A Caille

N Lyon

B Pereira

K Slim

M Robert

E Disse

Ruyet P Le

PhD Christelle Guillet
email: christelle.guillet@uca.fr

Bariatric surgery affects obesity-related protein requirements

Keywords: Non Esterified Fatty Acids, GH, Growth Hormone, IGF-1, insulin-like growth factor-1, CRP, C-Reactive Protein, IL-6, Interleukin obesity, nutrition, protein requirements, bariatric surgery, recovery

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

INTRODUCTION

Bariatric surgery is considered as one of the most effective treatment of clinically severe obesity, resulting in long-term body weight loss, control or remission of comorbidities (type 2 diabetes, hypertension, cardiovascular diseases, cancer) and improved quality of life and mortality [START_REF] Puzziferri | Long-term follow-up after bariatric surgery: a systematic review[END_REF][START_REF] Sjostrom | Review of the key results from the Swedish Obese Subjects (SOS) trial -a prospective controlled intervention study of bariatric surgery[END_REF] . Following bariatric surgery, weight loss is accompanied by a substantial loss of fat and fat-free mass implying many metabolic and functional consequences like reduced resting metabolic rate, fatty acid oxidation and skeletal integrity. In relation with nutrients deficit, these modification also increase the risk of weakness, fatigability and vulnerability to critical stress in the post-operative period, suggesting that nutritional intakes, especially protein intakes are key nutrients for protecting from these deleterious outcomes. Inappropriate protein intakes after bariatric surgery might contribute to a greater loss of fat-free mass. For instance, fat-free mass has been shown to be spared with high dietary protein intakes following caloric restriction [START_REF] Tang | Normal vs. high-protein weight loss diets in men: effects on body composition and indices of metabolic syndrome[END_REF][START_REF] Westerterp-Plantenga | Dietary protein, weight loss, and weight maintenance[END_REF] . In bariatric surgery patients, a better lean body mass preservation was reported when protein intakes was over 60g/d [START_REF] Moize | Protein intake and lean tissue mass retention following bariatric surgery[END_REF] , or when the ratio of protein to energy was higher than 20% [START_REF] Schollenberger | Impact of protein supplementation after bariatric surgery: A randomized controlled double-blind pilot study[END_REF] . In addition, previous studies have demonstrated severe postbariatric protein deficiency with a very high proportion of patients having dietary protein intakes below 60g/day even 4 months after bariatric surgery [START_REF] Schollenberger | Impact of protein supplementation after bariatric surgery: A randomized controlled double-blind pilot study[END_REF][START_REF] Andreu | Protein intake, body composition, and protein status following bariatric surgery[END_REF][START_REF] Verger | Nutritional and Protein Deficiencies in the Short Term following Both Gastric Bypass and Gastric Banding[END_REF] . The current guidelines for nutritional support in bariatric surgery patients state that protein intakes within meals, including supplementation, should be in the range of 60-120g/day. Many others programs recommend a range of 60 to 80 g/d of total protein intakes. However, these international recommendations are not based on studies specifically designed to assess protein requirement.

Recommendations are mostly based on clinical outcomes like plasma albumin alterations after bariatric surgery: albuminemia inferior to 35 g/L occuring in patients having a poor protein intake (< 60 g/d) [START_REF] Aills | ASMBS Allied Health Nutritional Guidelines for the Surgical Weight Loss Patient. Surgery for obesity and related diseases : official journal of the American Society for Bariatric Surgery[END_REF][START_REF] Bloomberg | Nutritional deficiencies following bariatric surgery: what have we learned?[END_REF] . Dietary protein needs in obese patients after bariatric surgery have yet to be defined with robust, internationally recognized and validated methods such as nitrogen balance, the only method used currently to assess average protein requirement in many different populations [START_REF] Rand | Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults[END_REF][START_REF]Protein and amino acid requirements in human nutrition[END_REF] . Therefore, the aim of this study was to determine the average protein requirement in obese patients using the classical nitrogen balance method before, three months and one year after bariatric surgery, in order to implement clinical data with accurate therapeutic targets aiming at limiting protein deficiency for etablishing suitable recommendations for morbidly obese patients with bariatric surgery.

MATERIAL AND METHODS

The trial was a longitudinal, prospective follow-up study of protein requirement of severely obese patients candidates for a sleeve gastrectomy (SG) or a Roux-en-Y gastric by-pass (RYGB) (Experimental group, n=21). Protein requirement in severely obese patients was also calculated and validated in a large cohort of obese patients not subjected to bariatric surgery (Validation group, n=106). The study protocol was conducted in accordance with the guidelines in the Declaration of Helsinki and was approved by the Ethical Committee of the Auvergne region (agreement no. AU 817; March 2010).

Populations of Patients

Experimental group

Thirty-three obese patients planned to undergo SG or RYGB procedures were recruited from the departments of clinical nutrition and digestive surgery of Clermont-Ferrand University Hospital (France), and from the department of digestive and general surgery of Edouard Herriot Hospital (Lyon, France). Adult men and/or women aged between 18 to 60 years, with a body mass index (BMI) higher than 40 kg/m², and having obtained the agreement of the medical staff for SG or RYGB, were eligible for the study. Among the 33 patients, five patients withdrew from the study during inclusion visit (Figure 1). Five patients were excluded because of taking drugs incompatible with the study (anti-inflammatory drugs). The remaining 23 patients, i.e. 16 patients scheduled for SG (13 women) and 7 patients scheduled for RYGB (5 women), were included in the study. After complete medical and biological assessment, no known neuromuscular disease, no progressive cardiovascular disease, no cancer, no uncontrolled hyperadrenocorticism and thyroid dysfunction, no severe infection or acute/chronic inflammatory disease in the 3 months prior to inclusion, and no treatment by corticosteroids, immunosuppressives, anabolics, growth hormone were detected. Included patients were not smoking more than 10 cigarettes per day, not drinking more than 2 alcoholic drinks per day and not practicing physical activity more than 5 hours per week. One patient operated on SG suffered from post-operated complications and dropped out 3 months after surgery. One patient had to be operated again on RYGB 10 months after SG and dropped out before the end of the study. Overall 21 patients were included in analysis for the entire study (Figure 1).

Validation group

The patients (n = 106) belonging to the Validation group were morbidly obese with a BMI > 40 kg/m². They were hospitalized in the clinical nutrition department of Clermont-Ferrand University Hospital (France) from October 2012 to December 2014 for the initial treatment of their weight management. All participants underwent a medical assessment which included biological analysis, anthropometric data, body composition assessment, evaluation of resting energy expenditure through indirect calorimetry. Exclusion criteria were chronic renal failure with a creatinine clearance < 60 ml/min using the Modification of Diet in Renal Disease (MDRD) equation [START_REF] Levey | A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group[END_REF] .

Study protocol

After inclusion, patients were investigated during three periods: before (M0), 3 months (M3) and 12 months (M12) after surgery. For each period, two nitrogen balance measurements (nitrogen intakes -nitrogen losses) were performed at two different levels of protein intakes ("high" and "low") at 15-day intervals. Each nitrogen balance lasted 3 days and were spaced by 6 days (d1 to d3 and d10 to d12). The day following the first nitrogen balance (d4), patients came to the laboratory for parameters measurement. Patients were weighted to the nearest 0.1 kg with the use of manual-weighing scale (Seca 709; Seca, Hamburg, Germany).

Height was measured to the nearest 0.5 cm with the use of a standardized wall-mounted height board. The metabolic, hormonal and inflammatory status of the patients were analysed from a blood sampling performed in the fasting state.

Body composition (fat and fat-free mass) was evaluated with total body water (TBW) measurement using whole body deuterium water dilution. Resting energy expenditure was measured using indirect calorimetry (Deltatrac®, Datex Instrument, Geneva, Switzerland).

Nitrogen intakes

Experimental group. For each nitrogen balance, nutritional intakes were assessed using a 3days food survey and Nutrilog software. Daily protein intakes in grams (gP) was converted to nitrogen intakes (gN), (1g of N corresponds to 6.25g of protein). The 3-days average nitrogen intakes were performed to calculate global daily nitrogen intakes. At d4, for each study period, the dietitians have analysed protein intakes of the first nitrogen balance. The food diary was started one day before the first nitrogen balance and was purchased at the last day of the second nitrogen balance to evaluate protein intakes compliance. If the dietitians considered these protein intakes as "low" (i.e. protein intakes < 0.8 g/kg/d), they advised the patient to increase their protein intakes of at least 0.20 g/kg/j for the next 9 days, including the period of the second nitrogen balance evaluation. If protein intakes at the first nitrogen balance period was considered as "high" (i.e. protein intakes > 1.2 g/kg/d), the patients had to decrease their protein intakes of at least 0.20 g/kg/j for the next 9 days, including the period of the second nitrogen balance evaluation.

Validation group. Estimation of daily protein intakes was provided for each patient by 5 dayfood records analysis previously to the patient stay in the department of clinical nutrition. A mean of daily protein intakes was then calculated for each patient, expressed per kg of body weight and then converted in nitrogen intakes. Then nitrogen loss measurements were based on urinary nitrogen loss and estimated losses using the accurate data from the experimental results.

Nitrogen excretion

Experimental group. Simultaneously with the 3-days food survey, nitrogen losses were assessed using a 3-day urines (3x24h) and 2-day stools collections (2x24h). Each day, a urine sample of 10 mL was collected from pooled urine after stirring and stored at -20°C for later use. 24-hour stools were collected in plastic bags, placed in sealed plastic boxes and stored at -20 °C for later use. Just before analysis, 24-hour feces were mixed and a sample was used for analysis. Concentration of nitrogen was measured by chemiluminescence (Antek 7000, Alytech, Juvisy Sur Orge, France) in urine samples and by refractometry method in faeces samples. Averages of 3-day nitrogen losses in urine and 2-day nitrogen losses in stools were calculated. Because of the extreme difficulty in measuring miscellaneous nitrogen losses, these sources were estimated at 0.008 g of nitrogen per kg of body weight per day as previously described [START_REF]Energy and protein requirements[END_REF] .

