Jerome Lelievre

M Josse

Pascal Marchet
email: pascal.marchet@unilim.fr

Structure and properties of (Na0.5Bi0.5)ZrO3 (NBZ) lead-free perovskite compound

Keywords: Lead-free perovskite, neutron diffraction, crystal structure, dielectric, relaxor

The structure of NBZ was studied by neutron diffraction. NBZ is monoclinic, (SG #11 P21/m) am = 5.696(8) Å, bm = 8.166(9) Å, cm = 5.790(4) Å and βm = 89.97(4)°. Being centrosymmetric, NBZ is neither piezoelectric nor ferroelectric. No phase transition was evidenced between 100 K and 923 K. NBZ presents a relaxor behavior below room temperature attributed to the mixed occupancy of the A-site by both Na + and Bi 3+ ions. The relative permittivity of NBZ appears noticeably stable (for 1 kHz -2.9% at -55 °C / 218K and +5.3% at 85 °C / 358 K compared to the 25 °C / 298 K value). NBZ appears thus as promising for the elaboration of capacitors requiring high thermal stability.

formula Pb(Zr1-xTix)O3. Indeed, PZT-based compositions are the dominant piezoceramics due to their high electromechanical properties and low cost. Because of restrictions about the use of lead-based materials related to health and environmental problems, a huge scientific effort started about 20 years ago in order to substitute PZT. Nowadays, only a few lead-free materials are considered as possible candidates to this substitution and most of them are of perovskite structure: (i) BaTiO3-based materials, (ii) Alkaline-niobates such as (K,Na)NbO3 (KNN) and (iii) alkaline-bismuth titanates like (Na0.5Bi0.5)TiO3 (NBT) or (K0.5Bi0.5)TiO3 (KBT) [1][2][3][4][5][6][7][8][9][10]. Nevertheless, the search for new lead-free compounds is still topical question, whether or not this compounds are ferroelectric/piezoelectric. In addition, perovskite compounds presenting mixed occupancy of A or B-site (e.g. NBT or PMN) could also present relaxor behavior and interesting dielectric properties.

In the case of titanates, the possibility of substituting titanium by zirconium was evidenced a long time ago. Indeed, PbTiO3, PbZrO3, BaTiO3 and BaZrO3 are perovskite compounds, even if their structure and space groups are different. Thus, this replacement is probably also possible in complex perovskite compound like NBT. Surprisingly, this possibility has only been barely considered. After the discovery of NBT in 1961, Smolenskii considered the possible occurrence of the (Na0.5Bi0.5)ZrO3 (NBZ) compound but reported only dielectric measurement without any XRD pattern [11]. In 1995, a letter devoted to the dielectric properties of NBT-NBZ-KBT-KBZ system suggested the occurrence of NBZ as a "not identified" compound [12]. It was not until 2007 that NBZ was evidenced for the first time and its XRD pattern reported [13,14]. NBZ was assigned to an orthorhombic structure (a = 4.379 Å, b= 7.489 Å and c = 7.078 Å), but the possible space group was not considered. The dielectric properties and conduction mechanisms were also studied, evidencing a grain boundary effect around 350°C (623 K), associated to non-Debye type dielectric relaxation [13,14]. A broad ferroelectric-paraelectric phase transition (diffuse phase transition) was reported at 425°C (698 K) [13]. The elaboration of NBZ powders and the densification mechanism of ceramics were studied and the orthorhombic space group Pnma (SG #62) was attributed to NBZ without any structural study [15,16]. Ferroelectric properties were reported for NBZ [17], but: (i) this property is conflicting with the centrosymmetric Pnma space group and (ii) the elliptic shape of hysteresis loop is typical of a lossy dielectric and not of a ferroelectric material [18]. The evolution of lattice parameters and dielectric properties in the (Na0.5Bi0.5)(Zr1-xTix)O3 solidsolution were later studied [19][20][21][22], confirming that zirconium can totally substitute titanium in such complex perovskites, like for KBT-KBZ system [23]. To summarize: (i) the existence of the NBZ perovskite compound is clearly established, (ii) the reported space group and ferroelectricity are questionable, (ii) the occurrence of phase transitions is questionable. Therefore, the aim of the present study is: (i) to determine the structure of NBZ, (ii) to study the dielectric properties for a large temperature range including low temperatures and (iii) to try to detect possible phase transitions below and above room temperature.

The NBZ compound was firstly synthetized in alumina crucible using Na2CO3 (Aldrich 99+ %), Bi2O3 (Puretech 99.975 %) and ZrO2 (Alfa Aesar, 99.7 %). A perovskite compound was obtained and XRD results evidenced a pattern similar to the one already reported [13][14]. Some secondary phases were also observed and assigned to Na2ZrO3, unreacted ZrO2.and eventually to doped bismuth oxide such as Na0.29Bi1.71O2.71. Similar result was obtained in platinum crucible thus excluding chemical reaction with crucible. Attempts to suppress this secondary phases by annealing were unsuccessful. As a consequence, the NBZ compound was finally synthetized by solid-state route using NaBiO3 (Alfa Aesar, 80 %) and ZrO2 (Alfa Aesar, 99.7 %). The raw materials were dried at 150 °C, weighted in stoichiometric amounts and calcined at 800 °C for 4 hours in alumina crucible. The weight losses associated to the volatilization of Na or Bi were checked as lower than 0.5 %. The XRD patterns (θ/2θ diffractometer, D8 Panalytical, Cu Kα1) of the obtained powder evidenced less secondary phases than for preliminary synthesis. (see supplementary materials, fig. S1). The crystallographic study of the powders has been led by using neutron diffraction (Leon Brillouin Laboratory, 3T2 diffractometer, Saclay, France). The powder (≈ 10g) has been placed in a vanadium can and the diffraction data has been collected between 4.5 and 121° at 20 °C (293 K) during 8 hours at a 1.2289 Å wavelength for incident neutrons. Structural refinements were performed using the Jana 2006 software [24]. A mixture of plasticizer and binder (polyethylene glycol and polyvinyl alcohol) was added to the powder in order to obtain suitable green pellets, then shaped by uniaxial pressing (250 MPa, 10 mm diameter matrix). Sintering has been carried out at 900 °C / 4hours. Dielectric measurements were performed between 80 K and 450 K using an impedance analyzer (HP 4194A, 100Hz -1MHz). Differential Scanning Calorimetry was performed on powders between 100 K and 350 K in order to search for phase transitions (DSC Q1000, TA Instruments) and by TG/DSC between 303 K and 1773 K (Netzsch STA 449F3). High temperature X-ray diffraction (HT-XRD) was performed between 300 K and 923 K in order to search for high temperature phase transitions.

As previously reported, powder neutron diffraction patterns (PND) evidenced that: (i) secondary phases Na2ZrO3 [25] and unreacted ZrO2 [26] are observed, (ii) the main peaks of the perovskite structure are clearly split and (iii) some extra peaks are observed, indicating a superstructure due to octahedra tilting [27][28][29][30]. Another unidentified compound is also observed by XRD, probably coming from Na or Zr -doped bismuth oxide like Na0.29Bi1.71O2.71, Bi7.38Zr0.62O12.31 or Bi7.38Na0.62O11.38. Considering the splitting of the main diffraction peaks of the perovskite phase, the structure could be either orthorhombic, monoclinic or triclinic. Thus, the structural investigation was undertaken based on the group / subgroup relationships associated to the different possible tilt systems and taking into account the Na2ZrO3 and ZrO2 secondary phases. The preliminary refinements quickly indicated a three tilts system, thus with possible space groups Pnma, C2/c, P1 or P21/m [31]. The former three groups gave results with quite poor quality factors or did not converge.

Refinement using the monoclinic P21/m were then performed, using as starting parameters the atomic positions of the ScRhO3 compound [32]. After refinement of the atomic positions, isotropic displacement parameters were refined. In a third step, anisotropic parameters for the (Na + /Bi 3+ ) pseudo-ion improved the results (see supplementary material, table 1), while the use of anisotropic parameters for other ions gave a non-meaningful solution. Therefore, anisotropy was only retained for (Na + /Bi 3+ ) pseudo-ion. The observed, calculated and difference curves for the final refinement are represented in fig. 1. The profile fit quality factors are satisfactory (Rp = 2.66%, wRp = 3.53%), indicating that the refinement with this space group is adequate.

