Odile Tresse
email: odile.tresse@oniris-nantes.fr

Hana Turonova

Romain Briandet

Ramila Rodrigues

Mathieu Hernould

Nabil Hayek

Alain Stintzi

Jarmila Pazlarova

Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions

Keywords: Campylobacter jejuni, biofilm, CLSM, oxidative stress, CosR

During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two welldescribed Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

Introduction

Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries [START_REF] Epps | Foodborne Campylobacter: infections, metabolism, pathogenesis and reservoirs[END_REF][START_REF] Golz | Relevance of Campylobacter to public health-the need for a one health approach[END_REF]. The resulting disease in humans, campylobacteriosis, is characterized by acute enteritis with the presence of blood and leukocytes in a stool, abdominal pain, and fever [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Lu | Examination of nanoparticle inactivation of Campylobacter jejuni biofilms using infrared and Raman spectroscopies[END_REF]. It is also associated with late onset complications such as Guillain-Barré syndrome, its variant Miller-Fisher syndrome [START_REF] Salloway | Miller-Fisher syndrome associated with Campylobacter jejuni bearing lipopolysaccharide molecules that mimic human ganglioside GD(3)[END_REF][START_REF] Nachamkin | Campylobacter species and Guillain-Barre syndrome[END_REF][START_REF] Kudirkiene | Phenotypic and genotypic characterizations of Campylobacter jejuni isolated from the broiler meat production process[END_REF], and inflammatory bowel diseases [START_REF] Kaakoush | Role of emerging Campylobacter species in inflammatory bowel diseases[END_REF]. The underlying molecular mechanisms responsible for its pathogenesis, persistence, and survival seem to be unique to Campylobacter as compared to other invasive foodborne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus). These features might result from high level of genomic polymorphism, restricted catabolic capacity, self-regulation and deregulation of genes, and other undefined survival routes.

The main reservoir of Campylobacter is the intestinal tract of birds and other endothermic animals, especially livestock. It is primarily isolated from poultry and, to a lesser extent, pork and beef. The infection of the human host is generally caused by the consumption of undercooked and mishandled poultry or by cross-contamination of cooking tools and fresh vegetables [START_REF] Butzler | Campylobacter, from obscurity to celebrity[END_REF][START_REF] Guyard-Nicodeme | Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board[END_REF]. A significant increase in the prevalence of campylobacteriosis cases has been observed over the past 5 years in the EU, based on quantitative epidemiological analyses from farms to retail outlets [START_REF] Efsa | European food safety authority, european centre for disease prevention and control: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010[END_REF][START_REF] Efsa | European food safety authority, European centre for disease prevention and control: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011[END_REF]. A baseline survey, conducted in 28 European countries in 2010, indicated that 71.2% of broiler batches and 75.8% of broiler carcasses were contaminated by Campylobacter [START_REF] Efsa | European food safety authority, European centre for disease prevention and control: analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU[END_REF]. These data were reinforced by an in-depth analysis over a 3-year period at the UK-wide level showing that in over 37 abattoirs (representing almost 90% of the total UK slaughter throughput), 79.2% of the slaughter batches were positive for Campylobacter [START_REF] Lawes | Investigation of prevalence and risk factors for Campylobacter in broiler flocks at slaughter: results from a UK survey[END_REF]. In addition, 87.3% of the broiler carcasses were contaminated by Campylobacter with 27.3% of them showing a load over 1000 cfu.g -1 [START_REF] Powell | The prevalence of Campylobacter spp. in broiler flocks and on broiler carcases, and the risks associated with highly contaminated carcases[END_REF]. In the USA, 168 pathogen-food combinations of 14 major pathogens across 12 food categories were compared [START_REF] Batz | Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation[END_REF]. The combination "Campylobacter-poultry" reached the first rank in terms of annual disease burden including illnesses, hospitalizations, deaths, and costs. Overall, these exhaustive data on Campylobacter contamination indicate that this microorganism can survive outside of its reservoir through breeding farms, slaughterhouses and food processing, defying environmental conditions, and human defense mechanisms.

The main pathogenic species, Campylobacter jejuni, has been isolated in more than 80% of the campylobacteriosis cases [START_REF] Moore | Campylobacter[END_REF]. Being an obligate microaerophilic bacterium, Campylobacter has to develop adaptation strategies Abbreviations: BFI, Biofilm index; CLSM, Confocal laser scanning microscopy; MAC, Microaerobic conditions; OEC, Oxygen-enriched conditions; OEC C , Cultivation in OEC; OEC A , Acclimation to OEC; ROS, Reactive oxygen species; TCS, Two-component system.

to survive oxidative conditions from food environments and macrophage attacks. It has been suggested that adhesion to surfaces and formation of biofilms could be one of the strategies used to maintain C. jejuni survival [START_REF] Nguyen | Role of attachment to surfaces on the prevalence and survival of Campylobacter through food systems[END_REF]. Moreover, the bacterium can be sheltered in mixed species biofilms [START_REF] Sanders | Culture and detection of Campylobacter jejuni within mixed microbial populations of biofilms on stainless steel[END_REF][START_REF] Ica | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms[END_REF]. C. jejuni can form three different types of biofilm: (i) a structure attached to an abiotic surface, (ii) aggregates floating in the liquid culture, or (iii) a pellicle formed at the gas/liquid interface [START_REF] Joshua | Biofilm formation in Campylobacter jejuni[END_REF]. Biofilm formation occurs within 48 h of cultivation with cell detachment becoming prominent after a prolonged cultivation time [START_REF] Sanders | Culture and detection of Campylobacter jejuni within mixed microbial populations of biofilms on stainless steel[END_REF][START_REF] Ica | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms[END_REF]. In line with other biofilm-producing foodborne bacteria, the substratum composition and its physicochemical properties influence the biofilm formation of C. jejuni [START_REF] Nguyen | Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces[END_REF]. These properties could play an important role in the early stages of biofilm formation when cells adhere to the surface. This assumption is supported by a variation in adhesion rates to inert surfaces, such as nitrocellulose membrane, glass, and stainless steel [START_REF] Joshua | Biofilm formation in Campylobacter jejuni[END_REF][START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF][START_REF] Gunther | The biofilm forming potential of bacterial species in the genus Campylobacter[END_REF]. The wide range of adhesion capability in Campylobacter spp. also raises the question of biological fitness among strains, in regards to their ability to attach irreversibly to a surface and initiate biofilm formation [START_REF] Joshua | Biofilm formation in Campylobacter jejuni[END_REF][START_REF] Sulaeman | Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test (R)[END_REF][START_REF] Teh | Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations[END_REF].

