the loading dose of amikacin for patients with severe sepsis and septic shock[END_REF][START_REF] Carlier | Population pharmacokinetics and dosing simulations of amoxicillin/clavulanic acid in critically ill patients[END_REF][START_REF] De Montmollin | Predictors of insufficient amikacin peak concentration in critically ill patients receiving a 25 mg/kg total body weight regimen[END_REF]. However, it is likely that the majority of Gram-negative rods involved in critical infections have a MIC below 8 mg/L.For a MIC of 4 mg/L, 96% of patients achieved a C 1h /MIC ratio ≥ 10 after a dose of 25 mg/kg in our simulations.

In conclusion, amikacin pharmacokinetic parameters values were similar to those previously reported. This study confirms recent results for the determination of the initial amikacin dose required in critically-ill patients. An empirical dose of 25mg/kg or more is needed to achieve the amikacinpharmacodynamic predictors of clinical efficacyin Gram-negative bacilli infections. Other studies are needed to prospectively evaluate these conclusions in order to improve the management of patients with severe infections.

  andSAPS-II[14]), 2 ventilatorrelated parameters (PEEP and PaO 2 /FIO 2 ratio) and 3 biochemical markers (serum albumin, total bilirubin and 4-hour creatinine clearance). Four-hour creatinine clearance was calculated using serum creatinine level and a 4-hour urine collection at the day of inclusion. This approach has been validated for monitoring renal function in critically-ill patients[20]. Missing values for tested covariates were imputedto the median value observed in the analysis population.Steps for selection of covariate model are described in Online Resource 1.

Title

Population pharmacokinetics of single-doseamikacin in critically-ill patients with suspected ventilator-associated pneumonia

Authors' contributions


Charles Burdet

MD, MPH Olivier Clermont

PhD Stéphane Bonacorsi

Cédric Laouénan

MD, MPH Edouard Bingen

MD Yannick Aujard

MD France Mentré

Agnès Lefort

MD, PhD Erick Denamur
email: erick.denamur@inserm.fr

MD, PhD; Cédric Laouénan

PhD ; Agnès Lefort

MD, PhD for the COLIBAFI Group

Michel Wolff

Loubna Alavoine

Xavier Duval

David Skurnik

Paul-Louis Woerther

Antoine Andremont

Etienne Carbonnelle

Olivier Lortholary

Xavier Nassif

Sophie Abgrall

Françoise Jaureguy

Bertrand Picard

Chu Avicenne

Véronique Houdouin

Chloé Lemaitre

Romain Basmaci

Chu Robert-Debré

Agnès Meybeck

Guilène Barnaud

Catherine Branger

Chu Louis-Mourier

Bruno Fantin

Claire Bellier

Frédéric Bert

Marie-Hélène Nicolas-Chanoine

Chu Beaujon

Bernard Page

Julie Cremniter

Jean-Louis Gaillard

Chu Ambroise-Paré

Bernard Garo

Séverine Ansart

Geneviève Herry-Arnaud

Didier Tandé

Brest Chu Brest

Jean-Claude Renet

Ze René

Renaud Bekolo

Roland Verdon

Leclercq

Claire De Gialluly

Jean-Marc Besnier

Laurent Mereghetti

Roland Quentin

Achille Kouatchet

Alain Mercat

Marie Laure Joly-Guillou

Catherine Dalebroux

Pascal Chavanet

Catherine Neuwirth

Camille Colliard

Martin Dary

Gilles Potel

Jocelyne Caillon

Françoise Leturdu

Jean-Pierre Sollet

Gaëtan Plantefève

C H Argenteuil

Argenteuil Agnès De Patureaux

Pierre Tattevin

Christine Amorin

Jérémy Glodt

Ludovic Lassel

Escherichia coli bacteraemia in children: age and portal of entry are the main predictors of severity

Keywords: Bacteraemia, Escherichia coli, Children, Portal of entry, Prognosis, Severity, Death, Intensive care units, Virulence

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

Severe sepsis in children remains a common cause of hospitalization [START_REF] Watson | The epidemiology of severe sepsis in children in the United States[END_REF]. Bacteraemia account for 25% of hospitalization for severe bacterial infections, and mortality rate is as high as 17% [START_REF] Watson | The epidemiology of severe sepsis in children in the United States[END_REF]. Few temporal changes in the distribution of bacterial species in bloodstream infections have been reported [START_REF] Laupland | Incidence, risk factors and outcomes of Escherichia coli bloodstream infections in a large Canadian region[END_REF]. In industrialized countries, Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli figure among the 5 more frequently isolated organisms [START_REF] Doit | Epidemiology of pediatric communityacquired bloodstream infections in a children hospital in Paris, France, 2001 to 2008[END_REF].

E. coli is a commensal bacteria of the gut [START_REF] Tenaillon | The population genetics of commensal Escherichia coli[END_REF]. Some strains can cause however intestinal or extra-intestinal infections due to specific virulence factors [START_REF] Kaper | Pathogenic Escherichia coli[END_REF]. The structure of E. coli population is mainly clonal [START_REF] Touchon | Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths[END_REF], with seven principal phylogenetic groups (A, B1, B2, C, D, E and F) and numerous clonal complexes [START_REF] Tenaillon | The population genetics of commensal Escherichia coli[END_REF][START_REF] Jaureguy | Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains[END_REF][START_REF] Gall | Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains[END_REF][START_REF] Wirth | Sex and virulence in Escherichia coli: an evolutionary perspective[END_REF][START_REF] Moissenet | Meningitis caused by Escherichia coli producing TEM-52 extended-spectrum beta-lactamase within an extensive outbreak in a neonatal ward: epidemiological investigation and characterization of the strain[END_REF]. Most clinical extra-intestinal pathogenic E. coli belong to the B2 phylogroup, and to a lesser extent to the D phylogenetic group [START_REF] Johnson | Extraintestinal pathogenic Escherichia coli: "the other bad E. coli[END_REF]. Some clones or clonal complexes have been linked to specific syndromes [START_REF] Nataro | Diarrheagenic Escherichia coli[END_REF][START_REF] Bidet | Combined multilocus sequence typing and O serogrouping distinguishes Escherichia coli subtypes associated with infant urosepsis and/or meningitis[END_REF].

In adults, mortality of E. coli bacteraemia has been reported to be as high as 13% in a recent study performed by our group [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF]. We showed that host factors and the portal of entry are the main drivers of mortality during E. coli bacteraemia. The only bacterial characteristic influencing the prognosis was the presence of virulence factor ireA, which was negatively associated with death.

Phylogenetic belonging of the strains was not a significant predictor.

