Effect of Enterococcus faecium NCIMB 10415 on Gut Barrier Function, Internal Redox State, Proinflammatory Response and Pathogen Inhibition Properties in Porcine Intestinal Epithelial Cells

In farm animals, intestinal diseases caused by Salmonella spp. and Escherichia coli may lead to significant economic loss. In the past few decades, the swine industry has largely relied on the prophylactic use of antibiotics to control gastrointestinal diseases. The development of antibiotic resistance has become an important issue both in animal and human health. The use of antibiotics for prophylactic purposes has been banned, moreover the new EU regulations further restrict the application of antibiotics in veterinary use. The swine industry seeks alternatives that are capable of maintaining the health of the gastrointestinal tract. Probiotics offer a promising alternative; however, their mode of action is not fully understood. In our experiments, porcine intestinal epithelial cells (IPEC-J2 cells) were challenged by Salmonella Typhimurium or Escherichia coli and we aimed at determining the effect of pre-, co-, and post-treatment with Enterococcus faecium NCIMB 10415 on the internal redox state, paracellular permeability, IL-6 and IL-8 secretion of IPEC-J2 cells. Moreover, the adhesion inhibition effect was also investigated. Enterococcus faecium was able to reduce oxidative stress and paracellular permeability of IPEC-J2 cells and could inhibit the adhesion of Salmonella Typhimurium and Escherichia coli. Based on our results, Enterococcus faecium is a promising candidate to maintain the health of the gastrointestinal tract.


Introduction
Intestinal diseases caused by Escherichia coli (E. coli) and Salmonella spp. may lead to significant economic loss in food-producing animals and may also pose a threat to human health as (1) both bacteria are zoonotic, (2) they may contaminate pork products in the food chain, and (3) they may develop resistance to antibiotics, thus contributing to the transmission of antimicrobial resistance [1][2][3].
To control gastrointestinal diseases, the swine industry has largely relied on the prophylactic use of antibiotics. Due to the growing concern about antibiotic resistance, the use of antibiotics as growth promoters were banned in the European Union in 2006. The new EU regulation (2019/6 of the European Parliament and of the Council of 11 December 2018 on veterinary medicinal products and repealing Directive 2001/82/EC) has come into force on 28 January 2022, further restricting the application of antibiotics in veterinary use [4]. However, according to the One Health concept, antimicrobial resistance is not only a concern for the veterinary sector, but it also affects humans and the natural environment that animals and humans share [5]. Any option that can reduce the spread

Cell Line and Culture Conditions
The IPEC-J2 epithelial cell line was a kind gift from Dr. Jody Gookin's Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA. The cells were grown and maintained in a complete medium consisting of 10 mL of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) in a 1:1 ratio. This was supplemented with 5% fetal bovine serum (FBS), 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 5 ng/mL epidermal growth factor (EGF) and 1% penicillin-streptomycin (Biocenter Ltd., Szeged, Hungary). Cells were cultured at 37 • C in a humidified atmosphere of 5% CO 2 [26]. Cells with passage numbers 49-52 were used for our experiments. For cell viability determination with the Neutral Red Uptake (NRU) method, cells were cultured onto a 96-well plate (Costar Corning Inc., Corning, NY, USA). For IL-6, IL-8 and intracellular ROS determination, cells were grown on 6-well culture plates (Costar Corning Inc., Corning, NY, USA). For adhesion inhibition, assays cells were seeded onto 24-well cell culture plates (Costar Corning Inc., Corning, NY, USA). For the measurement of paracellular permeability, cells were cultured on 12-well polyester membrane cell culture inserts (Costar Corning Inc., Corning, NY, USA). In each case, cells were cultured until confluency was reached.
In order to remove the remaining antibiotics before starting the treatment of IPEC-J2 cells with the different treatment solutions (described in Section 2.1) IPEC-J2 cells were washed twice with PBS then DMEM/F12 without antibiotics was added to each well, and cells were incubated for 30 min at 37 • C.

Neutral Red Uptake Assay for Cell Viability
The influence of different E. faecium bacterial suspension concentrations and different incubation periods on the viability of IPEC-J2 cells were tested with the neutral red uptake method based on the description of Repetto et al. [30]. E. faecium suspensions of different concentrations were prepared as described above. IPEC-J2 cells were seeded onto a 96well plate and incubated with E. faecium suspensions of different concentrations (10 8 , 10 6 , 10 4 CFU/mL) for 1, 2, 4 and 24 h, respectively (37 • C, 5% CO 2 ). Treatment with plain medium for 1 h was used as a control in the experiment. The viability of IPEC-J2 cells The influence of E. coli and S. Typhimurium suspensions applied in different concentrations and for different incubation periods was tested by our research group previously [27].

