M Carolina

MD Pardo Silva

PhD Omer T Njajou

MD Behrooz Z Alizadeh

MD Albert Hofman

PhD Jacqueline C M Witteman

Cornelia M Duijn

Dr A Cecile

PhD J W Janssens
email: a.janssens@erasmusmc.nl

PhD Cornelia M Van Duijn

gene mutations increase the risk of coronary heart disease in women

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

Introduction

Hereditary hemochromatosis is a genetic disorder characterized by iron overload [START_REF] Hanson | HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology[END_REF]. Iron overload in patients with hereditary hemochromatosis results in iron depots in the pancreas, liver, joints and heart. In the heart, such depots have been associated with coronary heart disease (CHD) and shortened life expectancy [START_REF] Pietrangelo | Hereditary hemochromatosis--a new look at an old disease[END_REF]. The most common cause of hereditary hemochromatosis are two common mutations in the HFE gene, C282Y and H63D [START_REF] Feder | A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis[END_REF][START_REF] Kotze | Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reversehybridization assay in the South African population[END_REF]. Over 80% of the patients with hereditary hemochromatosis are homozygous for the C282Y mutation, about 1% are homozygous for the H63D mutation and about 7% are compound heterozygous [START_REF] Hanson | HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology[END_REF]. In populations of European origin, an estimated 0.4% of the population is homozygous and 9% is heterozygous carrier of the C282Y mutation and 13% is homozygous and 2% heterozygous for the H63D mutation [START_REF] Hanson | HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology[END_REF].

Complications of iron overload may not only occur to homozygous carriers of the risk alleles. Compound heterozygotes and heterozygotes for the C282Y and H63D mutations also have a subtle increases in serum iron, serum ferritin and transferrin saturation [START_REF] Whitfield | Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins[END_REF][START_REF] Njajou | A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism[END_REF][START_REF] Burt | The significance of haemochromatosis gene mutations in the general population: implications for screening[END_REF][START_REF] Gurrin | HFE C282Y/H63D compound heterozygotes are at low risk of hemochromatosis-related morbidity[END_REF]. These slight changes suggest that heterozygotes are more likely to have a slow accumulation of iron, which may lead to pathology later in life. Iron deposits in the arterial wall trigger the low density lipoprotein cholesterol peroxidation and therefore contributes the formation of atherosclerotic lesions and to the inflammation leading to cardiovascular disease. [START_REF] Hanson | HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology[END_REF] [2] Some studies also showed that C282Y carriers had an increased risk of myocardial infarction, [START_REF] Feder | A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis[END_REF] and coronary heart disease (CHD). [START_REF] Kotze | Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reversehybridization assay in the South African population[END_REF] Similarly, smoking has also been associated to cardiovascular disease through increased inflammation, thrombosis, and oxidation of low-density lipoprotein cholesterol. [START_REF] Njajou | A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism[END_REF] Therefore, to properly disentangle the association between HFE mutations and cardiovascular disease requires taking into account the effect of smoking. Following this, previous work of our group also showed an increased risk of stroke among HFE carriers who smoked. [START_REF] Whitfield | Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins[END_REF] The aim of the present study was to examine the effect of the two common HFE C282Y and H63D mutations on incident CHD accounting for the effect of smoking. Since women are protected early in life from iron-related pathology by menstruation, we examined men and women separately.

Methods

Study population

The present analysis was performed within the Rotterdam Study, an ongoing populationbased study on the determinants of disease and disability in 7,983 subjects aged 55 years and older. Design, rationale and details of the study have been described previously [START_REF] Hofman | The Rotterdam Study: objectives and design update[END_REF]. The present analyses used baseline data (collected between 1990 and 1993) and follow-up morbidity and mortality data (collected until December 2001; mean follow-up of 8.3 years, standard deviation (SD) 2.7 years). The Medical Ethics Committee of the Erasmus MC University Medical Center approved the study protocol and all participants provided written informed consent.

Data collection

Baseline data collection

During home visits, a trained interviewer obtained information on health status, medical history, medication use and smoking status at baseline. Subsequently, participants were invited to the study center where they underwent an extensive clinical examination in which height, weight, systolic and diastolic blood pressures were measured. Body mass index (BMI) was computed as weight (kg) divided by height squared (m 2 ). Systolic and diastolic blood pressures were measured twice in sitting position, after 5 minutes rest, using a random-zero sphygmomanometer. The mean of the two measurements was used for the analysis.