Studies of E. coli bloodstream infections in children, especially in urinary-source bacteraemia, linked some virulence factors to the development of bacteraemia [START_REF] Bonacorsi | Comparative prevalence of virulence factors in Escherichia coli causing urinary tract infection in male infants with and without bacteremia[END_REF][START_REF] Cheng | Comparison of extended virulence genotypes for bacteria isolated from pediatric patients with urosepsis, acute pyelonephritis, and acute lobar nephronia[END_REF][START_REF] Mahjoub-Messai | Escherichia coli isolates causing bacteremia via gut translocation and urinary tract infection in young infants exhibit different virulence genotypes[END_REF]. They include adhesins, iron uptake systems, protectins and toxins, with differences according to the portal of entry. A higher content in virulence factors was thus reported in strains causing urosepsis when compared to strains causing urinary-tract infections without bacteraemia [START_REF] Cheng | Comparison of extended virulence genotypes for bacteria isolated from pediatric patients with urosepsis, acute pyelonephritis, and acute lobar nephronia[END_REF]. However, there is a lack of prospective studies taking into account both host and bacterial characteristics versus the clinical outcome of E. coli bacteraemia in children.

To better understand the pathophysiology and severity of E. coli bacteraemia in children, we a al sed the oho t of hild e -year-old of the COLIBAFI study [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF]. We compared bacterial characteristics (i) in bacteraemia from urinary vs. digesti e o igi , ii i hild e vs. >3-month-old, and (iii) in community-acquired urinary-source bacteraemia in children vs. adults. Then we searched for clinical and bacterial risk factors associated with the severity of E. coli bacteraemia in children.

MATERIALS AND METHODS

Study Design and Setting

This study is part of the COLIBAFI study, a large prospective observational study conducted in 15 French hospitals between January 2005 and November 2007 in which 1051 adults and 84 children with E. coli bacteraemia were enrolled. The methodology has been previously published [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF]. E. coli bacteraemia was defined as the presence of E. coli i asepti all filled lood ultu e ial s . The follow-up ended at hospital discharge, or at day 28 after the first positive blood culture. The study was approved by the institutional Ethics Committee (Hôpital Saint-Louis, Paris, France).

Clinical and Bacterial Characteristics

Clinical characteristics included age, gender, birth weight, preterm birth, immunodepression and origin of infection. Portal of entry, immunodepression and nosocomial bacteraemia were defined as in [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF]. Severity of bacteraemia was defined as the occurrence of death or transfer to ICU during follow-up.

Phylogenetic group of strains was determined by a quadruplex PCR method derived from the Clermont method (18), associated to allele specific PCRs allowing the delineation of 7 main phylogroups (i.e., A, B1, B2, C, D, E and F) [START_REF] Clermont | The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups[END_REF]. Strains exhibiting the A 1 genotype (chuA-, yjaA+, TspE4.C2-) (18) correspond to ST complex (STc) 10 according to the Achtman multilocus sequence typing (MLST) scheme [START_REF] Wirth | Sex and virulence in Escherichia coli: an evolutionary perspective[END_REF]. Among the D phylogroup strains, clonal group A (CGA) was identified as previously described [START_REF] Johnson | Rapid and specific detection of Escherichia coli clonal group A by gene-specific PCR[END_REF]. Ten main B2 phylogenetic subgroups (I to X) were detected by allele specific PCRs [START_REF] Gall | Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains[END_REF][START_REF] Clermont | Development of an allele-specific PCR for Escherichia coli B2 sub-typing, a rapid and easy to perform substitute of multilocus sequence typing[END_REF]. They correspond to STc 131, 73, 127, 141, 144, 12, 14, 452, 95 and 372, respectively [START_REF] Wirth | Sex and virulence in Escherichia coli: an evolutionary perspective[END_REF]. O-typing was performed by PCR [START_REF] Clermont | Determination of Escherichia coli O types by allele-specific polymerase chain reaction: application to the O types involved in human septicemia[END_REF][START_REF] Clermont | Animal and human pathogenic Escherichia coli strains share common genetic backgrounds[END_REF].

The presence of 20 extraintestinal virulence factors was tested by PCR [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF][START_REF] Johnson | Molecular epidemiology and phylogenetic distribution of the Escherichia coli pks genomic island[END_REF]

Statistical Methods

We compared bacterial characteristics i) in bacteraemia from urinary vs. digestive origin, ii) in children 3 vs. >3-month-old, iii) in children vs. adults presenting a community-acquired urinary-source bacteraemia. The cut-off of 3 months was chosen as the first 3 months of life correspond to a period of age with a known increased risk of E. coli bacteraemia and meningitis related to the impaired innate immunity in this period [START_REF] Houdouin | Association between mortality of Escherichia coli meningitis in young infants and non-virulent clonal groups of strains[END_REF][START_REF] Bachur | Bacteremia and meningitis among infants with urinary tract infections[END_REF]. For the third comparison, all adults from the COLIBAFI cohort presenting a community-acquired urinary-source bacteraemia were included.

Characteristics tested included phylogenetic group (from the results of the triplex PCR [START_REF] Clermont | Rapid and simple determination of the Escherichia coli phylogenetic group[END_REF], grouped as B2 vs. non-B2), virulence factors and PAIs, virulence and PAI scores, antimicrobial resistance, multidrug resistance, and resistance score. Comparisons between groups were performed using nonparametric tests (Wilcoxon or Fisher exact tests).

We searched for risk factors associated with severity (yes/no). Analyses were performed in all hild e a d i the -month-old subgroup. Clinical characteristics studied included gender, age ( 3 vs. >3-month-old), immunodepression, nosocomial infection and portal of entry. Prematurity and birth weight were also studied in 3-month-old infants. The same bacterial characteristics than those described above were analyzed. Variables achieving a p-value <0.20 in univariate logistic regression analysis were entered into a multivariate logistic regression analysis to identify risk factors of severity. Using a forward selection method, we obtained a final model in which all risk factors had a p-value <0.05. First order interaction was tested for significant variables. The model discrimination was assessed by the c-statistic and its 95% confidence interval (95%CI), and the model calibration was assessed by the Hosmer-Lemeshow goodness-of-fit test.

Analyses were performed with SAS v9.3 (SAS Institute Inc., Cary, NC). All tests were two-sided with a type-I error fixed to 0.05.

RESULTS

Clinical Characteristics

All 84 patients 18-year-old from the COLIBAFI cohort were included (Table 1). The most frequent portals of entry were the urinary tract (n=51, 66.2%) and the digestive tract (n=15, 19.5%).

Eight children (10.4%) were immunocompromized: 4 had a history of hemopathy, 2 had had solidorgan transplantation, 1 had neutropenia and 1 had a congenital immunodeficiency. Fourteen children (16.7%) were transferred to ICU, 8 (9.5%) died during follow-up. In total, 17 (20.2%) presented a severe bacteraemia (95%CI, 12.2% -30.4%).

Bacterial Characteristics

Three children had a polymicrobial bacteraemia.

Comparison of urinary vs. digestive origin

The aerobactin gene iucC, the papGII allele and the PAI I CFT073 were more frequent in strains responsible of bacteraemia from urinary origin than from digestive origin (p=0.003, p=0.03 and p=0.05, respectively). There was no difference in phylogroup repartition or antimicrobial susceptibility according to portal of entry (Table 2).