Experimental Setup
For our DCFH-DA, ELISA, FD4, adhesion assay experiments, IPEC-J2 cells were incubated for 1 h with the pathogen strain E. coli or S. Typhimurium, respectively. Control cells received plain DMEM/F12 medium. As a positive control, IPEC-J2 cells were mono-incubated with only E. coli (10 6 CFU/mL) or S. Typhimurium (10 6 CFU/mL), respectively. For pre-treatment assays, cells were pre-incubated with E. faecium for 1 h before the addition of the pathogen strain. For co-treatment experiments, the pathogen strain (E. coli or S. Typhimurium) and E. faecium were added at the same time to IPEC-J2 cells. In our post-treatment assay, IPEC-J2 cells were incubated with E. faecium for 1 h after the treatment with the pathogen strains (E. coli or S. Typhimurium). Bacterial infections were performed with E. coli or S. Typhimurium at a concentration of 10 6 CFU/mL. The applied tolerable pathogen concentration was based on our previous investigations [1]. E. faecium suspensions were applied either in a 10 7 or 10 8 CFU/ ml concentration, based on our cell viability experimental results. IPEC-J2 cells were also mono-incubated with E. faecium 10 8 and 10 7 CFU/mL. If further incubation was needed after the treatments, cells were washed with PBS and DMEM/F12 supplemented with antibiotics. Moreover, 1% penicillinstreptomycin was added to prevent the growth of bacteria. The applied treatment solutions in our experiments are summarized in Table 1, and Figure 1 shows the timeline of our experimental setup.

Determination of the Intracellular Redox Status of IPEC-J2 Cells
To evaluate the effect of E. faecium on the intracellular redox state of IPEC-J2 cells, DCFH-DA method was used. IPEC-J2 cells were challenged by E. coli or S. Typhimuriu respectively. E. faecium was added at either 10 8 CFU/mL or 10 7 CFU/mL 1 h before (p treatment), at the same time (co-treatment) or 1 h after (post-treatment) the indicator coli (10 8 CFU/mL) or S. Typhimurium (10 8 CFU/mL) strain was added. Moreover, the eff of E. faecium alone (applied in 10 8 CFU/mL or 10 7 CFU/mL) on the amount of intracellu reactive oxygen species were tested. As a negative control, cells treated with plain m dium were used. Cells treated with either E. coli or S. Typhimurium served as posit controls. After the treatment, the treatment solutions were discarded and plain mediu containing 1% penicillin-streptomycin was added.
For the detection, the cells were washed with PBS after 24 h, and DCFH-DA (Sigm Aldrich, Darmstadt, Germany) reagent (40 mM) was added to the cells. After one ho the reagent was removed. The cells were then washed twice with phenol-free pl DMEM/F12 (2 mL). Afterward, the cells were scraped and lysed. The lysed cells were th pipetted into an Eppendorf tube and centrifuged for 10 min at 4 °C at 4500 rpm. amount of 100 μL of supernatant from each sample was added to a 96-well plate. T Spectramax iD3 instrument was used to measure the fluorescence at an excitation wa length of 480 nm and an emission wavelength of 530 nm.

IL-6 and IL-8 Determination with ELISA
For the ELISA experiments, cells were seeded onto 6-well culture plates and pre-, , and post-treatments were performed as described in the experimental setup section. A ter the removal of the treatments solutions, IPEC-J2 cells were incubated with a cell cultu