As for all perovskite compounds, the structure corresponds to a network of tilted cornersharing [ZrO6] octahedra (fig. 1 b &c). The octahedra are tilted along the three directions, with a tilt system a -a + c -in Glazer notation (i.e. tilt system #9 [27]). Two kinds of octahedra are encountered, forming in phase tilted chains along the [010] direction and linked by O(3) and O(4). The first type includes Zr(1), O(2), O(3) and O(4) while the second one includes Zr(2), O(1), O(3) and O(4). These ZrO6 octahedra are slightly distorted with three different Zr-O distances. However, this distortion is small and these distances remains close to the mean value of 2.099 Å, with angles close to 90° (see supplementary material, table 1 andfig S2). The situation is different for the (Na + /Bi 3+ ) pseudo-ion. For sake of accommodation of (Na + /Bi 3+ ) pseudo ion, and the lone pair of the latter, the cuboctahedral 12 fold coordinated cavity is turned into an 8-fold coordinated polyhedron by means of octahedra tilting, with several bond lengths between 2.41 and 2.97 Å. Moreover, the (Na + /Bi 3+ ) pseudo-ion is represented by a flattened ellipsoid. These flattened ellipsoids have their larger directions in planes which are approximately {110} planes of the primitive perovskite cell forming 90 ° angles for the (Bi1/Na1) and (Bi2/Na2) sites. This effect probably comes from both compositional (Na/Bi) and spatial (different Na/Bi polyhedra) disorder on the A-site of the perovskite lattice induced by the mixed occupancy by two different ions, to which the lone-pair stereochemistry of Bi is adding. The chemical formula obtained by the refinement, (Na0.46Bi0.54)ZrO3, is only slightly different from the initial value (Na0.5Bi0.5)ZrO3. However, the chemical composition, calculated by using the mass fraction obtained by the refinement, indicates a Na/Bi ratio 0.61:0.44. This bismuth deficiency is probably due to its volatilization during synthesis, as frequently observed for other Bi-containing compounds. Furthermore, it must be noticed a trend to Na + and Bi 3+ ordering between the [Bi1/Na1] and [Bi2/Na2] sites: the [Bi1/Na1] ratio is 0.124:0.376 and 0.416:0.084 for [Bi2/Na2], against 0.5:0.5 initial value. This results indicates a tendency to Na enrichment for the first site and to the reverse for second one. Such Na/Bi ordering was reported in the homologous compound NBT [33][34][35]. Thus this phenomenon is not only observed in the case of NBZ. This ordering is correlated with mean displacement of (Na/Bi) ions since the direction of these displacements is different for the (Bi1/Na1) site which is Na rich and (Bi2/Na2) site which is Bi rich. Finally, these results clearly demonstrate that the correct space group of NBZ is P21/m and that the previous assignment to Pnma space group was unsuitable. As a consequence, and since this space group is centrosymmetric, NBZ can't be neither a ferroelectric nor a piezoelectric compound.

The XRD pattern of the studied ceramic pellet evidenced unreacted ZrO2.secondary phase and that Na2ZrO3 was eliminated during sintering (see supplementary materials fig. S1). However and because of the mixed occupancy of the A-site of the perovskite lattice by both Bi and Na-ions, some relaxor properties could be eventually observed. As a consequence, the relative permittivity ε' and dielectric losses tan(δ) of NBZ were investigated between 80 K and 450 K using frequencies ranging from 100 Hz to 1 MHz (Fig. 2). Higher temperatures were not investigated because of low densification of the sample. Several anomalies are observed: (i) between 100 K and 200 K, ε' is dependent on frequency and associated to a peak of tan(δ), (ii) from 250 K to ≈ 400 K, the relative permittivity remains quasi constant, (iii) above 400 K, ε' and tan(δ) increase quickly.

Between 100 K and 200 K, ε' is dependent on frequency and associated to a peak of tan(δ). This phenomenon is thus clearly associated to a relaxation process, as for the relaxor part of solid-solutions like BaTiO3-BiScO3 or BaTiO3-Bi(Zn2/3Nb1/3)O3, for which the chemical disorder in A-site or B-site of the perovskite lattice disrupts the long range ferroelectric order and changes the behavior to relaxor [36][37][38]. In the case of NBZ, this relaxation phenomena is probably related to the partial chemical ordering observed on the (Na/Bi) sites. The lack of epsilon peak is probably associated to the low temperature of the freezing of polar nano regions associated to local ordering. The relaxation phenomena are generally analyzed using the temperature tm of the maximum of the permittivity peak, which also corresponds to the inflexion point of the peak of tan(δ) curve. Since ε' doesn't present any maximum, the relaxation phenomena was analyzed using the temperature tm of the inflexion point of the tan(δ) curve for a given frequency (see supplementary materials, fig. S3). Both Arrhenius and Vogel-Fulcher fits to the frequency dispersion were considered [36]: = for Arrhenius and = ( ) for Vogel-Fulcher, where f is the frequency associated to the temperature Tm of the inflexion point of the tan(δ) curve, f0 the attempt jump frequency, Ea the activation energy, kb the Boltzmann constant and Tf the freezing temperature. (see supplementary materials fig. S4 for the results of the fit). For both cases, the correlation coefficient is close to unity indicating a good fit of the data (R² = 0.997 and 0.996). However, the attempt jump frequency obtained for Arrhenius fitting (f0 = 1.6 10 15 Hz) seems quite large compared to reported values (5.10 9 to 9.10 12 Hz) [36]. The Vogel-Fulcher fit considers dipole dynamic initiated above a freezing temperature, Tf, thus similar to dipolar glasses. This model is generally more appropriate for dielectric relaxors and Tf is the temperature below which thermal energy is no longer sufficient to permit dipolar cluster dynamics in relaxors [36]. The obtained results for the VF model (Ea = 0.17 eV, f0 = 6.8 10 11 Hz) are very similar to the one obtained for the BaTiO3-BiScO3 or BaTiO3-Bi(Zn2/3Nb1/3)O3 systems (Ea = 0.24 -0.26 eV, f0 = 10 11 -10 12 Hz) [36][37][38]. For the BaTiO3-BiScO3 system, this behavior, which also implies a similar trend of the dielectric properties, was attributed to a dipolar glass behavior associated to non-interacting or weakly coupled polar nanoregions [37]. In our case, similar phenomena could be due to the mixed occupancy of the A-site of the perovskite lattice by both Na + and Bi 3+ ions.

Above the relaxation range and between 250 K and ≈ 400 K, the relative permittivity remains quasi constant and the relative permittivity depends very few on frequency. At 300 K εr = 73.3 for 100 Hz, 84.1 for 1 kHz, 83.7 for 10 kHz, εr = 83.3 for 100 kHz and 82.7 for 1 MHz. This material appears thus as very suitable for dielectric applications, for which frequency stability is required. In additions, the thermal evolution is very limited: the relative change of ε' (using the EIA standard for temperatures) ∆ = at 1 kHz is -2.9% at -55 °C / 218K and +5.3% at 85 °C / 358 K compared to the 25 °C / 298 K value. The low values of the dielectric constant seems to confirm the absence of polar order, contrary to the parent compound (Na0.5Bi0.5)TiO3 (NBT) for which a long range ferroelectric order is observed, associated to high relative permittivity (εr ≈ 500 at 300 K [39]). The increase of ε' and tan(δ) above 400 K corresponds to the increasing conductivity of the material. Indeed, previous studies reported a hopping conduction mechanism associated to bismuth volatilization during sintering [13][14]. Such mechanism induces oxygen vacancies which increase conductivity of the material and the dielectric losses. However, this phenomenon appears at a lower temperature (around 400 K) than previously reported (around 300 °C i.e. 573 K) [14]. This difference could be due to the sintering process. Indeed, previous reports concern ceramic samples sintered under oxygen, while our samples were sintered in air. Therefore, oxygen losses are probably larger in our case and the hopping conductivity mechanism is thus triggered at a lower temperature.

