Introduction
Antibiotic growth promoters (AGPs), supplemented in animal feed at sub-therapeutic levels, have been widely used in animal production for decades. However, owing to the emergence of antimicrobial resistance in animals and humans, the European Union has banned the use of AGPs in feed since January 1, 2006 [9]. Development of alternative products and improved management are therefore necessary to deal with AGP removal from animal diets while achieving the same productivity. Probiotics are considered to be one possible alternative to AGPs in animal feeding owing to their health benefits [9,13]. Some proposed health effects of probiotics are relevant in the veterinary field, such as prevention and alleviation of diarrhea, modulation of the intestinal microbiota, and immunomodulatory function. Lactobacilli are important members of the commensal microbiota of both humans and animals, and have been demonstrated to be one of the main sources of probiotics [10]. The origin of the strain is an important factor to be considered since not all lactobacilli behave the same; even the same species of Lactobacillus originating from different hosts may vary [8,22,30]. Therefore, a microorganism strain used as a probiotic should preferably originate from the target host species, since the individual host-specific intestinal environment is conducive to the survival and colonization of the probiotic [17]. It is impossible to test large numbers of different lactobacilli in in vivo feeding trials owing to the vast species and strain diversity. For potential probiotics, proper in vitro studies should be performed to evaluate the probiotic properties before in vivo trials [1]. The commonly stated selection criteria for probiotic microbes involve several functional features, such as GI tract survival, adhesion, pathogen inhibition, and immunomodulation [24]. The use of viable microorganisms selected by in vitro methods may improve probiotic efficacy in vivo [4].
In this study, in an attempt to obtain probiotic strains of lactobacilli for commercial pig production, we isolated lactobacilli from the feces of piglets. Through a comprehensive comparison, a promising isolate, Lactobacillus reuteri LR1 strain, was selected for in vitro evaluation of its probiotic properties. The results suggest that L. reuteri LR1 has typical properties and functionality of a probiotic.
Materials and Methods
Bacterial Strains and Culture Conditions
Lactobacillus strains were isolated from the feces of a healthy 35-day-old weaned piglet (Duroc × Landrace × Large White) according to the method described by Lee et al. [20]. After overnight incubation, bacterial cells were collected by centrifugation and resuspended to the appropriate concentration in serum and antibiotic free DMEM/F-12 medium (Difco, USA) or in fresh MRS broth.
Five common pathogens of swine were used as indicator strains for antimicrobial activity assay. Enterotoxigenic Escherichia coli (ETEC, O149: K91, K88ac) was obtained from the China Institute of Veterinary Drugs Control. Salmonella enterica subsp. enterica serovar Choleraesuis (ATCC 13312) was obtained from Guangdong Microbiology Culture Center. Staphylococcus aureus was offered by the College of Animal Science, South China Agricultural University. Wild-type Streptococcus suis type 2 and Streptococcus suis type D strains were obtained from the Institute of Animal Health, Guangdong Academy of Agriculture Sciences. ETEC, Salmonella enterica, and Staphylococcus aureus were grown in Luria-Bertani (LB) broth (Guangdong Huankai, China) and the Streptococcus suis strains were cultured in BHI broth (Difco) at 37℃ for 16 h, respectively. The ETEC strain used in cell experiments was resuspended to the required concentration in DMEM/F-12 medium (serum and antibiotic free).
Bacterial cell concentrations of both ETEC and lactobacilli were determined by densitometry in preliminary experiments and confirmed by the plate counting method.
Acid and Bile Salt Tolerance Assay
Resistance to acid and bile salts was assessed as described by Lähteinen et al. [17] with minor modifications. The Lactobacillus isolates were exposed to either pH-adjusted MRS broth (pH 2.5), or to MRS broth containing 0.3% (w/v) Oxgall (Sigma-Aldrich, MD, USA) for 3 and 6 h. The number of viable bacteria was counted by plate count on MRS agar. As a control, samples were taken from the culture at 0 h and counted. The survival rate was expressed as the percentage of cells surviving low pH or bile compared with the control. All experiments were performed in triplicates.