Validation group. The relationship between 24-hour urinary urea and total nitrogen losses from the experimental group study was checked to estimate nitrogen balance in morbidly obese patients and to establish a new specific predictive equation of total nitrogen loss from daily urinary urea.

Nitrogen balance-Protein requirement

Nitrogen balance was calculated from the difference between average daily nitrogen intake and total nitrogen loss for each period of measurement [START_REF] Rand | Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults[END_REF][START_REF]Protein and amino acid requirements in human nutrition[END_REF] . Average protein requirement (APR) -i.e. average protein amount required to maintain a neutral nitrogen balance for a given population -for obese people before and after bariatric surgery, was calculated from the linear regression of 24-hour nitrogen balance related to protein intake in grams per kg of body weight per day [START_REF] Rand | Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults[END_REF][START_REF]Protein and amino acid requirements in human nutrition[END_REF] .

Blood parameters

Venous blood samples were collected for metabolites, hormones and inflammatory factors analyses. For triglycerides (TG), total-cholesterol (T-Chol), HDL-cholesterol (HDL-C), non esterified fatty acids (NEFA), glucose, insulin, albumin, prealbumin, adiponectin, leptin, growth hormone (GH), insulin-like growth factor-1 (IGF-1) and C-Reactive Protein (CRP) analysis, blood samples were collected into serum tubes and placed at room temperature for 20 minutes before centrifugation. For Interleukin 6 (IL-6), Tumor necrosis factor α (TNFα), unacylated and acylated ghrelin, blood samples were collected in EDTA-plasma tubes. For acylated ghrelin, para-hydroxymercuribenzoic acid in HCl (PHMB, 1mM in the final sample volume) was immediately added to plasma to prevent degradation of acylated ghrelin by protease. For Glucagon-like peptide-1 (GLP-1) collection, EDTA-plasma tubes with dipeptidyl peptidase IV inhibitor were used. After collection, all blood samples were centrifuged for 10 min at 4 °C at 4500 rpm. Plasma samples were immediately stored at -80 °C until analysis. For each metabolic exploration day, a total of 30 ml of blood was sampled.

Metabolites, hormones and inflammatory factors were measured by standard methods. Plasma glucose concentrations was determined using spectrophotometric method with hexokinase and G-6-PDH reagents (Abbott Diagnostics, Rungis, France) on an Architect® analyzer (Abbott Diagnostics, Rungis, France). Insulin concentration was measured using immunoreactive method with Bi-Ins-IRMA kit (Cisbio Bioassays, IBA, Gif/Yvette, France).

Adiponectin and leptin were determined by enzyme immunoassay kits (ELISA): Quantikine (R&D Systems, Abingdon, United Kingdom) and Biovendor (Heidelberg, Germany) respectively. T-Chol, TG and HDL-C concentrations were determined using spectrophotometric methods on an Architect® analyzer (Abbott Diagnostics, Rungis, France) with Abbott reactifs (Abbott Diagnostics, Rungis, France). LDL-Cholesterol (LDL-C) was calculated using Friedewald equation (Friedewald WT, 1972, Clin Chem, 4337382). NEFA concentration was determined via an enzymatic method (Wako® Chemicals GmbH Neuss, Deutschland) on a Pentra 400 analyzer (Horiba ABX, Montpellier, France). Albumin and prealbumin concentrations were determined using immunoturbidimetry on an analyzer Architect C8000 (Abbott Diagnostics, Rungis, France), with respectively antiserum reagents Albumin and Prealbumin (Diagam, Ghislenghien, Belgique). GH and IGF-1 were measured by immunoradiometry with respectively the kit HGH-RIACT and IGF1-RIACT (Cisbio Bioassays, Codolet, France). Acylated and unacylated ghrelin were measured using SPIBio ELISA kits: Ghrelin (human acylated) EIA Kit and Ghrelin (human unacylated) EIA Kit (Bertin Pharma, Montigny Bretonneux, France). Total GLP-1 was measured by EDI™ Total GLP-1 ELISA kit (Epitope Diagnostics, San Diego, CA USA). Ultrasensible CRP was determined by immunonephelemetry on an analyzer BN prospec (Global Siemens Healthcare Headquaters, Erlangen, Germany), with antiserum reagent CardioPhase hs CRP (Siemens Healthcare Diagnostics Products GmbH, Marburg, Allemagne). TNFα and IL-6 concentration were measured using ultrasensible ELISA kits: respectively Human TNF-alpha/TNFSF1A Quantikine HS ELISA Kit: HSTA00D and Human IL-6 Quantikine HS ELISA Kit: HS600B (R&D Systems, Minneapolis, MN, USA).

Body composition

Body composition was assessed by total body water (TBW) measurements using deuterium water dilution [START_REF] Schoeller | Total body water measurement in humans with 18O and 2H labeled water[END_REF] . 2 H2O (99.9 atom %) was obtained from Eurisotop® (Saint-Aubin, France).

The labeled water was sterilized before being drunk by the patient. After collection of baseline urine samples, an oral dose of deuterium oxide (0.07 g/ kg BW diluted in 40 mL of Evian water) was measured and administered to each participant. Then urine samples were collected 4 and 5 hours after deuterium water intake. All urine samples were stored at -20°C until completion of deuterium analysis by isotope ratio mass spectrometry. The fat-free mass compartment was calculated assuming a hydration level of 73.2%. Fat mass was obtained by difference with body weight and expressed as a percentage of body mass.

Resting energy expenditure

Measurements of O2 consumed and CO2 expired volumes were performed by indirect calorimetry (Deltatrac®, Datex Instrument, Geneva, Switzerland) over a period of 40 min, at rest, in thermoneutral conditions.

Statistics

Descriptive data, nitrogen intakes and losses are expressed as mean ± standard error of the mean (SEM). Statistical analysis for these data were conducted using NCSS 10 data analysis software. A repeated measures analysis of variance was used to assess the evolution over time of body weight, body mass index, body composition, resting metabolic rate, blood parameters, nitrogen intakes and losses. When a significant effect was detected, differences among individual means were assessed with Dunnett's two-sided multiple-comparison test with control post-hoc test to determine the difference. The threshold of statistical significance was set at P<0.05. Average protein requirements were expressed as mean [95% confidence interval]. Statistical analysis for APR were performed using Stata software, version 13 (StataCorp, College Station, TX, US). Linear regression between protein intakes and nitrogen balance was used to interpolate for APR determination. To take into account between and within subject variability due to several measures for a same subject, random-effects models for correlated data were performed rather than usual statistical tests which would be not appropriate due to the hypothesis of independence data not verified. The normality of residuals from these models was studied using the Shapiro-Wilk test. Then, the predicted values and 95% confidence interval were estimated according to these statistical models.

RESULTS

Patients characteristics

In the experimental group, 15 women and 6 men were included with a mean age of 43.2±2.2 years. Patients characteristics before and after bariatric surgery are shown in Table 1. At M3, patients lost a mean of 21.9% of fat mass and 10.3% of fat free mass. At M12, patients continued to loose weight with an additional mean loss of body weight of 15.3±1.7 kg (P<0.05 vs 3 months). The additionnal loss of fat mass and fat free mass at M12 in comparison to M3 was respectively 12.3±2.1kg and 3.5±1.1kg. Biological parameters, including glycemic, lipid, hormonal and inflammatory parameters, were significantly improved at M3 and M12 (Table 2).

In the validation group, 106 patients were included with a mean age of 45.6±1.3 years. The mean weight of the patients was 119.9±2.4 kg with a mean BMI of 43.4±7.1 kg/m². Body fat was 50.5±2.3kg and fat-free mass was 67.2±2.6kg. The resting metabolic rate in these patients was 2044±62 kcal/d. Fasting plasma glucose was 5.2±0.2 mmol/l, fasting plasma insulin was 18.7±1.4 mUI/l and the HOMA-IR index was 4.6±0.5. Plasma albumin was 37.5±0.3 g/l and prealbumin was 0.245±0.005 g/l.

Nitrogen intakes and losses before, 3 months and 1 year after bariatric surgery Before surgery, nitrogen intakes (12.31±0.65 g/d) were equilibrated with nitrogen losses (12.70±0.64 g/d) resulting in a not significantly different from zero and neutral mean nitrogen balance (-0.85±0.58 g/d) (Figure 2). At M3, nitrogen intakes were significantly reduced compared to the baseline intakes (9.47±0.63 g/d; 3 months after vs before surgery, P<0.05).

Nitrogen losses were also significantly reduced compared to the nitrogen losses at M0 (9.13±0.54 g/d; 3 months after vs before surgery, P<0.05). Mean nitrogen balance was equilibrated for most of the patients (-0.05±0.45 g/d) at M3. At M12, nitrogen intakes increased significantly compared to M3 (11.28±0.62 g/d, P<0.05). Nitrogen losses were not significantly increased compared to M3 (10.07±0.80 g/d, P=NS), leading to a mean nitrogen balance close to zero but positive (0.14±0.54 g/d) (Figure 2).

Determination of average protein requirement

In the experimental group, the equation of the relationship between protein intakes and nitrogen balance was used to determine minimum protein intake to reach nitrogen equilibrium, and consequently average protein requirement (APR) for three periods: M0, M3 and M12 (Figure 3). In the experimental group, at M0, APR was 0.76 [95%CI, 0.66-0.92] g/kg BW/d, a value not significantly different from the value obtained in the validation group (n=106 obese patients), i.e. 0.74 [0.70-0.80, P=NS] g/kg BW/d (Figure 4).