Concerning the polar properties, since the crystal structure is described in the centrosymmetric P21/m space group from 100 K to 900 K, the NBZ compound can present neither piezoelectricity nor ferroelectricity in this temperature range. Therefore, the previous assignment of ferroelectric properties for NBZ was incorrect [17]. Attempts to detect electrostrictive properties were unsuccessful. Furthermore, and since NBZ is not cubic and presents a three tilts system, one can suppose that phase transitions towards other symmetries could exist for this compound (change of tilt system and/or change of crystalline symmetry). Therefore, we investigated for such phase transitions by DSC (low temperature range) and TG/DSC and HT-XRD above room temperature (see supplementary materials fig. S5-7). Below room temperature, we didn't detect any phase transition by DSC. The absence of thermal event in the DSC results confirms that the low temperature anomaly of the dielectric constant comes only from a relaxation process, without any associated phase transition.

Above room temperature, apart from water desorption (330 K -400 K), a jump of the baseline is observed in the range 615 K -670 K (342-397°C). This event could be associated to a second order phase transition like the diffuse phase transition reported in the literature around 698 K (425° C) [13]. However, this event can't correspond to ferroelectric -paraelectric phase transition as previously reported [13], since the room temperature structure is non ferroelectric. Above room temperature, the HTXRD results only indicates a monotonous decrease of the monoclinic angle from β = 90.04(7)° at 303 K (30 °C) toward β = 90.03(6)° at 923 K (650 °C, fig. 3, standard deviation 0.002°). However, the phase transition reported at 698 K (425 °C) [13] was not detected and the splitting of the main peaks of the perovskite lattice persist. As a consequence and according to the group -subgroup relationships known for tilted perovskites [29], this event could correspond to a modification of tilt system (second order phase transitions) leading from P21/m (a -a + c tilt system) to C2/m (a 0 b -c -) or C2/c (a -b -b -) monoclinic space groups.

A third thermal event associated to weight loss (1 %) was also detected between 840 K and 1000 K (567 °C -727 °C). Because of weight loss, this event is probably associated to decomposition of the sample. Further investigation will be performed later in order to identify the existing phases for this temperature range. Finally, melting occurs at 1290 K (1017°C) associated to evaporation of liquid phase with a large weight loss (27.8%).

To sum up, the structure of NBZ was studied by neutron diffraction. The NBZ compound is monoclinic, with space group P21/m (SG #11) and lattice parameters am = 5.696(8) Å, bm = 8.166(9) Å, cm = 5.790(4) Å and β = 89.97(4)°. Since this space group is centrosymmetric, NBZ can present neither piezoelectric nor ferroelectric properties, contrary to what previous results reported. The study by DSC and HTXRD didn't evidence for any phase transition in the range 100 K -923 K. The diffuse phase transition previously reported at 662 K (425 °C) and assigned to ferroelectric-paraelectric phase transition was not detected. The dielectric study of NBZ evidenced a relaxor behavior below room temperature (150 -250K). The study of this relaxation process revealed an activation energy of 0.17 eV with attempt frequency f0 = 6.8 10 11 Hz. The appearance of this relaxation process is similar to the one observed in BaTiO3 based solid-solutions and can be attributed to some cationic disorder due to the mixed occupancy of the A-site of the perovskite lattice by both Na + and Bi 3+ ions. Because of the absence of phase transition, the relative permittivity of NBZ appears noticeably stable since a change of only -2.% for -55°C / 218 K and +5.3% for 85°C / 358K was measured compared to 25°C / 298 K value. Even if the relative permittivity is not very high (εr = 80.6 for 1 kHz), this compound appears thus as promising for the elaboration of capacitors requiring high thermal stability of their properties.

Figure Caption

Supplementary materials

Fig. 1 :

 1 Fig. 1: (a) Room temperature neutron diffraction pattern of NBZ powder and results of crystallographic refinement using the space group P21/m (SG #11) and taking into account secondary phases Na2ZrO3 and ZrO2 and representation of the obtained NBZ structure along the directions (b) [010] and (c) [100]

Figure 2 :

 2 Figure 2 : Evolution of (a) the relative permittivity and (b) the dielectric losses tan(δ) of NBZ as a function of the temperature for different frequencies (524.8 Hz, 1 kHz, 5.25 kHz, 10 kHz, 33.1 kHz, 69.2 kHz, 100 kHz, 331.1 kHz, 691.8 kHz and 1 MHz). The arrows indicate increasing frequencies. The details correspond to the range -55 °C / 218 K -85 °C / 358 K (EIA standard for temperatures).

Figure 3 :

 3 Figure 3 : Thermal evolution of the lattice parameters and of the β monoclinic angle of NBZ from room temperature 303 K (30°C) to 923 K (650°C) as determined by HTXRD (ap = am/√2, bp = bm/√2, cp = cm/2).

Fig

  Fig. S1: XRD pattern of NBZ powders and sintered pellet.

Fig. S2 :

 S2 Fig. S2: Distance and angles in NBZ as determined by structural refinement.

Fig. S3 :

 S3 Fig. S3: Determination of the temperature of inflexion point of tan(delta) curve.

Fig

  Fig. S4: Vogel-Fulcher and Arrhenius fitting of permittivity curves.

Fig. S5 :

 S5 Fig. S5: Low temperature DSC curves.

Fig. S6 :

 S6 Fig. S6: High temperature DSC curves.

Fig. S7 :

 S7 Fig. S7: temperature programmed XRD patterns of NBZ compound.

Acknowledgments: J. Lelievre wants to thanks the "Region Limousin" which granted its thesis scholarship and the Laboratoire Léon Brillouin, Orsay, France (LLB) for beam time and help in neutron diffraction experiments.


Clothilde Queiroux
email: clothilde.queiroux@gmail.com

Muriel Bonnet
email: muriel.bonnet@uca.fr

Taous Saraoui
email: taous.saraoui@uca.fr

Etienne Rifa
email: etienne.rifa@inra.fr

Cécile Moussard
email: cecile.moussard@uca.fr

Geneviève Gagne
email: genevieve.gagne@uca.fr

Céline Delbès
email: celine.delbes@inra.fr

Stéphanie Bornes
email: stephanie.bornes@uca.fr

Dialogue between Staphylococcus aureus SA15

Keywords:

published or not. The documents may come

Background

Staphylococcus aureus is an opportunistic human pathogen that can be responsible for food poisoning [START_REF] Loir | Staphylococcus aureus and food poisoning[END_REF]. Its pathogenic activity is due to the production of various enzymes and toxins. It can be found in different environments including milk and dairy products [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF]. In cheese, its level should not exceed 10 5 CFU.g -1 (European Community Regulation No. 852-853/2004).

Lactococcus garvieae is an ubiquitous LAB (Lactic Acid Bacteria) that can be found in various fermented foods including dairy products [START_REF] Callon | Stability of microbial communities in goat milk during a lactation year: molecular approaches[END_REF][START_REF] Villani | Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk[END_REF], in fish, ruminant or human microbiota [START_REF] Aguado-Urda | Utilization of lactose and presence of the phospho-β-galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources[END_REF] and can be associated with pathologies such as fish lactococcoses [START_REF] Eldar | Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases[END_REF][START_REF] Vendrell | Lactococcus garvieae in fish: A review[END_REF]. LABs such as Lactococcus lactis or Lactococcus garvieae are able to inhibit the proliferation of pathogens in cheese by the production of hydrogen peroxide [START_REF] Klebanoff | Control of the microbial flora of the vagina by H 2 O 2 -generating lactobacilli[END_REF], bacteriocins [START_REF] Gálvez | Bacteriocin-based strategies for food biopreservation[END_REF], by competition for nutrients [START_REF] Charlier | Acidification is not involved in the early inhibition of Staphylococcus aureus growth by Lactococcus lactis in milk[END_REF][START_REF] Haines | Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin[END_REF] or by acidification of the medium [START_REF] Charlier | Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives[END_REF][START_REF] Tatini | Production of staphylococcal enterotoxin A in cheddar and colby cheese[END_REF]. Depending on its concentration, hydrogen peroxide has a bactericide or a bacteriostatic effect on S. aureus [START_REF] Amin | Influence of catalase activity on resistance of coagulase-positive staphylococci to hydrogen peroxide[END_REF]. L. garvieae raises our interest because a specific dairy strain of this LAB, N201 strain, can inhibit S. aureus growth by the production of H 2 O 2 [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF][START_REF] Alomar | Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk[END_REF][START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF]. Moreover, it has a strong technological potential as a ferment for cheese production [START_REF] Fernández | Phenotypic, genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese[END_REF] and has almost no effect on acidification of the medium, compared to other LABs [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF]. The inhibitive properties of L. garvieae N201 were confirmed on 2 strains of S. aureus: a human pathogenic strain, MW2 [START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF] and a non-pathogenic dairy strain, SA15, isolated from Saint-Nectaire cheese [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF].