Comparison of ≤3 vs. >3-Month-Old Children

Child e -month-old (n=43, 51%) were more frequently males than patients >3-month-old

(

Children vs. Adults Community-Acquired Urinary-Source Bacteraemia

Community-acquired urinary-source bacteraemia concerned 45 children and 513 adults from the COLIBAFI cohort (Table 4). We observed more females (73.1% vs. 44.4%, p<0.001) and a higher rate of immunodepression in adults than in children (23.9% vs. 4.9 %, p=0.003). Severe bacteraemia was more frequent in adults (11.9% vs. 0%, p=0.01). There was no significant difference in phylogenetic groups (B2 vs. non-B2) nor in virulence scores, but 5 virulence factors were significantly more frequent in E. coli strains isolated from children (i.e., iucC, iha, papC, papGII and sat), whereas hra was significantly less frequent. Adult isolates had a higher resistance score (median [min-max] = 1

[0-5] vs. 1 [0-4], p=0.04).

Risk Factors of Severity

Results of the univariate and multivariate analyses to identify risk factors of severity in all the included children are reported

DISCUSSION

Few data are available about E. coli bacteraemia in children despite its frequency and severity. To our knowledge, this is the first report combining bacterial and host characteristics in E.

coli bacteraemia in children.

We found a high proportion of strains belonging to the B2 and D phylogroups. Main clonal groups delineated from the B2 strains were the II and IX clonal groups, representing 23.8% each of all the strains. They correspond respectively to the STc73 and STc95 [START_REF] Gall | Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains[END_REF][START_REF] Wirth | Sex and virulence in Escherichia coli: an evolutionary perspective[END_REF]. Strains belonging to STc131

(clonal group I) accounted for 3.6% in our study. We found a high proportion of CGA (STc69) among the D phylogroup strains, which accounted for 8.3% of all strains. All these CGA strains were resistant to cotrimoxazole. These results are similar to those obtained in adults. In a study of 300 uropathogenic E. coli, most common STc retrieved were STc73 (16.6%), STc131 (13.3%), STc69 (9%)

and STc95 (6.3%) [START_REF] Gibreel | Population structure, virulence potential and antibiotic susceptibility of uropathogenic Escherichia coli from Northwest England[END_REF]. We also observed a predominance of 4 specific O-types, i.e. O1, O2a/b, O6 and O7. These data indicate that some specific clones among the huge clonal diversity of the E. coli species (4) are involved in extraintestinal infections. At the opposite, a high diversity in the pattern of presence or absence of virulence genes was evidenced, with 61 observed combinations (Table S1).

Altogether, these data support the fact that extraintestinal virulence in E. coli is due to multiple combinations of virulence genes (6) arriving on specific genetic backgrounds [START_REF] Escobar-Paramo | A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli[END_REF]. However, some specific virulence factors have been associated to specific syndromes. In their study of 123 children, Cheng et al. showed that E. coli strains causing urinary-source bacteraemia harboured more frequently papGI, iutA, traT, focG, afa, bmaE, kpsMT III, rfc and cvaL than strains causing acute pyelonephritis or acute lobar nephronia [START_REF] Cheng | Comparison of extended virulence genotypes for bacteria isolated from pediatric patients with urosepsis, acute pyelonephritis, and acute lobar nephronia[END_REF]. Bonacorsi and colleagues found that, in infants younger than 90 days, the presence of hly and/or iroN, as was that of hly and/or antigen K1, was associated in strains causing urinary tract infections with bacteraemia [START_REF] Bonacorsi | Comparative prevalence of virulence factors in Escherichia coli causing urinary tract infection in male infants with and without bacteremia[END_REF]. This group also found that, in young infants, papGII, sfa/foc and hly were more frequent in strains isolated from urosepsis than from meningitis [START_REF] Nataro | Diarrheagenic Escherichia coli[END_REF][START_REF] Bidet | Combined multilocus sequence typing and O serogrouping distinguishes Escherichia coli subtypes associated with infant urosepsis and/or meningitis[END_REF]. Similarly, in a stud of i fa ts -month-old with bacteraemia, these authors concluded that papGII and tcpC were more frequent, but ibeA less frequent, in E. coli originating from urinary tract infection than from gut translocation [START_REF] Mahjoub-Messai | Escherichia coli isolates causing bacteremia via gut translocation and urinary tract infection in young infants exhibit different virulence genotypes[END_REF]. In the same line, we found that iucC, which belongs to the aerobactin operon as iutA, and papGII were more frequent in urinary-source bacteraemia (Table 2).

We took the opportunity of the large COLIBAFI cohort to compare community-acquired urinary-source bacteraemia between children and adults. We found that bacteraemia in children were less severe, but involved strains exhibiting a specific repertoire of virulence genes and being more resistant than those isolated in adults. Children are usually considered to be more susceptible to infections due to an immature immune system. This apparently contra intuitive result might be explained by comorbidities in the adult group (23.9% of adults were immunocompromized [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF], and only 4.9% of children). To our knowledge, no study compared the prevalence of resistant bacteria in children and in adults. Data of the European Antimicrobial Resistance Surveillance Network for the period 2005 -2007 show that about 55% of E. coli strains were resistant to aminopenicillins [START_REF]European Antimicrobial Resistance Surveillance Network -Annual Reports[END_REF]. The higher prevalence of resistance observed in our cohort of children (63.1%) might be explained by the difference in antibiotic consumption among children. In France in 2004, the number of antimicrobial prescriptions for respiratory infections was almost 3 times higher in infants <2.5-year-old than in adults [START_REF] Chahwakilian | Impact of the French campaign to reduce inappropriate ambulatory antibiotic use on the prescription and consultation rates for respiratory tract infections[END_REF]. This suggests that children have a higher selective pressure than adults, which could result in higher rates of infection with resistant bacteria.

In our study, we found a case-fatality rate of 9.5%, similar to that observed in a cohort of 2300 E. coli bacteraemia in Canada [START_REF] Laupland | Incidence, risk factors and outcomes of Escherichia coli bloodstream infections in a large Canadian region[END_REF]. No bacterial characteristic was associated with severity defined as death or transfer to ICU during follow-up. The severity outcome was driven by portal of entry and age. This observation was also made in adults, in whom we showed that host factors and the portal of entry outweighed bacterial determinants for predicting death from E. coli bacteraemia [START_REF] Lefort | Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia[END_REF]. Houdoui et al. e a i ed patie ts' ha a te isti s a d a te ial i ule e e ha is s i lethal and non-lethal E. coli meningitis [START_REF] Houdouin | Association between mortality of Escherichia coli meningitis in young infants and non-virulent clonal groups of strains[END_REF]. In thei et ospe ti e stud of hild e -month-old, no pre-onset clinical factor was significantly associated with a fatal outcome. The only bacterial characteristic significantly associated with lethality was the absence of the aerobactin gene iucC.

However, no multivariate analysis was performed.