A strong relationship between 24 hours urinary urea (mmol/24h) and total urinary nitrogen losses (g/24h) in the experimental population was established (r²=0.974) with urea consisting in 86±6% of total urinary nitrogen. So, the equation for estimating total urinary nitrogen loss from urinary urea in this specific population was: Total urinary nitrogen loss = (0.031 x urinary urea) + 0.43 where total urinary nitrogen loss is expressed in g/24h, urinary urea in mmol/24h. Then, to get an estimation of fecal nitrogen loss, an average of 8% of dietary nitrogen intake (~1g nitrogen/24h) can be applied, added with miscellaneous losses of 0.008g nitrogen/kgBW/d. Finally, when this new formula (urinary+fecal+miscellaneous nitrogen losses) was applied to the validation population, the protein requirement calculation was not different from that obtained after using McKenzie equation (0.79 [95%CI, 0.75-0.84] vs 0.74 [95%CI, 0.70-0.80] g/kg/d, p=NS). Thus, this simplified calculation can be used for the estimation of nitrogen balance in many other clinical conditions including morbidly obese patients. At M3, APR was measured at 0.62 [95%CI, 0.51-0.75] g/kg/d (Figure 3B) and at M12, APR was 0.87 [95%CI, 0.75-0.98] g/kg/d (Figure 3C).

DISCUSSION

This study is the first one to determine protein requirement in morbidly obese patients before, 3 and 12 months after surgery using a validated method such as nitrogen balance. Current protein recommendations after bariatric surgery are based on very few studies and do not rely on nitrogen balance at two nitrogen levels, whereas it is considered as the reference method for the assessment of protein needs in humans [START_REF]Dietary References Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids[END_REF] . The most recent recommendation was established by AACE/TOS/ASMBS guidelines in 2013 and set at 60 g/d [START_REF] Mechanick | Clinical practice guidelines for the perioperative nutritional, metabolic, and nonsurgical support of the bariatric surgery patient--2013 update: cosponsored by American Association of Clinical Endocrinologists, the Obesity Society, and American Society for Metabolic & Bariatric Surgery. Surgery for obesity and related diseases : official journal of the American Society for Bariatric Surgery[END_REF] . This recommendation was graded as D since there were not enough data to support the evidence and no consensus on the outcomes. Other proposed recommendations were 60 to 120 g/d in 2012 [START_REF] Heber | Endocrine and nutritional management of the post-bariatric surgery patient: an Endocrine Society Clinical Practice Guideline[END_REF] , > 60 g/d [START_REF] Moize | Protein intake and lean tissue mass retention following bariatric surgery[END_REF] , or 90 g/d [START_REF] Fried | Interdisciplinary European guidelines on surgery of severe obesity[END_REF] after gastric by-pass. All these recommendations were based on hypoalbuminemia (< 35 mg/l) [START_REF] Heber | Endocrine and nutritional management of the post-bariatric surgery patient: an Endocrine Society Clinical Practice Guideline[END_REF] or lean tissue mass retention [START_REF] Moize | Protein intake and lean tissue mass retention following bariatric surgery[END_REF] . No data have been reported on the mean protein requirement of morbidly adult obese using nitrogen balance method. This method remains the most robust, solid approach recognized by WHO for determining protein requirement. This method can be easily applied to patients with obesity and following bariatric surgery and allows exploration of the protein balance in the medium and long term, although it has some important shortcomings [START_REF]Energy and protein requirements[END_REF][START_REF] Millward | Methodological considerations[END_REF][START_REF] Waterlow | The mysteries of nitrogen balance[END_REF][START_REF] Young | Nutritional balance studies: indicators of human requirements or of adaptive mechanisms?[END_REF] . Alternative methods like indicator amino acid oxidation technique could have been used to evaluate protein requirements [START_REF] Elango | Indicator amino acid oxidation: concept and application[END_REF][START_REF] Millward | Identifying recommended dietary allowances for protein and amino acids: a critique of the 2007 WHO/FAO/UNU report[END_REF] but these must be adapted to the specificities of protein metabolism in obesity and after bariatric surgery, which still have to be fully investigated. Nitrogen balance measurement is criticized because nitrogen intakes are often over-estimated and nitrogen losses underestimated. In this study, nitrogen intakes were assessed using a 3-day food survey. As obese patients usually tend to underestimate their food intakes, it would lead potentially to an underestimation of the protein requirement. APR of morbidly obese patients at M0 (experimental group) was estimated as 0.76 [95%CI, 0.66-0.92] g/kgBW/d. To confirm these data in a larger cohort of morbidly obese patients (validation group; n=106), another cross-sectionnal study based on a single measurement approach was performed: a similar value of 0.74 [0.70-0.80] g/kgBW/d for average protein requirement was obtained confirming that APR of 0.76 g/kg/d for morbidly obese patients is higher than the APR proposed for nonobese healthy adults. In the 2007 FAO/WHO/UNU report [START_REF]Protein and amino acid requirements in human nutrition[END_REF] , protein requirement is given as 0.66 g/kg/d, with an approximately 95% confidence interval of 0.63 to 0.69 g/kg/d. Considering a higher body weight consisting in a larger fat mass, a lower APR could be expected. The higher APR per kg body weight in morbidly obese patients could be explained by a higher fat free mass in absolute value, but even when expressed per kg of lean mass in the obese patients, it is still different and higher than in the non-obese populations : it was 1.48 [95%CI, 1.29-1.79], 1.13 [95%CI, 0.93-1.37] and 1.43 [95%CI, 1.24-1.62] g/kgFFM/d, respectively at M0, M3 and M12, whereas extrapolations in non-obese adults considering FFM, protein requirement would be close to 1 g/kgFFM/d (considering 70 to 75% of FFM in non-obese adults and 0.66 g/kg/d as average protein requirement). This important observation confirms that in obese patients, protein requirement, whatever the expression mode, is higher than in non-obese adult population. Considering the difficulties to evaluate FFM in morbid obese patients, it would be thus more relevant and appropiate to express average protein requirement by body weight. On a metabolic point of view, the increased protein demand in morbid obesity could be related to metabolic alterations such as inflammation, insulin resistance, oxidative stress, which induced organs and tissues metabolic impairements and an increase in the production and the activity of a lot of proteins implicated in these perturbations [START_REF] Guillet | Impaired protein metabolism: interlinks between obesity, insulin resistance and inflammation. Obesity reviews : an official journal of the International Association for the Study of Obesity[END_REF] .

At M3, average protein requirement decreased to 0.62 [95%CI, 0.51-0.75] g/kg/d with no differences considering the types of surgery. This might be due to the massive changes in body composition after surgery, but also to the metabolic adaptations to low protein-energy intakes. Besides its significant effect on weight loss, bariatric surgery allows the improvement of obesity-related comorbidities, reduction of insulin-resistance and low grade inflammation [START_REF] Chang | The effectiveness and risks of bariatric surgery: an updated systematic review and meta-analysis, 2003-2012[END_REF][START_REF] Shah | Review: long-term impact of bariatric surgery on body weight, comorbidities, and nutritional status[END_REF][START_REF] Sjostrom | Lifestyle, diabetes, and cardiovascular risk factors 10 years after bariatric surgery[END_REF] which might contribute to a better protein efficiency compared to the metabolic state previous to surgery. Despite the massive weight loss during the first three months, a neutral nitrogen balance was found likely explained by the strong reduction of nitrogen losses in parallel with reduction in nitrogen intakes confirming adaptation of the body to lower nitrogen intakes [START_REF] Hector | Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise[END_REF][START_REF] Quevedo | Nitrogen homoeostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body leucine kinetics during a reduction from a high to a moderate protein intake[END_REF] . Nevertheless, the nitrogen balance method does not predict the orientation of metabolic fluxes (protein synthesis and degradation, interactions between muscle and splanchnic area) underlying the limitation of the method. Only few authors have evaluated changes in protein turnover after massive weight loss [START_REF] Henderson | Effects of adiposity and 30 days of caloric restriction upon protein metabolism in moderately vs. severely obese women[END_REF] which mainly explain body composition changes. After vertical banded gastroplasty, protein breakdown in patients decreased 3 months after surgery with no change in protein synthesis compared to before surgery, illustrating a slower protein turnover [START_REF] Van Gemert | Energy, substrate and protein metabolism in morbid obesity before, during and after massive weight loss. International journal of obesity and related metabolic disorders[END_REF] . After bariatric surgery, whatever the types of surgery, the susceptibility for protein malnutrition is very realistic not only as a result of massive weight loss, but also because the patients cannot consume several foods rich in protein [START_REF] Verger | Nutritional and Protein Deficiencies in the Short Term following Both Gastric Bypass and Gastric Banding[END_REF][START_REF] Verger | Micronutrient and Protein Deficiencies After Gastric Bypass and Sleeve Gastrectomy: a 1-year Follow-up[END_REF] . Nutritional deficiences in obese patients after bariatric surgeries have been pointed out, notably for protein intakes [START_REF] Verger | Nutritional and Protein Deficiencies in the Short Term following Both Gastric Bypass and Gastric Banding[END_REF][START_REF] Verger | Micronutrient and Protein Deficiencies After Gastric Bypass and Sleeve Gastrectomy: a 1-year Follow-up[END_REF] . In Verger et al. study, mean protein intakes 3 months after surgery were 0.38 g/kg/d after gastric bypass and 0.39 g/kg/d after sleeve gastrectomy [START_REF] Verger | Micronutrient and Protein Deficiencies After Gastric Bypass and Sleeve Gastrectomy: a 1-year Follow-up[END_REF] . Interestingly, Giusti et al. have observed a significant decrease in meat and vegetarian protein intakes at 1 mo after a Roux-en-Y gastric bypass and remained low until 1 y after surgery [START_REF] Giusti | Energy and macronutrient intake after gastric bypass for morbid obesity: a 3-y observational study focused on protein consumption[END_REF] . In our study, spontaneous protein intakes of the patients at M3 were 0.43±0.03 g/kg/d whatever the types of surgery. Unfortunately no distinction of protein source could be made. These values are inferior to the protein requirement evaluated 0.63±0.38 g/kg/d. In addition, 86% of our obese population do not reach the APR at M3. These observation emphasized the need of nutritional assessment and protein support of these patients after weight reducing surgery, especially during the phase of rapid weight loss. At M12, the average protein requirement (0.87 [95%CI, 0.75-0.98] g/kg/d) increased compared to the one observed at M3. Whereas weight and fat mass continued to decrease, the loss of FFM has been classically reported to be stabilised one year and up to at least 3 years after bariatric surgery [START_REF] Verger | Micronutrient and Protein Deficiencies After Gastric Bypass and Sleeve Gastrectomy: a 1-year Follow-up[END_REF][START_REF] Giusti | Energy and macronutrient intake after gastric bypass for morbid obesity: a 3-y observational study focused on protein consumption[END_REF] . The maintenance of FFM should involved an adaptation of protein metabolism. Only a few data is available on the evolution of protein metabolism after bariatric surgery. Tamboli et al. have observed higher 24h-urinary levels of 3-methylhistidine 12 months following bariatric surgery, illustrating an increase in muscle protein breakdown [START_REF] Tamboli | Body composition and energy metabolism following Roux-en-Y gastric bypass surgery[END_REF] .