Delpech et al. [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] showed that S. aureus had no effect on L. garvieae growth and H 2 O 2related gene expression. On the contrary, L. garvieae N201 impaired the capacity of both strains of S. aureus to deal with the presence of H 2 O 2 which led to growth deficiency [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF][START_REF] Alomar | Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk[END_REF][START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF].

In order to investigate the interaction between S. aureus and L. garvieae in oxidative stress-inducing culture conditions, genes involved in H 2 O 2 metabolism were first choice targets, whose expression has been monitored by Delpech et al. [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF][START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] in co-cultures of L. garvieae N201 and S. aureus SA15 or MW2.

In L. garvieae, superoxide dismutase sodA [START_REF] Chiang | Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria[END_REF][START_REF] Condon | Responses of lactic acid bacteria to oxygen[END_REF] and pyruvate oxidase poxB genes [START_REF] Murphy | Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures[END_REF][START_REF] Quatravaux | Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters[END_REF] are involved in H 2 O 2 synthesis. As they do not have any catalase, LAB generally degrade H 2 O 2 using alkyl hydroxyperoxidase (Ahp) [START_REF] Mishra | Why do bacteria use so many enzymes to scavenge hydrogen peroxide?[END_REF] or glutathione peroxidase (Gpx) [START_REF] Margis | Glutathione peroxidase familyan evolutionary overview[END_REF]. Thioredoxine reductase (Trx) are involved in response to Reactive Oxygen Species (ROS) [START_REF] Holmgren | Thioredoxin and glutaredoxin systems[END_REF][START_REF] Lu | The thioredoxin antioxidant system[END_REF].

In S. aureus, Catalase (KatA) and Alkyl hydroxyperoxidase (Ahp) play a role in H 2 O 2 degradation [START_REF] Cosgrove | Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus[END_REF][START_REF] Seaver | Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli[END_REF]. Ahp leads to a dual function in oxidative-stress resistance, environmental persistence and host-pathogen interaction [START_REF] Cosgrove | Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus[END_REF]. Amongst the targeted genes, dnaK is known to be involved in H 2 O 2 -resistance [START_REF] Rockabrand | An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H 2 O 2 resistance, and reductive division[END_REF][START_REF] Singh | Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus[END_REF], clpC has an important role in oxidative stress regulation, and ctsR is a transcriptional repressor of stress-genes [START_REF] Fiocco | Characterization of the CtsR stress response regulon in Lactobacillus plantarum[END_REF]. Moreover, the 2 latter genes, belonging to the dcw cluster involved in cellular division, might be modulated by H 2 O 2 -stress, leading to S. aureus growth impairment [START_REF] Hara | A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW[END_REF]. So, if LAB can modify this genes cluster expression, it could have an inhibiting effect on cellular division.

In addition to S. aureus growth modulation, L. garvieae may also have an effect on its virulence. Enterotoxins are the main toxins responsible for S. aureus food poisoning. Ninetyfour per cent of S. aureus isolated from cow milk have at least one enterotoxin-encoding gene [START_REF] Srinivasan | Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis[END_REF]. Amongst the different enterotoxins, enterotoxin C, encoded by sec4 gene, is the most frequently involved in food poisoning [START_REF] Cha | Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea[END_REF][START_REF] Chiang | PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan[END_REF][START_REF] Kérouanton | Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France[END_REF][START_REF] Wieneke | Staphylococcal food poisoning in the United Kingdom, 1969-90[END_REF]. Cretenet et al. [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] showed that this virulence-related gene expression can be modified by L. lactis in cheese. S. aureus virulence is under control of the agr system which is involved in regulating many stress response and virulence genes [START_REF] Dassy | Involvement of the accessory gene regulator (agr) in expression of type 5 capsular polysaccharide by Staphylococcus aureus[END_REF][START_REF] Janzon | The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus[END_REF]. agrA is a response regulator [START_REF] Peng | Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus[END_REF] and is able to induce hld, a -lysin gene [START_REF] Janzon | The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus[END_REF], and enterotoxin C encoding gene sec4 [START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF]. The agr system itself is controlled by several molecular intermediates such as SaeRS and SrrAB. SaeRS is a two-component system involved in response to environmental stress which could inhibit agr system [START_REF] Liang | Inactivation of a twocomponent signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus[END_REF] and also control virulence genes [START_REF] Giraudo | Phenotypic characterization and virulence of a saeagrmutant of Staphylococcus aureus[END_REF][START_REF] Steinhuber | Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors[END_REF]. SrrAB is also a two-component system activated in an anaerobic environment [START_REF] Fuchs | Anaerobic gene expression in Staphylococcus aureus[END_REF]. It would be involved in virulence gene control [START_REF] Ulrich | The staphylococcal respiratory response regulator SrrAB induces ica gene transcription and polysaccharide intercellular adhesin expression, protecting Staphylococcus aureus from neutrophil killing under anaerobic growth conditions[END_REF] in response to H 2 O 2 . Indeed, in the presence of H 2 O 2 , srrA is repressed in S. aureus [START_REF] Chang | Global transcriptome analysis of Staphylococcus aureus response to hydrogen peroxide[END_REF]. Under anaerobic conditions, SrrA represses agrA and hld expression [START_REF] Pragman | Characterization of Virulence Factor Regulation by SrrAB, a Two-Component System in Staphylococcus aureus[END_REF][START_REF] Yarwood | Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors ofStaphylococcus aureus[END_REF]. CodY, a regulatory protein involved in repressing virulence gene expression in S. aureus, is also involved in controlling agr system and virulence genes [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF].

The level of aeration can change the level of H 2 O 2 -production according to the LAB strain. Indeed, it has been demonstrated that the transcriptomes of L. lactis and L. garvieae are significantly modified by the aeration level [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF][START_REF] Dijkstra | Fermentation-induced variation in heat and oxidative stress phenotypes of Lactococcus lactis MG1363 reveals transcriptome signatures for robustness[END_REF]. Lactobacillus crispatus can produce H 2 O 2 in high aeration conditions but not in static conditions [START_REF]Growth inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus paracasei subsp. paracasei isolated from the human vagina -Ocaña -1999 -Pathogens and Disease -Wiley Online Library[END_REF]. Contrariwise, Lactobacillus delbrueckii subsp. bulgaricus can also produce H 2 O 2 in static conditions, even though the amount produced is lower than in high aeration conditions [59].

The aim of the present study was to compare the transcriptional response involved in the antagonistic interaction between S. aureus and different strains of L. garvieae. In addition to N201 strain isolated from raw milk Saint-Nectaire cheese and already well described [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF][START_REF] Alomar | Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk[END_REF], two other L. garvieae strains were selected for their different capacities to produce H 2 O 2 : a H 2 O 2 -producing strain, CL-1183 (VIVASET, Veterinary Faculty, Complutense University from Madrid) isolated in Brazil from the milk from buffalo cows affected by subclinical mastitis [START_REF] Aguado-Urda | Utilization of lactose and presence of the phospho-β-galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources[END_REF] as well as a H 2 O 2 -non producing strain, Lg2, a fish pathogenic strain isolated in Japan [START_REF] Morita | Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae[END_REF]. The expression of S. aureus genes related to oxygen metabolism, response to stress, cell division and virulence was measured as well as the expression of L. garvieae genes related to oxygen metabolism.

Methods

Strains and culture conditions

Strains Lactococcus garvieae N201, CL-1183 and Lg2 and Staphylococcus aureus SA15 were cultivated in Brain-Heart Infusion buffered to pH 7 using Potassium Phosphate (BHI, Biokar Diagnostic, Pantin, France) at 30°C and 37°C, respectively, under static condition. After 20 hours, cells were harvested at 3500 g during 15 min. Cell pellets were resuspended in 2 ml of BHI and syringed 5 times. Cell concentration was then determined using a Petroff-Hausser counting chamber. Fifty ml of buffered BHI, containing or not 4000 IU ml -1 of catalase from bovine liver (ref. C100, Sigma), were then co-inoculated at 10 7 cells per ml for L. garvieae and 10 6 cells per ml for S. aureus, or just with S. aureus at the same concentration in 250 ml Erlenmeyer flasks or in 50 ml Falcon tubes, as described previously [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF][START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF].

