# Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus

B Seridan Assis, P. Germon, A M Silva, S Even, J R Nicoli, Yves Le Loir

► To cite this version:

B Seridan Assis, P. Germon, A M Silva, S Even, J R Nicoli, et al.. Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus. Beneficial Microbes, 2015, 6 (6), pp.879 - 886. 10.3920/bm2015.0019. hal-05065131

HAL Id: hal-05065131

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# Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus

B. Seridan Assis¹,²,³, P. Germon⁴,⁵, A.M. Silva⁶, S. Even¹,², J.R. Nicoli³ and Y. Le Loir¹,²*

¹INRA, UMR 1253 STLO, 65 rue de Saint Brieuc, 35042 Rennes, France; ²Agrocampus Ouest, UMR1253 STLO, 85 rue de Saint Brieuc, 35042 Rennes, France; ³Departamento de Microbiologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270901, Brazil; ⁴INRA, UMR 1282 Infectiologie et Santé Publique, 37380 Nouzilly, France; ⁵Université François Rabelais de Tours, UMR1282, Infectiologie et Santé Publique, 37000 Tours, France; ⁶Departamento de Engenharia de Alimentos – Campus Sete Lagoas, Universidade Federal de São João del-Rey, Sete Lagoas, MG 36307-352, Brazil; yves.leloir@rennes.inra.fr

Received: 13 February 2015 / Accepted: 27 May 2015

© 2015 Wageningen Academic Publishers

## RESEARCH ARTICLE

### Abstract

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated *Lactococcus lactis* V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for *Escherichia coli* and for *Staphylococcus aureus*, two major mastitis pathogens, we investigated *L. lactis* V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. *L. lactis* V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with *E. coli* was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that *L. lactis* V7 inhibited the internalisation of both *E. coli* and *S. aureus* strains into bMEC, whereas it inhibited the adhesion of only one out of the two *S. aureus* strains and of none of the *E. coli* strains tested. Investigation of the bMEC immune response showed that *L. lactis* V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the *E. coli* strains. Altogether these features of *L. lactis* V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

**Keywords:** *Lactococcus lactis*, *Staphylococcus aureus*, *Escherichia coli*, mastitis, cell invasion, inflammatory response, mammary probiotics

### 1. Introduction

Bovine mastitis is an inflammation of the mammary gland often caused by bacterial infections. *Escherichia coli* (Suojala *et al.*, 2013), and *Staphylococcus aureus* (Voelk *et al.*, 2014), responsible for environmental mastitis and contagious mastitis, respectively, are major mastitis pathogens in bovine herds. Mastitis causes high economic losses in the dairy farming and industry (Le Maréchal *et al.*, 2011). Mastitis associated annual costs can reach up to 133 US$ for Gram-positive mastitis and 211 US$ for Gram-negative mastitis (Cha *et al.*, 2011) due to medicines,

veterinary charges, milk loss and premature culling. Despite improvements of sanitary conditions during milking, mastitis is far from controlled and it is the first cause of use of antibiotics in the dairy herd. Antibiotic treatments are not fully efficient, notably against *S. aureus* mastitis, and lead to the presence of antibiotic residues in milk, which may impair the industrial process (Le Maréchal *et al.*, 2011). They also contribute to the emergence and transmission of antibiotic resistance within the ecosystems, which include both commensal bacteria and opportunistic pathogens. There is thus a need for alternative strategies
for mastitis control preferably through preventive methods
to improve animal welfare.

Probiotics appear as an alternative option for mastitis control. Some lactic acid bacteria (LAB) have been studied and used as probiotics in contexts, such as vaginal and intestinal ecosystems, to maintain or restore a microbial balance and to avoid dysbiosis (Ducatelle *et al.*, 2014). Although, previous studies revealed the probiotic potential of LAB against bovine mastitis (Klostermann *et al.*, 2008), this option remains poorly documented.