Molecular mechanisms regulating biofilm formation of C. jejuni are still poorly understood. So far, genes described to be involved in the process include those responsible for cell motility (flaA, flaB, flaC, flaG, fliA, fliS, and flhA; [START_REF] Joshua | Biofilm formation in Campylobacter jejuni[END_REF][START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF][START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF][START_REF] Reuter | Biofilm formation by Campylobacter jejuni is increased under aerobic conditions[END_REF], cell surface modifications (peb4, pgp1, and waaF; [START_REF] Asakura | Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni[END_REF][START_REF] Naito | Effects of Sequential Campylobacter jejuni 81-176 Lipooligosaccharide core truncations on biofilm formation, stress survival, and pathogenesis[END_REF][START_REF] Frirdich | Peptidoglycan ld-Carboxypeptidase Pgp2 influences Campylobacter jejuni helical cell shape and pathogenic properties and provides the substrate for the dl-Carboxypeptidase Pgp1[END_REF], quorum sensing (luxS; [START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF]stress response (ppk1, spoT, cj1556, csrA, cosR, andcprS;[START_REF] Candon | Polyphosphate kinase 1 is a pathogenesis determinant in Campylobacter jejuni[END_REF][START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF][START_REF] Mclennan | Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide[END_REF][START_REF] Svensson | The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization[END_REF][START_REF] Gundogdu | The Campylobacter jejuni transcriptional regulator Cj1556 plays a role in the oxidative and aerobic stress response and is important for bacterial survival in vivo[END_REF][START_REF] Oh | Role of Alkyl Hydroperoxide Reductase (AhpC) in the biofilm formation of Campylobacter jejuni[END_REF]. It was found that biofilm formation is flagellum-mediated as the first step of the process-cellular adhesion-requires presence of flagella, although its functionality is not crucial for the biofilm initiation [START_REF] Svensson | Flagella-mediated adhesion and extracellular DNA release contribute to biofilm formation and stress tolerance of Campylobacter jejuni[END_REF]. Other components essential for development of biofilm structure are extracellular DNA (eDNA) and DNA-binding protein Dps, whose presence is required for proper formation of microcolonies and structuralization of biofilm [START_REF] Svensson | Flagella-mediated adhesion and extracellular DNA release contribute to biofilm formation and stress tolerance of Campylobacter jejuni[END_REF][START_REF] Brown | Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni[END_REF]. Genes regulating biofilm formation were not fully identified so far. Experiments using knock-out and knock-down mutants of various regulators revealed several genes influencing the process of biofilm formation. Except of aforementioned motility apparatus regulated by flhA [START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF], and functional quorum sensing luxS [START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF], other regulators involved mostly in stress response were found to be critical for biofilm formation. Interestingly, while mutants lacking genes responsible for oxidative stress response such as cj1556 and csrA were defective in biofilm formation [START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF][START_REF] Gundogdu | The Campylobacter jejuni transcriptional regulator Cj1556 plays a role in the oxidative and aerobic stress response and is important for bacterial survival in vivo[END_REF], knock-out/down of genes responsible for general stress response (spoT, ppk1, and cprS) resulted in increased biofilm formation suggesting that the process represents alternative pathway of stress defense in Campylobacter [START_REF] Candon | Polyphosphate kinase 1 is a pathogenesis determinant in Campylobacter jejuni[END_REF][START_REF] Mclennan | Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide[END_REF][START_REF] Svensson | The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization[END_REF]. Another regulator possibly involved in biofilm formation is gene cosR. This orphan twocomponent system (TCS) was recently discovered to be involved in the regulation pathway of ROS detoxification in C. jejuni [START_REF] Hwang | Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni[END_REF][START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. It was previously reported that CosR regulates transcription of 93 different genes in C. jejuni [START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF], it is overexpressed in sessile cells [START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF] and was already shown to influence biofilm formation by regulation of alkyl hydroperoxide reductase ahpC [START_REF] Oh | Role of Alkyl Hydroperoxide Reductase (AhpC) in the biofilm formation of Campylobacter jejuni[END_REF]. All these facts suggest that CosR might play significant role in biofilm formation of C. jejuni.

So far, analyses of pure cultures have mostly been carried out in an optimal growth atmosphere and were focused on the strain NCTC 11168 [START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF][START_REF] Ica | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms[END_REF]. Using colorimetric assessment methods (Crystal violet and Congo red assays) for biofilm detection in glass tubes, [START_REF] Reuter | Biofilm formation by Campylobacter jejuni is increased under aerobic conditions[END_REF] showed that aerobic cultivation enhanced C. jejuni NCTC 11168 biofilm. In a previous study, we have shown that the strain 81-176, grown under controlled oxygenenriched conditions (19% O 2 , 10% CO 2 , and 71% N 2 ), is able to overexpress membrane proteins involved in biofilm initiation and virulence process [START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF]. In this study, we compared the biofilm development of two C. jejuni strains responsible for human outbreaks (NCTC 11168 and 81-176), and the effect of dioxygen (O 2 ) on biofilm development. The usage of controlled atmosphere eliminated other factors possibly affecting biofilm formation. It was therefore possible to explore whether the increase of biofilm formation in aerobic conditions could be attributed solely to the level of oxygen and if the trend of enhanced biofilm formation is present in other strain of C. jejuni. This was evaluated, for the first time, using specific biofilm parameters (maximum height, biomass volume, and ultrastructure) from confocal laser scanning microscopy (CLSM) analyses. This non-invasive sensitive technique has been used previously to examine Campylobacter cell morphology and viability [START_REF] Chantarapanont | Direct microscopic observation and viability determination of Campylobacter jejuni on chicken skin[END_REF][START_REF] Lee | Expression of flagellin proteins of Campylobacter jejuni within microaerobic and aerobic exposures[END_REF][START_REF] Jang | Morphology and adhesion of Campylobacter jejuni to chicken skin under varying conditions[END_REF][START_REF] Ica | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms[END_REF] and bacterial interactions with live tissues [START_REF] Mooney | Invasion of human epithelial cells by Campylobacter upsaliensis[END_REF]. The CLSM has also been used for the detection of C. jejuni in mixed species biofilms [START_REF] Sanders | Culture and detection of Campylobacter jejuni within mixed microbial populations of biofilms on stainless steel[END_REF][START_REF] Ica | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms[END_REF]. In the present work, the impact of pretreatment and cultivation of cells in oxygenenriched conditions (OEC) on C. jejuni biofilm formation and its ultrastructural organization was investigated in comparison with cells cultivated in microaerobic conditions (MAC). In addition, analyses using an overexpressing cosR transformant were performed to determine the role of this regulator in C. jejuni biofilm development.

Results

Biofilm Development and Architecture

Two C. jejuni strains, NCTC 11168 and 81-176, were chosen in order to explore their biofilm formation capacities using CLSM with Syto 9 staining. This cell-permeable dye emits fluorescence after binding to nucleic acids and therefore allows the visualization of cells and any extracellular DNA present in the biofilm matrix. Both strains were able to form biofilm within 24 h of cultivation (Figure 1). At the initial stages of biofilm formation, cells gathered in clusters partially attached to the surface, forming finger-like structures. After 48 h, most of the biofilm mass remained attached to the bottom of the well. The biofilm structure evolved during the time of cultivation, increasing in both maximum height and biomass volume for both strains. However, 81-176 formed more biofilm than the NCTC 11168 strain (after 48 h: 233.33 ± 64.63 and 130.67 ± 14.70 µm, respectively, for the maximum height; 42. 3 × 10 5 ± 5.7 × 10 5 and0.4 × 10 5 ± 0.09 × 10 5 µm 3 , respectively, for the biomass volume; n = 3). In addition, unlike NCTC 11168, the biofilm of the 81-176 strain exhibited a pronounced open ultrastructure full of voids and channels, even after 96 h of incubation (data not shown). As growth rates of both strains were similar (µ max = 0.69 h -1 for NCTC 11168 and µ max = 0.67 h -1 for 81-176), these differences in biofilm formation cannot be explained by different growth abilities. During the experiment, no formation of pellicle or floating aggregates was observed probably due to the cultivation in static conditions.

Cell Motility in Biofilm

Motile cells, tracked using CLSM, were observed around or inside the biofilm structure after 48 h of cultivation (Supplementary Videos). However, the motility of cells differed according to their position in the biofilm structure. The highest number of motile cells was detected at the bottom of the well (Supplementary Videos 1, 3) moving more or less freely through the structure, while the motility and the number of motile cells decreased in the middle part of the biofilm (Supplementary Videos 2, 4). Furthermore, high number of motile cells was detected within the biofilm structure of 81-176 (Supplementary Videos 1, 2), whereas for NCTC 11168 the motile cells were detected mostly outside the biofilm (Supplementary Videos 3, 4).

Effect of Oxygen on Biofilm Formation of NCTC 11168 and 81-176 C. jejuni Strains

Two different approaches were used to evaluate the effect of subinhibitory oxygen concentration on biofilm formation of two strains with different biofilm forming ability (NCTC 11168 and 81-176). Firstly, biofilms were cultivated under controlled oxygen-enriched conditions (OEC c ) as described previously by [START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF]. In OEC, the same concentration of CO 2 (10%) as in MAC was maintained, while the O 2 concentration was increased to a sublethal level (19% O 2 in OEC vs. 5% in MAC). This enabled the evaluation of the effect of increased O 2 concentration on biofilm development of C. jejuni regardless of its capnophilic nature requiring increased concentration of CO 2 . Biofilm volume of both strains was significantly increased (P < 0.01) when cultivated in OEC c (Figure 2 and Supplementary Table 1). Incubation time and O 2 concentration had a significant effect (P < 0.01) on increased biomass production in OEC c when compared to MAC c . Interestingly, some significant differences in both maximum height and biomass volume (P < 0.01) remained between the two strains even after cultivation in OEC, with a greater biofilm development for 81-176 than for NCTC 11168, indicating that strain biology impacts biofilm formation (Supplementary Table 1). This was confirmed by formation of a denser compact biomass for NCTC 11168 biofilm while 81-176 induced more voids and open water channels across the biofilm.