Our study has several limitations. First, our prospective cohort included a rather small number of children. Bacteraemia was severe only in 17 children, thus diminishing the power to determine risk factors of severity. However, we obtained a model with good predictive capacity (cstatistic of 0.93). Second, our study was performed in 2005 -2007. Only 3 strains resistant to third generation cephalosporins were isolated. In the recent years, the prevalence of resistant bacteria has increased. A study performed in Sweden in 2010 showed that 2.9% of healthy children were colonized with extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. This figure rose to 8.4% in ill children [START_REF] Kaarme | Prevalence of extended-spectrum beta-lactamaseproducing Enterobacteriaceae in healthy Swedish preschool children[END_REF]. This could weaken our results, as bacteraemia due to ESBL-producing E. coli have been associated with an increased risk of mortality in adults [START_REF] Rodriguez-Bano | Community-onset bacteremia due to extended-spectrum beta-lactamase-producing Escherichia coli: risk factors and prognosis[END_REF]. Despite these limitations, our multicenter study with a prospective design is the first one focusing on E. coli bacteraemia in children. Further studies should be performed on larger cohorts to confirm our findings.

  72.1% vs. 41.5%, p<0.01) and less frequently immunocompromized (0.0% vs. 21.1%, p=0.002). There was no significant difference found in portals of entry. Severe bacteraemia were more frequently

	o se ed i hild e	-month-old (32.6% vs. . %, p= .	. Thi tee hild e	-month-old and 1
	children >3-month-old were transferred to ICU, hile	hild e	-month-old and 2 children >3-

month-old died during follow-up. Bacterial characteristics are reported in Table 3. Virulence score as highe i -month-old children (median [min-max] = 15 (5-18] vs. 10 [1-19], p=0.02), with 3 virulence factors being more frequently observed: neuC (K1 antigen, 55.8% vs. 26.8%, p=0.009), irp2 (100% vs. 87.8%, p=0.02) and fyuA (100% vs. 87.8%, p=0.02). Antimicrobial resistance score was lo e i -month-old infants (median [min-max] = 1 [0-4] vs. 1 [0-5], p=0.01), with lower rates of resistance to amoxicillin (51.2% vs. 75.6%, p=0.02) and cotrimoxazole (23.3% vs. 46.3%, p=0.04).

Table 5

 5

			. Because of some missing characteristics, the multivariate
	analysis was performed in 77 children among which 16 (20.8%) had a severe bacteraemia. Non-
	urinary-source bacteraemia (OR = 0.01, 95%CI = 0.001-. a d ei g -month-old (OR = 7.7, 95%CI
	= 1.4-42.8) were the only risk factors of severity. The c-statistic of the final model was 0.93 (95%CI =
	0.87-0.99). The p-value of the Hosmer-Lemeshow test was 0.93, showing no model misspecification.
	Among the	hild e	-month-old, the bacteraemia was severe in 14 (32.6%). Birth weight

was lower (median [min-max] = 1560g [595-3580] vs. 3285g [910-5090], p<0.01) and a preterm birth was more frequent (71.4% vs. . %, p< . i -month-old children with severe bacteraemia.

Child e -month-old with a severe bacteraemia had a more frequently non-urinary (92.9% vs. 17.2%, p<0.001) but digestive (50.0% vs. 10.3%, p<0.01) portal of entry than those without. Because of some missing characteristics, the multivariate analysis was performed in 41 children among which 13 (31.7%) had a severe bacteraemia. A non-urinary source of bacteraemia was the only risk factor associated with severity (OR = 72.0, CI95% = 7.2-796.9). Prematurity and birth weight were not found significantly associated with severity, nor any of the bacterial characteristics tested. The c-statistic for

TABLE 1 .

 1 Clinical and bacteriological characteristics of the 84 children with E. coli bacteraemia.

	Characteristic	n [%] 1
	Age, median (min-max), months	2.4 (0-204)
	Males	48 [57.1]
	Nosocomial infection 2	25 [30.1]
	Immunocompromized 2	8 [10.4]
	Portal of entry 2
	Urinary tract	51 [66.2]
	Digestive tract	15 [19.5]
	Respiratory tract	2 [2.6]
	Cutaneous	3 [3.9)
	Venous catheter	5 [6.5]
	Surgical site	1 [1.3]
	Not determined	7 [9.1]
	Two portals of entry	7 [9.1]
	Polymicrobial infection	3 [3.6]
	Phylogenetic group
	A	7 [8.3]
	B1	3 [3.6]
	B2	53 [63.1]
	C	3 [3.6]
	D	10 [11.9]
	F	8 [9.5]
	Antibiotic resistance
	Amoxicillin	53 [63.1]
	Amoxicillin + clavulanate	38 [45.2]
	Gentamicin	3 [3.6]
	Ofloxacin	3 [3.6]
	Cotrimoxazole	29 [34.5]
	3rd generation cephalosporins	3 [3.6]
	Multidrug resistance	3 [3.6]
	Resistance score, median (min-max)	1 (0-5)
	Virulence score, median (min-max)	12 (1-19)
	Notes:
	1 Except when otherwise notified.

2

Because of missing data, percentages are calculated based on available data.

TABLE 2 .

 2 Characteristics of the E. coli isolates according to the portal of entry (urinary-vs. digestivesource bacteraemia).

	Characteristic	Urinary tract (n=47)	n [%] 1 Digestive tract (n=11)	p
	Phylogenetic group
	B2 non-B2	31 [66] 16 [34]	5 [45.5] 6 [54.5]	0.3
	Extraintestinal virulence gene
	neuC (K1)	16 [34]	7 [63.6]	0.09
	sfa/soc	12 [25.5]	2 [18.2]	>.99
	iroN	25 [53.2]	5 [45.5]	0.7
	iucC	42 [89.4]	5 [45.5]	0.003
	iha	25 [53.2]	4 [36.4]	0.5
	papC	39 [83]	7 [63.6]	0.2
	papGII	38 [80.9]	5 [45.5]	0.03
	papGIII	2 [4.3]	2 [18.2]	0.2
	hlyC	17 [36.2]	2 [18.2]	0.3
	cnf1	7 [14.9]	2 [18.2]	>.99
	hra	26 [55.3]	6 [54.6]	>.99
	sat	31 [66]	4 [36.4]	0.09
	ire	22 [46.8]	4 [36.4]	0.7
	usp	31 [66]	7 [63.6]	>.99
	ompT	40 [85.1]	10 [90.9]	>.99
	ibeA	2 [4.3]	2 [18.2]	0.2
	fyuA	45 [95.7]	10 [90.9]	0.5
	irp2	45 [95.7]	10 [90.9]	0.5
	traT	38 [80.9]	9 [81.8]	>.99
	clbA	12 [25.5]	4 [36.4]	0.5
	clbQ	12 [25.5]	4 [36.4]	0.5
	Virulence score, median (min-max)	12 (1-18)	11 (3-15)	0.3
	PAI
	PAI I CFT073	14 [29.8]	0 [0]	0.05
	PAI II J96	7 [14.9]	2 [18.2]	>.99
	PAI III 536	12 [25.5]	2 [18.2]	>.99
	PAI IV 536	45 [95.7]	10 [90.9]	0.5
	PAI gimA	2 [4.3]	2 [18.2]	0.2
	PAI USP	31 [66]	7 [63.6]	>.99
	PAI pks	12 [25.5]	4 [36.4]	0.5
	PAI score, median (min-max)	2 (0-6)	2 (0-5)	0.8
	Antibiotic resistance
	Amoxicillin	34 [72.3]	6 [54.6]	0.3
	Gentamicin	1 [2.1]	0 [0]	>.99
	Ofloxacin	2 [4.3]	0 [0]	>.99
	Cotrimoxazole	19 [40.4]	4 [36.4]	>.99
	3rd generation cephalosporins	1 [2.1]	0 [0]	>.99
	Multidrug resistance	2 [4.3]	0 [0]	>.99
	Resistance score, median (min-max)	1 (0-4)	1 (0-2)	0.3
	Notes:
	1 Except when otherwise notified.
	PAI, pathogenicity-associated island

TABLE 3 .