Then, Erlenmeyer flasks were incubated for 6 h, 9 h or 24 h at 30°C under shaking condition (150 rpm), corresponding to the high level of aeration condition. The low level of aeration condition corresponded to static fully filled 50 ml Falcon tubes. At 0 h, 6 h, 9 h and 24 h, 1 ml of the cultures was removed to be syringed and serially diluted in Ringer's solution. After adequate mixing, 100 µl of each dilution were plated onto solid Baird Parker for S. aureus and BHI agar for L. garvieae for numeration. Colony-forming units were counted after overnight incubation at 37°C for S. aureus and 30°C for L. garvieae. Also, 1 ml was removed from the cultures for H 2 O 2 content analysis and 40 ml for RNA extraction. Cell pellets from 6 h, 9 h and 24 h cultures were resuspended in 200 µl of cold Tris-EDTA buffer and then frozen at -80°C. The experiment was carried out in triplicate within each of three independent biological replicates, for a total of 9 samples per time point. pH measurements were made with the rest of the cultures.

Quantitative analysis of H 2 O 2 in L. garvieae cultures supernatant

Hydrogen peroxide concentration was determined according to Batdorj et al. protocol [START_REF] Batdorj | Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens[END_REF] with slight modifications. Cells were harvested by centrifugation at 11500 g, 15°C for 10 min. One hundred µl of the sample supernatant were mixed with 100 µl of 4aminoantipyrine 4 mg.ml -1 (Sigma-Aldrich, St. Louis, Missouri, USA), 20 µl of watersaturated phenol (Sigma-Aldrich, St. Louis, Missouri, USA), 750 µl of phosphate buffer Na 2 HPO 4 /NaH 2 PO 4 0.1 M (pH 7) and 30 µl of horseradish peroxidase type VI-A (500 U.ml -1 in sodium phosphate buffer pH 6, Sigma-Aldrich, St. Louis, Missouri, USA). Sample was mixed by inverting the tube and OD was measured at 505 nm. Blank was done by replacing sample by sterile medium. H 2 O 2 concentration was determined using a standard curve performed with concentrations ranging from 0 to 3 mM. The minimal concentration that could be detected was 0.5 mM.

RNA extraction and DNase treatments

Cells in Tris-EDTA buffer were thawed out and 25 µl of 20% SDS, 500 µl of phenol (pH 4), 3.5 µl of β-mercaptoethanol and 600 mg of Zirconium beads were added. The cells were broken twice in a tissue homogenizer (Precellys ® 24, Bertin Technologies, Montigny-le-Bretonneux, France). Then, 200 µl of chloroform were added and mixed with the solution and sample was centrifuged at 11400 g for 20 min at 4°C. RNA extraction was performed using NucleoSpin RNA Midi kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) following the supplier's instructions. The RNA extracts were treated twice with DNAse I using an Ambion DNA-free kit following the supplier's instructions (Ambion, Inc., Austin, Texas, USA). The RNA extracts were quantified using a Nanodrop TM 2000C (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

Reverse Transcription and quantitative PCR

Reverse Transcription (RT) was performed using 0.5 µg of RNA twice treated with DNase, 5 µl of 10X buffer, 2 µl of 25X dNTP, 5 µl of random primers and 2.5 µl of retrotranscriptase (Applied Biosystems ® , Life Technologies, Foster City, California, USA).

The RT was done in a thermocycler (Techne ® Prime, Bibby Scientific, Stone, Staffordshire, UK) with the following parameters: 10 min at 25°C and 120 min at 37°C. Genes Of Interest (GOI) Ct were determined in quantitative PCR (qPCR) assays using 2.5 µl of cDNA suspensions 10-fold diluted in RNAse-free water and 10 µl of qPCR mix containing 1.25 µl of each primer (10 mM, Table 1, Table 2), 6.25 µl of qPCR Rotor Gene SybrGreen mix (Qiagen, Hilden, Germany) and 1.25 µl of RNAse-free water. The qPCR was performed according to the protocol on Rotor Gene Q (Qiagen, Hilden, Germany) with the following parameters: 4 min at 94°C, then for 35 cycles, 30 s at 94°C, 30 s at 55°C, 60 s at 72°C. Each reaction was performed in triplicate within each of three independent biological replicates, for a total of 9 reactions per time point.

Primers efficiencies were determined according to a 10-fold template dilution standard and were all ranging from 1.90 (~95%) to 2.293 (~115%) according to the following equation: Efficiency = 10^(-1/slope of calibration curve) [START_REF] Rasmussen | Quantification on the LightCycler[END_REF]. The primers used were previously described by others [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF]. One reference gene was used for L. garvieae data: tufB encoding the elongation factor Tu. Considering that the lag in growth phase reflects the inhibition of S. aureus by L. garvieae, we evaluated gene expression at identical time points corresponding to different population levels. Two reference genes whose expression was stable over time under the different conditions were used for S. aureus data: housekeeping genes recA, encoding the recombinase A, and hu, encoding a DNA-binding protein. The GOI expression was calculated according to the formula introduced by Hellemans et al. [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF].

Comparison of gene expression

Differential gene expression was evaluated by comparing expression between two conditions. First, the influence of aeration on gene expression in pure culture was evaluated for S. aureus. Then, the influence of different strains of L. garvieae (N201, CL-1183 and Lg2) on S. aureus SA15 gene expression was observed under high or low aeration condition.

Secondly, L. garvieae gene expression was compared between the different strains in co-culture with S. aureus SA15 under both aeration conditions. A gene was considered as differentially expressed when its expression was changed by at least a factor of 2.

Statistical analyses

Statistical analyses on microbial counts and H 2 O 2 concentrations were performed using R software [START_REF] Core | R: A language and environment for statistical computing[END_REF] by one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test. The gene expression statistical analyses were performed using R by ANOVA with Newmann-Keuls post-hoc test. Significance was declared at P<0.05.

Results

In order to investigate the interaction between S. aureus and L. garvieae in oxidative stressinducing culture conditions, we followed the growth of the strains and measured the amount of H 2 O 2 in two culture conditions i.e. under high and low aeration levels. The genes whose expression has been monitored by Delpech et al. [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF][START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] were chosen to compare the transcriptional response involved in the antagonistic interaction between S. aureus and different strains of L. garvieae. The expression of S. aureus genes related to oxygen metabolism, response to stress, cell division and virulence was measured as well as the expression of L. garvieae genes related to oxygen metabolism (Table 3).

Ability of L. garvieae strains to produce H 2 O 2 and to inhibit S. aureus growth

The effect of aeration level on microbial growth was tested by determining cellular concentrations of both microorganisms. In pure culture, S. aureus growth was almost identical under both high and low aeration conditions. However, the population count reached at 24h was 1.2 log CFU/ml lower under low aeration than under high aeration (Figure 1A). The 3 different strains of L. garvieae grew as well under high aeration level as under low aeration level independent of the presence of S. aureus ( Additional file 1 ). pH values remained stable (7.0 ± 0.2) in all cultures over the whole experiment (data not shown).

Detectable amounts of H 2 O 2 were produced exclusively under high aeration level by N201 and CL-1183 strains in co-culture with SA15. N201 strain produced from 1.327 mM ± 0.09 to 1.517 mM ± 0.18 of H 2 O 2 . The strain CL-1183 produced slightly more H 2 O 2 than the strain N201, from 1.663 mM ± 0.23 to 2.415 mM ± 0.34. While N201 H 2 O 2 -production peak was at 9 h, CL-1183 H 2 O 2 -production stayed stable after 9 h (Table 4).

Under high aeration, when co-cultivated with N201, SA15 growth was 2.1 log [CFU.ml -1 ] lower than in pure culture as early as 6 h with a maximal growth inhibition of 5.8 log [CFU.ml -1 ] at 24 h. SA15 growth co-cultivated with CL-1183 was 1.7 log [CFU.ml -1 ] lower compared to pure culture as early as 6 h. The maximal growth inhibition was observed after 24 h of co-culture (4.5 log [CFU.ml -1 ] lower). SA15 co-cultivated with Lg2 did not show any significant impairment in growth throughout the experiment compared to pure culture (Figure 1A).