We recently reported that *Lactobacillus casei* inhibits the invasion of bovine mammary epithelial cells (bMEC) by *S. aureus* (Bouchard *et al.*, 2013). *Lactococcus lactis* was previously shown to inhibit growth (Charlier *et al.*, 2008) and virulence expression in *S. aureus* *in vitro* and in cheese-making conditions (Cretenet *et al.*, 2011; Even *et al.*, 2009; Nouaille *et al.*, 2014). In the current study, we used tripartite interactions between bMEC and two major mastitis pathogens to investigate the probiotic potential of *L. lactis* V7, a LAB strain isolated from the bovine mammary gland. Its inhibitory potential against *S. aureus* and *E. coli* invasion into bMEC and its capacity to modulate the bMEC immune response under challenge with *E. coli* was investigated.

**2. Material and methods**

Isolation of *Lactococcus lactis* V7

LAB strains from bovine origin were sought considering
that LAB isolated from a bovine host might be better
adapted for a mammary probiotic use than other LAB
strains, e.g. LAB strains widely used in the dairy industry
and that underwent a so-called domestication process
(Passerini et al., 2010). During the year 2011, 68 lactating
cows of two Brazilian dairy farms were sampled by swab
of 1 cm² of teat skin, and collect of foremilk. The swabs
were placed in tubes containing 1 ml of sterile phosphate
buffered saline (PBS: sodium chloride 8 g/l, potassium
chloride 0.2 g/l, disodium hydrogen phosphate 1.44 g/l,
potassium dihydrogen phosphate 0.24 g/l) and stored on
ice until processing in the laboratory. Decimal dilutions of
swab samples were plated on M17 (Acumedia, Lansing,
MI, USA), and MRS (Acumedia) agar plates and incubated
for 48 h at 37 °C in aerobiosis and anaerobiosis to isolate
individual colonies. A total of 113 LAB candidates were
isolated and set in collection before further identification.
Species identification was carried out through 16S rRNA
(V3-V4 region) sequencing, exactly as described previously
(Otero et al., 2006). A great majority of the isolates
(n=98) were found to belong to the genus Enterococcus.
The remaining strains were Staphylococcus sp. (n=10),
Streptococcus uberis (n=1), Lactococcus garvieae (n=2),
and L. lactis (n=2). We further focused on the two L. lactis

strains, since all the other strains belong to species already
described as potentially pathogenic in dairy cows and/or as
faecal contaminants. Out of the two *L. lactis* strains, *L. lactis*
V7 was the strain which harboured the most promising
features in terms of adhesion capacities, when tested as
described previously by Bouchard *et al*. (2013).

Bacterial strains and culture conditions

Four strains of mastitis pathogens were used to assess
the probiotic potential of *L. lactis* V7 in terms of cell
invasion (i.e. adhesion and internalisation) inhibition
and immunomodulation on challenged bMEC. Two well-
characterised strains of *S. aureus* isolated from bovine
mastitis were used in this study: *S. aureus* RF122 (Herron-
Olson *et al*., 2007) and *S. aureus* Newbould 305 (hereafter
referred to as N305; Peton *et al*., 2014). These two well-
characterised strains differ by their pattern of virulence
factors and their invasion efficacy on bMEC (Peton *et al*.,
2014). *E. coli* strain P4 (Bramley, 1976) and *E. coli* K08,
a strain isolated from bovine mastitis in 2003 (available
at CIRM-BP, INRA Nouzilly, France, under reference
CIRMBP-548), belong to the same phylogenetic group A
and were kindly provided by Dr. Pierre Germon (UMR ISP,
INRA Nouzilly, France).

Subcultures prior to invasion assays were performed
overnight as follows: *L. lactis* strain V7 was grown in M17
broth medium at 30 °C without agitation; *S. aureus* strains
were grown in Brain Heart Infusion (BHI, Acumedia)
broth medium at 37 °C under agitation (180 rpm) and *E.
coli* strains were cultured in Luria Bertani broth medium
(LB D. Dutscher, Brumath, France) at 37 °C under orbital
shaking (180 rpm). Prior to invasion assays, subcultures
were washed once with PBS and suspended in DMEM (D.
Dutscher, Brumath, France) at different concentrations.
None of the strains displayed any loss of viability in DMEM
medium as determined by plate counting at 1 h, and 2
h time intervals (data not shown) corresponding to the
incubation times used in adhesion and internalisation assays
(see details below).