In the second approach, both strains were acclimatized to OEC (OEC a ) prior to biofilm formation in MAC. Acclimatized cells of both strains formed significantly larger biofilms than nonacclimatized ones after 24 h of cultivation, as expressed by the fold changes in maximum height and biomass volume values (Figure 3A). Conversely, the acclimatization of cells to OEC was no longer an advantage for biofilm formation after 48 h, as demonstrated by reduction of biofilm formation for both strains. This was also confirmed by statistical analyses, with the highest F-ratios of the interaction effect between "Incubation time" and "O 2 pretreatment, " showing higher variation in maximum height and biomass volume, than for the other factors (Supplementary Table 2).

In order to distinguish the effect of OEC prior to or during C. jejuni biofilm formation, biofilm development was compared between the cells acclimatized to OEC and the cells subjected to OEC during biofilm formation (OEC a and OEC c , respectively; Figure 3B). Although the fold change (OEC c /OEC a ) was not in favor of NCTC 11168 biofilm formation during the first 24 h, after 48 h both strains cultivated in OEC c showed enhanced biofilm formation with a marked difference in biomass volume for 81-176. This was confirmed statistically with a significant effect of OEC treatment (P < 0.0001) for O 2 treatment, and the interaction between "Incubation time" and "O 2 treatment" with the highest F-ratios (Supplementary Table 3). 2,3.

Role of cosR in Biofilm Development

A second copy of C. jejuni gene cosR and its promoter were inserted into the poorer biofilm-forming strain NCTC 11168 to determine its role in C. jejuni biofilm formation. This construction, with an ectopic copy of the cosR gene and its promoter, enabled to double the expression of the transcript level of cosR in the cells (Supplementary Figure 1) in a same manner as in the cosR-overexpressing strain obtained by [START_REF] Hwang | Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni[END_REF] and used by [START_REF] Oh | Role of Alkyl Hydroperoxide Reductase (AhpC) in the biofilm formation of Campylobacter jejuni[END_REF]. Then, the parental NCTC 11168 strain and the cosR overexpressing strain, namely transformant (TrfcosR), were compared for their ability to adhere to an inert surface and to develop a biofilm (Figure 4). Using the BioFilm Control Ring Test R , a significantly higher BFI was obtained (P = 0.0007) for the transformed strain, indicating its greater ability to adhere to inert surfaces (Figure 4A). In addition, using the crystal violet assay, the transformant showed enhanced biofilm formation after 24 and 48 h (P = 0.0006 and 0.02, respectively) but not after 72 h (P > 0.05) when compared with its parental strain (Figure 4B). The CLSM observations and biofilm analyses indicated that the transformant formed significantly more (P < 0.01) biofilm than its parental strain (Figure 5, Supplementary Table 4). In addition, the maximum height and biomass volume reached by the transformant was not significantly different from those obtained with the strongest biofilm-forming strain 81-176 (Supplementary Table 5). These data showed that the presence of two genes encoding cosR significantly enhanced biofilm development in MAC (592.7-times higher biomass volume after 24 h). Interestingly, this was correlated with the formation of an open biofilm ultrastructure with voids and water channels similar to the one described for 81-176 (Figures 1,5A). Comparison of genomic sequences using xBASE2 [START_REF] Chaudhuri | xBASE2: a comprehensive resource for comparative bacterial genomics[END_REF] showed that the cosR gene (cj0335c and cjj0379c, respectively) and its flanking regions are 100% identical in NCTC 11168 and 81-176. Both strains carry the exact same form of the gene. Therefore, some other mechanisms, related to the cosR sequence and its flanking regions, for regulating the C. jejuni biofilm formation, should exist. Moreover, unlike the two wild strains, an increased O 2 concentration during cultivation did not promote biofilm formation of the transformant (Figure 5). These data indicate that a second ectopic copy of cosR enhanced biofilm development by promoting a complex architecture of C. jejuni biofilm irrespective of O 2 demand. Nevertheless, further experiments should be performed to evaluate cosR transcript level and CosR expression throughout all phases of biofilm development.

Discussion

As the leading cause of bacterial foodborne diseases, whose incidence has been significantly increasing during the recent years in Europe [START_REF] Efsa | European food safety authority, European centre for disease prevention and control: analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU[END_REF][START_REF] Efsa | European food safety authority, european centre for disease prevention and control: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010[END_REF][START_REF] Efsa | European food safety authority, European centre for disease prevention and control: the European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011[END_REF], this pathogen has to adapt and survive environmental conditions outside and inside its main hosts, particularly oxidative stress. In this study, we have shown that C. jejuni can form biofilm in static conditions with a clearly defined finger-like structure. Our observation is consistent with previous studies indicating that C. jejuni could develop monospecies biofilms [START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF][START_REF] Asakura | Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni[END_REF][START_REF] Corcoran | Influence of growth conditions on diverse polysaccharide production by Campylobacter jejuni[END_REF][START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF][START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF][START_REF] Hanning | Campylobacter jejuni as a secondary colonizer of poultry biofilms[END_REF][START_REF] Mclennan | Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide[END_REF][START_REF] Sanders | Temperature and nutrient effects on Campylobacter jejuni attachment on multispecies biofilms on stainless steel[END_REF][START_REF] Gunther | The biofilm forming potential of bacterial species in the genus Campylobacter[END_REF]. Both examined strains were able to produce a biofilm, although their maximum height, biomass volume, and ultrastructure differed significantly between the two strains. In previous studies, stronger adhesion to an inert surface was observed for 81-176 than for NCTC 11168 [START_REF] Gunther | The biofilm forming potential of bacterial species in the genus Campylobacter[END_REF][START_REF] Sulaeman | Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test (R)[END_REF][START_REF] Teh | Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations[END_REF]. Although the adhesion strength could not be fully correlated to the capability of bacterial species to form biofilms, biofilm initiation is crucial to anchor the embryonic core of the biofilm. Our qualitative and quantitative data indicated that 4,5.

NCTC 11168 formed a thin but compact multilayered biofilm without achieving a more complex organization during the time of incubation. In contrast, the 81-176 strain was able to form a thick biofilm with an open ultrastructure composed of voids and channels. This kind of heterogeneous structure is considered to be the signature of a mature biofilm. It enhances the formation of convective flows bringing nutrients to cell aggregates and draining metabolic waste from cells in these aggregates [START_REF] Donlan | Biofilms: survival mechanisms of clinically relevant microorganisms[END_REF]. The heterogeneity of the 81-176 biofilm was confirmed by tracking the motile cells within the C. jejuni 81-176 biofilm. In contrast to many other bacteria, C. jejuni is able to maintain the expression level of genes responsible for cell motility and flagella biosynthesis when grown in biofilms [START_REF] Joshua | Biofilm formation in Campylobacter jejuni[END_REF][START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF][START_REF] Asakura | Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni[END_REF][START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF]. In our study, we observed the presence of motile, less motile and sessile cells, indicating that the biofilm is composed of cells in different physiological states. Due to the biofilm organization, different cell phenotypes coexist in the structure and therefore a wide range of cells can be found in the biofilm, from dormant to motile cells. As in nature (ex vivo or in vivo) C. jejuni cells may encompass various physiological states, biofilm could be considered as a model of mixed subpopulations of C. jejuni which could be found in food products, food-processing plants, in poultry gut, or human digestive tract.