 3 Ba te ial ha a te isti s i the vs. >3-month-old groups.

	Characteristic

Acknowledgements

We are grateful to Cécile Gateau for her technical help in the genotyping of the E. coli strains and to Jorge Blanco from the University of Santiago de Compostela, Lugo, Spain for serotyping of some strains.

1 Except when otherwise notified. PAI, pathogenicity-associated island.

Conflicts of Interest and Source of Funding

All authors reported no conflict of interest.

Meetings

This work has partly been reported at the 32nd Réunion interdisciplinaire de chimiothérapie antiinfectieuse (RICAI), held in Paris, France on 21 -22 November 2012 (poster 548). Because of missing characteristics, the multivariate analysis was performed in 77 children.


Romain Bodeux

Dominique Michau
email: d.michau@icmcb-bordeaux.cnrs.fr

Michaël Josse

Mario Maglione

Dielectric properties of tetragonal tungsten bronze films deposited by RF magnetron sputtering

Keywords: TTB, PVD, semiconductor oxide, interfaces, Schottky barrier, ferroelectric

Tetragonal tungsten bronze (TTB) films have been synthesised on Pt(111)/TiO 2 /SiO 2 /Si substrates from Ba 2 LnFeNb 4 O 15 ceramics (Ln = La, Nd, Eu) by RF magnetron sputtering. X-ray diffraction measurements evidenced the multi-oriented nature of films with some degrees of preferential orientation along (111). The dependence of the dielectric properties on temperature and frequency has been investigated. The dielectric properties of the films are similar to those of the bulk, i.e.,  ~ 150 and  ~ 10 -6  -1 cm -1 at 1 MHz and room temperature. The films exhibit two dielectric anomalies which are attributed to Maxwell Wagner polarization mechanism and relaxor behaviour. Both anomalies are sensitive to post-annealing under oxygen atmosphere and their activation energies are similar E a ~ 0.30 eV. They are explained in terms of electrically heterogeneous contributions in the films.

Introduction

In the search for improved electronic functionalities, perovskite oxides with the formula ABO 3 are widely studied because of their versatile properties allowing for many applications. Chemical substitution allows for the tuning of their physical properties like magnetization and polarization [1].

Thus, perovskites could be compatible with the development of a new generation of Random Access Memories (RAMs), in which the superposition and coupling of ferromagnetism and ferroelectricity would be used. Such materials, simultaneously exhibiting these two properties, are known as multiferroics. One can achieve multiferroism in perovskites by inducing a structural distortion and polarization with appropriate substitution at the A site, while magnetism is induced from the B site cations, as in BiMnO 3 [2]. However such multiferroic perovskites are rare, and it is important to investigate others materials families, particularly by relying on an increased range of cationic substitutions.

In this view, tetragonal tungsten bronzes (TTB) are particularly attractive, because of the diversity of cationic sites they offer. Their general formula is (A1) 4 (A2) 2 (C) 4 (B1) 2 (B2) 8 O 30 , and their polyhedral linking can be related to the perovskite structure, through appropriate rotations of "perovskite columns" [3]. The TTB structure is a framework of edge-sharing BO 6 octahedra in which three types of interstices (A1, A2 and C) are occupied by a variety of cations. Those in A1 occupy a pentagonal channel while those in A2 occupy a square channel. Generally, the smallest interstices (C) are empty. The most studied TTB-type systems are Ba 2 NaNb 5 O 15 (BNN) [4,5] and Sr 1-x Ba x Nb 2 O 6 (SBN) [6], which are candidates for the development of optoelectronic devices. In addition they show remarkable ferroelectric properties such as spontaneous polarization P S ~ 40 µC/cm 2 at room temperature. TTB compounds were also prepared by inserting trivalent rare earth ions [7][8][9][10]. Their dielectric behaviour is rather complex, indicating a large flexibility of their physical properties upon chemical substitution, and is related to subtle structural modifications. For example, it was reported that the ferroelectric/relaxor behaviour of Ba 2 LnNb 3 Ti 2 O 15 (Ln = rare earth) is driven by the size of barium and rare earth ions [11,12]. The compounds with a larger size difference between the cations occupying A1 and A2 tends to be normal ferroelectric while a smaller size difference is associated with a relaxor behaviour.

In order to induce magnetic properties in ferroelectric materials, TTB-type compounds, paramagnetic Fe 3+ cations were also introduced in the octahedral framework, for example by a double substitution mechanism 2Ti 4+ -> Fe 3+ + Nb 5+ [START_REF] Castel | Synthèse de Nouveaux Matériaux Multiferroïques au Sein de la Famille des Bronzes Quadratiques de Formule Ba 2 LnFeNb 4 O 15[END_REF][START_REF] Josse | [END_REF][15][16][17][18][19][20]. It was demonstrated that the ferroelectric and magnetic properties of the Ba 2 LnFeNb 4 O 15 TTBs are directly influenced by the nature of the rare earth, despite the extrinsic origin of the magnetic properties [START_REF] Josse | [END_REF]. A correlation appears between cell volume, dielectric ordering temperature and magnetization, which are driven by the size of the rare earth. Moreover the Ba 2 LaFeNb 4 O 15 niobate displays a relaxor behaviour [START_REF] Castel | Synthèse de Nouveaux Matériaux Multiferroïques au Sein de la Famille des Bronzes Quadratiques de Formule Ba 2 LnFeNb 4 O 15[END_REF] while Nd, Sm and Eu substituted niobates constitute a family of room-temperature composite multiferroics. It is thus interesting to transfer these original dielectric properties to TTB films and this is the main goal of the present work.

TTB-type films have already been synthesized by various methods, such as RF magnetron sputtering [21,22], sol-gel [23], PLD [24], and MOCVD [25]. Reproducing the chemical complexity of TTB-type materials is the main challenge of thin film growth, along with the transfer of physical properties from bulk to films. It was nevertheless reported in few investigations that TTB films can display approximately the same physical properties as those observed in the bulk. Concerning RF magnetron sputtering, Antsigina et al. reported that the Ba 0.5 Sr 0.5 Nb 2 O 6 films are ferroelectric, although with a diffuse phase transition [26]. Michau et al. reproduced the relaxor behaviour of Ba 1+x/2 LaNb 5-x Ti x O 15 TTBs, as observed in ceramics [27].