Species-Level Identification of Isolates
Isolates were identified by 16S rRNA gene sequencing. The PCR primer pair was as follows: 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’; 1492R: 5’-GGTTACCTTTGTTACGACTT-3’. Amplified products were sequenced by BGI Tech Solutions Co., Ltd (China). The Basic Local Alignment Search Tool program was used to compare sequences against the GenBank database.
Antimicrobial Activity
Antibacterial activity was tested by the agar well diffusion assay method as described by Lee et al. [20]. Cell-free supernatant of the Lactobacillus isolates (150 μl), prepared by filtration through a 0.22 μm filter membrane, was respectively added into the Oxford cup (pore diameter 7 mm). The plates were incubated at 37℃ for 24 h, and then the diameters of the inhibition zones were measured. All experiments were performed in triplicates.
IPEC-1 Cells
Intestinal porcine epithelial cells (IPEC-1) were kindly provided by Prof. Guoyao Wu (Texas A&M University, USA). IPEC-1 cells were grown at 37℃ in a 5% CO2, 95% air humidified incubator in DMEM/F-12 supplemented with 100 mg/l streptomycin (Fluka Chemie GmbH, Switzerland), 100 mg/l penicillin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 10% fetal bovine serum (Life Technologies Ltd., USA). In different experiments, IPEC-1 cells were grown on Transwell filters, tissue culture plates, or glass coverslips as described below, and cultured for 10 days after confluency in medium without fetal calf serum to allow differentiation.
Adhesion Assay
The adherence of isolated strains to IPEC-1 cells was tested by two different methods: agar plating and microscopy examination.
Plate counting was performed according to the method described by Kaushik et al. [12] with slight modifications. One milliliter of the suspension of the Lactobacillus isolates (approx. 1 × 108 CFU/ml) was added to the differentiated IPEC-1 cells. After incubation for 2 h, bacterial cells adhering to IPEC-1 cells were counted by plating on MRS agar. The original suspension of the Lactobacillus isolates was also counted as a control. The adherence was expressed as the percentage of the bacteria cells adhering to IPEC-1 cells compared with the control.
Microscopy examination was performed as described by Kotzamanidis et al. [15] with slight modifications. The differentiated IPEC-1 cells on glass coverslips were co-incubated with a 3 ml L. reuteri LR1 suspension (1 × 108 CFU/ml) for 2 h. Then, IPEC-1 cells were washed with PBS, fixed with methanol, stained with Giemsa solution (Merck, Germany), and finally sealed with resin (Sigma-Aldrich). Samples were observed with a Zeiss AxioScope A1 microscope (Carl Zeiss, Germany) at 400× magnification. The adhesive capacity was scored as non-adhesive when fewer than five bacteria adhered to 100 cells and adhesive when six to 40 bacteria adhered to 100 cells, whereas strong adhesion occurred with more than 40 bacteria adhering to 100 cells [15].
Inhibition of Pathogen Adhesion
Competitive exclusion of pathogens was assessed according to the methods described by Zanello et al. [32] with minor modifications. Two different approaches were used in adhesion inhibition assays. In the first approach, the IPEC-1 cells were preincubated with L. reuteri LR1 (1 × 108 or 2 × 108 CFU/ml) for 2 h, followed by co-incubation with ETEC (108 CFU/ml). In the second approach, both L. reuteri LR1 (1 × 108 or 2 × 108 CFU/ml) and ETEC (1 × 108 CFU/ml) were added to IPEC-1 cells simultaneously. As a control, IPEC-1 cells were treated with ETEC (1 × 108 CFU/ml) alone. After incubation for 1.5 h, all IPEC-1 cells were washed four times to remove unbound bacteria. Adhering ETEC cells were measured by plate count on LB agar. The adhesion (%) was expressed as the percentage of the adhering ETEC population in the presence of L. reuteri LR1 compared with the control. Each assay was performed in triplicates.