Although C. jejuni is sensitive to increased concentrations of oxygen, absence of oxygen in anaerobic conditions induces cell death. C. jejuni requires a basal amount of available oxygen to maintain the processes essential for respiration and multiplication [START_REF] Kelly | Complexity and versatility in the physiology and metabolism of Campylobacter jejuni[END_REF]. The availability of dissolved oxygen is therefore one of the main environmental parameters for the survival of C. jejuni. Previous studies showed that aerobic conditions enhanced biofilm formation of the strain NCTC 11168 [START_REF] Asakura | Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni[END_REF][START_REF] Reuter | Biofilm formation by Campylobacter jejuni is increased under aerobic conditions[END_REF]. In those studies, biofilms grown in glass tubes or in 24 well plates were detected by crystal violet or Congo red after exposition to air and air supplemented with CO 2 . These colorimetric assays showed enhancement of biofilm formation under the oxidative stress, but could not predict whether and how the biofilm structure would change. The controlled O 2 gaseous conditions, respecting the capnophilic nature of C. jejuni, and the use of CLSM allowed us not only to quantify the amount of biofilm, but also to evaluate any structural changes caused by increased oxygen concentration. In accordance to our expectations, we did observe an increased biofilm formation for both strains under OEC. Moreover, the data obtained using CLSM suggest that the response to an increased O 2 level is strain-dependent. Although the biofilm formation for both strains was enhanced, the ultrastructures were remarkably different. The poorer biofilm forming NCTC 11168 produced more voluminous biofilm without increasing its thickness and without switching to a maturation phase as observed for 81-176. As the physiological state of cells may correspond to their close environment, the cell response to environmental conditions could differ according to its location in the biofilm structure. The formation of voluminous flat biofilm may be beneficial for NCTC 11168 under OEC, as a smaller area, and therefore a smaller number of cells, is exposed to the malignant effect of oxygen. On the other hand, the 81-176 strain increased in both biofilm volume and height, keeping the porous ultrastructure of the biofilms produced under MAC. It seems like the strain disregards the negative effects of an increased oxygen level and is supported to multiply and form a mature biofilm composed of mixed subpopulations of cells. The biofilm organization may therefore offer a favorable oxygen tuning niche for C. jejuni. These findings indicate that oxygen growth requirements of C. jejuni are not as fastidious when cells are organized in biofilm. Consequently, the paradigm of fastidious requirements for C. jejuni growth [START_REF] Jones | The Campylobacter conundrum[END_REF][START_REF] Park | The physiology of Campylobacter species and its relevance to their role as foodborne pathogens[END_REF] should be reconsidered according to the cell physiological state and cell population cooperation.

Unlike well-studied aerobes, C. jejuni lacks specific and global regulators involved in oxidative stress resistance, such as SoxRS, OxyR, or RpoS [START_REF] Garenaux | Role of the Cj1371 periplasmic protein and the Cj0355c twocomponent regulator in the Campylobacter jejuni NCTC 11168 response to oxidative stress caused by paraquat[END_REF]. C. jejuni carries two Fur homologs, Fur, and PerR (peroxide stress regulator), which regulate iron homeostasis and contribute to the oxidative stress response [START_REF] Van Vliet | The role of iron in Campylobacter gene regulation, metabolism and oxidative stress defense[END_REF]. Recently, [START_REF] Hwang | Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni[END_REF][START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF] have suggested that the orphan TCS Cj0355c could be involved in the oxidative stress response and named it CosR. The protein product of cosR shares 60% amino acid identity with Hp1043, a TCS response regulator element from the close relative Helicobacter pylori. Deletion of hp1043 induced death of H. pylori in the same way as it has been observed for cosR and C. jejuni [START_REF] Stahl | Identification of essential genes in C. jejuni genome highlights hyper-variable plasticity regions[END_REF]. However, the hp1043 gene has been successfully substituted by C. jejuni cosR [START_REF] Muller | Regulation of expression of atypical orphan response regulators of Helicobacter pylori[END_REF] suggesting that CosR exhibits similar biological functions to Hp1043.

In this study, the essential gene cosR was overexpressed in the poorer biofilm-forming strain, NCTC 11168, in order to investigate the role of this TCS in biofilm formation and structuring. In our study, the adhesion of cells to inert surfaces was correlated to the biofilm formation detected by Crystal violet and analyzed by CLSM. All three different detection techniques led to the same conclusion. The significantly greater adhesion to an inert surface and the increased biofilm formation of the transformant (TrfcosR) revealed that the expression of this gene is connected to biofilm formation. This was also confirmed by the CLSM experiments, which showed a much greater thickness and volume of the transformant's biofilm under MAC than those observed for the parental strain. This result is in accordance with the previously published work describing increased expression of CosR in C. jejuni NCTC 11168 biofilm as compared to planktonic counterparts [START_REF] Kalmokoff | Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation[END_REF], although [START_REF] Oh | Role of Alkyl Hydroperoxide Reductase (AhpC) in the biofilm formation of Campylobacter jejuni[END_REF] observed decrease of biofilm formation in strain overexpressing cosR. This discrepancy might be explained by looking at the structure of biofilms of parental strain and the transformant. Interestingly, the ultrastructure of the TrfcosR biofilm was found to be more similar to the one described for 81-176 than the parental strain, showing an open organization with pores and channels across the structure. Unexpectedly, when the transformant was cultivated under OEC, the maximum height and biomass volume of the biofilm were not higher than when the biofilm was produced under MAC. Nevertheless, the values still remained higher than those of the parental strain. These data indicate that cells overexpressing cosR were not stimulated by the higher O 2 concentrations to enhance the biofilm formation. Thus, CosR seems to be crucial for initiation of the maturation phase of C. jejuni biofilm development. This might be the reason of arisen discrepancy between our work and the one published by [START_REF] Oh | Role of Alkyl Hydroperoxide Reductase (AhpC) in the biofilm formation of Campylobacter jejuni[END_REF]. The authors used Mueller Hinton broth and higher temperature of cultivation. These factors were previously found to increase the biofilm formation of C. jejuni [START_REF] Reeser | Characterization of Campylobacter jejuni biofilms under defined growth conditions[END_REF]. The usage of supportive cultivation conditions in combination with enhanced initiation of biofilm maturation caused by overexpression of CosR might result in earlier dispersal of cells and microcolonies from mature biofilm. Such acceleration of dispersal would result in reduction of biofilm mass attached to the surface of the well and cosR transformant would therefore seem to be less biofilm forming. This is in accordance with our observation of the dramatic biovolume decrease after 48 h of cultivation (from 600to 16-times more biofilm mass than the parental strain) observed for the transformant. Nevertheless, further experiments should be performed in order to confirm or refuse this hypothesis.

The regulator CosR was initially identified as a potential regulator of ROS scavengers by promoting or repressing genes encoding KatA, AhpC, and SodB in C. jejuni [START_REF] Hwang | Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni[END_REF][START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. It was also differently expressed after a superoxide stress induced by paraquat [START_REF] Garenaux | Role of the Cj1371 periplasmic protein and the Cj0355c twocomponent regulator in the Campylobacter jejuni NCTC 11168 response to oxidative stress caused by paraquat[END_REF]. Binding to the promoter of luxS, CosR might also be contributing to a quorum sensing system [START_REF] Hwang | Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni[END_REF]. Recently, it was also demonstrated that CosR is involved in the expression of the antibiotic efflux pump CmeABC in C. jejuni [START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. In this study, role of CosR in the maturation of C. jejuni biofilm, independently of oxidative stress, adds a new element in favor of its pleiotropic function in the main metabolic processes allowing the survival of C. jejuni in response to environmental stresses.

In contrast to its highly restricted catabolic capacity, C. jejuni is able to develop strategies to survive environmental oxidative stress using O 2 as an advantage for biofilm development. As C. jejuni is equipped to withstand oxidative stress through cooperation of subpopulations within a biofilm, further analyses are required to assess if this feature could explain the survival of this emerging pathogen in slaughterhouses, after evisceration, during food processing, or during macrophage attack. In addition, these findings advocate further studies to analyze quiescent, dormant and sessile C. jejuni cells and cell cooperation in response to environmental stresses, to identify the underlying cellular and molecular mechanisms supporting the persistence and resistance of this mysterious pathogen.