Hypertension was defined as systolic blood pressure higher than 160 mm Hg, diastolic blood pressure higher than 100 mm Hg or the use of medication indicated to treat high blood pressure (hypertension grades 2 and 3) [START_REF]1999 World Health Organization-International Society of Hypertension Guidelines for the Management of Hypertension. Guidelines Subcommittee[END_REF]. Serum glucose, total cholesterol, high-density lipoproteins (HDL) cholesterol and C-reactive protein levels, as a marker of inflammation, were determined using an automated enzymatic procedure [START_REF] Van Gent | Cholesterol determinations. A comparative study of methods with special reference to enzymatic procedures[END_REF][START_REF]Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III)[END_REF]. Diabetes was diagnosed based on the use of medication, and/or a random or post-load glucose levels higher than 11.1 mmol/L [START_REF]Report of the expert committee on the diagnosis and classification of diabetes mellitus[END_REF]. Iron and ferritin levels and transferrin saturation were determined in subgroup as previously described [START_REF] Njajou | A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism[END_REF].

Follow-up assessment

During follow-up, information on fatal and non-fatal cardiovascular endpoints was obtained from the general practitioners and hospital records. Two research physicians and a cardiologist independently reviewed all information and classified all the events according to the International Classification of Diseases, 10 th edition (ICD-10) [START_REF] Who | International Statistical classification of diseases and related health problems[END_REF]. Incident CHD was defined as the occurrence of non-fatal myocardial infarction (ICD-10 code I21), revascularization procedure (percutaneous transluminal coronary angioplasty or coronary artery bypass graft) and cardiac death. Cardiac death was defined as death caused by myocardial infarction or other ischemic heart disease (ICD-10 codes I20 -I25), sudden cardiac death (ICD-10 code I46), cardiac arrhythmias (ICD-10 code I49), sudden death undefined (ICD-10 code R96) or death from heart failure (ICD-10 code I50).

Genotyping of HFE mutations

C282Y and H63D mutations were genotyped in a random sample of 3,798 individuals as described in details previously [START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF]. Baseline characteristics did not differ between randomly selected and non-selected participants.

Statistical analysis

From the 3,798 individuals who had C282Y and H63D genotype available, 3,435 had complete information on cardiovascular risk factors at baseline and were included in the analyses. There were only 10 homozygotes for the C282Y and 80 homozygotes for the H63D mutation among which 2 and 11 cases of incident CHD respectively. Previous studies on the HFE mutations and iron parameters showed that serum iron, serum ferritin and transferrin saturation were similar for C282Y and H63D heterozygotes [START_REF] Whitfield | Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins[END_REF][START_REF] Njajou | A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism[END_REF]. Therefore, we pooled carriers of C282Y or H63D mutations as HFE carriers. Differences in baseline characteristics between the HFE carriers and non-carriers were tested using chi-squared statistics (for categorical variables) or t-tests (for continuous variables). Variables that were not normally distributed, are presented as median with interquartile range and analyzed using the Mann-Whitney-U test. CHD incidence rates were calculated as number of events per 1,000 personyears. Risks of incident CHD were quantified as hazard ratios (HRs) from Cox proportional hazards models using age as time scale [START_REF] Korn | Time-to-event analysis of longitudinal follow-up of a survey: choice of the time-scale[END_REF]. Survival time was calculated from age at baseline to age at event. HRs were calculated crude and adjusted for sex, smoking status, hypertension, BMI, total serum cholesterol, HDL cholesterol, diabetes mellitus and CHD at baseline. Further, HRs were additionally adjusted for C reactive protein, to evaluate the role of inflammation in the relationship between HFE mutations and incident CHD. The association between HFE mutations and the risk of incident CHD was investigated overall and in sex and smoking subgroups given that both are associated with CHD and the clinical manifestations of iron overload in our study [START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF]. Proportionality of all models was tested by Schoenfeld residuals [START_REF] Schoenfeld | Partial residuals for the proportional hazards regression model[END_REF]. P-values lower then 0.05 were considered statistically significant.

Results

Genotypes and allele frequencies were in Hardy-Weinberg equilibrium (C282Y: CC 88.1%, CY 11.6%, YY 0.3%, p = 0.41; H63D: HH 73.2%, HD 24.5%, DD 2.4%, p = 0.25). At baseline, women had lower prevalence of smoking, hypertension and myocardial infarction and a higher mean BMI compared to men. Baseline characteristics by genotype are presented in Table 1. Carriers and non-carriers of the HFE mutations did not significantly differ in risk factors for cardiovascular disease with three exceptions in women. In women, HFE carriers had higher median body mass index (27.0 versus 26.6 kg/m 2 ; p=0.05), higher median Creactive protein levels than non-carriers (2.0 versus 1.7 mg/dL; p = 0.05) and a lower frequency of diabetes mellitus (10% versus 13%; p=0.05). HFE carriers had higher levels of iron and ferritin and higher transferrin saturation than non-carriers (Table 2). This difference was statistically significant for both iron levels and transferrin saturation in men and women.