Bacterial concentrations in subcultures were estimated
by spectrophotometric measurements at 600 nm with a
VWR V-1200 spectrophotometer. One unity of absorbance
was equivalent to 7.7×10⁸ cfu/ml for *L. lactis*, 5.7×10⁸
and 7.4×10⁸ cfu/ml for *E. coli* P4 and K08, respectively, and to
1.7×10⁸ and 2.6×10⁸ cfu/ml for *S. aureus* RF122 and N305,
respectively. They were further confirmed by determination
of bacterial population using the micromethod (Baron *et
al*., 2006). Bacterial populations (cfu/ml) were determined
on Mannitol Salt Agar (MSA, D. Dutscher) after a 24 h
incubation at 37 °C (for *S. aureus*), LB Agar (LBA, D.
Dutscher) after a 24 h incubation at 37 °C (for *E. coli*) and
on M17 agar (Acumedia) incubated for 24 h at 30 °C (for
*L. lactis*).
# Adhesion and internalisation assays

## Mammary epithelial cells and culture conditions

The established bMEC MAC-T line (Nexia Biotechnologies, North York, ON, Canada) has been widely used for invasion assays and thus was retained for this study. MAC-T cells were cultured in T75 cell culture flasks in DMEM containing 10% heat-inactivated foetal calf serum (FCS), 100 U/ml penicillin, 10 µg/ml streptomycin, and 5 µg/ml insulin (D. Dutscher). Cells were incubated at 37 °C in a humidified incubator with 5% CO₂. They were cultured to a confluent monolayer, treated with 0.05% trypsin (Gibco-BRL, Grand Island, NY, USA), and suspended in fresh culture medium at a concentration of 2×10⁵ cells/ml. For adhesion and internalisation assays, cells were then seeded in 12-well plates (2×10⁵ cells/well) and incubated overnight at 37 °C in 5% CO₂ to obtain a confluent monolayer.

## Adhesion assay

Adhesion assays were conducted essentially as described previously (Bouchard et al., 2013), with slight modifications. Briefly, confluent monolayers of MAC-T cells were prepared in 12-well plates as described above. The interaction was carried out with *L. lactis* V7, *E. coli* (P4 and K08 strains) and *S. aureus* (RF122 and N305 strains) suspended in 1.0 ml of fresh DMEM to achieve a Ratio of Interaction (ROI) of 2,000:1 for *L. lactis* V7 and a Multiplicity of Infection (MOI) of 100:1 for *E. coli* and *S. aureus*. Both ROI (for *L. lactis* V7) and MOI (for pathogens) were chosen based on previous works by Bouchard et al. (2013). Bacterial adhesion was measured 1 h post-infection. After washing 4 times the cells on plates with PBS, the monolayer was treated with 0.05% trypsin, centrifuged for 5 min at 800×g and lysed with 0.01% Triton. The bacterial population that adhered to these cells was determined by colony counting, on M17 agar, McConkey agar (Acumedia) and MSA for *L. lactis*, *E. coli*, and *S. aureus* respectively, from serial decimal dilutions of the lysed cells. Adhesion of the pathogens alone was used as a reference. For adhesion inhibition assay, *L. lactis* V7 and the pathogenic strain were co-incubated for 1 h prior to cell monolayer treatment and bacteria counting.

## Internalisation assay

Internalisation assay was performed following the procedure described previously (Bouchard et al., 2013). Confluent monolayers of MAC-T cells, prepared as described above, were washed twice with PBS and incubated with a 1 ml suspension of *E. coli* (P4 or K08) or *S. aureus* (RF122 or N305) and/or *L. lactis* V7 in DMEM at an MOI of 100:1 for *E. coli* and *S. aureus* and an ROI of 2,000:1 for *L. lactis*.

Internalisation of *L. lactis* V7 alone was measured 2 h post-interaction. After contact, MAC-T cells were washed 4 times with PBS and then incubated for 2 h in 1.0 ml of DMEM supplemented with 100 µg/ml of gentamicin to kill the extracellular bacteria. After this incubation, the cells were lysed and the population of internalised *L. lactis* was determined as described for adhesion assay.