In the present work, films were deposited on Pt(111)/TiO 2 /SiO 2 /Si substrate by RF magnetron sputtering from Ba 2 LnFeNb 4 O 15 ceramic targets (Ln = La, Nd and Eu). The Ba 2 LnFeNb 4 O 15 TTBs (Ln = La, Pr, Nd, Sm, Eu, Gd) form a rich and versatile family of materials in which original behaviours such as in situ generated multiferroic composites and ferroelectric to relaxor crossovers are encountered [START_REF] Castel | Synthèse de Nouveaux Matériaux Multiferroïques au Sein de la Famille des Bronzes Quadratiques de Formule Ba 2 LnFeNb 4 O 15[END_REF][START_REF] Josse | [END_REF][15][16][17]. After their initial study in polycrystalline form, these materials were grown as single-crystals [28][29][30] and in the present work, as thin films.

For a better understanding of the context of this study, it is worth recalling some observations concerning the chemical composition of Ba 2 LnFeNb 4 O 15 , extracted from previous ceramic and singlecrystal studies. The TTB matrix is reluctant to accommodate the smaller rare earths in the square channels, yielding in some cases to their partial accommodation. In such cases, the TTB framework electroneutrality is maintained by subsequent adjustments of the Fe/Nb ratio. The growth of Ba 2 LnFeNb 4 O 15 single crystals confirmed this mechanism [28][29][30]. Chemical strain and distortions induced by smaller reare earths lead to the evolution of the BLnFN compositions and the occurrence of secondary phases, i.e. LnNbO 4 ferguonite and BaFe 12 O 19 barium ferrite [START_REF] Castel | Synthèse de Nouveaux Matériaux Multiferroïques au Sein de la Famille des Bronzes Quadratiques de Formule Ba 2 LnFeNb 4 O 15[END_REF][START_REF] Josse | [END_REF] The excess of lanthanide oxide reacts with niobium oxide to form fergusonite. To maintain electrical neutrality of the TTB framework, the Fe/Nb ratio is modified in the TTB framework. The subsequent excess of iron oxide reacts with a small amount of barium oxide to form barium ferrite. Thus the ceramics obtained from the stoichiometric composition are composites materials, the multiferroic properties of which can be controlled by chemical and crystal-chemical means [16,17].

In this study, the films were grown from targets with the Ba 2 LnFeNb 4 O 15 composition, in order to evaluate the opportunity to transfer this composite multiferroic system to thin films.

Following chemical and structural characterizations, we have investigated their dielectric properties by impedance spectroscopy over a broad temperature and frequency range.

Experiment Details

Three different films types were grown on the Pt(111)/TiO 2 /SiO 2 /Si(100) (PtS) substrates from stoichiometric Ba 2 LnFeNb 4 O 15 targets with Ln = La, Nd and Eu by RF magnetron sputtering (Plassys PM450 -13.56 MHz) [27]. The films are referred to as BLnFN where Ln = La, Nd or Eu. The targets were prepared through a solid state route from stoichiometric mixtures of BaCO 3 , Fe 2 O 3 , Nb 2 O 5 and Ln 2 O 3 and were sintered at 1300°C [START_REF] Josse | [END_REF]. The PtS substrates were purchased from INOSTEK and a part of them was protected during the films deposition in order to access the bottom electrode. The deposition parameters were optimized regarding both the stabilization of TTB structure and the crystalline quality. The films were deposited at 650 °C, 40 mTorr plasma pressure (O 2 = 10 % + Ar = 90 %), target to substrate distance at d = 70 mm and power 100 W i.e. 5 W/cm 2 . X-ray diffraction data in the -2 Bragg-Brentano geometry were recorded on a Philips XPert pro diffractometer (Cu K 1 radiation,  = 1.54056 Å) with 10° < 2< 80° and step = 0.02°. The film thickness was measured using a Veeco Dektak6M stylus profile mechanical profilometer. Deposition time (5 hours) was adjusted to obtain films thickness about 1 µm. The films compositions were quantified using the electron probe micro-analysis (EPMA) SX-100 apparatus from CAMECA, without calibration.

Electron beam, which typically has probe 150 nm and energy 5 keV, provided a lateral resolution close to 300 nm. The films' compositions were checked by Rutherford Backscattering Spectrometry (RBS) of an alpha particles beam_(He 2+ , 2 MeV, 15 nA) obtained from a HVEE Singletron, at the AIFIRA facility (Centre d'Etudes Nucléaires de Bordeaux-Gradignan, CENBG). Calibration from Ba, La, Nd, Eu, Fe, Nb, Pt, Ti and Si pure targets allowed for an accuracy below 5 %. Pt Circular dots were deposited ex-situ as a top electrode by dc sputtering through a metal mask at room temperature on the TTB layer. The dot geometry defined the capacitors area: S = 0.1 mm 2 . The dielectric properties of capacitors were measured in the metal/insulator/metal configuration, as a function of frequency (100 Hz -1 MHz) and temperature (80 K -420 K), using a Hewlett-Packard 4194 impedance analyser with an ac excitation amplitude of 100 mV.

Results and Discussion

Chemical characterization of Ba 2 LnFeNb 4 O 15 thin films

The compositions transferred from the target to the film were investigated by means of Electron-Probe Micro-Analysis (EPMA) and Rutherford BackScattering (RBS). Since oxygen content can hardly be evaluated by these methods, only the cationic ratios are reported here. We obtained the same Ba:Ln:Fe:Nb ratios for La, Eu and Nd based films, i.e 2:1:1:15.5 by EPMA and 2:1:1:12.5 by RBS.

These ratios have to be compared with the 2:1:1:4 ratios associated to the Ba 2 LnFeNb 4 O 15 formula. On one hand, the experimental findings show that the Ba:Ln and Ba:Fe ratios remain quite consistent with the target composition. On the other hand, it appears that Ba:Nb, Ln:Nb and Fe:Nb ratios are much lower than expected i.e. there is a Nb excess in the films. This suggests a difference of sputtering yield between Nb atoms and the other metallic atoms, as was also observed in other studies [27,31,32].

XRD confirmed that the grown films retained the TTB structure, as shown by the XRD patterns of three BLnFN/PtS films with Ln = La, Nd and Eu and the BNdFN target in Fig. 1a and Fig. 1b, respectively. In the absence of secondary phases as detected by XRD, the excess of Nb observed in the films could be ascribed to an amorphous phase, the most likely still being niobium oxide Nb 2 O 5 [32]. This phase has a low permittivity ( = 40) and should appear as an additional impedance source [33].

However the absence of Nb-rich regions in EPMA mappings does not supports its formation. Thus we can suggest that the niobium excess may be located within the TTB structure itself, with the accommodation of [Nb-O-Nb] chains in its pentagonal channels, as observed by [34] for example.

Further investigations dedicated to the study of Nb-content in sputerred TTB thin films are in progress.