Determination of the Intracellular Redox Status of IPEC-J2 Cells
To evaluate the effect of E. faecium on the intracellular redox state of IPEC-J2 cells, the DCFH-DA method was used. IPEC-J2 cells were challenged by E. coli or S. Typhimurium, respectively. E. faecium was added at either 10 8 CFU/mL or 10 7 CFU/mL 1 h before (pretreatment), at the same time (co-treatment) or 1 h after (post-treatment) the indicator E. coli (10 8 CFU/mL) or S. Typhimurium (10 8 CFU/mL) strain was added. Moreover, the effect of E. faecium alone (applied in 10 8 CFU/mL or 10 7 CFU/mL) on the amount of intracellular reactive oxygen species were tested. As a negative control, cells treated with plain medium were used. Cells treated with either E. coli or S. Typhimurium served as positive controls. After the treatment, the treatment solutions were discarded and plain medium containing 1% penicillin-streptomycin was added.
Intracellular ROS was measured using 2 , 7 -dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye (Sigma-Aldrich, Budapest, Hungary). DCFH-DA is oxidized to the highly fluorescent form dichloro-fluorescein (DCF) by the intracellular ROS [31]. With this method, the overall oxidative stress is measured in cells, since various free radicals are capable of oxidizing the DCFH-DA.
For the detection, the cells were washed with PBS after 24 h, and DCFH-DA (Sigma-Aldrich, Darmstadt, Germany) reagent (40 mM) was added to the cells. After one hour, the reagent was removed. The cells were then washed twice with phenol-free plain DMEM/F12 (2 mL). Afterward, the cells were scraped and lysed. The lysed cells were then pipetted into an Eppendorf tube and centrifuged for 10 min at 4 • C at 4500 rpm. An amount of 100 µL of supernatant from each sample was added to a 96-well plate. The Spectramax iD3 instrument was used to measure the fluorescence at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

IL-6 and IL-8 Determination with ELISA
For the ELISA experiments, cells were seeded onto 6-well culture plates and pre-, co-, and post-treatments were performed as described in the experimental setup section. After the removal of the treatments solutions, IPEC-J2 cells were incubated with a cell culture medium and cell supernatants were collected after 6 h. IL-6 and IL-8 secretion were determined by porcine-specific ELISA kits (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer's instructions.

Paracellular Permeability Measurements/Assay
The effect of E. faecium and E. coli or S. Typhimurium on the paracellular permeability of IPEC-J2 cells was evaluated with tracer dye FD4 (Sigma-Aldrich, Darmstadt, Germany). Cells were seeded onto 12-well membrane inserts. Prior to treatments, TEER values were measured to check the development of a differentiated confluent monolayer. Mono-, pre-, co-, and post-treatments were performed as described in the section's experimental setup. After treatment, the cells were washed with PBS and FD4 (dissolved in phenol-free DMEM/F12 medium) at a final concentration of 0.25 mg/mL was added to the apical layer cells. To the basolateral chamber, phenol-free DMEM/F12 medium was added. Cells were incubated at 37 • C (5% CO 2 ). Samples of 100 µL were taken from the basolateral chamber after 24 h. The fluorescent signal was measured with a Spectramax iD3 instrument using 485 nm excitation and a 535 nm emission wavelength.

Adhesion Inhibition Assay
In order to evaluate the inhibitory effect of E. faecium on E. coli or S. Typhimurium adhesion to IPEC-J2 cells, E. faecium was added at 10 8 CFU/mL 1 h before (pre-treatment), at the same time (co-treatment) or 1 h after (post-treatment) the indicator E. coli or S. Typhimurium strain was added. As the control, cells treated with only E. coli or S. Typhimurium were used. IPEC-J2 cells were incubated for 1 h and then were washed to remove unbound bacteria. The lysis of cells was performed with 500 µL 0.1% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany). Viable E. coli and S. Typhimurium counts were determined by serial dilution and plating on ChromoBio Coliform (for E. coli) or ChromoBio Salmonella Plus Base (for S. Typhimurium) agar. ChromoBio Coliform and ChromoBio Salmonella Plus Base selective agars were purchased from Biolab Zrt. (Budapest, Hungary). Adhesion was calculated as the control percentage. Adhering E. coli and S. Typhimurium was normalized to the control.

Statistical Analysis
Data were tested for normality of distribution and statistical analysis was performed with the R 4.0.4 software package. The data are given as mean values ± S.E.M (n) where n refers to the number of parallel measurements. Differences between means were evaluated by one-way analysis of variance (ANOVA) with a post hoc Tukey's test when data were of normal distribution and homogeneity of variances was confirmed, or a Kruskal-Wallis nonparametric test. A p value of <0.05 was accepted to indicate statistical significance. The exact statistical comparisons are indicated in the text and in the appropriate figure legends.

Cell Viability Assay
In order to determine the effect of E. faecium suspensions on the viability of IPEC-J2 cells, the neutral red uptake method was used. E. faecium suspensions of a 10 8 CFU/mL concentration significantly reduced the viability of IPEC-J2 cells when they were applied for 4 and 24 h ( Figure 2). Any other treatment concentrations and treatment times did not cause any significant change in the viability of IPEC-J2 cells as compared to the control. The cytotoxic effect of E. coli and S. Typhimurium were previously tested, the optimal treatment concentrations were found to be 10 6 CFU/mL and the optimal treatment time was set to 1 h [1].