For internalisation inhibition assays, *L. lactis* and each of the pathogens were simultaneously added to the cells for 2 h. Pathogens internalisation was measured 2 h post-infection following an additional 2-h incubation step with DMEM supplemented with gentamicin (100 µg/ml). MAC-T monolayers were then washed four times with PBS, treated with trypsin, centrifuged for 5 min at 800×g, and lysed in 0.01% Triton. *E. coli* and *S. aureus* populations were determined as described above and for adhesion assays. For the internalisation assay, *E. coli* or *S. aureus* alone was used as a reference. Internalisation rates were then defined as the internalised *E. coli* or *S. aureus* population in the presence of *L. lactis* relative to the internalised *E. coli* or *S. aureus* population in the reference experiment.

# Quantitation of CXCL8 cytokine

Challenge of pluripotent stem (PS) cells with *E. coli* was shown to induce a pro-inflammatory response (Roussel et al., in press). To evaluate the pro-inflammatory effect of *L. lactis* V7 in a mastitis context, the production of CXCL8, a cytokine involved in the first step of the inflammatory response, was investigated on PS cells challenged with *E. coli*.

## Mammary epithelial cells and culture conditions

To assess the capacities of *L. lactis* in terms of immunomodulation, we used the newly described bMEC line PS (Roussel et al., 2015). The MAC-T cells indeed hardly modify their interleukin genes expression pattern in reaction to contact with pathogens or PAMPs whereas the PS cells were shown to significantly react to stimulation (Roussel et al., 2015). PS cells (INRA, Tours, France) were cultured with DMEM-F12 advanced (D. Dutcher) containing 10 ng/ml of insulin-like growth factor-1 (Peprotech, Rocky Hill, NJ, USA), 5 ng/ml of fibroblast growth factor (Peprotech), 5 ng/ml of epidermal growth factor (Sigma-Aldrich), 1 µg/ml of hydrocortisone (Sigma-Aldrich), 20 mM of Hepes buffer (D. Dutcher), and 2 mM of glutamine (Gibco-BRL). Plates were incubated at 37 °C in a humidified chamber with 5% of CO₂. After trypsin treatment, PS cells were counted in a Malassez chamber, and 2.5×10⁵ cells were transferred into a 24-well plate and incubated for 72 h at 37 °C.
CXCL8 determination by ELISA

Stimulation of PS cells with *L. lactis*, *E. coli*, or both species and subsequent measurement of CXCL8 production were performed essentially as described in Roussel *et al.* (2015). PS cells were seeded at $10^5$ cells/well in a 24-well plate and incubated for 72 h at 37 °C (until cells formed a confluent layer). Cells were then cultured overnight (16 h) in fresh stimulating medium without growth factors (PS stimulation medium). Cells were then washed twice with HBSS (Hank's Balanced Salt Solution; Sigma-Aldrich, Saint-Quentin Fallavier, France) and stimulated with either *L. lactis* V7 at a ROI of 100:1 or *E. coli* P4 or K08 at a MOI of 10:1 or both species. The MOI (for *E. coli*) was set according to Roussel *et al.* (2015) and the ROI (for *L. lactis* V7) was chosen lower than the ROI used for adhesion and internalisation experiments because of the reportedly good responsiveness of PS cells to bacterial stimuli (P. Germon, personal communication). Cells were incubated for 3 h, washed with HBSS and incubated in PS stimulation medium supplemented with 10 µg/ml of gentamicin, for 21 h, completing 24 h of infection. After this incubation, the supernatant was collected and stored at -20 °C until use. Concentration of CXCL8 in the supernatant was measured by ELISA as previously described (Rainard *et al.*, 2008).

Statistical analysis

All experiments were performed in triplicate. Statistical analysis was done using the R software (R Core Team, Vienna, Austria). Data were analysed using the Student t test.

3. Results

Adhesion and internalisation test of *Lactobacillus lactis*

Adhesion and internalisation tests were carried out to characterise the potential invasion capacities of *L. lactis* V7 strain into bMEC (MAC-T cells). Experiments with MAC-T cells and *L. lactis* V7 were conducted in a two-step procedure (assay of adhesion and internalisation) as described in materials and methods. The results showed that, when *L. lactis* V7 was incubated at a concentration of $5×10^8$ cfu/ml (100%) with MAC-T cells (at ROI 2,000:1), the number of adhered bacterial cells was $8.27×10^5$ cfu/ml (0.165%), while the number of internalised cells was only $8.03×10^4$ cfu/ml (0.016%).