XRD results are quite similar for the three studied rare earth, the observed peaks position corresponding to polycrystalline, multi-oriented TTB phases, and matching with the targets' XRD M" spectra displays a small peak at 1 kHz and a large peak at 70 kHz, which are related to the two semicircles observed in Fig 2a . Thus, impedance data can be modelled and analyzed using an equivalent circuit with two parallel R 1 C 1 and R 2 C 2 elements, in series, each corresponding to one of the observed semicircles. As temperature decreases from 350 K to 300 K, Both M" peaks shift toward lower frequencies. Their intensities remain almost constant indicating that the effective permittivity for each response is unchanged. Permittivities   and  and conductivities  1 and  2 can be extracted from relations of complex impedance and R 1 C 1 and R 2 C 2 elements [3]. We obtained   and  On the one hand   is close to the permittivity of the corresponding BNdFN ceramic [START_REF] Josse | [END_REF] and can be identified as the permittivity of bulk, i.e.  b =  2 . The higher conductivity of bulk,  b =  2 ~ 10 -6  -1 .cm -1 at 300 K, indicates the presence of semiconductive grains. The analysis of  b shows that the bulk conductivity is thermally activated, and follows a straightline relationship in an Arrhenius scale, with a characteristic activation energy E a ~ 0.30 eV. On the other hand the high value of   excludes a dielectric response from an amorphous Nb 2 O 5 oxide, and should involve a mechanism of barrier layer capacitors which will be further discussed later. This observation thus supports the formation of a single phased film. The complex permittivity * = ' + j" was extracted from impedance measurements. Dielectric loss tan  and conductivity ' are related to the complex permittivity through the relations tan "/' and  = "( is the angular frequency), respectively. The frequency dependence of dielectric permittivity and losses for BNdFN films were investigated at various temperatures and are shown in Fig 3a and3b, respectively. At high temperatures and low frequencies, the permittivity reaches a value ' =  1 = 800 and drops to ' =  2 = 150 at higher frequencies. The relaxation in ' is accompanied by a peak in tan , which shifts to lower frequencies when the temperature decreases (Fig 3b). Below 200 K, only the bulk response is visible over the whole frequency range, while permittivity and losses become smaller, i.e. ' ~ 100 and tan 2%. The temperature dependence of the relaxation indicates a thermally activated mechanism. The relaxation frequency follows an Arrhenius law with Ea ~ 0.30 eV (Table 1). Ion jump polarization may explain this relaxation mechanism, as it is frequently found in polycrystalline dielectrics encompassing ion vacancies. However, activation energy similar to that of the bulk resistivity corroborates a relaxation governed by the bulk, and thus Maxwell Wagner (MW) type contributions [START_REF] Jonscher | Dielectric Relaxations in Solids[END_REF]. Indeed, MW relaxation is significant in samples with semiconductive grains because of the formation of barrier layer capacitors in the region close to the metallic electrodes [START_REF] Bodeux | [END_REF] and/or the grain boundaries [38]. Stable space charges regions called depletion layers are formed at interfaces and lead to high capacitive and resistive effects. The barrier layers act as an extrinsic source of impedance connected in series to the bulk. Given R 1 >> R 2 and C 1 >> C 2 , their contributions on the dielectric response are visible on characteristic ranges of frequency, i.e. = (RC) -1 and the barrier layers dominate at low frequency while the bulk appears at high frequency. In this case, the time constant follows approximatively  = R 2 C 1 . Thus, assuming that C 1 is nearly constant with the temperature, the time constant is essentially driven by the conductivity of grains. , 4b and4c, respectively. At high frequencies, i.e. f = 1 MHz, the three films display similar bulk permittivities. As the frequency decreases below 10 kHz, a large increase of ' is observed for BNdFN and BEuFN films, accompanied by a peak in tan and a relaxation in '. The permittivity remains constant over the whole frequency range for BLaFN film. The conductivity for both BNdFN and BEuFN films is higher than for the BLaFN film. Temperature-dependent studies of the BEuFN film revealed that the relaxation is also thermally activated, with Ea = 0.33 eV, close to the activation energy found in BNdFN film (Table 1). Thus, it appears that the rise of the conductivity is due to the same phenomenon in both BNdFN and BEuFN films. This phenomenon could be related to the one observed in corresponding ceramics and singlecrystals, in which the insertion of smaller rare earths (i.e. Nd or Eu instead of La) leads to and offstoichiometry that could involve oxygen losses, which can be induced during the processing [START_REF] Josse | [END_REF]30,31]. It was reported that the leakage current of oxide films could be reduced by activated oxygen annealing [39]. Considering these results, and in order to evidence an effect of oxygen vacancies, the BNdFN film was ex-situ annealed under oxygen atmosphere at the deposition temperature. XRD (not shown) confirmed the preservation of the TTB structure and the absence of structural changes following the post-annealing process. For both the as-deposited and the post annealed BNdFN films, frequency dependence of the dielectric constant and the conductivity are shown in Fig 5a and5b, respectively. The relaxation disappears after the post-annealing process, while the film displays a lower conductivity, which drops from  ~ 5.10 -7  -1 .cm -1 to  ~ 8.10 -8  -1 .cm -1 at 10 kHz. This is similar to the conductivity of bulk ceramics [START_REF] Josse | [END_REF], and in agreement with the higher conductivity of as deposited samples being induced by a slight oxygen deficiency. In the range [5 kHz -1 MHz], the conductivity increases with the frequency (Fig 5b), and can be described by the universal dielectric response  =  dc +  ac  n (dc is the dc conductivity of bulk,  ac is a constant and n is a parameter having a value between 0 and 1) [START_REF] Jonscher | Dielectric Relaxations in Solids[END_REF]. The bulk dc conductivity is visible at intermediate frequencies on the as deposited sample while the bulk ac conductivity dominates at higher frequencies. For the annealed sample the dc conductivity is not visible in this frequency range and its response is described only by the frequency-dependent bulk ac conductivity  ~ n . This type of variation is often observed in disordered systems and is attributed to hopping transport between localized states close to Fermi level [START_REF] Emin | Electronic and Structural Properties of Amorphous Semiconductors[END_REF]. In the investigated sample, the statistical distribution of Fe 3+ and Nb 5+ within the octahedral framework could trigger such a mechanism [START_REF] Abdelkafi | [END_REF]. The temperature dependence of the dielectric properties was investigated on a post annealed BNdFN film. The frequency-dependence of the dielectric permittivity is shown at various temperatures in Fig 6 . At 100 K, ' is constant over the whole frequency range and corresponds to bulk permittivity. Upon increasing the temperature from 100 K to 260 K, the permittivity increases from 100 to 150. When raising the temperature to 420 K, the permittivity further increases at lower frequencies, as the capacitive response of the barrier layer appears. Meanwhile, the permittivity remains almost constant at higher frequencies, whatever the temperature. 3) increasing of the apparent permittivity due to the effect of barrier layer capacitors at higher temperatures. In addition, the dielectric anomaly in (T) is transferred into a peak in tan (T). This peak shifts to higher temperatures as frequency increases. From several frequency scans, we conclude that this dielectric anomaly is not related to a static phenomenon, but is of purely dynamic origin.