In patients following a Roux-en-Y gastric bypass, urinary urea nitrogen excretion increased 12 months after surgery [START_REF] Giusti | Energy and macronutrient intake after gastric bypass for morbid obesity: a 3-y observational study focused on protein consumption[END_REF] . Alongside these changes, protein synthesis might also increase to balance the elevation in protein degradation and nitrogen losses in order to ensure the slowing down of FFM loss. Even if the mechanisms of protein metabolism regulation in bariatric surgery have yet to be elucidated, an adaptation of protein metabolism must be seriously considered to explain the higher level of protein requirement at 12 months after surgery. Some studies indicate that the maintenance of weight loss is achieved among subjects assigned to a higher protein proportion in the diet (25% of energy consumed) after weight loss induced by a low-calorie diet [START_REF] Larsen | Diets with high or low protein content and glycemic index for weight-loss maintenance[END_REF] . A mean daily of protein intake of 64.1±2.9 g/d (58.01±14.23 g/d in our study) at 12 months following bariatric surgery has been reported in a previous cohort of 50 patients. This study has shown that a larger protein intake is associated with a better preservation of lean mass after bariatric surgery even 12 months postoperatively [START_REF] Moize | Protein intake and lean tissue mass retention following bariatric surgery[END_REF] .

In addition, loss of fat free mass is associated with weight regain [START_REF] Vink | The effect of rate of weight loss on long-term weight regain in adults with overweight and obesity[END_REF] due to a diminished resting metabolic rate [START_REF] Hopkins | Modelling the associations between fat-free mass, resting metabolic rate and energy intake in the context of total energy balance[END_REF] , but also due to the triggering effect of fat free mass loss to promote an increased energy intake in an attempt to restore FFM to an optimal level as refered to the so called "collateral fattening" concept [START_REF] Dulloo | Passive and active roles of fat-free mass in the control of energy intake and body composition regulation[END_REF] . This suggests the importance to adapt protein intakes to the needs of the patients after surgery to avoid notably excessive loss of FFM. The quality of dietary protein is also of importance for FFM improvement as we demonstrated recently beneficial effects of soluble milk proteins to preserve muscle and lean body mass in old subjects [START_REF] Gryson | Four-month course of soluble milk proteins interacts with exercise to improve muscle strength and delay fatigue in elderly participants[END_REF][START_REF] Gryson | Fast proteins" with a unique essential amino acid content as an optimal nutrition in the elderly: growing evidence[END_REF] .

In conclusion, the present study demonstrated an increased protein requirement in severe and morbid obese subjects. Since most of obese patients spontaneously do not reach the protein intake targets, it is particularly relevant to reconsider protein intake in the monitoring of morbidly obese subjects in pre-and post-operative periods, especially with regard to potential a protein deficit with underestimated consequences such as sarcopenia or immune deficit or post-operative complications. Further studies investigating the impact of protein supplementation in patients receiving bariatric surgery with definite protein targets should then be proposed. The estimation of protein requirements in patients with morbid obesity and following bariatric surgery needs to be precise and validated by similar and alternative methods in order to address new specific recommandations for severely and morbidly obese patients for the regular monitoring of this population, but also on a long term basis after bariatric surgery.

ACKNOWLEDGMENTS

This study was supported by research funds from Lactalis ® financial and by French National

TABLES AND FIGURES

  Spontaneous protein intakes were estimated at 93.21±21.59 g/d (0.80±0.21 g/kg/d), 45.57±16.41 g/d (0.46±0.13 g/kg/d) and 58.01±14.23 g/d (0.71±0.18 g/kg/d) respectively at M0, M3 and M12 after bariatric surgery.

  program for Clinical Research (PHRC). The authors gratefully acknowledge all the medical staff from the Clinical Nutrition and the Digestive Surgery departments of the CHU of Clermont Ferrand and Lyon, the nurses and technician from the Human Nutrition Laboratory, and the Biochemical department of Lyon civil Hospitals for their skillful medical and technical assistance. CG contributed to the design of the study, data analysis, data interpretation, and writing of the report. AM contributed to the design of the study, study oversight, recruitment of participants, data collection, biological analysis, data analysis, data interpretation, and writing of the report. AMD, AB and NF contributed to study oversight, recruitment of participants, data collection. BP contributed to statistical analysis, data interpretation, and writing of the report. KS and MR contributed to the recruitment of participants and surgical procedures. ED and ML contributed to the design of the study, the recruitment of participants, study oversight, data analysis, and data interpretation. PLR contributed to the data interpretation. CL, MM and NF contributed mainly to the data collection and interpretation of the "Validation group" and to the recruitment of participants, study oversight, and data interpretation of the "Experimental group". YB had the idea for the study and contributed to the design of the study, study oversight, data interpretation and writing of the report. All authors read and approved the final manuscript. Sources of Support: French National Program for Clinical Research (PHRC) and research funds from Lactalis ® . Disclosure of interests: CG, AM and YB have received research funding from Lactalis. PLR is employed by Lactalis Group.

Figure 2 .

 2 Figure 2. Nitrogen intakes, losses and balance before, 3 months and 1 year after bariatric

Figure 3 .

 3 Figure 3. Relationship between nitrogen balance and protein intakes, and determination of

Figure 4 .

 4 Figure 4. Determination of average protein requirement in an additional group of obese

Figure 1 . 33 2 28 Figure 2

 133282 Figure 1.

Table 1 .

 1 Body composition characteristics before, 3 months and 12 months after bariatric surgery.

		Baseline	M3	M12
	Body weight (kg)	119.1±5.1	100.0±5.1 *	84.8±5.4 *#
	Body mass index (kg/m²)	43.9±1.4	36.8±1.5 *	31.0±1.6 *#
	Body fat mass (kg)	57.5±3.2	44.9±3.5 *	32.6±3.7 *#
	Body fat mass (%)	48.4±1.3	44.6±1.4 *	37.6±2.5 *#
	Body fat free mass (kg)	61.1±3.1	54.8±2.7 *	51.4±3.0 *#
	Body water (L)	44.7±2.3	40.1±2.0 *	37.6±2.2 *#
	Resting metabolic rate (kcal/d)	1953±97	1657±90 *	1549±86 *#
	Data are mean±SEM (based on isotopically labeled water measurements).
	Baseline: before; M3: 3 months after surgery; M12: 12 months after surgery
	* significant differences vs baseline, P<0.05
	# significant differences between M12 and M3, P<0.05

Table 2 .

 2 Biological characteristics of the patients before, 3 months and 12 months after, bariatric surgery.Data are mean±SEM Baseline: before; M3: 3 months after surgery; M12: 12 months after surgery IGF-1: Insulin Growth Factor-1; GH: Growth Hormone; GLP-1: Glucagon Like peptide 1; IL-6: Interleukine-6 * significant differences vs baseline, P<0.05 # significant differences between M12 and M3, P<0.05

		Baseline	M3	M12
	Fasting plasma glucose (mmol/L)	6.1±0.3	5.3±0.2 *	5.1±0.2 *
	Fasting plasma insulin (mUI/L)	14.3±2.2	7.0±1.2 *	5.4±1.1 *
	HOMA-IR index	4.0±0.7	1.7±0.3 *	1.3±0.3 *
	Triglycerides (mmol/L)	1.37±0.10	1.08±0.08 *	0.99±0.08 *
	Total-cholesterol (mmol/L)	4.88±0.25	4.20±0.19 *	4.60±0.16 #
	HDL-cholesterol (mmol/L)	1.14±0.05	1.08±0.03	1.41±0.06 *#
	LDL-cholesterol (mmol/L)	3.12±0.24	2.62±0.19	2.87±0.23
	NEFA (µmol/L)	627.9±43.7	737.3±48.1	483.2±58.9 #
	Albumin (g/L)	40.2±0.6	40.4±0.7	39.7±0.7
	Prealbumin (g/L)	0.25±0.01	0.20±0.01 *	0.23±0.01 *
	Adiponectin (µg/mL)	5.2±0.7	6.7±0.6	10.5±1.1 *#
	Leptin (ng/mL)	53.4±4.1	23.4±3.2 *	18.7±4.4 *
	Acylated ghrelin (pg/mL)	17.0±3.5	8.1±3.1	23.9±7.6 #
	Unacylated ghrelin (pg/mL)	132.6±17.8	94.0±29.1 *	102.3±21.5
	IGF-1 (µg/L)	161.2±12.2	136.2±15.9 *	165.6±14.4 #
	GH (mUI/L)	1.5±0.5	5.3±1.4	14.2±3.5 *#
	GLP-1 (µg/mL)	1.78±0.42	1.30±0.19	0.92±0.15
	High sensitive C-reactive protein	6.63±1.32	6.38±2.55	2.70±1.00
	(mg/L)
	IL-6 (pg/mL)	2.36±0.29	2.13±0.20	1.35±0.16 *#