A similar trend was observed within smoking strata, although the differences were not statistically significant (Data not shown). Figure 1 shows that females HFE carriers tend to have higher C-reactive protein levels than non-carriers, particularly among current or former smokers (p=0.08). In men, no differences in C-reactive protein levels between HFE carriers and non-carriers were observed.

During follow-up, 483 participants developed incident CHD. Men had higher CHD incidence than women (25.3 versus 11.8 cases/1,000 person years, p <0.001; Table 3). HFE 4). Exclusion of the homozygous and compound heterozygous carriers did not change the results (Data not shown). The interaction term HFE status * smoking status was not statistically significant.

Discussion

We have found that the HFE mutations are associated with higher risk of incident CHD in women. C-reactive protein, a strong predictor of CHD risk was not associated with HFE mutations in women with current or former smoking history. Our study was embedded in a large follow-up study of Caucasian individuals aged 55 and older. This long follow-up allows us to study outcomes that are the result of lifelong exposures and express at older ages like cardiovascular disease associated with iron overload.

A previous report based on a smaller sample of the Rotterdam study (n = 342) showed that HFE genotypes, also those who were heterozygous, were associated with higher levels of transferrin saturation and serum iron levels, albeit that levels in those homozygous for the mutations are exponentially higher [START_REF] Njajou | A population-based study of the effect of the HFE C282Y and H63D mutations on iron metabolism[END_REF]. Previous work of our group also showed an increased risk of stroke among HFE carriers who smoked [START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF]. Altogether, these results are in line with the mechanism linking HFE mutations and smoking to cardio-and cerebrovascular disease through damage to vessel wall and inflammation [START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF]. The underlying mechanism linking HFE and CHD is thought to be through iron-related oxidative stress and the subsequent damage to vessel wall and inflammation [START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF][START_REF] Zacharski | Reduction of iron stores and cardiovascular outcomes in patients with peripheral arterial disease: a randomized controlled trial[END_REF]. Such mechanism is also supported by endothelial dysfunction observed in normal subjects after receiving intravenous iron sucrose reported recently [START_REF] Zheng | Iron sucrose augments homocysteineinduced endothelial dysfunction in normal subjects[END_REF], as well as the positive association between markers of oxidative stress and iron therapy [START_REF] Bayes | Oxidative stress, inflammation and cardiovascular mortality in haemodialysis--role of seniority and intravenous ferrotherapy: analysis at 4 years of follow-up[END_REF]. Despite the solid grounds for this association, our data showed that CRP levels were not an intermediate factor in this association. A previous study on the association of chronic inflammation and hemochromatosis phenotype revealed that CRP levels did not change between homozygotes for the C282Y mutations with high or low iron stores [START_REF] Beutler | Chronic inflammation does not appear to modify the homozygous hereditary hemochromatosis phenotype[END_REF]. Most likely, blood CRP level is a biomarker for generalized inflammation not specific to the association HFE and CHD. How and through which early intermediate inflammatory pathways HFE is related to inflammation, remains to be determined. Another issue to be explained is the fact we only found significant evidence for association of HFE and CHD in women and not in men. While there is an ongoing discussion of the validity of subgroup testing by sex in genetic studies [START_REF] Patsopoulos | Claims of sex differences: an empirical assessment in genetic associations[END_REF], there are reasons to separate men and women for HFE. First, phlebotomy is the key strategy for prevention of hemochromatosis and women are therefore naturally protected due to their menstruation [START_REF] Allen | Ironoverload-related disease in HFE hereditary hemochromatosis[END_REF].

While this is a common explanation for sex-differences in CHD risk associated with HFE mutations, in our study an increased risk was found in female carriers rather than in male carriers. This may be related to the study design. We studied elderly people and therefore male carriers may have been selected out from the population due to early mortality related to HFE. In women, the gene effect is likely to express clinically later as they are protected by menstruation up to menopause. Second, it also should be noted that the major risks factors for CHD are smoking, body mass index, diabetes and lipid profile as supported by the effect of these co-variates when adjusting HR for CHD in women and men. The extent of CHD risk conferred by these factors is of such magnitude that the genetic predisposition associated with HFE carrier status is rapidly outweighed as seen in Table 4 where it is shown that greater CHD risk is associated with smoking status rather than with HFE mutations. Given the higher prevalence of smoking (current and former) among men compared to women at baseline, it is likely that the modest effect of HFE mutations on the risk of CHD is only apparent among women.