Inhibitory potential of *Lactobacillus lactis* against
*Escherichia coli* and *Staphylococcus aureus*

The inhibitory potential of *L. lactis* V7 on the invasion capacities of *E. coli* or *S. aureus* into MAC-T cells was determined as described in materials and methods. *E. coli* or *S. aureus* were able to internalise into the MAC-T cells within a 2 h period (Figure 1). The conditions used to test the influence of *L. lactis* on the pathogen adhesion were a co-incubation of MAC-T cells with *L. lactis* at ROI of 2,000:1 ($5×10^8$ cfu/ml) and *E. coli* or *S. aureus* at MOI of 100:1 ($2.5×10^7$ cfu/ml) for 2 h. In these conditions, the adhesion mean values were $5.71×10^4$ cfu/ml for *E. coli* P4, $8.74×10^5$ cfu/ml for *E. coli* K08, $1.64×10^5$ cfu/ml for *S. aureus* RF122, and $1.47×10^6$ cfu/ml for *S. aureus* N305. Of note, the higher adhesion observed in N305 compared to RF122 is in accordance with previous results (Peton *et al.*, 2014). A significant reduction of the adhesion rate was observed for *S. aureus* N305 only whose adhesion was reduced by 75% (Figure 1A), suggesting that the adhesion

<figure><img src="image_2.png" /><figcaption>Figure 1. Adhesion and internalisation (%) of *Escherichia coli* or *Staphylococcus aureus* strains into MAC-T cell cultures with or without *Lactococcus lactis* V7. (A) Adhesion of *E. coli* P4 and K08 and *S. aureus* RF122 and Newbould 305 (N305) to MAC-T cells, without or with co-incubation with *L. lactis* V7 for 1 h. (B) Internalisation by *E. coli* P4 and K08 and *S. aureus* RF122 and N305 strains into MAC-T cells, without or with co-incubation with *L. lactis* V7 for 2 h. Data are presented as mean ± standard deviation (n=3). \* *P*<0.02.</figcaption></figure>
inhibition mediated by *L. lactis* V7 is strain-dependent. The effect of *L. lactis* co-incubation upon internalisation was also investigated using a MOI of 100:1 for the pathogenic strain and a MOI of 2,000:1 for *L. lactis* V7. In these conditions, the internalisation mean values were 1.31×10⁴ cfu/ml for *E. coli* P4, 6.63×10³ cfu/ml for *E. coli* K08, 2.83×10⁶ cfu/ml for *S. aureus* RF122, and 1.51×10⁷ cfu/ml for *S. aureus* N305. *L. lactis* V7 reduced the internalisation rates of all the *E. coli* and *S. aureus* strains tested on MAC-T cells (Figure 1B). This inhibition was higher for the *S. aureus* strains (80 and 88% for RF122 and N305, respectively) when compared to the *E. coli* strains (45 and 55% for P4 and K08, respectively).

## CXCL8 production in pluripotent stem cell culture challenged with *Escherichia coli*

Production of CXCL8, an important chemokine produced during the first steps of inflammation and involved in neutrophils recruitment, was measured to evaluate the effect of *L. lactis* on PS cells infected with *E. coli* P4 and K08 at MOI 10:1. PS cell cultures were first stimulated by the *E. coli* strains and *L. lactis*, alone. An analysis of variance (ANOVA) showed that there was significant difference between groups of different challenges (P<7.09×10⁻⁹). The CXCL8 production was shown to be normally distributed by Komogorov-Smirnov and by Shapiro-Wilk tests. Then, the T-test was applied, showing that PS cells produced higher levels of CXCL8 when challenged with both *E. coli* strains (~2,000 pg/ml) than in the control condition without any challenge (P<0.01) (Figure 2). *L. lactis* V7 at ROI 100:1 also significantly stimulated CXCL8 production by PS cell culture (P<0.05), although to a level (~500 pg/