Materials and Methods

Bacterial Strains and Culture Conditions

All experiments were performed using three strains of C. jejuni: two well-documented clinical isolates 81-176 and NCTC 11168 purchased from general collections, and a cosR overexpressing transformant built for this study as described below. All strains were subcultured from the stock stored at -80 • C by cultivation on Karmali agar plates (Oxoid, UK) at 42 • C for 48 h in MAC (5% O 2 , 10% CO 2 , and 85% N 2 , namely MAC). Grown colonies were inoculated onto Karmali agar plates and incubated either for 24 h at 42 • C in MAC, or for 42 h in oxygen-enriched conditions (19% O 2 , 10% CO 2 , and 71% N 2 , namely OEC) to allow acclimation of cells to oxidative stress. The OEC were previously described as a sublethal atmosphere not repressing growth of C. jejuni NCTC 11168 and 81-176 [START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF]. The gas conditions were maintained using hermetic stainless steel jars vacuum flushed and then filled with commercially purchased gas mixture. The process was repeated two times to minimize air residua in the cultivation atmospheres. The growth rates of all tested strains were determined from cultivation in BHI (Merck, Germany) in MAC using plate counts in triplicates with the appropriated decimal dilution.

Construction of the cosR Overexpressing Strain

For the construction of the cosR overexpressing strain, the cj0355c (cosR) gene was amplified from the strain NCTC 11168 using PCR primers Cj0355c F and Cj0355c R (Supplementary Table 6). The positions of the forward and reverse primers were chosen upstream and downstream of cosR within the folB (start position at 325186) and fdxB (end position at 323902) genes, respectively, to ensure that cosR was under the control of its own promoter. The PCR product was purified using the Qiagen PCR purification kit (Toronto, ON, Canada) and then cloned into the pRRK-1 plasmid [START_REF] Reid | Identification of Campylobacter jejuni genes involved in the response to acidic pH and stomach transit[END_REF]. The cloning step was achieved using the Clontech In-Fusion ™ PCR cloning kit (Mountain View, CA, USA). Briefly, the primers were designed with 15 bp extensions that allow recombination with the nucleotides flanking the XbaI restriction site on the pRRK vector. The recombinant pRRK + cosR plasmid was transformed into Fusion-Blue competent cells and positive transformants were selected on LB agar plates supplemented with Km. The cloned plasmid with the cosR gene was extracted from the grown transformants, purified, and sequenced to confirm the absence of point mutations. The plasmid was then naturally transformed into C. jejuni NCTC 11168 grown to mid-log phase. Following incorporation of the cosR into the chromosome was achieved by heterologous recombination. The location of the inserted gene was determined by amplifying three possible insertion sites on the chromosome using the ak233, ak234, ak235, and AR56 primers (Supplementary Table 6). The expected PCR product size was detected using the ak234 and AR56 primers indicating that cosR was inserted downstream of cj0431. The NCTC 11168 + cosR + Km R strain is henceforth referred to as the cosR overexpression transformant or, for simplicity, the "transformant" or "TrfcosR." The growth rates of the parental NCTC11168 strain and the transformant were similar (µ max = 0.69 and 0.72 h -1 , respectively). The overexpression of cosR was validated using quantitative RT-PCR after RNA extraction according to [START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF] with the following modifications. The quantity of total RNA was assessed using a Nanodrop 2000 (Thermo Fisher Scientific, Courtaboeuf, France), and the integrity of the RNA was verified with an Experion ™ Automated Electrophoresis Station (BioRad) using the Experion RNA StdSens Analysis Kit (BioRad) according to the manufacturer's guidelines. Absence of DNA in the samples was confirmed by PCR with primers targeting flaA (Supplementary Table 6). Only high quality RNA samples without DNA contamination were used in qRT-PCR assays.

Adhesion to an Inert Surface

The adhesion capability of C. jejuni strains was determined using the BioFilm Ring Test R (BioFilm Control, France) as described previously by [START_REF] Sulaeman | Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test (R)[END_REF]. Briefly, each culture was pelleted and resuspended in buffered peptone water, the OD 600nm was adjusted to 1 and suspension was used for inoculation of plate wells. After 2 h of cultivation under MAC at 42 • C, adhesion was determined by measuring BFI (Biofilm Index) using the BioFilm Control developed software. The BFI correlates to the number of magnetic microbeads detected after well magnetization. The BFI was calculated by subtracting the BFI of blank control from the BFI of the sample. The assay was repeated three times with three technical replicates for each independent culture.

Biofilm Formation

The crystal violet biofilm assay was used to determine the amount of biofilm produced by C. jejuni. The protocol was adapted from that described by [START_REF] Djordjevic | Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation[END_REF]. Briefly, 2 ml of C. jejuni suspension was inoculated in 24-well sterile microtitre plates. Each plate was incubated statically for 24, 48, or 72 h at 42 • C in MAC. After cultivation, planktonic cells were washed out and biofilm was stained with 1% crystal violet solution. The crystal violet bound to the biofilm was then eluted using 99% ethanol and the absorbance of the eluate was measured at 595 nm.

Qualitative (ultrastructural) and quantitative data (maximum thickness and biomass volume) of C. jejuni biofilm were measured on biofilm produced in 96-well polystyrene microtitre plates with a µ clear R base (thickness of 190 ± 5 µm; Greiner Bio-one, Germany). Prior to the inoculation of microtitre plates, grown cells were transferred from Karmali plates into BHI, washed once and resuspended in sterile BHI to final OD 600nm = 0.8 ± 0.05. The suspension was then loaded onto the microtitre plate in triplicates for each strain (250 µl per well). The plates were incubated in MAC or OEC for 4-5 h at 37 • C allowing C. jejuni cells to adhere to the substratum. After that, the bacterial suspension was carefully replaced with 250 µl of sterile BHI and microtitre plates were incubated for the next 24 and 48 h at 37 • C in MAC or OEC, depending on the experiment. The µ clear R base material allows diffusion of gas molecules into the liquid media and therefore ensures formation of biofilm that is attached to the bottom of the well and not floating on the air-liquid interphase. Finally, wells containing biofilm were stained using 50 µl of Syto 9 solution (Invitrogen, USA) diluted in BHI to the final concentration of 2 µl/ml. The Syto 9 is cellpermeable dye intercalating with DNA and therefore staining the cells and the eDNA of biofilm matrix. All biofilms were observed using confocal laser scanning microscope (CLSM) as described below. For each condition, three independent replicates were analyzed.

Confocal Laser Scanning Microscopy (CLSM) Image Acquisition

Each well of the microtitre plates was scanned using the inverted Leica SP2 AOBS confocal laser scanning microscope (LEICA Microsystems, Germany) at 400 Hz with a 40x/0.8 water immersion objective lens Leica HCX Apo. The fluorophore Syto 9 was excited with a 488-nm argon laser. The whole well area was inspected to verify the presence of biofilm, then the most representative place was scanned providing a stack of horizontal planar images (512 × 512 pixels representing an area of 375 × 375 µm) with a z-step of 1 µm. At least one stack of horizontal planar images was acquired for each replicate.

Image Analysis

The stacks obtained from the microscopic observations were processed using Imaris 7.6.4 software (Bitplane, Switzerland). Images representing an aerial view of biofilm structure were rendered using the Easy 3D view with the auto-adjustment function to correct pixel intensities. Numerical data and 3D models of the biofilm structures were generated using the surface generator function of the Measurement Pro module with the minimal threshold set at 40 for the green channel (Syto 9). Only objects bigger than 10 voxels were included in the analysis. Biofilm development was normalized according to height (thickness determined from z-stacks as the last image showing consecutive signal from biofilm structures) and biomass volume (cell abundance).

Statistical Analyses

The numerical data obtained from Imaris were processed with STATGRAPHICS Centurion 16.1.11 software (StatPoint, Inc., Herndon, VA, USA) with the maximum height (biofilm thickness) and the biomass volume (cell abundance) as explanatory values. For all variance analyses, ANOVAs were performed to determine the individual effect of each factor and potential interacting effects with the confirmation of a normal distribution for each data set.

Assay variations were excluded from interacting effects, as they were not significantly different at the first order. The significance level was selected at 99%, consequently an effect was considered significant if its P-value was lower than 0.01. All Fratios were based on the average residual squared error. When the transformant (TrfcosR) was used, a multiple comparison using the Scheffé method was implemented in ANOVAs to classify the significant variations (at 95% confidence) according to the strains.

For cell adhesion to an inert surface and crystal violet biofilm assays, significant differences were determined using two-sided Student's t-test comparisons at a 95% significance level with the confirmation of a normal distribution for each data set.