Because of their role in iron overload and their impact on CHD risk, HFE mutations have been of interest in public health [START_REF] Burt | The significance of haemochromatosis gene mutations in the general population: implications for screening[END_REF][START_REF] Burke | Public health strategies to prevent complications of hemochromatosis[END_REF][START_REF] Burke | Contribution of different HFE genotypes to iron overload disease: a pooled analysis[END_REF][START_REF] Cogswell | Screening for hemochromatosis. A public health perspective[END_REF][START_REF] Khoury | Genetics and public health: a framework for integration of human genetics into public health practices[END_REF][START_REF] Khoury | Population screening in the age of genomic medicine[END_REF][START_REF] Whitlock | Screening for hereditary hemochromatosis: a systematic review for the U.S. Preventive Services Task Force[END_REF], For long, they have been viewed as a model for genetic testing. A genetic-based strategy for prevention of iron overload and its complications has been investigated after the identification of the two common mutations in the HFE gene [START_REF] Burke | Hereditary hemochromatosis: gene discovery and its implications for population-based screening[END_REF]. The small effect size, the lack of consistency in the association between HFE mutations and cardiovascular disease and mortality [START_REF] Coppin | Longevity and carrying the C282Y mutation for haemochromatosis on the HFE gene: case control study of 492 French centenarians[END_REF][START_REF] Beutler | The C282Y mutation does not shorten life span[END_REF][START_REF] Beutler | Penetrance of 845G--> A (C282Y) HFE hereditary haemochromatosis mutation in the USA[END_REF][START_REF] Van Aken | No increase in mortality and morbidity among carriers of the C282Y mutation of the hereditary haemochromatosis gene in the oldest old: the Leiden 85-plus study[END_REF], and the possible effect modification by environmental factors such as smoking [START_REF] Coppin | Longevity and carrying the C282Y mutation for haemochromatosis on the HFE gene: case control study of 492 French centenarians[END_REF][START_REF] Roest | Heterozygosity for a hereditary hemochromatosis gene is associated with cardiovascular death in women[END_REF][START_REF] Beutler | The C282Y mutation does not shorten life span[END_REF][START_REF] Beutler | Penetrance of 845G--> A (C282Y) HFE hereditary haemochromatosis mutation in the USA[END_REF][START_REF] Van Aken | No increase in mortality and morbidity among carriers of the C282Y mutation of the hereditary haemochromatosis gene in the oldest old: the Leiden 85-plus study[END_REF][START_REF] Njajou | Mutations in the hemochromatosis gene (HFE) and stroke[END_REF] implies that the benefit of such screening program varies between different subgroups, imposing further difficulties to the use of genetic testing for population-based screening. The results of our study confirm these previous findings and do not support genetic screening on HFE mutations. C-reactive protein (mg/L) † 2.0 (0.9-3.7) 1.7 (0.9-3.3) 0.05 1.9 (0.9-3.9) 1.9 (0.9-3.

  mutations were associated with an increased risk of incident CHD in women (HR crude [95% CI] = 1.3 [1.0 to 1.8]), but not in men (HR crude [95% CI] = 0.9 [0.7 to 1.2]). Further adjustment by co-variates including smoking status lead to an increase in the risk of CHD in women (HR adjusted [95% CI] = 1.7 [1.2 to 2.4]), but not in men (HR adjusted [95% CI] = 0.9 [0.7 to 1.2]). Additionally to HFE status, history of myocardial infarction, diabetes mellitus at baseline and smoking were important risk factors for CHD in women. In men, serum cholesterol level and high-density lipoprotein level, diabetes mellitus and history of myocardial infarction were key risk factors for CHD. This increased CHD risk associated with HFE mutations in women was statistically significant in current smokers (HR = 3.1, 95% CI 1.4 to 7.1) and never smokers (HR = 1.8, 95% CI 1.1 to 2.8), but not in former smokers (HR = 1.3, 95% CI 0.7 to 2.4; Table

Figure 1 .