Under low aeration level and in co-culture with N201, CL-1183 or Lg2, SA15 growth was lower than in pure culture as early as 6 h (0.6 log [CFU.ml -1 ], 0.9 log [CFU.ml -1 ] or 0.6 log [CFU.ml -1 ], with a maximal growth inhibition at 24 h (2.5 log [CFU.ml -1 ], 3.5 log [CFU.ml -1 or 1.6 log [CFU.ml -1 ]], respectively) (Figure 1A).

To determine the involvement of H 2 O 2 produced by N201 and CL-1183 in growth inhibition of SA15, co-cultures of SA15 with these 2 strains were performed under high aeration level in presence of catalase (Figure 1B). No detectable amount of H 2 O 2 was observed at any time. S. aureus cell concentration was not significantly different in presence of catalase compared to control (Figure 1A vs. Figure 1B) during both exponential and stationary phases. Statistical analysis, using ANOVA, showed that in presence of catalase there was no significant difference of SA15 count after 24 h in co-culture with CL-1183 compared to pure culture. In contrary, SA15 cell concentration in co-culture SA15/N201 was still 0.8 log [CFU.ml -1 ] lower than in pure culture. These results showed that the inhibition of SA15 growth by N201 and CL-1183 was partially or completely suppressed by catalase.

Effect of L. garvieae strains on S. aureus genes expression under high aeration level (Additional file 2)

Whatever the L. garvieae strain cultivated with SA15, none of the H 2 O 2 -response genes tested was up-regulated. When SA15 was cultivated with L. garvieae N201 or CL-1183, katA was down-regulated, at 9 h and 24 h with N201 (3.7 and 3.0-times, respectively), or at 6 h and 9 h with CL-1183 (3.9 and 3.2-times, respectively). When cultivated with N201, ahpF expression in SA15 was not modified in comparison with pure culture, whereas in co-cultures SA15/CL-1183 and SA15/Lg2, it was down-regulated (2.1-times at 6 h and 2.9-times at 24 h, 6.9-times at 24 h, respectively). sodA was down-regulated at 6 h, 9 h and 24 h in co-cultures with N201 (2.2, 4.4 and 3.9-times, respectively) or at 24 h (2.2-times) with CL-1183. No change in expression of katA and sodA was observed with Lg2.

L. garvieae N201 induced an up-regulation of the three SA15 stress-response genes tested at 9 h; clpC, ctsR and dnaK were 2.0-times, 3.7-times and 2.5-times more expressed than in pure culture, respectively. When cultivated with CL-1183, only dnaK was 2.8-times up-regulated at 6 h, while clpC and ctsR expressions were not affected. Conversely, in coculture SA15/Lg2, clpC and dnaK were 3.9-times and 19.8-times down-regulated respectively, while ctsR did not show any significant difference in its expression.

In both SA15/N201 and SA15/CL-1183 co-cultures, mraW cell division gene was strongly down-regulated at 6 h and 9 h (7.0-times and 20.3-times, and 20.0-times and 12.0times, respectively). In co-culture SA15/Lg2, mraW expression was not affected. contrasted patterns of expression. At 9 h, in both SA15/N201 and SA15/CL-1183 co-cultures, agrA was up-regulated (4.0-times with N201 and 3.4-times with CL-1183), while srrA was down-regulated (7.1-times or 6.4-times with N201 or CL-1183 respectively). codY expression was down-regulated 4.1-times at 24 h by N201 and 3.6-times at 6 h by CL-1183. saeS expression was 2.4-times lower at 24 h in co-culture with N201 than in pure culture, whereas it was not affected by CL-1183. hld expression remained stable until 24 h when SA15 was in co-culture with N201 or CL-1183. In co-culture SA15/Lg2, at 24 h, agrA, codY and hld expressions were all down-regulated (3.0-times, 2.6-times and 3.9-times, respectively), while saeS and srrA expressions remained stable.

In co-cultures SA15/N201 and SA15/CL-1183, amongst the 2 enterotoxins-encoding genes tested, only sec4 expression was modified at 24 h, with a 4.1-times and a 9.0-times down-regulation, respectively. sel2 expression was not modified whatever the SA15 culture conditions were. In co-culture SA15/Lg2, no enterotoxin gene expression was affected.

Effect of L. garvieae strains on S. aureus genes expression under low aeration level (Additional file 3)

Under low aeration level, in presence of CL-1183, the H 2 O 2 -response gene ahpF was 4.2-times up-regulated at 9 h, whereas in presence of Lg2, sodA was 6.5-times downregulated at 24 h. All the stress-response genes tested were up-regulated at 9 h (3.4-times for clpC, 4.0-times for ctsR and 2.1-times for dnaK) with Lg2. No change in stress-response genes expression was observed when SA15 was cultivated with N201 or CL-1183.

Concerning the cell division gene mraW, its expression was not modified, whatever the strain cultivated with SA15. Amongst virulence-related regulator genes, agrA was up-regulated in all three cocultures at different stages of growth. It was the only virulence-related regulator gene differentially regulated by N201 (6.0-times up-regulated at 9 h). In the same way, this gene was 4.1-times up-regulated at 9 h by CL-1183, and 2.5-times up-regulated at 24 h by Lg2. codY was 2.0-times down-regulated at 6 h but 2.0-times up-regulated at 24 h with CL-1183, while codY and srrA were 2.1-and 2.7-times down-regulated at 9 h and 24 h, respectively with Lg2.

The enterotoxin gene sec4 was up-regulated in both N201/SA15 and CL-1183/SA15 co-cultures, (3.5-and 2.7-times, at 6 h and 9 h respectively, with N201, and 2.5-times at 9h with CL-1183). sel2 was 3.5-times up-regulated at 9 h only in the presence of CL-1183.

Effect of aeration on L. garvieae strains (Additional file 4)

To evaluate the strain-specific effect of aeration on L. garvieae, expression of 5 H 2 O 2 -related genes of them (trxB1, ahpC, gpx, poxB and sodA) was monitored and compared between the 3 L. garvieae strains in co-culture with SA15. Overall, most differential expressions concerned H 2 O 2 -degradation genes. Differences between H 2 O 2 -producing and non-producing strains were essentially related to ahpC: both N201 and CL-1183 overexpressed this gene under low aeration (5.6-times at 24 h in N201, 4.0-times at 6 h and 6.5-times at 9 h in CL-1183). Conversely, Lg2 overexpressed ahpC (4.7-times) at 9 h under high level of aeration. Both CL-1183 and Lg2 overexpressed trxB1 under high level of aeration (2.9-times at 6 h in CL-1183 and 2.1-times 24 h in Lg2). Amongst H 2 O 2 -synthesis genes, only CL-1183 overexpressed poxB (4.1-times at 9 h under low aeration).

Discussion

The aim of the study was to compare the transcriptional response involved in the antagonistic interaction between S. aureus and different strains of L. garvieae. Three strains of L. garvieae from different origins were used: N201, a dairy-isolated strain known to be a S. aureus inhibiting strain; CL-1183, a strain isolated from milk from buffalo cows suffering from mastitis; and Lg2, a fish pathogenic strain. Under high aeration condition, two of them were shown to be able to produce detectable amount of H 2 O 2 (N201 and CL-1183), whereas the third one (Lg2) was not.