FIGURE 1 |

 1 FIGURE 1 | C. jejuni NCTC 11168 and 81-176 biofilm architecture and development are different after incubation for 24 and 48 h in MAC (5% O 2 , 10% CO 2 , 85% N 2 ). The CLSM images represent an aerial view of biofilm structures with the shadow projection at the bottom. The structures were visualized using Syto 9, an intercalating agent staining the nucleic acids.

FIGURE 2 |

 2 FIGURE 2 | Oxygen enhances biofilm development of C. jejuni NCTC 11168 and 81-176 after incubation for 24 and 48 h. (A) The CLSM images represent an aerial view of the biofilm structures in OEC (19% O 2 , 10% CO 2 , 71% N 2 ) with the shadow projection at the bottom. (B) The effect

  1

  .

FIGURE 3 |

 3 FIGURE 3 | Oxygen-enriched conditions enhanced biofilm development prior to and during biofilm formation of C. jejuni. Fold changes (FC) of biofilm development of C. jejuni strains NCTC 11168 (black bars) and 81-176 (white bars) as expressed by maximum height and biomass volume. (A) FC representing biofilm formation of cells acclimatized

FIGURE 4 |

 4 FIGURE 4 | The NCTC 11168 cosR overexpressing transformant enhanced cell adhesion to inert surface and biofilm formation in comparison with its parental strain. Adhesion to an inert surface (A) and biofilm formation (B) of C. jejuni NCTC 11168 (white bars) and the cosR overexpressing transformant (TrfcosR) (black bars) strains. Bacterial adhesion was determined after 2 h by calculating the BioFilm Index (BFI) using the

Frontiers in Microbiology | www.frontiersin.org

July 2015 | Volume 6 | Article 709

Acknowledgments

The work was financially supported by Secalim (INRA/Oniris, Nantes, France), the Czech Science Foundation Grant No. 14-23597S, LD 14097-COST-CZ, 7AMB15FR012 Project Barrande, and the Cost BacFoodNet FA1202. We would like to thank the Micalis Unit (INRA Jouy-en-Josas, France) for material support and advice with special thanks to Julien Deschamps, who helped us to acquire the CLSM images.

Author Contributions

HT, RB, and OT conceived and designed the study. NH, MH, and AS built and validated the transformant. HT, RR, and RB performed the experimental work. HT prepared the manuscript and RB and OT contributed to the final manuscript.

Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.

2015.00709

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Krzysztof Dudek
email: k.dudek@if.uz.zgora.pl

Julio Andrés

Iglesias Martinez

Muamer Kadic

Krzysztof K Dudek

Julio A Iglesias

Martínez Muamer

Kadic K K Dudek

K K Dudek

J A Iglesias Martínez

Variable Dual Auxeticity of the Hierarchical Mechanical Metamaterial composed of Re-entrant Structural Motifs

Keywords: auxetic, mechanical metamaterials, re-entrant metamaterials

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Mechanical metamaterials [1,2,3,4,5,6,7,8,9,10,11,12,13] are rationally-designed structures capable of exhibiting a plethora of atypical mechanical properties that are rarely observed in the case of naturally-occurring materials. Some of the most commonly studied of such properties are negative Poisson's ratio [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29] (auxetic behaviour), negative stiffness [30,31,32,33,34] and negative compressibility [35,36]. Over the last thirty years, it has been demonstrated that devices utilising materials exhibiting such characteristics can be used in the case of a variety of applications including protective devices [37,38], sports equipment [39] as well as biomedical [40,41] and vibration damping devices [42,43,44]. One of the most studied of these unusual mechanical properties seems to be auxetic behaviour where the keen interest of researchers in this property stems from the fact that auxetic materials often exhibit high indentation resistance [45], wave attenuation [46,47] and many other features that are useful in the case of various applications. The commercial appeal of auxetic mechanical metamaterials resulted in a broad range of studies focused on different types of mechanical structures. One of the most commonly investigated classes of such systems are truss-lattice-based auxetic mechanical metamaterials.

In recent years, auxetic mechanical metamaterials in the form of truss-lattice structures have been a subject of numerous studies [48,49,50,51,52,53] with a particular emphasis on the design of efficient protective materials. This stems from the fact that in addition to their ability to exhibit the negative Poisson's ratio, such systems are also very light in comparison to many other auxetic mechanical metamaterials. This, in turn, is of great significance when designing lightweight protective devices that can exhibit significant energy absorption similarly to their heavier counterparts while at the same time they do not significantly increase the mass of the vehicle/object that they protect. One of the earliest and most famous examples of such structures corresponds to the so-called arrow-head mechanical metamaterial that can be used both as a 2D [49] and 3D [50,51,52,53] system characterised by the negative Poisson's ratio. Thanks to the success of this and other similar re-entrant mechanical metamaterials [54,55,56,57,58], over the years, it has been possible to observe a large number of truss-lattice auxetic mechanical metamaterials that are typically working based on a similar hinging mechanism of mutually-connected truss elements. Nevertheless, despite the numerous advantages of standard truss-lattice auxetic mechanical metamaterials, they normally share several limitations. Most importantly, such structures typically correspond to a specific set of mechanical properties that cannot be significantly altered once the structure is manufactured. In fact, a similar limitation often applies to a vast majority of other known mechanical metamaterials. However, in recent years, researchers reported a few approaches that allow to overcome this shortcoming. It seems that one of the most promising approaches allowing to observe numerous different mechanical properties and deformation patterns, without the need of significantly changing or rebuilding the system, corresponds to hierarchical mechanical metamaterials.

Hierarchical mechanical metamaterials [59,60,61,62,63,64,[START_REF] Cho | Proc. Natl. Acad. Sci[END_REF][START_REF] Dudek | [END_REF] are a class of structures composed of multiple structural levels having their own geometry that typically can deform irrespective of each other. This, in some cases, allows the system to exhibit quantitatively very different mechanical properties without the need of being reconstructed. This, in turn, significantly enhances the applicability of hierarchical structures. In principle, the concept of hierarchical mechanical metamaterials is not limited to a specific type of mechanical metamaterials and can be also applied in the case of truss-lattice systems. In the literature, there are several examples of such structures that are based on re-entrant honeycombs [63] as well as other structural motifs [48]. Nevertheless, this direction of studies is still in its infancy and many new studies have to be conducted in order to better utilise the potential of these structures as well as to fully understand the underlying physics. One of such relatively unexplored aspects related to this class of systems corresponds to the possibility of controlling the band gap formation [START_REF] Cho | Proc. Natl. Acad. Sci[END_REF]67] that would allow to construct tunable vibration insulators or sensors for different ranges of frequencies. Another aspect corresponds to the control over the mechanical properties (e.g. auxeticity) of the hierarchical structure. In this case, despite the success of several projects and their commercial appeal, there are still relatively few known efficient hierarchical mechanisms in comparison to standard non-hierarchical mechanical metamaterials.

In this work, a novel hierarchical mechanical metamaterial composed of truss-lattice elements is proposed and analysed from the point of view of its mechanical properties. More specifically, it is demonstrated that depending on the thickness of different groups of its hinges, the considered structure can follow different deformation patterns. In addition, depending on which hierarchical level is deforming, the whole structure can exhibit a different type of auxetic behaviour that corresponds to a unique deformation mechanism. This results in a dual auxetic structure where the interplay between the two auxetic mechanisms determines the evolution of the system. Finally, it is also presented that depending on which hierarchical level of the structure is deforming, one can observe a drastically different phonon dispersion of the system that also leads to a very different band gap formation.

Model

Design

The main objective of this work is to design a hierarchical mechanical metamaterial capable of exhibiting versatile magnitudes of the negative Poisson's ratio that can be adjusted by a small variation in the design parameters with an emphasis on the thickness of hinges. To this aim, the considered model corresponds to the two-dimensional hierarchical mechanical metamaterial composed of truss-lattice elements as shown in Fig. 1.