 1 Figure 1. Median C-reactive protein (CRP) levels and interquartile range by HFE

Table 1 . Baseline characteristics by sex and HFE carrier status

 1

	Women

  Values are means (standard deviations) for continuous variables and numbers (percentages) for categorical variables. P-Values were obtained by t-test for continuous variables, χ2 for categorical variables, and Mann-Whitney U test for C-reactive protein. † Median (interquartile range). HDL = High density lipoprotein

							8)	0.72
	Myocardial infarction at baseline	66	(11%)	152 (13%)	0.18	147 (25%)	232 (24%)	0.74

Acknowledgements

The Rotterdam Study is supported by the Erasmus Medical Center and Erasmus University Rotterdam, the Netherlands Organization for Scientific Research (NWO), the Netherlands Organization for Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry of Health, Welfare and Sports, the European Commission (DGXII), the Municipality of Rotterdam, and the Centre for Medical Systems Biology (CMSB). This work was supported by an Erasmus University Rotterdam grant to Dr. Omer T. Njajou and by a ZonMW grant to Dr. Behrooz Z. Alizadeh. The contributions of the general practitioners and pharmacists of the Ommoord district to the Rotterdam Study are greatly acknowledged. We are grateful to JM Vergeer, JR Oskamp and WM Luijten for their help in the genotyping.


Chatzipanagiotou

Prof Stylianos Chatzipanagiotou

MD Konstantina Papavasileiou

Eleni Papavasileiou

Angelina Tseleni-Kotsovili

Sotiris

Bersimis

Chryssoula Nicolaou

Anastassios Ioannidis

Prof. Dr Stylianos Chatzipanagiotou

Comparative antimicrobial susceptibility of biofilm versus planktonic forms of 3 Salmonella enterica strains isolated from children with gastroenteritis. 4 5

Keywords: Antimicrobial resistance, biofilms, planktonic bacteria, Salmonella enterica, gastroenteritis Antimicrobial resistance, biofilms, planktonic bacteria, Salmonella enterica, 54 gastroenteritis. 55 56 57

Comparative antimicrobial susceptibility of biofilm versus planktonic forms of strains isolated from children with gastroenteritis

Order of Authors: Konstantina Papavasileiou, MD; Eleni Papavasileiou, PhD; Angelina Tseleni-Kotsovili, PhD; Sotiris Bersimis, PhD; Chryssoula Nicolaou, PhD; Anastassios Ioannidis, PhD; Stylianos Chatzipanagiotou, PhD Abstract: Purpose. In the present study 194 S. enterica strains, isolated from infected children, belonging to various serotypes were investigated for their ability to form biofilms and the biofilm forms of the isolated strains were compared to their corresponding planktonic forms with respect to the antimicrobial susceptibility. Methods. For the biofilm forming strains the Minimal Inhibitory Concentration for Bacterial Regrowth from the Biofilm (MICBR) of nine clinically applicable antimicrobial agents, was determined, and the results were compared to the respective MIC values of the planktonic forms. Results. A hundred and nine S. enterica strains out of 194 (56%) belonging to 13 serotypes were biofilm forming. The biofilm forms showed increased antimicrobial resistance versus the planktonic bacteria. The highest resistance rates of the biofilm bacteria were observed with respect to gentamicin (89.9%) and ampicillin (84.4%) and the lowest rates with respect to ciprofloxacin and moxifloxacin (2.8% for both). Conclusions. A remarkable shift of the MICBR50 and MICBR90 toward resistance was observed in the biofilm forms as compared to the respective planktonic forms. Further studies are required to elucidate the biofilm antimicrobial resistance mechanisms of the bacterial biofilms and their contribution to the therapeutic failure in infections with in vitro susceptible bacteria.

Response to Reviewers: Dear Mr. Alex van Belkum, we have taken into consderation all the reviewer's comments and we have revised the manuscript acordingly. There is one exception with respect to the introduction comments g. and h. were we rewrote the text, but not in the proposed way because this would alter the essence. We have also corrected the references. Although the mechanisms are poorly understood, there is evidence that the biofilm associated 68 resistance should be related to modified nutrient environments, leading to suppression of 69 growth rate within the biofilm, interaction between exopolymer matrices and the antimicrobial, 70 as well as the development of biofilm / attachment specific phenotypes [START_REF] Kunin | Culture of the surfaces of urinary catheters to sample urethral 226 flora and study the effect of antimicrobial therapy[END_REF][START_REF] Nickel | Tobramycin resistance of 229 Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter materials[END_REF][START_REF] Gristina | Adhesive colonisation of 232 biomaterials and antibiotic resistance[END_REF][START_REF] Costerton | Practical measures to control 234 device-related bacterial infections[END_REF].

71

In comparative antimicrobial susceptibility studies, many common gram-negative and gram-72 positive bacterial pathogens produce biofilms showing significantly higher antimicrobial 73 resistance rates than their planktonic state. Most of these studies have largely focused on 74 Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa [START_REF] Olson | Biofilm bacteria: formation and 236 comparative susceptibility to antibiotics (2002)[END_REF].