Gilles Nevez
email: gilles.nevez@univ-brest.frsolene.legal@univ-brest.fr

Philippe M Hauser
email: philippe.hauser@chuv.ch

Solène Le Gal

Pneumocystis jirovecii

Keywords: Pneumocystis jirovecii, Pneumocystis Pneumonia, Ascomycete, Airborne acquisition and transmission

P. jirovecii is the species of the Pneumocystis genus infecting specifically humans. It has a worldwide distribution. It is not routinely cultivable. It does not have a proven environmental niche. It is acquired and transmitted from host-to-host via the airborne route. It shows a strong tropism for the lungs. This pathogen constitutes a unique phenomenon in medical mycology. Ascospores contained in asci and/or asci themselves represent the transmitted infectious stages. P. jirovecii's cytoplasmic membrane does not comprise ergosterol that is replaced by cholesterol. For this reason, this fungus is resistant to amphotericin B and azoles. The first genome sequence of P. jirovecii was published in 2012 (8,1 Mbp, 3898 coding genes) DISEASE FACTS: P. jirovecii is an opportunistic fungus which causes a life-threatening pneumonia in immunocompromised patients (Pneumocystis pneumonia, PCP). PCP is the most frequent AIDS-defining disease in developed countries. Its incidence in this context is about 400,000 cases with ca. 50,000 deaths. Torpid infections may occur in patients with acute or chronic bronchopulmonary diseases, primary infected infants, pregnant women, and health care workers in contact with infected patients. These colonized patients and PCP patients represent sources of the infection in the community and hospitals. The first line for treatment is sulfamethoxazole-trimethoprim (SMX-TMP). The combination of SMX-TMP with echinocandins deserves to be evaluated. Chemoprophylaxis remains essential but isolation measures of infected patients within the hospitals are required.

o

  containing ascospores (indicated by the arrow, Wright Giemsa stain, X 1000) B. Asci (Toluidine blue stain, X 400) C. Cluster of P. jirovecii organisms (indicated by the arrow, Wright Giemsa stain, X 100) D. Asci (Indirect two-step IFA, anti-ascus antibodies, MONOFLUO Kit P. jirovecii Bio-Rad, Variation of major surface glycoproteins (MSGs) o Masking of beta-1,3-D glucan o Lack of chitin o Lack of outer chain of N-mannans on MSGs mitosis leading to an ascus with eight ascospores (4) -Released ascospores (5)

  Literature 1. Choukri, F. et al. (2010). Quantification and Spread of Pneumocystis jirovecii in the Surrounding Air of Patients with Pneumocystis Pneumonia. Clinical Infectious Diseases 51, 259-265 2. Cissé, O.H. et al. (2012) De novo assembly of the Pneumocystis jirovecii genome from a single bronchoalveolar lavage fluid specimen from a patient. MBio 4, e00428-12 3. Cushion, M. et al. (2010) Echinocandin Treatment of Pneumocystis Pneumonia in Rodent Models Depletes Cysts Leaving Trophic Burdens That Cannot Transmit the Infection. PLoS ONE 5, e8524. 4. Edman, J. et al. (1988) Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi.

	Nature 334:519-22
	5. Hauser, P.M. & Cushion, M.T. (2018) Is sex necessary for the proliferation and
	transmission of Pneumocystis? PLoS Pathogens 14, e1007409.
	6. Le Gal, S. et al. (2012) A cluster of Pneumocystis infections among renal transplant
	recipients: molecular evidence of colonized patients as potential infectious sources of
	Pneumocystis jirovecii. Clinical Infectious Diseases 54, e62-71
	7. Le Gal, S. et al. (2015) Pneumocystis jirovecii in the air surrounding patients with
	Pneumocystis pulmonary colonization. Diagnostic Microbiology and Infectious
	Disease 82, 137-142
	8. Ma, L. et al. (2016) Genome analysis of three Pneumocystis species reveals adaptation
	mechanisms to life exclusively in mammalian hosts. Nat. Commun. 7, 10740.
	9. Rabodonirina, M. et al. (2004) Molecular evidence of interhuman transmission of
	Pneumocystis pneumonia among renal transplant recipients hospitalized with HIV-
	infected patients. Emerging Infectious Diseases 10, 1766-1767
	10. Stringer, J.R. et al. (2009) Spelling Pneumocystis jirovecii. Emerging Infectious
	Diseases. 15, 506
	TAXONOMY AND CLASSIFICATION:
	KINGDOM: Fungi
	PHYLUM: Ascomycota
	SUB-PHYLUM: Taphrinomycotina
	CLASS: Pneumocystomycetes
	ORDER: Pneumocystidales
	FAMILY: Pneumocystidacease
	GENUS: Pneumocystis


N Jarrige

G Cazeau

G Bosquet

J Bastien

F Benoit

E Gay

Effects of antimicrobial exposure on the antimicrobial resistance of Escherichia coli in the digestive flora of dairy calves

Keywords: E. coli, antimicrobial resistance, antimicrobial use, dairy calf

Veal calves are often identified as reservoirs for antimicrobial resistant Escherichia coli (E. coli). This production is closely linked with dairy production, as young calves -mostly males -are collected from dairy farms to enter the fattening process. The aim of this prospective study was to explore the factors on dairy farms that favour the selection of antimicrobial resistance (AMR) in the digestive E. coli strains of young calves and to assess whether the resistance levels and selection pressure were the same for males and females. The exposure of calves to antimicrobials was investigated through three factors: antimicrobial treatment of calves; feeding of calves with milk from cows treated with antimicrobials; and the consumption of colostrum from cows treated with antimicrobials at dry-off.

The study design involved 100 dairy farms. A calf of each sex was selected from birth on each farm. Information on the calves' exposure to antimicrobials was collected daily and calves were sampled (rectal swab) two weeks after birth, then seven weeks after birth for females only. Laboratory analyses included culture on two distinct media: a non-selective medium (identifying dominant flora) and a medium containing ceftiofur to select the extended-spectrum beta-lactamase (ESBL) phenotype. Susceptibility testing was performed on an E. coli strain from each medium. Generalised linear models were used to assess associations between the resistance of E. coli strains and antimicrobial exposure. A set of 280 swabs from healthy calves were analysed. In dominant

INTRODUCTION

The commensal and pathogenic nature of Escherichia coli associated with its great genomic plasticity makes it a central species in the emergence and spread of antimicrobial resistance (AMR). The epidemic dissemination of extended-spectrum beta-lactamase (ESBL) enterobacteriaceae in humans, animals and the environment is particularly worrying.

Among food production animal species, calves are considered to be reservoirs of AMR strains. Data from the French monitoring network for antimicrobial resistance in bacteria from diseased animals [START_REF] Anses | Resapath -French surveillance network for antimicrobial resistance in pathogenic bacteria of animal origin, 2018 annual report[END_REF] indicate that E. coli strains isolated from neonatal calf diarrhoea carry most of the resistances observed in the beef sector. On dairy farms where calves are born, some of the females are kept in the farm for herd renewal, while the other females and most of the males are sold for fattening around the age of 15 days. Calves from different farms are grouped together in batches and fattened on specialised farms to be slaughtered at the age of 5 to 6 months. Antimicrobial treatments are numerous during the fattening process [START_REF] Jarrige | Quantitative and qualitative analysis of antimicrobial usage in white veal calves in France[END_REF] and AMR monitoring programmes and epidemiological studies have measured the high prevalence of E. coli harbouring resistances and ESBL in calves at slaughterhouses [START_REF] Haenni | Comparative prevalence and characterization of ESBL-producing Enterobacteriaceae in dominant versus subdominant enteric flora in veal calves at slaughterhouse, France[END_REF]. These veal calves are directly introduced into the food chain and could expose human health through meat consumption. However, it has also been evidenced that by the time they arrive at fattening farms, these calves are already carrying many resistant E. coli in their digestive tract. Major resistances concern amoxicillin, tetracycline and streptomycin. ESBL E. coli from subdominant flora is also frequent [START_REF] Gay | Antimicrobial Usages and Antimicrobial Resistance in Commensal Escherichia coli From Veal Calves in France: Evolution During the Fattening Process[END_REF]. Calves therefore appear to acquire AMR very early in their life, even on their farm of birth [START_REF] Berge | Animal and farm influences on the dynamics of antibiotic resistance in faecal Escherichia coli in young dairy calves[END_REF].

The European Food Safety Authority (EFSA) has investigated this issue and identified two putative sources of calves' exposure to antimicrobials or their residues on dairy farms. First, the colostrum from cows treated with antimicrobials at the beginning of their dry period can be administrated to calves. Second, the milk from cows treated with antimicrobials during their lactation (waste milk, prohibited for sale) that can also be used to feed calves during the withdrawal period in order to limit economic losses (EFSA Panel on Biological Hazards [START_REF] Efsa Panel On | Risk for the development of Antimicrobial Resistance (AMR) due to feeding of calves with milk containing residues of antibiotics[END_REF]. The EFSA report finally recommended avoiding feeding calves colostrum and milk containing residues of antimicrobials that could select for antimicrobial-resistant bacteria, particularly those selecting for resistance to high-priority critically important antimicrobials (CIAs).