Analysis of Cytokines
IPEC-1 cells were seeded in six-well plates (4 × 105 cells/well). The differentiated cells were exposed to ETEC (108 CFU/ml) in the presence or absence of L. reuteri LR1 (108 CFU/ml), or to L. reuteri LR1 (108 CFU/ml) alone. In the meantime, IPEC-1 cells were incubated in DMEM/F-12 medium (serum and antibiotic free) without bacteria as a control. After incubation for 1.5 h, the cell culture supernatants were collected for quantitative analysis of porcine cytokine IL-6 and IL-10 using a commercial ELISA kit (R&D Systems, USA). The abundance of mRNA encoding the cytokines IL-8, IL-6, TNF-α, and IL-10 was determined by quantitative real-time PCR (qPCR). The cells were lysed with Trizol Reagent (Sigma-Aldrich) and total RNA was extracted using a PrimeScript RT Reagent Kit (Takara, China). The qPCR was performed using a CFX Connect Detection System with iTaq Universal SYBR Green Supermix (Bio-Rad). Each cytokine gene was normalized to β-actin, yielding a relative transcript level. The primer pairs are described in Table 1. The PCR conditions were 95℃ for 10 min, followed by 40 amplification cycles (95℃ for 30 sec, 60℃ for 30 sec, 72℃ for 20 sec). All analyses were carried out in triplicates.
Table 1.Primers used for qRT-PCR in this study.
In Vitro Determination of Tight Junction Permeability
The transport of FD-4 (fluorescein isothiocyanate dextrans with an average molecular mass of 4,400 Da; Sigma-Aldrich) across the IPEC-1 cell monolayer was studied according to the method described by Kowapradit et al. [16] with minor modifications. IPEC-1 cells were seeded into 12-well Transwell insert chambers (0.4 μm pore size; Corning Life Sciences, USA) and allowed to differentiate for 10 days after reaching confluency. The cell monolayers were exposed to ETEC (108 CFU/ml) in the presence or absence of L. reuteri LR1 (108 CFU/ml), or to L. reuteri LR1 (108 CFU/ml) alone, in the apical compartment. A control experiment was carried out in the same way using medium without bacteria. The FD-4 solution (100 μl) was added to the apical side of the monolayers. Samples (100 μl) were collected from the basolateral side every 1 h until 6 h and replaced with an equal volume of fresh medium. The amount of FD-4 was determined using a Thermo Scientific Varioskan Flash Multimode Reader (Thermo Scientific, USA). Calibration curves were prepared with gradient FD-4 solution. Permeability was expressed as cumulative transport with time. Experiments were performed in triplicates.
Localization of Tight Junction Protein ZO-1
Localization of ZO-1 was performed according to the method described by Roselli et al. [23] with minor modifications. IPEC-1 cells were seeded on glass coverslips in 6-well plates and allowed to differentiate. The cells were exposed to ETEC (108 CFU/ml) in the presence or absence of L. reuteri LR1 (109 CFU/ml), respectively. A control experiment was carried out in the same way using medium without bacteria. After incubation for 2 h, the cells were wished with PBS, fixed with 4% paraformaldehyde, and treated with rabbit polyclonal anti-ZO-1 antibody (Cell Signaling Technology, USA), and then the cells were incubated with Alexa Fluor 647-labeled mouse anti-rabbit antibody (Cell Signaling Technology, USA). Finally, the cells were stained with fluorescent Hoechst 33258 (Life Tech., USA) followed by sealing with coverslips with anti-fluorescence quenching agent (Life Tech., USA). Samples were observed using a Carl Zeiss LSM 710 laser scanning confocal microscope.
Statistical Analysis
All data are presented as the mean ± SD. The significance of differences was evaluated by one-way ANOVA followed by Dunnett’s test. Significance was set at p < 0.05. All statistical analyses were performed with SPSS software (ver. 18.0).
Results
Screening and Identification of Swine-Derived Lactobacillus Isolates
A total of 106 putative Lactobacillus isolates were initially selected for screening as potential probiotic strains. Of these candidate strains, 90 were identified as gram-positive. All gram-positive strains were tested for viability in acid and bile salt conditions; however, only 16 isolates showed notable tolerance to low pH and bile (Table S1).