Effect of Enterococcus faecium on the Intracellular Redox State of IPEC-J2 Cells Challenge by Salmonella Typhimurium and Escherichia coli
In order to characterize the intracellular redox state of the IPEC-J2 cells, the DCF DA method was used. Treatment with S. Typhimurium caused an increase in the fluor cence compared to the control ( Figure 3). All three treatment combinations (i.e., pre-tre ment, co-treatment and post-treatment with S. Typhimurium and E. faecium in two diff ent concentrations) resulted in a decreased amount of ROS. When IPEC-J2 cells we treated with only E. faecium 10 8 CFU/mL and 10 7 CFU/mL, a decrease in fluorescence cou be observed compared to the control.
Treatment with E. coli caused an increase in the fluorescence compared to the cont ( Figure 4). The pre-treatment with E. faecium significantly reduced the amount of reacti oxygen species in the cells compared with samples only treated with E. coli. Both appli concentrations (10 8 CFU/mL and 10 7 CFU/mL) of E. faecium resulted in a significant d crease in reactive oxygen species. The same could be observed in the case of co-treatmen and post-treatments.

Effect of Enterococcus faecium on the Intracellular Redox State of IPEC-J2 Cells Challenged by Salmonella Typhimurium and Escherichia coli
In order to characterize the intracellular redox state of the IPEC-J2 cells, the DCFH-DA method was used. Treatment with S. Typhimurium caused an increase in the fluorescence compared to the control ( Figure 3). All three treatment combinations (i.e., pre-treatment, co-treatment and post-treatment with S. Typhimurium and E. faecium in two different concentrations) resulted in a decreased amount of ROS. When IPEC-J2 cells were treated with only E. faecium 10 8 CFU/mL and 10 7 CFU/mL, a decrease in fluorescence could be observed compared to the control.   pre-treatment with E. faecium 10 8 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ7 PRE: pretreatment with E. faecium 10 7 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ8 CO: co-treatment with E. faecium 10 8 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ7 CO: co-treatment with E. faecium 10 7 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ8 POST: post-treatment with E. faecium 10 8 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ7 POST: post-treatment with E. faecium 10 7 CFU/mL + S. Typhimurium 10 6 CFU/mL. Data are shown as means with standard deviations, n = 6/group. ** p ≤ 0.01; *** p ≤ 0.0001.