<figure><img src="image_3.png" /><figcaption>**Figure 2.** CXCL8 production (pg/ml) by pluripotent stem (PS) cell cultures challenged with *Escherichia coli* strains P4 and K08 in the presence or not of *Lactococcus lactis* V7. CXCL8 production of PS cell cultures alone (PSCell) and in the presence of *L. lactis* V7, *E. coli* P4, *E. coli* K08, or a co-culture of *L. lactis* V7 and *E. coli* P4 or K08. Data are presented as mean ± standard deviation (n=3). \* P<0.03 when compared to cell with P4 or K08; \# P<0.05 when compared to PSCell.</figcaption></figure>

ml) lower than that of *E. coli* challenged cells. The presence of *L. lactis* V7 appeared to highly stimulate the increase of CXCL8 production (~3,500 and 4,000 pg/ml) in PS cell cultures when cells were simultaneously challenged with either of the *E. coli* strains (Figure 2). The T-test allowed us to see that there was not a significant difference between each of the *E. coli* strains effects on PS cell production of CXCL8 (P>0.05).

## 4. Discussion

In this study, we investigated *L. lactis* V7, a strain isolated from a cow teat in Brazil, with regard to its probiotic potential in terms of inhibition of pathogens adhesion on and internalisation into bMEC by both *E. coli* and *S. aureus* strains, and stimulation of the production of pro-inflammatory cytokines by bMEC.

*L. lactis* is naturally found in many different niches and presents a wide ecological distribution, which ranges from vegetal origins (sugar cane plants, mung bean sprouts, mustard) to the human vaginal flora and the gut of trouts (for review, see Cavanagh *et al.*, 2015). However, *L. lactis* is most famous for its association with the fermented food process and especially with the milk environment and fermented milk products. The intensive use of some *L. lactis* strains in the dairy industry has led to the domestication of this bacterium to the milk environment. Such domestication results in deep modifications such as genome reduction, gene decay, and horizontal acquisition of genes providing a better adaptation to the milk environment (Passerini *et al.*, 2010, 2013a,b). Some authors hypothesise that some strains are so specialised that they can no longer occupy non-dairy niches (Kelly *et al.*, 2010). Here, *L. lactis* V7 was isolated from dairy ruminant, and might thus harbour genetic determinants associated to the animal host adaptation as well as to the milk habitat. Investigations on *L. lactis* V7 genome through genome sequencing and analysis and on *L. lactis* V7 ability to grow in milk would be of great interest but were not addressed in this study.

*L. lactis* V7 was able to adhere in relatively high amounts to MACT-T cells (1.58×10⁵ cfu/ml), but its internalisation was low (1.93×10³ cfu/ml). The value found for *L. lactis* V7 adhesion is similar to that previously reported for *L. casei* strains (Bouchard *et al.*, 2013) and other LAB strains (unpublished data) onto MAC-T cells. Similar levels were also reported for other lactobacilli using different mammary cell lines (Garriga *et al.*, 2014; Vizoso-Pinto *et al.*, 2007). *L. lactis* V7 was also shown to internalise into bMEC at a rate comparable to that obtained for another *L. lactis* strain (unpublished data). The *L. lactis* V7 internalisation rate was however lower than that of other LAB species (unpublished data), which can be considered as an advantage since it considerably limits the risk of tissue invasion by the probiotic candidate.
L. lactis V7 was able to inhibit the internalisation of pathogens frequently associated to mastitis, E. coli and S. aureus, in high percentages ranging from 45 to 88% of inhibition. These values are similar to those previously observed with L. casei in bMEC cell lines (Bouchard et al., 2013). Interestingly here, the inhibition of internalisation did not correlate with the inhibition of the adhesion onto the bMEC, except for S. aureus N305, suggesting that L. lactis V7 has a wider range of internalisation inhibition against mastitis pathogens and that different features contribute to these inhibitory potentials. Adhesion capacities and inhibition of adhesion is a criterion often used in the selection of LAB candidates against the intracellular pathogen Listeria monocytogenes (Garriga et al., 2014; Lavilla-Lerma et al., 2013; Nakamura et al., 2012). Since S. aureus and L. monocytogenes internalise using different mechanisms, it is likely that the LAB inhibition of a pathogen invasion into its host cell is mediated by various mechanisms.

The ROI (2,000:1) and MOI (100:1) used in this work were chosen based on previous works on L. casei capacity to inhibit S. aureus adhesion and internalisation into bMEC (Bouchard et al., 2013). It would be interesting to test L. lactis V7 at different ROI to check whether it maintains its inhibitory potential against cell invasion by pathogens at lower ROI.