The identification of these 16 isolates was undertaken by sequencing of the whole 16S rRNA gene. By comparing the 16S sequences against the GenBank database, 15 isolates were identified as the species Lactobacillus reuteri, and the remaining isolate was identified as Lactobacillus fermentum.
The antimicrobial activity of the 16 isolates was investigated against common pathogens including ETEC, S. enterica subsp. enterica serovar Choleraesuis, Staph. aureus, Strep. suis type 2, and Strep. suis type D. The inhibition zones of the 16 isolates against the pathogens are shown in Table S2. Some strains (e.g., LR1, LR4, LR5, and LR10) inhibited the pathogenic bacteria broadly, whereas others, such as LR11 and LR7, showed weak or no inhibition of the pathogenic bacteria.
The adhesion capacity of a probiotic strain is crucial for an extended residence time in the host’s GI tract. The adhesion capacity of the 16 isolates to porcine jejunal epithelial cell line IPEC-1 was very variable (data not shown). Some strains, such as LR1, LR4, LR9, LR12, LR14, and LR15, were strongly adhesive, whereas other strains exhibited moderate or low adhesion.
By comprehensive comparisons of acid and bile tolerance, antibacterial activity, and adhesion capacity of the 16 isolates, the strain LR1, identified as L. reuteri, was selected for further study of its probiotic properties. The 16S rRNA sequence of L. reuteri LR1 strain has been deposited in the GenBank database (Accession No. KT205306).
Probiotic and Functional Properties of L. reuteri Strain LR1
Adhesion. The adhesion of L. reuteri LR1 to IPEC-1 cells was observed by light microscopy with Gram staining (Fig. 1). L. reuteri LR1 showed excellent good adherence capacity.
Fig. 1.Adherence of L. reuteri strain LR1 to IPEC-1 cells. (A) IPEC-1 cells; (B) Adherence of L. reuteri LR1 to IPEC-1 cells. The magnification is 400×.
Competitive exclusion against ETEC adhesion to IPEC-1 cells. The capability of L. reuteri LR1 to inhibit ETEC adherence to IPEC-1 cells was determined (Fig. 2). In comparison with IPEC-1 cells simultaneously co-incubated with L. reuteri LR1 and ETEC, pre-incubation of IPEC-1 cells with L. reuteri LR1 before ETEC exposure significantly inhibited ETEC adherence to the IPEC-1 cells (59.3% adherence vs. 78.9% for L. reuteri LR1:ETEC = 1:1; 28.2% adherence vs. 39.4% for L. reuteri LR1:ETEC = 2:1; Fig. 2). A higher concentration of L. reuteri LR1 more efficiently inhibited ETEC adherence to IPEC-1 cells (39.4% adherence vs. 78.9% (Fig. 2A, simultaneous incubation of IPEC-1, ETEC, and LR1); 28.2% adherence vs. 59.3% (Fig. 2B, pre-incubation of IPEC-1 with LR1, followed by incubation with ETEC)).
Fig. 2.The effect of L. reuteri LR1 on ETEC adhesion to IPEC-1. A: Cells were treated simultaneously with ETEC and different concentrations of L. reuteri LR1 for 1.5 h. B: Cells were pre-incubated with different concentrations of L. reuteri LR1 for 2 h, followed by ETEC exposure for 1.5 h.
Immunological effects of L. reuteri LR1 on IPEC-1 cells challenged with ETEC. To investigate the effects of L. reuteri LR1 on the inflammatory processes of IPEC-1 cells caused by ETEC challenge, we analyzed the gene expressions of cytokines IL-6, IL-8, TNF-α, and IL-10 (Fig. 3A). When IPEC-1 cells were incubated with ETEC alone, the mRNA expression of cytokines IL-8, TNF-α, and IL-10 increased significantly (p < 0.05) compared with untreated IPEC-1 cells (control). However, when IPEC-1 cells were incubated with ETEC simultaneously with L. reuteri LR1, the transcript levels of proinflammatory cytokines IL-6 and TNF-α were significantly down-regulated (p < 0.01) compared with the cells challenged with ETEC alone. There was no significant effect on IL-8 expression. In addition, L. reuteri LR1 significantly increased the transcript levels of the anti-inflammatory cytokine IL-10 (p < 0.01) compared with cells challenged with ETEC alone.