The considered system is a two-level hierarchical structure composed of truss-lattice elements having the thickness of l 3 (see Fig. 1(a)). Level 1 of the structure corresponds to parallelogram-like elements connected to each other at vertices similarly to the well-known rotating squares [23] or rotating rhombi [68] systems that had been reported to be able to exhibit auxetic behaviour. On the other hand, level 0 elements assume the form of two arrow-head units that share one edge having a length denoted as a. It is important to remember that as mentioned in the Introduction, mechanical metamaterials consisting of arrow-head-based structural elements are known in the literature and, as shown in diagrams presented in Fig. 1(b), can exhibit a strong auxetic behaviour. This means that both level 0 and level 1 of the considered hierarchical system correspond to very different deformation mechanisms that can lead to the entire system exhibiting a negative Poisson's ratio. For the considered model, such observation is of great significance since the two hierarchical levels can deform independently. This means that level 1 blocks can rotate with respect to each other while retaining their shape. Thus, such a process can occur without hinging of truss-lattice beams corresponding to level 0 elements. Conversely, it is possible to observe hinging of level 0 elements without the rotation of level 1 blocks. In the case of this model, the hinging of level 0 elements is quantified by a change in the value of θ (see Fig. 1(a)). On the other hand, the rotation of level 1 blocks is quantified by a variation in the value of γ. As a result, once the structure is manufactured and specific geometric dimensions are well-defined, the magnitudes of angles θ and γ provide full information about the configuration assumed by the structure assuming no flexure of trusslattice elements.

The factor that determines the extent of the deformation of level 0 and level 1 of the structure is the relative thickness of hinges connecting structural elements corresponding to different hierarchical levels. These hinges are denoted as d 1 -d 5 in Fig. 1(a) and in general can assume very different values. As demonstrated schematically in Fig. 1(b), it is expected that upon changing the relative thickness of hinges connecting truss-beams corresponding to level 0 and level 1 elements, it should be possible to increase the extent of deformation of one hierarchical level over another. In the extreme case, if the difference in the thickness of the two groups of hinges was very considerable, it could be even possible to observe an isolated deformation either of level 0 or level 1 of the system. Such possibility is shown in Fig. 1(b) where in the first scenario one can see the deformation of the structure corresponding solely to the rotation of level 1 blocks while in the second case the deformation corresponds exclusively to hinging of level 0 elements. However, in many situations, it can be expected that the mixture of the two deformation mechanisms could be observed.

At this point, it should be also noted that the two types of truss-lattice beams present within the system have a very specific shape. Namely, both ends of such beams having the length of either a or b (see Fig. 1(a)) are significantly narrower than their central parts. This particular aspect of the design of the considered system allows ensuring that its deformation will occur primarily via the relative hinging of truss-lattice beams and that their flexure will be negligible.

Simulations

To assess the mechanical properties of the considered system, all models are deformed via the compression along the y-axis as schematically indicated by means of black arrows in Fig. 1(b). Furthermore, since only a single unit-cell is taken into account, in order to describe the behaviour of the system, it is assumed that periodic boundary conditions are imposed [69] on the left and right edge of the unit-cell. These boundary conditions ensure that the two edges retain the form of a straight line throughout the deformation process. In order to simulate the behaviour of the structure, Finite Element Method (FEM) simulations were conducted through the use of the COMSOL Multiphysics software. In order to obtain realistic results, the nonlinear geometry feature was enabled while it was assumed that the material itself was isotropic. More specifically, the respective properties of the material used for the considered model were set to be the following for all conducted simulations. Youngs's modulus: 4 GPa, Poisson's ratio: 0.4 and density: 1200 kg m -3 . These material properties correspond to some of the resins [70] that could be used in order to 3D print the considered structure. The remaining geometric parameters used in the simulations were set to be the following: a = 3.5 cm, b = 1.5 cm, θ(t = 0) = 46.24 • (initial value), γ(t = 0) = 10 • (initial value), l 1 = 5.5 mm, l 2 = 4 mm, l 3 = 2 mm.

At this point, it should be mentioned that in this work, only three variations of the considered system are being studied instead of a hypothetical larger number of possible scenarios. The reason for this is the fact that there are only three types of the behaviour of the analysed structure that are significantly different from each other. Namely, the two deformation processes corresponding solely to the hinging of level 0 elements or the rotation of level 1 blocks as well as the hybrid case associated with the combination of these two deformation mechanisms. Of course, one could consider a larger number of scenarios but all of them would exhibit the behaviour that is qualitatively the same as for the already considered cases.

d 1 [mm] d 2 [mm] d 3 [mm] d 4 [mm] d 5 [mm] d 6 [mm]

Results and Discussion

In order to assess the ability of the considered system to exhibit versatile deformation patterns and different values of the negative Poisson's ratio, three different configurations of the considered model were selected. These configurations named case 1, case 2 and case 3 are almost identical with the only difference between them being the thickness of hinges d 1 -d 6 . Specific values of these parameters are provided in Table 1.

Controllable auxetic behaviour

As shown in Fig. 2, the three different versions of the considered system exhibit very distinct deformation patterns. According to Fig. 2(a), the structure named case 1 deforms primarily via the rotation of level 1 blocks while level 0 elements do not undergo the hinging process. This stems from the fact that as specified in Table 1, hinges within the arrow-head-shaped structural elements (in particular the hinge having its thickness defined as d 1 ) are in this case very thick in comparison to hinges connecting level 1 blocks (primarily hinges having their thickness defined as d 2 and d 5 although all hinges with the exception for the hinge having the thickness of d 1 affect the rotation of level 1 elements). A very different behaviour can be observed in the case of the structure named case 2 where level 1 blocks seemingly do not rotate while level 0 elements undergo a hinging process to a significant extent. This very different deformation pattern can be explained by the fact that in this scenario, some of the hinges connecting level 1 blocks are very thick. On the other hand, hinges connecting truss beams within arrow-head structural elements are very thin. Finally, it is also important to note that as demonstrated in the case of the deformation of the structure referred to as case 3, it is possible to observe the simultaneous rotation of level 1 elements and hinging of level 0 structural motifs. For better clarity, all three of these deformation mechanisms are schematically shown in Fig. 2(c). At this point, it is worth mentioning that in the hypothetical scenario where all of the hinges present within the system would have the same thickness, the behaviour of the structure would resemble that of the system referred as case 3 (see Supplementary Information). It is also worth noting that the three cases considered in this work represent the only types of the mechanical behaviour that are qualitatively different from each other. Any other combination of the values d 1 -d 6 than those already considered will result with one of the three deformation mechanisms described in the main text.

In addition to the possibility of conducting the qualitative comparison of the bahaviour of the three types of the considered model, it is also possible to conduct the quantitative analysis where the variation in specific geometric parameters would be analysed. As shown in Fig. 3, the above qualitative discussion matches the variation in angles θ and γ that quantify the deformation of level 0 and level 1 respectively. More specifically, one can note that angle θ changes significantly for case 2 and case 3 structures (see Fig. 3(a)) where both of these cases correspond to a significant hinging of level 0 elements. On the other hand, for the structure named case 1, it is possible to see a very small change in this parameter. Conversely, as presented in Fig. 3(b), the opposite trend can be seen in the case of the variation in the angle γ that defines the extent of rotation of level 1 elements.

As described above, the change in the thickness of some of the hinges can significantly change the deformation patterns exhibited by the structure during the compression. However, from the point of view of potential applications, it is essential to determine the ability of the considered structure to exhibit a negative Poisson's ratio. As shown in Fig. 2(b), all of the considered versions of the analysed system exhibit auxetic behaviour throughout the deformation process. This in turn is very important since it shows that a change in the deformation pattern does not affect the ability of the structure to exhibit a negative Poisson's ratio. Nonetheless, it does not mean that its magnitude does not change. According to Fig. 2(b), the Poisson's ratio corresponding to the case 1 structure assumes very low values that reach the magnitude of approximately -2.5. On the other hand, a significantly less negative value of the Poisson's ratio can be observed in the case of the deformation of the system referred to as case 2. For this structure, the magnitude of the Poisson's ratio initially assumes the value of -0.5 and becomes gradually lower as the vertical compression of the system continues. Furthermore, in the last of the analysed scenarios, the structure named case 3 exhibits the intermediate range of the negative Poisson's ratio with its values changing at the interval approximately between -0.6 and -0.7. One can note that the Poisson's ratio, in this case, is more similar to case 2 than the case 1 system. This can be explained by the fact that for this type of structure, the hinging of level 0 elements is more dominant than the rotation of level 0 elements (see Fig. 2(a) and Fig. 3). However, this does not mean that the intermediate case cannot exhibit the Poisson's ratio that is more similar to the auxeticity exhibited by the case 1 system should the thicknesses of its hinges be appropriately adjusted.