75

Little is known about the response to antimicrobials of the biofilm forms of the Salmonella 76 enterica serotypes, which are worldwide the most common cause of acute, selflimited 77 gastroenteritis, usually not requiring treatment [10,11]. However, the majority of Salmonella 78 enterica strains are able to form biofilms and to synthesize cell surface components, that help 79 them survive in hostile or suboptimal environments [START_REF] Zogaj | The multicellurar 243 morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as 244 the second component of the extracellular matrix[END_REF], and express resistance to multiple 80 antimicrobials. This ability contributes to their persistence in the host after the acute phase of 81 infection (carrier state), the dissemination of the organism over a long period of time in the 82 environment and its transmission to new individuals [START_REF] Prouty | Biofilm formation and interaction with 246 the surfaces of gallstones by Salmonella spp[END_REF]. Regarding these properties 83 Salmonella enterica could serve as a very good model for the study of the comparative 84 response to antimicrobials between the planktonic and their respective biofilm forms.

85

The present study aimed to detect the production of biofilms by clinical strains of S. enterica 144

The antimicrobial resistance rates of the planktonic and the biofilm bacteria are given in table 145 2. The biofilm forms showed increased antimicrobial resistance versus the planktonic 146 bacteria. The highest resistance rates of the biofilm bacteria were observed with respect to 147 gentamicin (89.9%) and ampicillin (84.4%) and the lowest rates with respect to ciprofloxacin 148 and moxifloxacin (2.8% for both).

149

Since the P-value for all antimicrobials was less than 1%, all the differences were assumed to 150 be statistically significant at 99.0% (at least) confidence level (table 2).

151

forms as compared to the respective planktonic forms (table 3). The MICBR 50 lay in the 153 upper limit corresponding to susceptibility only for cefotaxime, imipenem and the two 154 quinolones (ciprofloxacin and moxifloxacin), while the MICBR 90 was above this limit 155 (intermediate or resistant) for all the antimicrobials tested.

156

In the present study a significant difference in antimicrobial susceptibility was found between 157 the planktonic and biofilm forms of the S. enterica strains isolated from clinical cases with 158 gastroenteritis, the biofilm forms showing increased resistance rates. Despite this statistically 159 significant increase in resistance, quinolones and the broad spectrum β-lactams cefotaxime 160 and imipenem showed the best antimicrobial activity against the biofilm forms, having 161 MCBR 50 s in the level of susceptibility (table 3). Although the methodology followed regarding

  a major concern in human and veterinary medicine. They consist of 60 growing microorganisms intimately associated with each other, producing an extracellular 61 polymeric substance (ESP) consisting of carbohydrate (exopolysaccharide) adhering to 62 synthetic or biological surfaces [1-4]. The encased sessile microorganisms bear quite distinct 63 properties from those growing independently or as planktonic populations in liquid media. One 64 of the most important properties of the biofilm associated bacteria in clinical medicine is the 65 markedly enhanced resistance to antimicrobial agents, through protection by the ESP, leading 66 to multidrug resistance and therapeutic failure.