Effect of E. faecium on IL-6 and IL-8 Production of IPEC-J2 Cells Provoked by E. coli or S. Typhimurium
Infection of intestinal epithelial cells with S. Typhimurium significantly induced the secretion of IL-6 compared to the controls (i.e., non-infected cells) ( Figure 5). In comparison, treatment with only the probiotic strain did not result in a significant change in IL-6 secretion, even if E. faecium was applied at a concentration of 10 8 CFU/mL or 10 7 CFU/mL. The pre-treatment with E. faecium 10 8 CFU/mL caused a significant decrease in IL-6 production as compared to the IL-6 secretion induced by S. Typhimurium. However, the co-treatment of S. Typhimurium and E. faecium at 10 8 CFU/mL did not alter the IL-6 secretion compared to the IL-6 secretion evoked by S. Typhimurium. The pre-treatment and the co-treatment with E. faecium 10 7 CFU/mL failed to significantly decrease IL-6 secretion compared to the IL-6 production induced by S. Typhimurium. secretion of IL-6 compared to the controls (i.e., non-infected cells) ( Figure 5). In comparison, treatment with only the probiotic strain did not result in a significant change in IL-6 secretion, even if E. faecium was applied at a concentration of 10 8 CFU/mL or 10 7 CFU/mL. The pre-treatment with E. faecium 10 8 CFU/mL caused a significant decrease in IL-6 production as compared to the IL-6 secretion induced by S. Typhimurium. However, the cotreatment of S. Typhimurium and E. faecium at 10 8 CFU/mL did not alter the IL-6 secretion compared to the IL-6 secretion evoked by S. Typhimurium. The pre-treatment and the cotreatment with E. faecium 10 7 CFU/mL failed to significantly decrease IL-6 secretion compared to the IL-6 production induced by S. Typhimurium.   faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of E. coli. E. faecium was added in 10 8 CFU/mL or in 10 7 CFU/mL concentration. Control: plain cell culture medium treatment; Ec: E. coli 10 6 CFU/mL; Ef 10^8 PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10^7 PRE: pre-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL; Ef 10^8 CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10^7 CO: co-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL. Data are shown as means with standard deviations, n = 6/group. * p ≤ 0.05.
Infection of IPEC-J2 cells with S. Typhimurium also increased the secretion of IL-8 ( Figure 7). Treatment with the probiotic strain itself did not result in a significant change in IL-8 secretion, regardless of the applied concentration. Pre-treatment and co-treatment with E. faecium, applied at a concentration of 10 8 CFU/mL, significantly reduced the secretion of IL-8 compared to the amount of IL-8 secretion when IPEC-J2 cells were challenged by S. Typhimurium. Pre-treatment and co-treatment with E. faecium, applied at a concen- Figure 6. Induction of IL-6 secretion of IPEC-J2 cells after stimulation with E. coli and E. faecium. E. faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of E. coli. E. faecium was added in 10 8 CFU/mL or in 10 7 CFU/mL concentration. Control: plain cell culture medium treatment; Ec: E. coli 10 6 CFU/mL; Ef 10ˆ8 PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 PRE: pre-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ8 CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 CO: co-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL. Data are shown as means with standard deviations, n = 6/group. * p ≤ 0.05. Infection of IPEC-J2 cells with S. Typhimurium also increased the secretion of IL-8 ( Figure 7). Treatment with the probiotic strain itself did not result in a significant change in IL-8 secretion, regardless of the applied concentration. Pre-treatment and co-treatment with E. faecium, applied at a concentration of 10 8 CFU/mL, significantly reduced the secretion of IL-8 compared to the amount of IL-8 secretion when IPEC-J2 cells were challenged by S. Typhimurium. Pre-treatment and co-treatment with E. faecium, applied at a concentration of 10 7 CFU/mL, failed to decrease the IL-8 secretion in comparison to the secretion observed when cells were treated with S. Typhimurium itself. CFU/mL; Ef 10^8 CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; co-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL. Data are shown as means ard deviations, n = 6/group. * p ≤ 0.05.
Infection of IPEC-J2 cells with S. Typhimurium also increased the secre (Figure 7). Treatment with the probiotic strain itself did not result in a signific in IL-8 secretion, regardless of the applied concentration. Pre-treatment and co with E. faecium, applied at a concentration of 10 8 CFU/mL, significantly reduce tion of IL-8 compared to the amount of IL-8 secretion when IPEC-J2 cells were by S. Typhimurium. Pre-treatment and co-treatment with E. faecium, applied a tration of 10 7 CFU/mL, failed to decrease the IL-8 secretion in comparison to th observed when cells were treated with S. Typhimurium itself.  faecium. E. faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of S. Typhimurium. E. faecium was added in 10 8 CFU/mL or in 10ˆ7 CFU/mL concentration. Control: plain cell culture medium treatment; St: S. Typhimurium 10 6 CFU/mL; Ef 10ˆ8: E. faecium 10 8 CFU/mL; Ef 10ˆ7: E. faecium 10 7 CFU/mL; Ef 10ˆ8 PRE: pre-treatment with E. faecium 10 8 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ7 PRE: pre-treatment with E. faecium 10 7 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ8 CO: co-treatment with E. faecium 10 8 CFU/mL + S. Typhimurium 10 6 CFU/mL; Ef 10ˆ7 CO: co-treatment with E. faecium 10 7 CFU/mL + S. Typhimurium 10 6 CFU/mL. Data are shown as means with standard deviations, n = 6/group; *** p ≤ 0.0001. IL-8 secretion was induced significantly by E. coli compared to the control cells ( Figure 8). Pre-treatment and co-treatment with E. faecium, applied at a concentration of 10 8 CFU/mL further increased the secretion of IL-8. The pre-treatment and co-treatment with E. faecium, applied at a concentration of 10 7 CFU/mL, failed to cause any significant effect on IL-8 secretion.