Vibrational properties

In addition to the possibility of controlling the deformation pattern and the extent of the exhibited auxeticity, it is also interesting to check whether different types of the considered model allow observing some differences in terms of the band gap formation and frequencies of vibrations that can propagate through the system. To achieve it, FEM simulations were conducted by means of the COMSOL Multiphysics software. In the case of each of the structures, to generate the results, Floquet periodic boundary conditions were implemented in the x-and y-directions assuming two-dimensional unit-cells. All of the design parameters were set to be identical as in the case of mechanical testing.

Based on Fig. 4, one can note that the phonon dispersion graphs corresponding to systems called case 1 and case 2 are very different. Most importantly, in the case of the first system, within the presented range of frequencies, there are no band gaps. On the other hand, the phonon dispersion associated with the case 2 structure corresponds to a band gap for frequencies approximately in the range between 0.5 kHz and 1.3 kHz. This, in turn, is very interesting as it indicates that a small change in the thickness of the hinges may cause the appearance of a considerable band gap where its size can be further modified by adjusting the relative thickness of the hinges. This means that the considered system can in general undergo a transition from the structure with no band gaps to the system working as a vibration damper where specific frequencies are not transmitted. Of course, in the current version of the proposed model such transition cannot occur in an active manner since the change in the thickness of hinges would require reconstructing the system or subjecting it to a post-manufacturing treatment that would alter some of its dimensions. However, this is not the only approach making it possible to change the behaviour of the considered system from the characteristic typical for the case 1 system to the behaviour of the case 2 structure and vice versa. This can be achieved for example by constructing hinges from another material corresponding to a very different thermal expansion coefficient than the rest of the system. Such a solution could allow changing the behaviour of the structure at different temperatures. Another approach could correspond to the use of electromagnets / magnets [53] embedded in specific parts of the unit-cell. The mutual interaction of such inclusions could adjust the effective stiffness of specific hinges within the system and as a result, would allow for the active transition of the structure. At this point, we would like to also emphasise the fact that according to the results presented in Fig. 4, the band gap formation can be controlled by the relative thickness of hinges within the system with similar results being observed for other hierarchical mechanical metamaterials known in the literature [67]. However, even though this topic does not belong to the scope of this work, the band gap formation could also be potentially influenced by the deformation of the system. Nevertheless, this aspect of the behaviour of the considered structure would have to be thoroughly analysed before reaching any conclusions.

In this work, we decided to focus on two specific configurations that deform via two different deformation mechanisms, i.e. the hinging of level 0 elements and the rotation of level 1 blocks. As demonstrated in Fig. 4, these two cases result in drastically different phonon dispersion relationship, where for the structure referred to as case 2, a significant band gap can be observed. At the same time, for the structure referred to as case 1, no band gab can be seen within the considered range of frequencies. If one was to consider the hybrid scenario that incorporates the two types of the deformation mechanisms, i.e. the system called case 3, the size of the band gap would be reduced relative to the structure referred to as case 2. This stems from the fact that as demonstrated in Fig. 4(a), the rotation of level 1 blocks does not lead to the formation of band gaps.

All of this is very important since, in this work, it is shown that the proposed model can exhibit versatile deformation patterns depending on the interplay between the two auxetic mechanisms present within the system. It is also shown that a modification in the thickness of different groups of hinges can significantly change the extent of the auxetic behaviour exhibited by the system. Both of these results are not commonly observed for standard mechanical metamaterials and can have multiple applications. More specifically, if one was to construct the active version of the considered system (e.g. by using mutuallyinteracting inclusions in the form of electromagnets to modify the effective stiffness of hinges), then it would be possible to significantly change auxeticity of the structure without the need of reconstructing the system. This would allow to construct highly-efficient protective devices that could adjust the extent of their auxeticity to a specific cause of the deformation. The concept presented in this work could be also very interesting from the point of view of biomedical devices. More specifically, if one was to construct the structure composed of multiple unit-cells corresponding to a different deformation pattern, then in general, it would be possible to arbitrarily modify the shape of the entire structure. If the mismatch in the Poisson's ratio between different parts of the system would be significant, it would be possible to observe considerable shape-morphing similarly to the concept proposed in [START_REF] Dudek | [END_REF] (in the aforementioned study, it was demonstrated that an initial shape of the metamaterial subjected to the mechanical deformation can be significantly modified depending on the ratio in the Poisson's ratio associated with different parts of the structure). The possibility of exhibiting controllable shape-morphing could be used in the design of stents in order to better support specific parts of the blood vessel that are particularly weak. Finally, it is important to emphasise the fact that the results presented in this work are not limited to a specific scale. In fact, the proposed model can be even constructed at the microscale.

Conclusion

It is shown that the considered hierarchical structure can follow very different deformation patterns depending on the variation in the thickness of its hinges. This means that the proposed system can utilise very different deformation mechanisms which is very atypical and highly beneficial in comparison to typical mechanical metamaterials. Furthermore, in this work, it is presented that the considered model can exhibit a very broad range of the negative Poisson's ratio that can be controlled by adjusting the relative stiffness of hinges. This result, which originates from the interplay of two independent auxetic mechanisms present within the structure, indicates that in theory, the auxeticity of the system can be adjusted depending on the specific application. This, in turn, could prove to be very useful in the design of highly-efficient protective devices. Finally, it should be emphasised that different configurations of the considered system may result in a very distinct behaviour of the structure in terms of frequencies of waves that can be transmitted through the analysed metamaterial. In fact, it is demonstrated that even a very small change in the parametric design of the system may lead to a significantly different band gap formation that can be useful in the design of tunable vibration dampers or sensors.

Figure 1 :

 1 Figure 1: The unit-cell of the structure analysed in this work from the point of view of its mechanical properties. a) A diagram of the unit-cell of the system with the definition of all of the dimensions and geometric parameters of the structure. b) Different deformation patterns exhibited by the considered system in the case of the isolated deformation of the level 0 or level 1 elements of the hierarchical structure.

Figure 2 :

 2 Figure 2: Deformation patterns and Poisson's ratio of the three versions of the considered model. a) Deformation of the three analysed cases subjected to the compression along the y-axis. For each of the examples, the black outline in the background corresponds to the initial shape of the structure. The extent of mechanical deformation is graphically enhanced by a factor of 2 to visually emphasise the difference between different deformation mechanisms. The deformation processes corresponding to structures named case 1, case 2 and case 3 are also presented as animations provided in the form of the Supplementary Video 1, Supplementary Video 2 and Supplementary Video 3. b) Engineering Poisson's ratio for all types of the system. c) Conceptual diagrams showing differences between the three types of possible deformation patterns.

Figure 3 :

 3 Figure 3: Variation in the angles describing the deformation of level 0 and level 1 of the considered hierarchical structure.

Figure 4 :

 4 Figure 4: Phonon dispersion for the two types of structures considered in this work. The inset provided on panel (a) schematically depicts the first Brillouin zone corresponding to the considered model. Specific points presented at the centre as well as at the edge and corners of the first Brillouin zone indicate high-symmetry points that were used in the conducted analysis in order define directions in which the vibrational properties of the system were investigated.

Table 1 :

 1 The thickness of hinges corresponding to three types of configurations of the considered model.

	case 1	1.6	0.6	8.0	4.0	0.6	4.0
	case 2	0.2	2.5	0.4	0.2	0.4	0.2
	case 3	0.8	1.6	1.6	1.4	1.6	0.8

Acknowledgements K.K.D. acknowledges the support of the Polish National Science Centre (NCN) in the form of the grant awarded as a part of the SONATINA 5 program, project No. 2021/40/C/ST5/00007 under the name "Programmable magneto-mechanical metamaterials guided by the magnetic field". This research was funded by the Polish Minister of Education and Science under the program "Regional Initiative of Excellence" in 2019-2023, project No. 003/RID/2018/19, funding amount PLN 11 936 596.10.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of Interest

The authors declare no conflict of interest.