The aim of the present longitudinal study was to explore on dairy farms the factors associated with the selection of antimicrobial resistance among E. coli strains in the digestive flora of calves of two weeks of age, and to verify whether the resistance and selection pressure were the same in males and females. The assumption was that males and females could be treated differently on farms according to their future destination (fattening or herd renewal). The hypotheses included examining the influence of three potential modes of exposure of calves to antimicrobials: (i) antimicrobial treatment given directly, (ii) consumption of milk from cows treated with antimicrobials, (iii) consumption of colostrum from cows having received antimicrobial treatment at dry-off.

MATERIALS AND METHODS

The study design involved a cohort of 100 dairy farms. We selected ten voluntary veterinary practitioners spread over the main cattle production areas in France. Each veterinarian was responsible for choosing ten farms: six farms that usually feed their calves with milk from cows treated with antimicrobials and four farms never doing so. All the farms selected had to meet the following inclusion criteria: being dairy farms with at least 40 cows, having at least five cows close to calving, usually selling their males for veal calf production and keeping their females for herd renewal.

At each farm, two calves -one male and one female -were studied from birth to two weeks of age for males (corresponding to their departure to a fattening farm) and from birth to seven weeks for females (before completing the weaning). Farmers completed questionnaires on farm characteristics (herd size, main breed, average milk production per cow, geographical area), calf feeding (type of milk, distribution system) and the selected calves' characteristics (breed, estimated birth weight, conditions of birth). They also completed daily questionnaires on the antimicrobial exposure of selected calves: i) consumption of colostrum during the first 48 hours of life and the antimicrobial treatments used at dry-off for the cow that supplied the colostrum, ii) consumption of milk from cows treated with antimicrobials, whether during the cow's treatment or the withdrawal period (date, antimicrobial used and route of administration), iii) the antimicrobial treatments given to the calf (date of treatment, antimicrobial used).

All these calves were sampled at the age of 15 days (rectal swab) and females were sampled again seven weeks after birth. The sample were undertaken by veterinarians. Sick calves at sampling time were excluded from the study. The digestive flora of diseased calves can be considerably modified and it was estimated that their inclusion in that study, focused on commensal E. coli, could bias the results. If the calf was sick at sampled time, another calf was selected at birth in the farm to restart the protocol. Rectal swabs were sent to the veterinary laboratory selected for the study within 24 hours of sampling and processed upon arrival. They were directly plated in parallel: i) onto MacConkey agar (bioMérieux, Marcy l'Etoile, France) for the culture of the dominant flora and ii) onto selective ChromID ESBL agar containing ceftiofur (bioMérieux) for the selection of ESBL from the subdominant flora. After incubation at 37°C for 24 hours, one presumptive E. coli colony was arbitrary selected from each plate and isolates were identified through matrix-assisted laser desorption ionization using a time-of-flight (MALDI-TOF) mass spectrometer [START_REF] Van Veen | High-throughput identification of bacteria and yeast by matrixassisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories[END_REF]). If the isolate was not identified as E. coli, another colony was selected and identified. The process was repeated twice if needed.

Antimicrobial susceptibility was tested using the disc diffusion method on Mueller-Hinton agar and results were interpreted according to the breakpoints recommended by the veterinary section of the Antibiogram Committee of the French Society of Microbiology (CA-SFM) (http://www.sfm-microbiologie.org/). The antimicrobials used were seven beta-lactams (amoxicillin, amoxicillin/clavulanic acid, cefalotin, ceftiofur, cefquinome, cefoxitin and ertapenem) and nine non-beta-lactam antimicrobials (tetracycline, streptomycin, gentamicin, kanamycin, florfenicol, colistin, trimethoprim-sulfamethoxazole, nalidixic acid and enrofloxacin).

Florfenicol was tested for epidemiological purposes using the breakpoints assigned for Pasteurella spp.

Ertapenem is not authorised in veterinary medicine, but the molecule was tested considering its importance for human health. Synergy between the amoxicillin/clavulanic acid and ceftiofur discs was used to detect the presence of an ESBL phenotype, according to CA-SFM recommendations. Cefoxitin was used to highlight the AmpC phenotype. Multidrug resistance (MDR) was defined as resistance to at least three antimicrobial molecules of distinct categories, using the following seven molecules as markers of the antimicrobial categories: amoxicillin, ceftiofur, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, enrofloxacin and florfenicol.

The resistance ratio for each antimicrobial tested was calculated as the number of animals harbouring resistant E. coli divided by the total number of animals tested. Comparisons of resistance ratios between males and females at the age of 15 days and comparisons of resistance ratios for females at different stages (15 days versus seven weeks after birth) were made using a simple Chi-squared test. The significance level was set to p≤ 0.05. Logistic models were used to evaluate associations between antimicrobial resistance and exposure factors. The models have been implemented for samples taken from 15 days calves whose males are directly sent to fattening workshops and slaughtered around 5-6 months. For E. coli isolated from dominant flora, resistance was the dependent variable and was taken into account in various forms in different models: i) resistance to each antimicrobial tested (antimicrobials for which resistance ratios were less than 1% were not tested in the modelling process), ii) multidrug resistance, iii) the ESBL phenotype. For E. coli isolated from sub-dominant flora, only the ESBL phenotype was tested.

In order to select the variables to include in the models, univariate analyses were first performed. Variables concerning calves, their birth, and their feeding conditions were tested: sex of the calves, birth weight, calf breed (dairy, cross breed, mixed), farms geographical areas (4 classes), lactation rank, birth area (separate and clean or not), type of milk given to the calves (dairy milk or powder), milk distribution system used (individual, collective) and contact between calves. Three antimicrobial exposure variables were also tested: feeding with colostrum from cows treated with antimicrobials at dry-off, feeding with milk from cows treated with antimicrobials, and antimicrobial treatment of the calf.

To take into account that two calves were studied per farm and that each veterinarian had included ten farms in the sample, mixed models were used with two variables introduced as random effects (the farm and the veterinarian).

The significant variables at the univariate steps were introduced in multivariate models.

Exposures were considered globally, all antimicrobials taken together. The odds ratio (OR) and 95% confidence intervals were calculated. The associations were considered significant at p≤ 0.05.

In this work, several antimicrobials were studied jointly each having its own prevalence of resistance.

Antimicrobial exposures practices had also specific frequencies. It was then difficult to determine the optimal sample size. However, by including 182 calves in the study, it was possible to identify, with a power of 80%, an OR of 2.2, out of a proportion of 40% of resistance in the unexposed, with as many exposed as unexposed units. Statistical analyses were performed with the R software (R Core Team, 2017, R Foundation for Statistical Computing, version 3.4.0).

RESULTS

One hundred farms located in the main French dairy areas were monitored from April to September 2017. The average herd size was 84 cows, with an average milk production of 8,303 L per lactation. The first male and female calves born on the farm and meeting the inclusion criteria were included in the cohort. These calves were mainly Prim'holstein (61%), Montbéliarde (19%) or crossbred (15%). The calves with diarrhoea at the time of sampling were removed from the statistical analyses, which finally focused on 185 calves: 90 males and 95 females.

Exposure of calves to antimicrobials

All the calves received colostrum within 48 hours of their birth. For 65% of calves, the colostrum came from a cow treated with antimicrobials at dry-off. For 71% of them, the time between the cow's treatment and the distribution of colostrum (mean of 64 days) was compliant with the time defined in the summary of product characteristics (SPC) for the antimicrobial used. Cow treatments at dry-off mostly included 1 st -generation cephalosporins (35% of the calves concerned), penicillins (29%), aminoglycosides (11%), macrolides (2%) and 4 th -generation cephalosporins (<1%).

The exact antimicrobial treatments of calves were recorded daily. By two weeks after birth, 22% of the calves had already received at least one antimicrobial treatment. These treatments mostly contained aminoglycosides (11% of the total calves), penicillins (8%), polypeptides, i.e. colistin (6%), amoxicillin with clavulanic acid (4%) or trimethoprim with sulfonamides (3%). Other antimicrobial classes (macrolides, tetracyclines and phenicols) were rarely used (less than 1% of the calves). By seven weeks old, 31% of the female calves had been treated at least once by antimicrobials.

By two weeks after birth, one-third of the calves (31%) had consumed, at least once, milk from cows treated with antimicrobials, whether during the treatment or withdrawal periods. The milk came from cows treated by the intramammary route (50% of the calves concerned), by the parenteral route (14%) or both of these routes (36%). Cow treatments contained aminoglycosides (21% of the calves consumed milk from cows treated with this antimicrobial class), tetracycline (16%), polypeptides, i.e. colistin (13%) or penicillins (11%). By seven weeks old, nearly 40% of the females had consumed milk from cows treated with antimicrobials.

For these three antimicrobial exposures (colostrum from cow treated with an antimicrobial at dry-off; antimicrobial treatment of the calf; consumption of milk from cows treated with antimicrobials), no significant difference was found according to the sex of the calves.

Resistance of E. coli strains in dominant flora

A strain of E. coli was isolated from the dominant flora for each of the 280 samples (185 samples at 15 days and 95 samples at 7 weeks). For 15-day-old calves, the proportion of E. coli resistance in dominant flora was high for streptomycin (70%), tetracycline (68%), amoxicillin (64%) and kanamycin (55%) (Table 1). Resistance proportions for CIAs were low: 3% for ceftiofur, 2% for cefquinome and 3% for enrofloxacin. There was no significant difference between the resistance proportions observed for males and females. For females, resistance was lower at 7 weeks old than at 15 days old for almost all the antimicrobials and significant for the following ones: amoxicillin (23.2% vs. 64.2% at 15 days old), tetracycline (29.5% vs. 66.3%), nalidixic acid (1.1% vs. 14.7%), trimethoprim-sulfamethoxazole (4.2% vs. 27.4%) and the three aminoglycosides (streptomycin 26.3% vs. 66.3%, kanamycin 17.9% vs. 52.6% and gentamicin 1.1% vs. 9.5%) (Table 1).