Effect of E. faecium on the Adhesion of S. Typhimurium and E. coli to IPEC-J2 Cells
E. faecium was able to inhibit the adhesion of both E. coli and S. Typhimurium in all treatment combinations (Figure 9). When IPEC-J2 cells were challenged by E. coli, pretreatment with E. faecium had the highest inhibitory effect, followed by co-treatment, while post-treatment showed the lowest inhibitory effect. E. coli adhesion was 26.2% in the case of pre-treatment, 27.8% in the co-treatment assay and 37.6% in the post-treatment. When IPEC-J2 cells were exposed to S. Typhimurium, only a minor difference could be found in the effect of adhesion between the different treatment (pre-, co-and post-) conditions. S. Typhimurium adhesion was 12.9% in the case of pre-treatment, 11.2% in the cotreatment assay, and 12.3% for the post-treatment. E. faecium was added 1 h before (pre-treatment) or at the same time (co-treatment) of the addition of E. coli. E. faecium was added in 10 8 CFU/mL or in 10 7 CFU/mL concentration. Control: plain cell culture medium treatment; Ec: E. coli 10 6 CFU/mL; Ef 10ˆ8 PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 PRE: pre-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ8 CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 CO: co-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL. Data are shown as means with standard deviations, n = 6/group. * p ≤ 0.05; ** p ≤ 0.01.

3.4.
Effect of E. faecium on the Adhesion of S. Typhimurium and E. coli to IPEC-J2 Cells E. faecium was able to inhibit the adhesion of both E. coli and S. Typhimurium in all treatment combinations (Figure 9). When IPEC-J2 cells were challenged by E. coli, pretreatment with E. faecium had the highest inhibitory effect, followed by co-treatment, while post-treatment showed the lowest inhibitory effect. E. coli adhesion was 26.2% in the case of pre-treatment, 27.8% in the co-treatment assay and 37.6% in the post-treatment. When IPEC-J2 cells were exposed to S. Typhimurium, only a minor difference could be found in the effect of adhesion between the different treatment (pre-, co-and post-) conditions. S. Typhimurium adhesion was 12.9% in the case of pre-treatment, 11.2% in the co-treatment assay, and 12.3% for the post-treatment.  Figure 9. Inhibitory effect of E. faecium on E. coli and S. Typhimurium adhesion to coli and S. Typhimurium adhesion inhibitions were determined upon incubation added 1 h before (pre-treatment), at the same time (co-treatment) and 1 h after (pos addition of E. coli and S. Typhimurium, respectively. E. faecium was added in 10 8 CF 10 6 CFU/mL; Ef PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Ty CFU/mL; Ef CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Typhimuriu Ef POST: post-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Typhimurium 10 ues are presented as means ± SEs of four independent experiments. *** p ≤ 0.0001 co ment with S. Typhimurium. *** p ≤ 0.0001 compared to treatment with E. coli.

The Effect of E. faecium on Paracellular Permeability of IPEC-J2 Cells Challen and S. Typhimurium
After 24 h of pathogen exposure, the epithelial cell layer was partially d fluorescence intensity measured in the basolateral compartment significa (compared to the untreated control samples) when IPEC-J2 cells were t Typhimurium ( Figure 10) or E. coli (Figure 11). The treatment with E. faecium different concentrations (10 8 CFU/mL or 10 7 CFU/mL), did not result in th fluorescence intensity (Figure 10). Pre-treatment, co-treatment and post-tre faecium significantly decreased the presence of FD4 tracer in the basolat when cells were exposed to S. Typhimurium (Figure 10). The same effec served when IPEC-J2 cells were challenged by E. coli (Figure 11). E. coli and S. Typhimurium adhesion inhibitions were determined upon incubation with E. faecium added 1 h before (pre-treatment), at the same time (co-treatment) and 1 h after (post-treatment) the addition of E. coli and S. Typhimurium, respectively. E. faecium was added in 10 8 CFU/mL. Ec: E. coli 10 6 CFU/mL; Ef PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Typhimurium 10 6 CFU/mL; Ef CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Typhimurium 10 6 CFU/mL; Ef POST: post-treatment with E. faecium 10 8 CFU/mL + E. coli or S. Typhimurium 10 6 CFU/mL. Values are presented as means ± SEs of four independent experiments. *** p ≤ 0.0001 compared to treatment with S. Typhimurium. *** p ≤ 0.0001 compared to treatment with E. coli.