A slight production of the pro-inflammatory cytokines CXCL8 was observed with L. lactis V7 alone and the increase in CXCL8 production was greater when PS cells were co-incubated with L. lactis V7 and E. coli strains, suggesting that L. lactis V7 induces a slight inflammatory response in PS cells and that co-incubation with E. coli and L. lactis V7 has a synergistic rather than cumulative effect on CXCL8 production. CXCL8 is produced in inflammatory conditions, and plays a role in the inflammatory response, and in neutrophils recruitment (Rainard et al., 2008). Another L. lactis strain (DCP3147) was also shown to enhance the expression of CXCL8 gene (together with IL-6 gene, as determined by RT-qPCR) when injected in a cow teat (Beecher et al., 2009). In addition, the intramammary injection of L. lactis DCP3147 was able to treat bovine mastitis as efficiently as antibiotics (Klostermann et al., 2008). The CXCL8 stimulation by L. lactis V7 we report here might thus be considered an interesting trait to be further investigated for the development of mammary probiotics.

In this work, adhesion and internalisation experiments were carried out using MAC-T, which is the model cell line for bMEC and which was used in several previous studies to investigate cell invasion by mastitis pathogens such as E. coli (Silva et al., 2014), Streptococcus dysgalactiae (Song et al., 2004) or S. aureus (Kerro-Dego et al., 2012). Although MAC-T cells were also used to investigate the

transcriptional response to interactions with pathogens or components of pathogens (Ibeagha-Awemu et al., 2008; Jin et al., 2014), recent works developed at INRA Tours (P. Germon's team) question the use of MAC-T cells for immunomodulation experiments since they hardly respond to stimulation (e.g. with E. coli LPS) in terms of chemokines production. The characterisation of PS, the bMEC line used here, was recently completed and provides a better tool to investigate the bMEC immune response to both probiotic (this work) or pathogenic bacteria (Roussel et al., 2015).

In conclusion, we showed here that L. lactis V7 was able to inhibit the internalisation of E. coli and S. aureus strains into bMEC. Moreover, L. lactis V7 alone was shown to trigger an immunomodulation on bMEC through a slight increase in CXCL8 production. When co-incubated with E. coli, it significantly amplified the induction of the pro-inflammatory cytokine CXCL8 compared to the induction obtained with E. coli alone. These features could be of interest for the development of a probiotic strategy against mastitis. Quantitation of other inflammatory cytokines is however required to reinforce these promising results. Inhibition of pathogens internalisation can reduce the risk of tissue invasion, infection dissemination and persistence of the pathogens in the mammary gland. In addition, a stronger inflammatory response can enhance the clearance of the pathogen out of the mammary gland. Such property is of particular interest in the case of pathogens inducing a low inflammatory response. These promising results nevertheless require further experiments. To better understand the immunomodulatory effects of L. lactis V7, chemokine profiling has to be completed by quantifying other types of chemokines (through RT-qPCR, when ELISA reagents are not available for bovine chemokines). Similarly, the safety of L. lactis V7 strain has to be investigated on an intermediate in vivo model such as a mouse model before being experimented in the target species (Notebaert and Meyer, 2006). Mouse model of experimental mastitis might also help validating L. lactis V7 protective effect in experimentally induced mastitis before validation in field conditions (Breyne et al., 2015).

## Acknowledgements

We thank Mr. Philippe Langella and Mr. Florien Chain (INRA, Jouy-en-Josas, MICALIS) for their support. The work was supported by Coordenação de Aperfeiçoamento do Pessoal de Ensino Superior (CAPES/Brazil) and Institut National de la Recherche Agronomique (INRA/France). Bianca Seridan was recipient of a PhD fellowship from Coordenação de Aperfeiçoamento do Pessoal de Ensino Superior (CAPES/Brazil) and of an Excellence Eiffel fellowship (No. 748798C; Campus France) from the French government. Yves Le Loir is the recipient of a PVE grant (71/2013) from Programa Ciências sem Fronteiras. Part of this work was financially supported
by the French National Research Agency (ANR) project NABAB (ANR-08-ALIA-11) and the INRA-funded GISA project (Ruminflame).

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