L. garvieae strains and aeration level effect on S. aureus SA15 growth and on H 2 O 2 -and stress-responses

Under high aeration level, only L. garvieae N201 and CL-1183 inhibited S. aureus growth. CL-1183 down-regulated S. aureus H 2 O 2 -degradation genes (ahpF and katA) and N201 and Lg2 only down-regulated katA or ahpF respectively. This could mean that SA15 lost its ability to deal with H 2 O 2 -stress and suggest that the H 2 O 2 detoxification occurred predominantly via KatA. These results are in accordance with those obtained by Cosgrove et al. [START_REF] Cosgrove | Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus[END_REF], showing that S. aureus ahpC-katA mutant was more sensitive to H 2 O 2 than the katA mutant. They also found out that, Ahp would have less affinity for H 2 O 2 than KatA and that it could be an alternative solution for H 2 O 2 -degradation when KatA was not functional. In our study, despite N201 only down-regulated katA and not ahpF, AhpF did not play its compensatory role in H 2 O 2 detoxification. This result can be explained by the fact that KatA was responsible for detoxifying high levels of H 2 O 2 , whereas AhpC was responsible for the removal of low levels of H 2 O 2 [START_REF] Cosgrove | Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus[END_REF]. This could be correlated with the large amounts of H 2 O 2 detected in the co-cultures SA15/N201. The down-regulation of katA, at 9 h and 24 h, can be explained by the fact that srrA was also down regulated. Mashruwala and Boyd [START_REF] Mashruwala | The Staphylococcus aureus srrAB Regulatory system modulates hydrogen peroxide resistance factors, which imparts protection to aconitase during aerobic growth[END_REF] found that srrAB mutant strain had decreased transcription of genes encoding for H 2 O 2 resistance factors as katA. They also reported that SrrAB positively influenced H 2 O 2 resistance during periods of high O 2 dependent respiratory activity, but not when cellular respiration was diminished as a result of lower O 2 availability. S. aureus SA15 genes involved in stressresponse (clpC, ctsR, dnaK) were all up-regulated by N201 and only dnaK was up-regulated by CL-1183, whereas clpC and dnaK were down-regulated by Lg2. These results indicated that in presence N201 and CL-1183, SA15 detected an oxidative stress as a result of H 2 O 2 production by these two L. garvieae strains. However, the up-regulation of these genes did not allow SA15 to fight against this stress, although precedent studies reported that S. aureus clpC or dnaK mutants growth was impaired in the presence of H 2 O 2 stress [START_REF] Singh | Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus[END_REF][START_REF] Chatterjee | Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death[END_REF][START_REF] Krüger | Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance[END_REF] and that a basic level of expression of dnaK was sufficient in response to this stress [START_REF] Singh | Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus[END_REF]. All these data highlighted the complexity of the stress response machinery and the important role of clpC, ctsR and dnaK genes. Moreover, while mraW gene was not differentially expressed in presence of Lg2, it was repressed at 6 h and 9 h by N201, confirming results obtained by Delpech et al. [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF], and by CL-1183 in our study, when S. aureus growth was inhibited. The repression of mraW by N201 and CL-1183 was positively correlated with inhibition of SA15 growth. These results are supported by Cretenet et al. [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] showing that L. lactis was able to inhibit ftsH, ftsL and ftsZ genes also involved in cellular division. Interestingly, several studies reported that inhibition of cell division protein is a promising approach for antistaphylococcal therapy [START_REF] Singh | FtsZ inhibition: A promising approach for antistaphylococcal therapy[END_REF][START_REF] Kim | Madurahydroxylactone, an Inhibitor of Staphylococcus aureus FtsZ from Nonomuraea sp. AN100570[END_REF].

Catalase treatment partially reduced SA15 growth inhibition by N201, confirming the result obtained by Delbes-Paus et al. [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF], whereas the inhibition by CL-1183 was completely suppressed. Oogai et al. [START_REF] Oogai | Staphylococcus aureus SrrAB affects susceptibility to hydrogen peroxide and co-existence with Streptococcus sanguinis[END_REF] observed that in presence of catalase, the S. aureus MW2 growth inhibition by Streptococcus sanguinis was completely suppressed. These observations confirmed the role of H 2 O 2 produced by LAB in growth inhibition of S. aureus. Our study demonstrated that under high level of aeration, the inhibition of S. aureus by L. garvieae involved strain specific mechanisms. Indeed, CL-1183 inhibited SA15 growth mainly by H 2 O 2 production whereas inhibition due to N201 may involve the combined action of H 2 O 2 and other antagonistic mechanism.

Under low aeration condition, S. aureus growth was almost identical to that measured under high aeration condition, except after 24 hours, when we observed a 1,2 log reduction in the S. aureus population level that could be due to the depletion of oxygen. Ledala et al. [START_REF] Ledala | Influence of iron andaeration on Staphylococcus aureus growth, metabolism, and transcription[END_REF] showed that the growth rate of S. aureus was independent of oxygen limitation over 12 hours although its metabolome was significantly affected. S. aureus growth was slightly inhibited by the three L. garvieae strains, although none of them could produce detectable amounts of H 2 O 2 (Figure 1A). This suggests that there was another inhibitory mechanism involved.

These results match those obtained with N201 in the previous studies [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF][START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF][START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF]. Delbes-Paus et al. [START_REF] Delbes-Paus | Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community[END_REF] have shown that in milk, even if H 2 O 2 was not detected, S. aureus growth was inhibited by L. garvieae N201. However, no clear hypothesis to explain the mechanism of the anti-staphylococcal activity under low aeration could be drawn from the gene expression data. However, the up-regulation of stress-response genes in the presence of Lg2 was observed. Indeed, clpC, ctsR and dnaK genes, were all up-regulated at 6 h in co-culture with Lg2, suggesting that Lg2 triggered a stress on SA15 which led to a growth inhibition.

L. garvieae strains and aeration level effect on S. aureus SA15 virulence gene expression

Under high level of aeration, L. garvieae N201 reduced S. aureus virulence-related genes expression confirming results obtained by Delpech et al. [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF]. Cretenet et al. [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] and Queck et al. [START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF] have shown that the agr system, involved in the regulation of genes linked to S. aureus virulence, was repressed by L. lactis even if L. lactis does not produce H 2 O 2 in this condition. Molecular intermediaries SaeRS and SrrAB are involved in controlling the agr system [START_REF] Liang | Inactivation of a twocomponent signal transduction system, SaeRS, eliminates adherence and attenuates virulence of Staphylococcus aureus[END_REF]. We showed srrA expression was repressed in co-culture with N201 and CL-1183, when H 2 O 2 was produced as already observed by Chang et al. [START_REF] Chang | Global transcriptome analysis of Staphylococcus aureus response to hydrogen peroxide[END_REF]. Moreover, Majerczyk et al. [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF] have shown that a codY mutant could derepressed agr system. In our conditions, codY was repressed by CL-1183 at 6 h explaining agrA up-regulation at 9 h, whereas repression of codY at 24 h by Lg2 cannot explain agrA down-regulation suggesting that another regulator might be involved. Moreover, at 24 h, despite SA15 growth was not modified, Lg2 caused a down-regulation of three virulence-related regulator genes tested in this study (agrA, codY, hld). sec4 and sel2 are two enteroxin-encoding genes found in S. aureus SA15 [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF]. Although enteroxin C-encoding gene sec4 is under the control of the agr system [START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF], agrA expression was only induced by N201 and CL-1183 and sec4 was repressed under high aeration level as shown by Delpech et al. [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF]. Our data showed the repression of virulence associated genes and of enterotoxin encoding genes as well as simultaneous S. aureus growth inhibition in the presence of N201 or CL-1183, indicating that these 2 strains not only inhibited SA15 growth but also potentially attenuated its virulence.

Under low aeration condition, S. aureus over-expressed agrA and enterotoxinencoding genes sel2 and sec4 in presence of N201 and CL-1183, whereas Lg2 did not induce any modification. In presence of Lg2, agrA was up-regulated at 24 h. This result can be explained by the fact that SA15 srrA gene was down-regulated, at the same time. The same observation was reported by Yarwood et al. [START_REF] Yarwood | Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors ofStaphylococcus aureus[END_REF]. They showed that transcription of RNAIII from the agr locus was inversely dependent on expression of srrAB. Inherently, despite SA15 agrA up-regulation by Lg2, no modification in enterotoxin genes expression was observed. Yarwood et al. [START_REF] Yarwood | Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors ofStaphylococcus aureus[END_REF] also reported that the SA15 srrB mutant growth was significantly slower in anaerobic condition, which is in accordance with SA15 growth inhibition in presence of Lg2.

L. garvieae strain responses to low and high levels of aeration in co-culture with S. aureus SA15

It seemed that the three strains of L. garvieae differed essentially in their ability to degrade H 2 O 2 . Presence of H 2 O 2 would not depend only on H 2 O 2 -synthesis, but also on H 2 O 2 -degradation by L. garvieae strains, which is an original mechanism, and in accordance with the results obtained by Delpech et al. [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF].