Tetragonal tungsten bronzes are known to exhibit a series of dielectric anomalies, especially at low temperature [42]. For the greater part, they are explained by either ferroelectric or relaxor behaviour, which could be driven by the size of the ions inserted in the TTB framework [43,44]. As indicated by Fig 7, the frequency dispersion excludes a normal ferroelectric behaviour. For a better insight into the films dielectric behaviour, pyroelectric and piezoeletric measurements have to be considered since such signals are expected from relaxor compounds. However, it is important to note that in ceramics, a diffuse dielectric anomaly is observed at low temperature for all compounds Ba 2 LnFeNb 4 O 15 with Ln = La, Pr, Nd, Sm and Eu [START_REF] Castel | Synthèse de Nouveaux Matériaux Multiferroïques au Sein de la Famille des Bronzes Quadratiques de Formule Ba 2 LnFeNb 4 O 15[END_REF][START_REF] Josse | [END_REF][15][16][17]. Thus one may consider that these dielectric anomalies originate from the same phenomenon in both ceramics and films. In fact, these anomalies were observed in the BLaFN and BEuFN films in temperature ranges similar to those of ceramics. However, because of a restricted frequency range, no reliable fit of the experimental data using the Vogel-Fulcher relationship could be performed, and thus no accurate Vogel-Fulcher freezing temperature T VF could be determined. Thus, the activation energy was extracted from an Arrhenius fit, and found similar in all films, E a ~ 0.30 eV (table 1), indicating that the relaxation mechanism in our BLnFN films is relatively independent from the size of rare earth ions. Although anomalies appear quite similar, there is some difference in activation energies of ceramics, i.e, E a = 0.17 eV in BEuFN ceramics (table 1) and the activation energies varies with the size of rare earth. Michau et al. showed in a previous paper that the relaxor behaviour of Ba 1+x/2 LaNb 5-x Ti x O 15 films was driven by the (Ba+La)/(Nb+Ti) cationic ratio [27]. Thus, the deviation from the stoichiometry in our films may also act as major parameter on their activation energies. One may wonder if oxygen vacancies are involved in these low temperature anomalies. For such a purpose, more detailed studies were performed on Nd films, to obtain some insights on the role of oxygen content. The temperature dependence of the dielectric constant and the dielectric loss for the as-deposited and post annealed films are compared in Fig 8a and8b, respectively. The dielectric anomaly is visible in both as deposited and post annealed samples. However it is more pronounced following the post annealing, suggesting that oxygen vacancies are not responsible for the dielectric anomaly observed at low temperature but may hinder it. This dielectric anomaly may be related to offcenter barium or rare earth ions displacements for example, which could be influenced by some oxygen vacancies. Thus, the dielectric anomaly observed at low temperature is likely related to the TTB phase stabilized in the films, and further investigation is needed to assess the mechanisms involved in its apparition.

Conclusion

Tetragonal tungsten bronze films from Ba 2 LnFeNb 4 O 15 (Ln = La, Nd and Eu) ceramic targets were deposited by RF magnetron sputtering on Pt/TiO 2 /SiO 2 /Si substrates. The dielectric properties are similar to those of the bulk with  ~ 150 and  ~ 10 -6  -1 .cm -1 at 1 MHz and at room temperature. Two dielectric anomalies with similar activation energies were observed in the films with an obvious effect of post annealing under oxygen for one of them. It can be concluded that the high temperature anomaly results from a MW-type mechanism while the low-temperature anomaly is likely related to a relaxor behaviour which is also observed in ceramics. Further developments of this work include the investigation of the mechanisms involved in the dielectric response of the films at low temperature, and its comparison to similar responses observed in bulk samples, whether ceramics or single-crystals.

Fig. 1 .

 1 Fig 1b are practically invisible in the films and the (111) reflection is the most intense. This indicatesthat the films present some degree of preferential orientation along (111), as the I (111) /I (420) intensity ratio is close to 1 while it should be smaller than 0.13 for a fully disoriented film. The intensity ratios vary for each film, meaning that the growth orientation is significantly affected by the composition. To go further, a special attention was paid to impedance spectroscopy analysis, in order to highlight the contribution of TTB phase to the dielectric properties of films

Fig. 2 .

 2 Fig. 2. (Color online) Complex impedance plot at 350 K (a) and M'' spectroscopy plot at two

Fig. 3 (

 3 Fig. 3 (Color online) Frequency dependence of (a) the effective dielectric constant ' and (b) the dielectric loss tan  for the BNdFN film at various temperatures. Inset shows the MW relaxation frequency in an Arrhenius law

Fig. 4 (

 4 Fig. 4 (Color online) Frequency dependence of (a) the effective dielectric constant ', (b) the dielectric loss tan  and (c) the conductivity ' for the BLnFN films (Ln = La, Nd and Eu) at room temperature

Fig. 5 (

 5 Fig. 5 (Color online) Frequency dependence of the dielectric constant ε' (a) and the conductivity  ' (b) for both as-deposited and post annealed BNdFN films at room temperature

Fig. 6 (

 6 Fig. 6 (Color online) Frequency dependence of the dielectric constant ε' for post annealed BNdFN film at various temperatures

Fig. 7 (

 7 Fig. 7 (Color online) Temperature dependence of the dielectric constant ε' (a) and the dielectric loss tan  (b) for the post annealed BNdFN film at various frequency. Inset shows the relaxation frequency in an Arrhenius law.

Fig. 8 (

 8 Fig. 8 (Color online) Temperature dependence of the dielectric constant ε' (a) and the dielectric loss tan  (b) for both as-deposited and post annealed BNdFN films at 1 MHz

. The chemical equilibrium can be depicted as follows: Ba 2 LnFeNb 4 O 15  Ba 2-x/8 Ln 1-x Fe 1-3x/2 Nb 4+3x/2 O 15-x/8 + x LnNbO 4 + 3x/24 BaFe 12 O 19 .

Table 1 .

 1 Activation energies E a of the Maxwell-Wagner relaxation and relaxor behaviour at low temperature versus BLnFN (Ln = La, Nd and Eu) as deposited, the post annealed BNdFN films and the BEuFN ceramic.

		Maxwell-Wagner E a (eV)	Relaxor E a (eV)
	BLaFN	-	0.31 (0.07)
	BNdFN	0.30 (0.03)	-
	BNdFN	-	0.34 (0.04)
	post-ann.
	BEuFN	0.33 (0.04)	0.30 (0.05)
	BEuFN	-	0.17 (0.02)
	ceramic
	The frequency dependence of ' , tan and ' for Pt/BLnFN/PtS stacks (with Ln = La, Nd and
	Eu) are shown in Fig 4a

Acknowledgments

We thank S. Sorieul from CENBG in Bordeaux for the RBS measurements. This project was supported by the French Agency for Research under contract ANR CROCRODIEL (ANR-09-JCJC-