Fig. 3.The effect of L. reuteri LR1 on cytokine gene expression in IPEC-1 cells challenged with ETEC. (A) Effect of L. reuteri LR1 on cytokine mRNA transcripts in ETEC-challenged IPEC-1 cells. Cells were incubated with ETEC alone, or simultaneously with ETEC and L. reuteri LR1, for 1.5 h. (B) L. reuteri LR1 up-regulated the secretion of IL-10 in IPEC-1 cells challenged with ETEC. In this panel, protein levels are shown. The control was basal medium (serum free) alone.
At the protein level, ELISA results confirmed that L. reuteri LR1 up-regulated significantly the secretion of IL-10 (p < 0.01) and decreased IL-6 (p < 0.05) expression in IPEC-1 cells treated with L. reuteri LR1 simultaneously with ETEC, compared with treatment with ETEC alone (Fig. 3B).
L. reuteri LR1 protected the integrity of cellular junctions challenged with ETEC. The transport of FD-4 into the culture medium was measured to investigate the protective effect of L. reuteri LR1 on the tight junction permeability of IPEC-1 cells challenged with ETEC. Table 2 shows that the FD-4 concentration in both the control and the L. reuteri LR1-treatment-only groups was not significantly different from 1 to 6 h. When IPEC-1 cells were incubated with different concentrations of ETEC in the presence or absence of L. reuteri LR1, the permeation of FD-4 was increased in comparison with the control. However, for each ETEC concentration, co-incubation with L. reuteri LR1 and ETEC reduced the flux of FD-4 compared with ETEC-treatment alone.
Table 2.aResults are shown as the mean ± SD (n = 3). bLR1, L. reuteri strain LR1.
L. reuteri LR1 decreases ETEC-induced tight junction ZO-1 disruption. To investigate the effect of L. reuteri LR1 on ETEC-induced tight junction disruption, we performed an immunolocalization of the tight junction ZO-1 protein. Uninfected IPEC-1 cells showed a uniform distribution of ZO-1 a round the cell boundaries (Fig. 4A). When IPEC-1 cells were exposed to ETEC, the cell-cell contacts were consistently broken (Fig. 4B), resulting in tight junction disruption. However, when IPEC-1 cells were treated with ETEC simultaneously with L. reuteri LR1, a relatively small disruption of tight junctions was observed in comparison with treatment with ETEC alone (Figs. 4B and 4C).
Fig. 4.L. reuteri LR1 inhibited the destruction of ZO-1 induced by ETEC. (A) IPEC-1 cells without bacteria; (B) IPEC-1 cells infected with ETEC; (C) IPEC-1 cells simultaneously treated with ETEC and L. reuteri LR1. Loss of cell-cell contact and dissociation from cell membranes are indicated by arrows.
Discussion
Lactobacilli are a major component of the commensal microbial flora in both the small and large intestinal tract of vertebrates, and L. reuteri is one of the dominant species [22]. In this study, the 16 Lactobacillus isolates were predominantly L. reuteri, which was in accordance with previous results [17]. L. reuteri, as one of the main probiotics, has been studied extensively and shown to possess broad-spectrum probiotic efficacy in various hosts [3].
Resistance to gastric conditions and bile salts in in vitro tests is frequently suggested in the evaluation of the probiotic potential of an individual strain. Our results suggest that L. reuteri LR1 can tolerate low pH (pH 2.5) and grow on MRS broth containing 0.3% bile salts, indicating that the isolate possesses the potential to survive in the GI tract. This is in line with previous studies of L. reuteri of swine origin [19,25].