  67

  86 serotypes isolated from children with gastroenteritis and to compare the antimicrobial 87 susceptibility of planktonic versus biofilm forming bacteria. 88 year period (2006-2008) 194 S. enterica strains were collected from children 93 with gastroenteritis, either hospitalized or attended at the outpatient clinic. The age of the 94 children balanced from 1-14 years. The isolation and serological identification of S. enterica. 95 was performed by conventional methods. 96 Biofilm formation was detected by the use of silicone disks (Folio C6 0,25 mm, 97 NOVATECH; new biotechnology for life ZI ATHELIA III-VOIE ANTIOPE 13705 LA CIOTAT 98 CEDEX-FRANCE) modified as described previously [14]. The silicone disks were cut in 99 similar size (4-5 mm) and weight (25-30 mg) through an in-house invented spacer 100 construction and were placed into tubes, weighed at a scale and left overnight under UV 101 irradiation for sterilization. Tubes containing 2,5 ml trypticase soya broth (TSB) were 102 inoculated with Salmonella strains and incubated for 72 hours at 30 ο C. The content was then 103 poured off and the tubes were washed 3 times with distilled water and air dried in a laminar 104 flow for 24 hours. The tubes containing the silicone disks with the attached bacteria were 105 weighed once more and the difference in weight showed the presence of biofilms. 106 Antimicrobial susceptibility of the planktonic bacterial forms was perfomed by means of 107 determination of the minimal inhibitory concentration (MIC). MIC was determined using two 108 methods: a) the automatic system VITEK 2 (bioMérieux SA, 69280 Marcy-l'Etoile, France) 109 and b) the standard broth dilution method according to guidelines of the Clinical Laboratory 110 Standards Institute (CLSI)[15]. The antimicrobials included were those of importance in the 111 clinical practice: ampicillin, coamoxiclav, cefuroxime, cefotaxime, gentamicin, imipenem, 112 cotrimoxazole, ciprofloxacin and moxifloxacin. 113 The strains producing biofilms were further tested for their antimicrobial susceptibility by 114 determination of the Minimal Inhibitory Concentration for Bacterial Regrowth from the biofilm 115 (MICBR) using a modified broth macrodilution method according to the guidelines of the 116 Clinical laboratory Standards Institute (CLSI) [15]. 117 Silicone disks coated with the biofilm forming Salmonella strains were prepared in tubes as 118 described above, omitting the last step (air-drying). Serial dilutions of the antimicrobials in 5 Mueller -Hinton broth, corresponding to the concentrations used for the MIC determination 120 regarding the planktonic bacteria, were prepared and poured into the silicone disk containing 121 tubes. The antimicrobial containing tubes were then incubated at 35 o C for 24 h. The growth 122 of planktonic bacteria was visualized by the development of turbidity in the medium. The 123 MICBR was defined as the lowest concentration inhibiting the growth in the medium as 124 observed by a complete clarity. An aliquot of the medium from the tubes with the lowest 125 antimicrobial concentration showing a turbidity indicating bacterial growth, was subcultured in 126 blood and Mc Conkey agar medium in order to check the purity of the grown Salmonella 127 population. 128 The statistical analysis was performed using the statistical package SPSS for Windows 129 (version 15.0) in order to disclose any significant differences between the percentages of 130 antimicrobial susceptibility of the planktonic and the biofilm bacterial forms. The analysis was 131 done by applying an appropriate hypothesis test concerning the difference between the 132 proportions of two samples. The normal approximation to the binomial distribution was used. serotypes among the S. enterica isolates and their respective ability to form 140 biofilms are shown in table 1. Biofilm formation was detected in 109 out of 194 Salmonella 141 strains (56%) included in the study. For all the positive strains the difference in weight before 142 and after incubation of the silicone disk was >50 mg, while in the negative strains the absence 143 of biofilm formation was indicated by differences of less than 1 mg.

  162the antimicrobial susceptibility testing of the biofilm bacteria is not a standardized consensus 163 one, the results arising are clear and more or less in agreement with previous reports, 164 referring to Salmonella, as well as to other bacterial species (Escherichia coli, Staphylococcus 165 epidermidis, Pseudomonas aeruginosa) using in-house biofilm formation and biofilm 166 antimicrobial susceptibility detection methods[START_REF] Olson | Biofilm bacteria: formation and 236 comparative susceptibility to antibiotics (2002)[END_REF][START_REF] Ashby | Effect of antibiotics on non-254 growing cells and biofilms of Escherichia coli[END_REF][START_REF] Duguid | Effect of biofilm culture upon the 256 susceptibility of Staphylococcus epidermidis to tobramycin[END_REF][START_REF] Evans | Susceptibility of bacterial biofilms to 259 tobramycin: role of specific growth rate and phase in the division cycle[END_REF][START_REF] Evans | Susceptibility of Escherichia coli and 262 Pseudomonas aeruginosa biofilms to ciprofloxacin: effect of specific growth rate 263[END_REF]. However the detection of biofilm 167 formation as such, does not predict in all cases a possible clinical therapeutic failure (or 168 clinical resistance). According to the hitherto reports, this phenomenon seems not to pertain 169 to all bacterial species[START_REF] Olson | Biofilm bacteria: formation and 236 comparative susceptibility to antibiotics (2002)[END_REF]. In veterinary infections caused by Pasteurella multocida, 170 Mannheimia haemolytica and Haemophilus somnus no difference in susceptibility was found 171 between the planktonic and the biofilm forms, and the treated animals responded well to most complexity in prediction of the therapeutic outcome, the latter probably depending on a variety 174 of factors conditioned by the nature of the antimicrobial, the bacterial species properties and 175 the specific biofilm features. 176 In the present report Salmonella enterica served practically as a very good model for the 177 study of biofilm formation and antimicrobial susceptibily, first because about half of the strains 7 Soon after the beginning of the "golden" antibiotic era in 1940, the emergence of the 182 antimicrobial resistance arose, which incited intensive research and major developments in 183 the field of the antimicrobial chemotherapy. However, despite the progress in the revelation of 184 most of the antimicrobial resistance mechanisms, the discovery of new drugs and the 185 improvement and standardization of the antimicrobial susceptibility testing using consensus 186 methodologies, the problem of the antimicrobial "clinical resistance" resulting in many cases 187 in therapeutic failure is still of major concern. 188 In clinical practice the MIC assay is the gold standard and the best way to select potentially 189 effective antimicrobial agents for the rational treatment of infections [4, 20]. The setting of the 190 antimicrobial breakpoints taking into consideration the pharmacokineticpharmacodynamic 191 properties of the drugs, besides clinical trials, is based on the use of planktonic bacterial 192 forms, a fact that does not correspond to the in vivo infectious disease pathogenesis, as in 193 some cases other factors like the formation of biofilms might be involved. 194 In all the hitherto published antimicrobial susceptibility studies dealing with biofilm bacterial 195 forms, the applied methodology varies, because standardized techniques have not yet been 196 established. However, all reported results are completely in agreement with each other, 197 indicating that human pathogenic biofilm forming bacteria bear significantly increased 198 antimicrobial resistance properties, compared to their corresponding planktonic forms. In the 199 present study, testing of antimicrobial susceptibility (or resistance) referred to the ability of 200 already formed biofilms to grow and generate planktonic forms. In other reports, biofilm 201resistance was found to be related to the low growth rates within the biofilm mass[START_REF] Gilbert | Biofilm susceptibility to antimicrobials[END_REF][22][START_REF] Gilbert | Influence of growth rate on susceptibility to 271 antimicrobial agents: biofilms, cell cycle and dormancy, and stringent response[END_REF][START_REF] Prosser | Method of evaluating effects of 274 antimicrobials upon bacterial biofilms (1987)[END_REF].202This versatile reaction of biofilms against the antimicrobials is noticeably impeding the search 203 for novel anti-biofilm acting antimicrobial agents[START_REF] Gilbert | Microbial quality assurance: a guide towards relevance 278 and reproducibility of inocula[END_REF]. 204 The development of new consensus methods for the determination of the antimicrobial 205 susceptibility of biofilm forms in biofilm forming bacteria seems to be a major research 206 challenge in infectious disease therapeutics for the management of infections that are difficult 207 to treat. The present study involving S. enterica, a biofilm forming microorganism, was 208 conducted to demonstrate the ability of bacterial biofilms to escape in vitro the action of the 209 commonly used antimicrobial agents. Further studies are warranted to elucidate the biofilm 8 antimicrobial resistance mechanisms and their contribution to therapeutic failure in infections 211 with in vitro susceptible bacteria.