The Effect of E. faecium on Paracellular Permeability of IPEC-J2 Cells Challenged by E. coli and S. Typhimurium
After 24 h of pathogen exposure, the epithelial cell layer was partially disrupted. The fluorescence intensity measured in the basolateral compartment significantly increased (compared to the untreated control samples) when IPEC-J2 cells were treated with S. Typhimurium (Figure 10) or E. coli (Figure 11). The treatment with E. faecium alone, in two different concentrations (10 8 CFU/mL or 10 7 CFU/mL), did not result in the alteration of fluorescence intensity ( Figure 10). Pre-treatment, co-treatment and post-treatment with E. faecium significantly decreased the presence of FD4 tracer in the basolateral chamber, when cells were exposed to S. Typhimurium (Figure 10). The same effect could be observed when IPEC-J2 cells were challenged by E. coli ( Figure 11).
(compared to the untreated control samples) when IPEC-J2 cells were treated with S. Typhimurium (Figure 10) or E. coli (Figure 11). The treatment with E. faecium alone, in two different concentrations (10 8 CFU/mL or 10 7 CFU/mL), did not result in the alteration of fluorescence intensity (Figure 10). Pre-treatment, co-treatment and post-treatment with E. faecium significantly decreased the presence of FD4 tracer in the basolateral chamber, when cells were exposed to S. Typhimurium (Figure 10). The same effect could be observed when IPEC-J2 cells were challenged by E. coli (Figure 11).

Discussion
The present study aims to elucidate the effect of E. faecium on the inflammatory response, internal redox state and barrier function of the intestinal epithelium. In addition, the adhesion inhibiting effects of E. faecium on S. Typhimurium and E. coli were investigated. In order to examine the capability of the probiotic strain to modify the epithelial response to a pathogenic challenge, epithelial cells were incubated with E. faecium and either E. coli or S. Typhimurium. Our hypothesis was that E. faecium might (1) reduce the secretion of proinflammatory cytokines, (2) decrease the amount of reactive oxygen species, (3) improve epithelial integrity and (4) inhibit the adhesion of pathogenic bacteria. Figure 11. Effect of E. faecium on the paracellular permeability of IPEC-J2 cells treated with E. coli. E. faecium was added 1 h before (pre-treatment), at the same time (co-treatment) and 1 h after (posttreatment) the addition of E. coli. Detection of the FD4 dye was performed 24 after the treatment of E. coli. Control: plain cell culture medium treatment; Ec: E. coli 10 6 CFU/mL; Ef 10ˆ8 PRE: pre-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 PRE: pre-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ8 CO: co-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 CO: co-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ8 POST: post-treatment with E. faecium 10 8 CFU/mL + E. coli 10 6 CFU/mL; Ef 10ˆ7 POST: post-treatment with E. faecium 10 7 CFU/mL + E. coli 10 6 CFU/mL. Data are shown as means ± SEs of three independent experiments; *** p ≤ 0.0001 compared to treatment with E. coli.

Discussion
The present study aims to elucidate the effect of E. faecium on the inflammatory response, internal redox state and barrier function of the intestinal epithelium. In addition, the adhesion inhibiting effects of E. faecium on S. Typhimurium and E. coli were investigated. In order to examine the capability of the probiotic strain to modify the epithelial response to a pathogenic challenge, epithelial cells were incubated with E. faecium and either E. coli or S. Typhimurium. Our hypothesis was that E. faecium might (1) reduce the secretion of proinflammatory cytokines, (2) decrease the amount of reactive oxygen species, (3) improve epithelial integrity and (4) inhibit the adhesion of pathogenic bacteria.

Inflammatory Response
Intestinal epithelial cells play a major role in activating the adaptive immune response upon pathogen infection, mostly by producing various cytokines [32][33][34][35][36][37][38]. In our experiments, both IL-6 and IL-8 secretion were significantly increased when IPEC-J2 cells were challenged by E. coli or S. Typhimurium, respectively. These findings agree with previous studies that also demonstrated an increase in IL-6 or IL-8 upon pathogen challenge [20,32]. The pre-treatment with E. faecium in a concentration of 10 8 CFU/mL could abrogate the increase in both IL-6 and IL-8 secretion, while the co-incubation with E. faecium applied at a concentration of 10 8 CFU/mL could also significantly decrease the secretion of IL-8 when an inflammatory response was evoked by S. Typhimurium. Salmonella-induced IL-8 secretion was decreased by probiotic strains Lactobacillus reuteri ATCC 53608 and Bacillus licheniformis ATCC 10716, which agree with our finding, that probiotics may attenuate the proinflammatory cytokine response upon pathophysiological challenge [17]. When IPEC-J2 cells were challenged with E. coli, the pre-and co-incubation with 10 8 CFU/mL E. faecium either did not show any effect on the production of proinflammatory cytokines (IL-6) or unexpectedly, further increased their secretion (IL-8). Others, however, found that the E. coli induced IL-8 elevation was reduced by E. faecium co-incubation [20,24]. This inconsistency might be due to differences in the mode of action of various probiotic strains [17].