A major property of probiotics is an inhibitory effect on the growth of pathogenic bacteria. The present results indicate that L. reuteri LR1 could inhibit common intestinal pathogenic bacteria, in agreement with previously published reports on other L. reuteri strains [25]. L. reuteri can produce a broad-spectrum antimicrobial substance, reuterin [26], which has activity against pathogens, including gram-positive and gram-negative bacteria, fungi, and protozoa [29]. In addition, pathogen growth inhibition can be caused by the acidity of the culture. The inhibition of ETEC is of special interest in the swine industry because ETEC is the primary cause of diarrhea in neonatal and post-weaning piglets and results in great economic losses [6].
The ability to adhere to host intestinal mucosa is considered an important selection criterion for potential probiotics. Adherence to the intestinal mucosa is associated with stimulation of the immune system and is also crucial for colonization [11]. Porcine IPEC-1 has been widely used as an in vitro model system to study the interaction of bacteria with enterocytes [14]. The observations from the present study showed that L. reuteri LR1 generally adhered well to IPEC-1, which agreed with previously published report on other swine-derived L. reuteri strains [17]. L. reuteri LR1 could reduce ETEC adhesion to IPEC-1. Probiotic adhesion may interfere with the adherence of pathogens, exerting a barrier against pathogen colonization through competitive exclusion mechanisms [28]; mechanisms like coaggregation with the pathogen or secretion of inhibitory factors could also be involved in the inhibition [18].
Beneficial immunomodulatory properties are a highly desirable characteristic of probiotic lactobacilli. Several studies have investigated the effects of probiotics on intestinal cell lines in vitro [14,23]. Here, L. reuteri LR1 was observed to possess regulatory activity of early immune responses by preventing the increase of IL-6 and TNF-α caused by ETEC, and by mediating induction of a higher level of the anti-inflammatory cytokine IL-10. These results were consistent with a previous study [23]. We also found that L. reuteri LR1 itself exhibited a very weak capacity to induce expression of proinflammatory cytokines IL-8 and IL-6. It is possible that L. reuteri LR1 activated innate responses in IPEC-1 cells. Dibner and Richards [5] showed that probiotic bacteria could transiently trigger innate signal transduction and proinflammatory cytokine gene expression in the intestinal epithelium in the early stages of bacterial colonization. There is a correlation between in vitro and in vivo immunomodulatory properties of LAB, and the result of the in vitro cytokine-modulation profile of the studied LAB can be used to predict immunomodulatory activities in vivo and serve as a useful primary indicator for potential probiotic strains [7].
The tight junction plays a fundamental role in maintaining membrane barrier function and integrity through interaction of the ZO-1 proteins with transmembrane proteins such as claudin, occludin, and JAMs [27]. We observed that ETEC caused widespread disruption of cell-cell contact. Yu et al. [31] found that lipopolysaccharide increased the paracellular permeability and tight junction disruption of Caco-2 cells. Our results show that L. reuteri LR1 could reduce ETEC-induced membrane barrier disruption by maintaining the correct localization of ZO-1. Western blot analysis of ZO-1 also indicated that L. reuteri LR1 inhibited the destruction of ZO-1 protein between ETEC-induced IPEC-1 cells (unpublished data). In addition to inhibition of adhesion, the mechanism could include direct interaction of L. reuteri LR1 with the ZO-1 proteins. The cell-wall-component lipoteichoic acids of lactobacilli can enhance ZO-1-associated intestinal epithelial barrier integrity through activation of protein kinase C, via the TLR2 signaling pathway [2].
In conclusion, L. reuteri strain LR1 possessed common probiotic properties, including acid and bile tolerance, inhibition of pathogenic bacteria, and adhesion capacity towards porcine enterocytes. In addition, L. reuteri LR1 could inhibit ETEC adhesion to IPEC-1 cells, modulate the immune response, and strengthen epithelial barrier function. These results indicate that L. reuteri LR1 possesses desirable probiotic properties and may be a suitable candidate for probiotic products. Animal feeding trials are needed to confirm the findings of our in vitro study.
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