Table 1 . Distribution of serovars and biofilm production among the Salmonella enterica 303 strains isolated from children with gastroenteritis.

 1

	304
	305
	306

Table 3 . MIC 50 and MIC 90 of the antimicrobials for the planktonic and MICBR 50 and 337 MICBR 90 of the biofilm forms of Salmonella enterica isolates.

 3

	338
	339
	340
	Antimicrobial	mg/l		mg/l		Breakpoints (susceptible)
		planktonic	biofilm	planktonic	biofilm
		MIC 50	MICBR 50	MIC 90	MICBR 90
	Ampicillin	≤ 2	≥ 64	≥ 32	≥ 64	≤ 8
	Coamoxiclav	≤ 2	32	4	64	≤ 8
	Cefuroxime	4	32	8	64	≤ 8
	Cefotaxime	≤ 1	8	≤ 1	64	≤ 8
	Gentamicin	≤ 1	16	≤ 1	32	≤ 4
	Imipenem	≤ 1	4	≤ 1	8	≤ 4
	Cotrimoxazole	≤ 20	160	≤ 20	320	≤ 40
	Ciprofloxacin	≤ 0,25	1	≤ 0,25	2	≤ 1
	Moxifloxacin	≤ 0,25	1	1	2	≤ 1

antimicrobials. This diversity in biofilm response to antimicrobials demonstrates the

(56%) were biofilm producers in vitro, and second because most of the strains in their

planktonic forms bore antimicrobial susceptible phenotypes (table 2), thus giving conspicuous

differences in the biofilm phenotypes.

References

Fierer J, Swancutt M. Non-typhoid Salmonella: a review (2000) Curr Clin Top Infect

Dis 20: 134-157.

(using normal approximation).

Salmonella enterica serovar number of strains biofilm positive

S. enteritidis 143 78

S. typhimurium 27 17

S. abony 3 0

S. oranienburg 3 3

S. II 2 1

S. muenchen 1 1

S. montevideo 1 1

S. lagos 1 1

S. goldcoast 1 0

S. blockley 1 1

S. thompson 1 1

S. newport 1 1

S. virchow 1 1

S. bardo 1 0

S. stanley 1 0

S. miami 1 1

S. haifa