In high aeration level, Lg2 overexpressed two H 2 O 2 degradation genes ahpC and trxB at 9 h and 24 h respectively. The non-detection of H 2 O 2 could be explained by the absence of production of H 2 O 2 or by its degradation by AhpC and TrxB. The latter mechanism could explain why, under low aeration, despite CL-1183 overexpressed an H 2 O 2 -synthesis gene, poxB, no detectable H 2 O 2 was observed. Indeed, at the same time, this strain overexpressed the H 2 O 2 degradation gene ahpC. Our data showed that the importance of the inhibition varies depending on the presence or absence of H 2 O 2 . Indeed, inhibition can occur while H 2 O 2 is not detected. As regards other potential explanations for the growth inhibition, the only putative bacteriocin identified in the L. garvieae N201 genome was homologous to garvieaecin Q (GarQ, data not shown), a class IId bacteriocin [START_REF] Tosukhowong | Garvieacin Q, a Novel Class II Bacteriocin from Lactococcus garvieae BCC 43578[END_REF]. In view of the results of previous experiments on L. garvieae N201 [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF], the inhibition was probably caused neither by garvieaecin Q nor by a protein, nor by a lipid, and nor by a polysaccharide. In the present study, we evidenced L. garvieae strain-specific response in S. aureus gene expression which suggests that molecular mechanisms involved in the inhibition could differ between L. garvieae strains. Further investigations are needed to elucidate these mechanisms. A detailed analysis of strains physiology is needed to evaluate the potential role of nutritional competition in the inhibition. A global transcriptomic approach of the interaction would shed some light on the metabolic dialogue between these strains, and more particularly the potential involvement of quorum-sensing.

Conclusions

Our data evidenced an impact of the aeration condition on the interaction between SA15 and L. garvieae strains.

Under high level of aeration, only N201 and CL-1183 produced detectable amounts of H 2 O 2 and consequently inhibited SA15 growth. Both L. garvieae strains activated SA15 stress-response genes and inhibited the expression of several genes involved in H 2 O 2degradation , virulence and cellular division . A catalase treatment partially or completely suppressed this inhibition and no H 2 O 2 was detected. Lg2 strain did not produce any detectable amount of H 2 O 2 and did not inhibit SA15 growth. However, as N201 and CL-1183, Lg2 inhibited SA15 virulence-related genes and stress-response related genes.

Under low aeration level, none of the L. garvieae strains produced any detectable amount of H 2 O 2 . N201 and CL-1183 were still able to inhibit SA15 growth, as well as, Lg2.

While N201 and CL-1183 induced the up-regulation of the SA15 enterotoxin-encoding genes and related regulator agrA, Lg2 caused up-regulation of SA15 stress-related genes and downregulated virulence-related regulators genes.

The antagonistic properties of L. garvieae against S. aureus were strain-and aeration level-dependent. Under high aeration, they involved H 2 O 2 production by two out of the three L. garvieae tested strains. This study provides new insights into microbial interactions mechanisms and shows the importance of investigating strain-specific effects. recA a Recombinase A Housekeeping gene [START_REF] Valihrach | Impact of normalization method on experimental outcome using RT-qPCR in Staphylococcus aureus[END_REF] agrA Accessory gene regulator A Virulence-related regulator [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] ahpF Alkyl hydroperoxidase F H 2 O 2 -response [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF] clpC Clp proteinase C Stress-response [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] ctsR Transcriptional repressor of stress-genes Stress-response [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] codY Transcriptional repressor Virulence-related regulator [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF] dnaK Chaperone protein DnaK Stress-response [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] hld Deltahemolysin Virulence-related regulator [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] katA Catalase H 2 O 2 -response [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] mraW S-adenosyl methyltransferase Cell division [START_REF] Delpech | Staphylococcus aureus transcriptomic response to inhibition by H 2 O 2 -producing Lactococcus garvieae[END_REF] saeS Histidine protein kinase Virulence-related regulator [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] sec4 Enterotoxin C Enterotoxin [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] sel2 Enterotoxin L Enterotoxin [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] sodA Superoxide dismutase H 2 O 2 -response [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] srrA Staphylococcal respiratory response Virulence-related regulator [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF] a Reference genes. [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] ahpC Alkyl hydroxyperoxide reductase H 2 O 2 -degradation [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] gpx Glutathione peroxidase H 2 O 2 -degradation [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] poxB Pyruvate oxidase H 2 O 2 -synthesis [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] sodA Superoxide dismutase H 2 O 2 -synthesis [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] trxB1 Thioredoxin reductase H 2 O 2 -degradation [START_REF] Delpech | New insights into the anti-pathogenic potential of Lactococcus garvieae against Staphylococcus aureus based on RNA sequencing profiling[END_REF] a Reference gene.

Declarations

Ethics approval and consent to participate

Figure 1 .

 1 Figure 1. Effect of L. garvieae strains on S. aureus SA15 growth. (A) Under different levels of aeration, (B) Under high aeration level in presence of catalase (4000 IU/ml). a, b, c, d, e represent groups determined with LSD test, a same letter indicates values not

Table 2 .

 2 Targeted L. garvieae genes.

	Gene Description	Category	Primers references
	tufB a Elongation factor Tu	Housekeeping gene

Table 3 .

 3 Differentially expressed S. aureus genes in co-culture with 3 L. garvieae strains (N201, CJ-1183 and Lg2), summary table.For detailed values, see additional files 2 and 3.

	High aeration		6 h			9 h			24 h
	Genes	N201 CL-1183 Lg2	N201 CL-1183 Lg2	N201 CL-1183 Lg2
	Up-regulated	-	dnaK	-	clpC	agrA	-	-	-	-
					cstR
					dnaK
					agrA
	Down-regulated sodA	ahpF	-	katA	katA	-	katA	ahpF	ahpF
		mraW	katA		sodA	mraW		sodA	sodA	clpC
			mraw		mraW	srrA		codY	sec4	dnaK
			codY		srrA			saeS		agrA
								sec4		codY
										hld
	Low aeration		6 h			9 h			24 h
	Genes	N201 CL-1183 Lg2	N201 CL-1183 Lg2	N201 CL-1183 Lg2
	Up-regulated	-	-	-	agrA	ahpF	clpC	-	codY	agrA
					sec4	agrA	cstR
						sec4	dnaK
						sel2
	Down-regulated	sec4	codY	-	-	-	codY	-	-	sodA
										srrA

Table 4 .

 4 H 2 O 2 concentration in L. garvieae co-culture with S. aureus SA15, under high aeration a, b represent groups determined with LSD test, a same letter indicates values not significantly different (p-value < 0.05 by ANOVA1) through one column.

	ND = not detected
					751
		Hydrogen peroxide concentration (mM)
	Time (h)	0	6	9	24
	SA15	ND	ND	ND	ND
	SA15 + N201	ND	1.343 a	1.517 a	1.327 a
	SA15 + CL-1183 ND	1.663 a	2.415 b	2.409 b
	SA15 + Lg2	ND	ND	ND	ND
	N201	ND	1.455	1.549	1.105
	CL-1183	ND	1.257	1.317	1.148
	Lg2	ND	ND	ND	ND

Acknowledgements

The authors acknowledge Mr Mar Blanco for the gift of Lactococcus garvieae strain CL-1183, as well as Masahira Hattori and Hidetoshi Morita for the gift of Lactococcus garvieae strain Lg2 and Maryline Bornes and Kate James for English proofreading.

Funding

This work was funded by IUT Clermont-Ferrand and University Clermont-Auvergne.

Availability of data and materials



All data generated or analysed during this study are included in this published article [and its supplementary information files].

Consent to publish

Not applicable

Competing interests

The authors declare that they have no competing interests.

Authors' Contributions

CQ performed the RT-qPCR, analyzed genes expression, ran statistical analyses and was a major contributor in writing the manuscript. MB contributed to cultivate the different strains, performed the quantitative analysis of H 2 O 2 in L. garvieae cultures, the RNA extraction, DNase treatment and the RT-qPCR, and was also a contributor in writing the manuscript. TS cultivated the different strains, performed the tests with added catalase, and contributed in writing the manuscript. PD helped in RT-qPCR, genes expression analysis and improvement of the manuscript. PV contributed to cultivate the different strains and to extract the RNA. ER contributed to statistical analyses. CM contributed to the adjustment of the method of quantitative analysis of H 2 O 2 . GG read and corrected the manuscript. SB and CD conceived the study. CD contributed to genes expression analysis and to the structuring and improvement of the manuscript. SB contributed to cultivate the different strains, performed the RNA extraction and was also a contributor in writing the manuscript.

Additional materials



Additional file 1: Table 5