Response to Oxidative Stress
Here, E. coli and S. Typhimurium were used to induce oxidative stress in IPEC-J2 cells. The exact mechanism of how E. coli and Salmonella exert their oxidative stress-inducing effect is obscure, but pathogens may produce oxygen to generate an aerobic environment, thus establishing oxidative stress conditions in the intestines [8]. To confirm the antioxidant effect of the application of E. faecium as a pre-treatment, co-treatment, and post-treatment, we determined the capacity of the treatment methods for the alleviation of ROS production. E. coli and S. Typhimurium induced an intracellular ROS burst in IPEC-J2 cells. Pre-, co-, and post-treatment with E. faecium applied in either 10 8 CFU/mL or 10 7 CFU/mL remarkably reduced ROS generation induced by E. coli or S. Typhimurium, respectively. This finding indicates that E. faecium could alleviate the oxidative stress caused by E. coli and S. Typhimurium. Interestingly, certain probiotics have been shown to mitigate induced ROS production, and that pre-treatment of IPEC-J2 cells with L. plantarum ZLP001 reduced the ROS burst evoked by H 2 O 2 in IPEC-J2 cells [8], supporting the potential beneficial effect of probiotics on ROS generation.

Pathogen Adhesion
The inhibition of pathogen adhesion is one of the most important properties in how probiotics may exert their beneficial effects. The ability of different probiotic strains to inhibit pathogen adhesion has been studied extensively [39,40]. Our results confirm that probiotics can inhibit pathogen adhesion. However, in our experiments, the inhibition effect of E. faecium was independent of the time of addition. Significant adhesion inhibition was observed in the case of all three treatment conditions, similar to other recent reports [41]. Our finding that post-treatment could also inhibit the adhesion of both E. coli and S. Typhimurium indicates that E. faecium was able to disrupt established pathogen colonization.

Epithelial Barrier Function
One mode of action of probiotics is likely the strengthening of the epithelial barrier [7]. E. coli and S. Typhimurium can disrupt this barrier integrity. The enhancement of intestinal barrier function by probiotics has been intensely investigated [7]. In our experiments, the FD4 method was used to assess the changes in the integrity and permeability of the epithelial barrier. In our experiments, E. faecium alone had no significant effect on the amount of FD4 dye measured in the basolateral compartment. This result agrees with studies showing that the use of probiotics alone does not affect the integrity and permeability of the epithelial barrier [21,42,43]. However, other in vitro studies showed that the application of probiotic bacteria alone might enhance the barrier function [44][45][46]. Interestingly, E. coli or S. Typhimurium's induced pathophysiological challenge resulted in a significant increase in the amount of FD4 dye measured in the basolateral compartment, indicating that these strains were able to disrupt the integrity of the barrier, in line with previous findings [47]. Lipopolysaccharides or bacterial metabolites might be responsible for the disruption of the epithelial barrier [21]. Pathogens might also induce the apoptosis of enterocytes, which results in increased TEER values, indicating that the barrier function has been damaged. We suggest that E. faecium might be able to counteract the increased FD4 flux. Studies on Caco-2 and T84 cells have shown that probiotic bacteria could prevent the barrier disrupting effects of E. coli [42,48]. Our experiments showed that pre-treatment, co-treatment, and post-treatment with E. faecium could also prevent the damaging effects on barrier integrity induced by E. coli or S. Typhimurium, and significantly reduce the FD4 flux.
Taken together, the treatment of IPEC-J2 cells with E. faecium has multiple beneficial effects on cell integrity, paracellular permeability and intracellular ROS production, proinflammatory cytokine secretions, and the adhesion of Salmonella Typhimurium and Escherichia coli. Therefore, we suggest that E. faecium is a promising probiotic candidate for both human and animal use. The use of this strain as a probiotic also addresses the challenge of finding alternative treatments that can strengthen gastrointestinal health without the use of antibiotics. Furthermore, our in vitro model proved to be a useful tool to examine the effects of promising probiotics and other alternative substance candidates in future investigations. Funding: This research was funded by project no. TKP2020-NKA-01 and has been implemented with the support provided by the National Research, Development and Innovation Fund of Hungary, financed under the Tématerületi Kiválósági Program 2020 (2020-4.1.1-TKP2020) funding scheme. Further funding was received from the European Union and co-financed by the European Social Fund (grant agreement no. EFOP-3.6.3-VEKOP-16-2017-00005, project title: "Strengthening the scientific replacement by supporting the academic workshops and programs of students, developing a mentoring process").

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: All data that supports the above-detailed findings can be obtained from the corresponding author upon request.