Almost a third (32%) of isolates from 15-day-old calves had an MDR profile. The most frequent MDR associations (75% of multidrug resistant strains) combined amoxicillin, tetracycline and trimethoprimsulfamethoxazole resistances. For 7-week-old females, MDR was less common (8%).

The ESBL phenotype was identified for 2% of E. coli strains isolated from calves' dominant flora. There was no ESBL phenotype among samples from 7-week-old females.

Resistance of E. coli strains in sub-dominant flora

An E. coli strain was isolated on selective medium for 21% of the samples (n=58/280). Most of them (93.5%) were confirmed as having an ESBL profile after phenotypic analysis. One isolate was confirmed as AmpC.

By two weeks after birth, 22% of the calves were carrying ESBL E. coli in their digestive subdominant flora, with equivalent levels for males and females. The proportions of co-resistance were very high for tetracycline (82.5%), streptomycin (>85.0%) and kanamycin (72.5%), and lower for gentamicin (10.0%) and enrofloxacin (15.0%) (Table 2). The proportion of ESBL E.coli detected on selective medium decreased slightly over time from 22% at 15 days to 19% for 7-week-old females. Almost half of females with ESBL E. coli at 15 days old were still positive at 7 weeks old.

Association between antimicrobial resistance and exposure to antimicrobials

The univariate analyses carried out on the variables concerning calves and their conditions of birth and feeding, did not highlight specific factors having impact on the resistance of antimicrobials studied. Only sparse associations were observed. Considering these findings, the very low levels of resistance of some antimicrobials (not allowing many explanatory variables to be tested), it was decided to favor a model restricted to the main exposure variables in order to use similar models for all the antimicrobials tested. Consequently, four bimodal explanatory variables were kept in the final models: sex of the calf (whose impact assessment was a study objective) and three antimicrobial exposure variables (feeding with colostrum from cows treated with antimicrobials at dry-off; feeding with milk from cows treated with antimicrobials; and antimicrobial treatment of the calf). The implementation of mixed models to take into account the presence of two animals from the same farm or the involvement of the same veterinarian for ten farms was tested but the models showed little or no effect for random variables and prevented from reaching convergence for some of the models. It was verified that removing the random effects had no impact on the results for the other exposure indicators studied, they were therefore removed from the final models to help convergence and to keep the same model for all the antimicrobials.

No statistical association was identified between calves' consumption of colostrum from cows treated with antimicrobials at dry-off and proportions of resistant E. coli strains, whatever the resistance indicator used.

Similarly, no models revealed a link between the ESBL phenotype and calves' antimicrobial exposure on farms for either dominant or subdominant flora.

Feeding calves with milk from cows treated with antimicrobials was associated with significantly increased resistance in the dominant flora from 15-day-old calves for the following antibiotics: amoxicillin (OR=2.6[1.3; 5.6]), streptomycin (OR=2.7[1.3; 6.2]), gentamicin (OR=4.0[1.2; 14.5]), kanamycin (OR=3.0[1.5; 6.0]), tetracycline (OR=2.6[2.3; 5.8]) and trimethoprim-sulfonamides (OR=2.2 [1.1; 4.4]) (Table 3). The presence of MDR E. coli strains was also significantly associated with consumption of milk from treated cows (OR=2.3 [1.2;

4.7]).

Treating calves with antimicrobials was also associated with increased resistance to amoxicillin (OR=2.9[1.3; 7.2]), gentamicin (OR=4.6[1.3; 16.2]), florfenicol (OR=5.0[2.0; 12.6]) and trimethoprim-sulfonamides (OR=5.5[2.6; 12.2]) (Table 3). The presence of MDR E. coli at 15 days old was greatly increased by treatment of the calf (OR=6.0 [2.8; 13.3]).

DISCUSSION

Study results highlight that feeding calves with milk from cows treated with antimicrobials and treating calves themselves with antimicrobials increase faecal E. coli resistance to many antimicrobials and increase E. coli MDR phenotypes. The results also show time trend on resistance since an overall decrease in resistance burden was observed in female calves between the age of 15 days and seven weeks after birth.

From a methodological point of view, one of the strengths of the study lies in the daily data collection, which guarantees the high precision of data collected. The location of the farms reflected the main dairy production areas in France, and the farm characteristics were consistent with national data in terms of breed and milk production. The mean herd size of study farms was higher than the national mean, but this does not impact the observed relationships between resistance and antimicrobial exposure.

On the other hand, it must also be considered that in a field study context, the number of calves to which the study relates is still limited. It should be taken into account in the interpretation of the model results that certain relationships between the exposures studied and the AMR may not have been identified due to a lack of power. In the statistical modelling process, it was not possible to integrate random effects to avoid convergence issues. That could lead to an overestimation of significance. However, for risk factors and antimicrobials for which there was a significant effect, the p-values resulting from the models are clearly lower than 0.05, which allows being confident about the significance of the results obtained.

The study was designed to highlight associations between exposure and resistance, and did not estimate the prevalence of resistance or antimicrobial exposure since the farms were not randomly selected but chosen according to their practice concerning the feeding of calves with milk from treated cows. Nevertheless, the ratio between the six farms that usually fed their calves with milk from cows treated with antimicrobials and the four farms that never did seems to be realistic according to field veterinarians. The highest levels of resistance in the E. coli of dominant flora were for aminoglycosides, tetracyclines and penicillins. These results are consistent with those of a previous study using quite different methods of analysis, i.e. by determination of minimal inhibitor concentrations (CLSI 2013) and EUCAST epidemiological cut-offs [START_REF] Duse | Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves[END_REF].

The results of our study largely suggest a correlation of resistance for the four antimicrobials with higher levels of resistance and common patterns over time. This correlation is also supported by the modelling results, which highlighted very similar odds ratios for these antimicrobials. This situation could be linked to different phenomena: i) these antimicrobials are the most used in veterinary medicine causing significant selection pressure, ii) some are frequently used jointly for their synergistic effect (aminoglycosides and penicillins), iii) common resistance mechanisms with the acquisition by E. coli of one or more resistance genes carried by transferable mobile structures [START_REF] Poirel | Antimicrobial Resistance in Escherichia coli[END_REF].

The correlation between resistances was also highlighted through the MDR results in the models. The choice was made to consider gentamicin, among aminoglycosides in the MDR definition, whose impact on human health is significant. This choice may have led to an underestimation of the MDR rates compared to those which would have been obtained by choosing streptomycin, widely used in veterinary medicine.

Our study also showed a sharp decrease in resistance during the first weeks of life for females. For some antimicrobials, this decrease was not statistically significant but it could linked to an insufficient statistical power, the prevalence for these antimicrobials being low (<10%) and most below 5% for calves at 15 days. Some publications [START_REF] Berge | Assessing antibiotic resistance in fecal Escherichia coli in young calves using cluster analysis techniques[END_REF][START_REF] Pereira | In vivo selection of resistant E. coli after ingestion of milk with added drug residues[END_REF][START_REF] Pereira | Impacts of feeding preweaned calves milk containing drug residues on the functional profile of the fecal microbiota[END_REF] have also stressed a peak of resistance towards the second week of life and then a decrease, probably resulting from the natural evolution of the intestinal microbiota of calves in connection with the maturation of their digestive system until weaning.

The present study did not reveal any difference in antimicrobial exposure according to the sex or destination of the calves (fattening for males and herd renewal on the same farm for most of the females). The resistance rates for antimicrobials tested were equivalent for males and females and very similar to those found on fattening farms [START_REF] Gay | Antimicrobial Usages and Antimicrobial Resistance in Commensal Escherichia coli From Veal Calves in France: Evolution During the Fattening Process[END_REF].

Strains with an ESBL phenotype were rare (2%) among dominant flora but frequently identified (22%) using selective medium. However, the proportions of resistance found in our study were far below those observed in calves on arrival at fattening farms after departure from dairy farms (respectively 3% and 68%) [START_REF] Gay | Antimicrobial Usages and Antimicrobial Resistance in Commensal Escherichia coli From Veal Calves in France: Evolution During the Fattening Process[END_REF]. Two hypotheses may explain such a disparity: i) practices changed between the two studies (a new French decree in 2016 led in particular to a reduction in the use of 3 rd -and 4 th -generation cephalosporins), ii) the other production steps between dairy farms and fattening farms (i.e. batching centres) have an influence on resistance selection.

Until it develops its own ability to resist disease, the calf is entirely dependent on the immunity acquired by colostrum consumption [START_REF] Barrington | Bovine neonatal immunology[END_REF][START_REF] Mcguirk | Managing the production, storage, and delivery of colostrum[END_REF]. This contains high concentrations of antibodies that provide temporary and passive immunity against infections. Our study reported a widespread distribution of colostrum on the farms surveyed, reflecting the farmers' good knowledge of the importance of this practice. Most of calves received colostrum from cows for which the drying period after treatment was in accordance with the SPCs. Finally, no link was reported between consumption of colostrum from cows treated with antimicrobials at dry-off and the resistance levels of faecal E. coli from calves. This corroborates and supplements the findings of a previous study that focused on penicillin and aminoglycoside treatments because they were the only antimicrobials used in dry cow therapy in Sweden [START_REF] Duse | Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves[END_REF]. Maximum Residue Limit (MRL) assessments and the scientific literature have also shown that levels of antimicrobial residues in colostrum are low and decrease in keeping with the duration of the dry period [START_REF] Johnson | Persistence of antibiotics in milk from cows treated late in the dry period[END_REF][START_REF] Oliver | Residues in colostrum following antibiotic dry cow therapy[END_REF][START_REF] Rangel-Lugo | Prevention of antibiotic residues in veal calves fed colostrum[END_REF][START_REF] Hausler | Hot topic: investigating the risk of violative meat residues in bob veal calves fed colostrum from cows treated at dry-off with cephapirin benzathine[END_REF]. However, EFSA has suggested that further studies are needed on antimicrobial residues in colostrum and the thresholds at which selection for AMR occurs in calves (EFSA Panel on Biological Hazards (BIOHAZ) et al., 2017).

In our study, many calves were treated with antimicrobials during their first two weeks of life. The antimicrobials used corresponded globally to the French recommendations of good practices for the use of antimicrobials, particularly the use of colistin, penicillins and aminoglycosides as first-line treatments. No critically important antimicrobials were reported to have been used to treat calves. This is not surprising because since 2016 in France, the use of these molecules is subject to the result of a prior susceptibility test.

The results showed that the E. coli strains from treated calves had higher levels of resistance for amoxicillin, gentamicin and trimethoprim-sulfonamide, but also for florfenicol which was rarely used on these farms. The use of one antimicrobial selects resistance to this antimicrobial but also frequently participates in the coselection of other resistances. This is because some resistance genes are genetically linked, being carried by mobile genetic supports such as plasmids [START_REF] Carattoli | Resistance plasmid families in Enterobacteriaceae[END_REF][START_REF] Meunier | Plasmid-borne florfenicol and ceftiofur resistance encoded by the floR and blaCMY-2 genes in Escherichia coli isolates from diseased cattle in France[END_REF].

The impact of antimicrobial treatment on resistance has previously been demonstrated on a macroscopic scale by linking antimicrobial consumption and resistance levels in different countries and animal species [START_REF] Chantziaras | Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries[END_REF]. Available studies on calves have either been experimentation-based [START_REF] Berge | Field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal Escherichia coli in dairy calves[END_REF][START_REF] Bosman | Antimicrobial resistance in commensal Escherichia coli in veal calves is associated with antimicrobial drug use[END_REF] or have focused on calves on fattening farms [START_REF] Catry | Effect of Antimicrobial Consumption and Production Type on Antibacterial Resistance in the Bovine Respiratory and Digestive Tract[END_REF], making comparisons with our study limited. However, they have generally shown that an increase in resistancethough sometimes transient-was associated with the use of antimicrobials in calves.

Nevertheless, although neonatal diarrhoea is a common disease, the routine use of antimicrobials cannot be recommended in calves without systemic illness (normal appetite for milk, no fever) [START_REF] Constable | Antimicrobial use in the treatment of calf diarrhea[END_REF][START_REF] Berge | Targeting therapy to minimize antimicrobial use in preweaned calves: effects on health, growth, and treatment costs[END_REF]. Providing better care and healthier conditions for calves or using oral rehydration therapy, originally developed in human medicine, may also be helpful [START_REF] Victora | Reducing deaths from diarrhoea through oral rehydration therapy[END_REF].

The most striking result of this study is the negative impact of the distribution of milk from cows treated with antimicrobials on the AMR of calves' commensal digestive flora. There is no regulation prohibiting the distribution of this milk to calves in France. This practice is most often carried out for economic reasons, to limit the losses linked to this non-marketable milk [START_REF] Brunton | A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales[END_REF][START_REF] Rollin | The cost of clinical mastitis in the first 30 days of lactation: An economic modeling tool[END_REF]. Our study did not identify the frequency of this practice, the sample being selected on this criterion, but veterinarians encountered no difficulty in finding such farms even though they had to include six farms giving this milk to calves, which shows that this practice is quite common.

In our study, the consumption of milk from treated cows increased the resistance of E. coli to a broad range of antimicrobials (amoxicillin, aminoglycosides, tetracycline, trimethoprim-sulfonamide association). It would have been beneficial to distinguish cow treatments according to whether they were intramammary or parenteral. This was not possible in the modelling process considering the very small number of parenteral route treatments.

The effect of milk consumption from treated cows on streptomycin resistance has already been evidenced in Sweden and Spain [START_REF] Duse | Risk factors for antimicrobial resistance in fecal Escherichia coli from preweaned dairy calves[END_REF][START_REF] Maynou | Feeding of waste milk to Holstein calves affects antimicrobial resistance of Escherichia coli and Pasteurella multocida isolated from fecal and nasal swabs[END_REF]. These studies also found effects for quinolones and fluoroquinolones. Other experimental protocols have also demonstrated an impact on C3G/4G [START_REF] Brunton | A longitudinal field trial assesing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves[END_REF][START_REF] Pereira | In vivo selection of resistant E. coli after ingestion of milk with added drug residues[END_REF]. This was not the case in our study conducted under field conditions, where these antimicrobials were not used (in the case of fluoroquinolones) or rarely used (in the case of cephalosporins) to treat cows on the selected farms.

CONCLUSION

This study highlights the impact of feeding calves with milk from cows treated with antimicrobials and the impact of calves' antimicrobial treatments on the AMR of commensal E. coli in their digestive flora. It therefore appears important to consider the impact of these resistant bacteria in calves and their potential dissemination to other bacteria or other species -including humans -via their environment, contact with animals or consumption of their meat. Conversely, feeding calves with colostrum from cows treated with antimicrobials at dry-off does not appear to induce an increase in resistance levels of the commensal digestive flora. The impact of this practice also depends on the period of time between the date of the cow's antimicrobial treatment at dry-off and consumption by the calf of the treated cow's colostrum, which in our study often complied with the recommended periods. Consumption by calf of colostrum from cow treated with antimicrobial at dry-off; 2 Consumption by calf of milk from cow treated with antimicrobial; 3 Calf treated with at least one antimicrobial treatment. 4 Multidrug resistance: resistance to at least three antimicrobial molecules among seven tested (amoxicillin, ceftiofur, gentamicin, tetracycline, trimethoprimsulfamethoxazole, enrofloxacin and florfenicol).

Table 1 :

 1 Proportion of resistant E. coli strains isolated from the dominant flora (rectal swab) of healthy calves sampled at 15 days and 7 weeks after birth on dairy farms. 462

	Antimicrobials	Breakpoints (mm: S≥/R<)	15 days Male Female n=90 n=95 (%) (%)	Total n=185 (%)	p of comparison, male 15 days versus female 15 days	7 weeks Female n=95 (%)	p of comparison, female 15 days versus female 7 weeks
	Amoxicillin	21/14	63.3	64.2	63.8	1.00	23.2	<0.01
	Amoxicillin/clavulanic acid	21/14	6.7	6.3	6.5	0.76	2.1	0.28
	Cefalotin	18/12	6.7	4.2	5.4	0.52	1.1	0.39
	Cefoxitin	22/15	3.3	2.1	2.7	0.67	0.0	0.50
	Ceftiofur	21/18	2.2	3.2	2.7	1.00	1.1	0.62
	Cefquinome	22/19	2.2	2.1	2.2	1.00	1.1	1.00
	Ertapenem	28/26*	0.0	0.0	0.0	1.00	1.1	1.00
	Streptomycin	15/13	73.3	66.3	69.7	0.26	26.3	<0.01
	Gentamicin	18/16	4.4	9.5	7.0	0.25	1.1	0.02
	Kanamycin	17/15	57.8	52.6	55.1	0.55	17.9	<0.01
	Tetracycline	19/17	70.0	66.3	68.1	0.53	29.5	<0.01
	Nalidixic acid	20/15	10.0	14.7	12.4	0.38	1.1	<0.01
	Enrofloxacin	19/19	4.4	2.1	3.2	0.43	0.0	0.50
	Florfenicol	19/15 †	12.2	14.7	13.5	0.67	7.4	0.16
	Trimethoprim-sulfamethoxazole	16/10	28.9	27.4	28.1	0.87	4.2	<0.01
	Colistin	18/15	0.0	0.0	0.0	1.00	0.0	1.00

* Human breakpoints. † Breakpoints for Pasteurella spp. 463

Table 2 :

 2 Proportion of co-resistant ESBL E. coli strains isolated from the subdominant flora (selective culture medium containing ceftiofur) of healthy calves sampled at 15 days and 7 weeks after birth on dairy farms (rectal swab).

				15 days		7 weeks
	Antimicrobials	Breakpoints (mm: S≥/R<)	Male Female n=20 n=20 (%) (%)	Total n=40 (%)	Female n=18 (%)
	Streptomycin	15/13	85.0	85.0	85.0	66.7
	Gentamicin	18/16	15.0	5.0	10.0	16.7
	Kanamycin	17/15	70.0	75.0	72.5	50.0
	Tetracycline	19/17	85.0	80.0	82.5	88.9
	Nalidixic acid	20/15	40.0	40.0	40.0	22.2
	Enrofloxacin	19/19	15.0	15.0	15.0	11.1
	Florfenicol	19/15 †	40.0	35.0	37.5	22.2
	Trimethoprim-sulfamethoxazole	16/10	50.0	55.0	52.5	33.3
	Colistin	18/15	0.0	0.0	0.0	0.0

† Breakpoints for Pasteurella spp.

Table 3 :

 3 Results from generalised linear models relating significant associations between antimicrobial resistance of commensal E. coli isolates from the dominant flora of dairy calves (sampled at 15 days after birth by rectal swab) and antimicrobial exposure of calves on farms, all antimicrobial classes combined.Significant associations are in bold; *Reference class; **OR [95% CI] (p): odd ratio with 95% confidence interval and p value.

	Explanatory variables
	Modality (number of calves)

Acknowledgements

The authors would like to thank all the farmers and veterinarians involved in the study, Christelle Philippon for data input and Marisa Haënni and Agnese Lupo for their advice as bacteriologists.

Financial support

This work was supported by the EcoAntibio 2012-2017 action plan (grant 2015-206) implemented by the French Ministry for Agriculture. The funding organisation was not involved in the study design, data collection, analysis and interpretation or in writing the article.

Declarations of interest

None to declare.