Adhesion of Lactobacillus reuteri strain Lr1 to equine epithelial cells and competitive exclusion of Clostridium difficile from the gastro-intestinal tract of horses

Abstract

Lactobacillus reuteri Lr1, isolated from healthy horses, remained viable after 2 h at pH 2.0 and in the presence of 1.5 % (w/v) bile. Strain Lr1 survived passage through the equine gastro-intestinal tract (GIT). However, no viable cells of L. reuteri Lr1 were detected on the third day after administration, suggesting that the strain did not colonise the GIT for longer than two days. Strain Lr1 adhered to non-viable, but not to viable, buccal epithelial cells in vitro. Adherence of strain Lr1 to buccal epithelial cells increased 25 % after treatment of the bacterial cells with pepsin. Treatment with pronase prevented the adhesion to epithelial cells. This suggested that specific proteins on the cell surface of L. reuteri Lr1 are involved in adhesion to epithelial cells. Strain Lr1 aggregated with Clostridium difficile C6, isolated from the GIT of a horse that died from severe colic. Adherence of C. difficile C6 to epithelial cells declined from 60 % to 3 % when challenged with L. reuteri Lr1 and the number of viable clostridia decreased tenfold during dosage. Red blood cell, haemoglobin and haemocrit levels were significantly (P ≤ 0.05) lower after dosage with L. reuteri Lr1. Cholesterol and glucose levels were mildly elevated for one day during dosage, but decreased significantly thereafter to levels similar than before dosage. Genes encoding adhesion to collagen, production of aggregation substances, cytolysin and β hemolysin III, resistance to vancomycin A, B and C, and gelatinase activity were not detected, suggesting that L. reuteri Lr1 is a potential probiotic that may be used to control C. difficile cell numbers in the GIT.

Introduction

Many strains of lactic acid bacteria (LAB) are well known for their probiotic properties and are commercially available (Dicks and Botes 2010). Survival at pH below 3.0, tolerance to bile salts of 0.3 to 1.5 % (w/v), and adherence to epithelial cells and mucus are considered important probiotic properties (Dicks and Botes 2010; Dicks et al. 2014). Probiotic strains have been known to stimulate the functioning of epithelial cells, increase the production of immunoglobulins, and decrease the production of inflammatory cytokines (Kopp-Hoolihan 2001; Parvez et al. 2006). Little is known about the immunological effect LAB have on horses. Botha (2011) showed that direct contact of probiotic cells with equine epithelial cells may stimulate the immune response in horses. A few studies showed that bacteriocins (ribosomally translated antimicrobial peptides) of LAB may elicit an immune response (reviewed by Dicks and Botes 2010).

Yuyama and co-workers (2004) reported alleviation of diarrhoea when horses were fed probiotic strains belonging to the species Lactobacillus equi, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus salivarius. Bernard and co-workers (2011) claimed that an equine strain of L. reuteri isolated from the stratified squamous epithelium of the stomach and mucosa of the colon inhibited the growth of Salmonella spp. Similar findings were reported by Ward et al. (2004), but with a combination of strains ascribable to Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Enterococcus faecium. Botha and co-workers (2012) reported the invasion of Lactobacillus equigenerosi Le1 into buccal epithelial cells. When the authors administered L. equigenerosi Le1 to horses at 1 × 10⁹ CFU/50 kg body weight per day, white blood cell numbers and aspartate aminotransferase levels remained unchanged. However, a slight decrease in glucose, lactate, cholesterol, and urea levels was reported during the week following administration with L. equigenerosi Le1 (Botha et al. 2012).

Clostridium difficile survives in faeces for up to four years from the first infection, is almost always associated with equine colic, and was isolated from foals with acute diarrhoea (Båverud et al. 2003). Preliminary studies (Botha 2011) have shown that L. reuteri Lr1, isolated from a healthy horse, inhibited the growth of C. difficile C6, isolated from a horse that died from acute colic.

In this study we report on the ability of L. reuteri Lr1 to compete with C. difficile for adhesion to epithelial cells, to survive in the equine gastro-intestinal tract (GIT), and to compete efficiently with Clostridium spp. in the GIT. The safety of L. reuteri Lr1 was evaluated by screening for the presence of genes encoding virulence factors such as adhesion to collagen, production of aggregation substances, cytolysin and β hemolysin III, resistance to vancomycin, and gelatinase activity. Changes in blood cell numbers, cholesterol, glucose, lactate, urea, and aspartase aminotransferase were monitored during and after dosage with L. reuteri Lr1.

Materials and methods

Horses

Four healthy crossbreed horses, between three and 16-years-old, each weighing between 450 kg and 500 kg and stabled at the Welgevallen experimental farm of the University of Stellenbosch, were included in this study. Permission to perform the research was granted by the Ethical Committee (ethical clearance number 2009B03002). The horses were stabled in separate quarters and were fed high quality lucern, hay, and a commercial energy-rich feed supplement twice a day. The horses were dewormed four weeks before the onset of the experiment.

Bacterial strains and growth conditions

Lactobacillus reuteri Lr1 was isolated from horses that received a balanced diet (Botha 2011). The strain was maintained by culturing in MRS broth (Biolab, Biolab Diagnostics, Midrand, South Africa) at 37 °C. Clostridium difficile C6 was isolated from the GIT of a horse that died from severe colic (Botha et al. 2012) and was maintained by culturing in Differential Reinforced Clostridial Medium (DRCM; Merck, Darmstadt, Germany). Incubation was at 37 °C in anaerobic flasks with gas generating envelopes (Anaerobic system BR0038B, Oxoid Ltd., Basingstoke, Hants, UK).

pH and bile salt tolerance of L. reuteri Lr1

Three sets of test tubes, each containing 10 ml MRS broth (Biolab), were prepared. The first set of medium was adjusted to pH 2.0, 2.5, 3.0, 4.0, and 5.0, using 1 M HCl. The second set was adjusted to the same pH values, but with concentrated DL-lactic acid. The pH of the media was determined after autoclaving and readjusted by adding either HCl or DL-lactic acid. The third set of the medium was supplemented with 0.5 %, 1.0 % and 1.5 % (w/v) Oxbile (Oxoid). Each of the test tubes was inoculated with 100 μl of an 18 h-old culture of L. reuteri Lr1 in MRS broth. After 2 h of incubation at 37 °C, the cultures were serially diluted in sterile saline (0.8 %, w/v, NaCl), plated onto MRS agar and incubated at 37 °C. Colonies were counted after 24 h of incubation.

Cell surface hydrophobicity of L. reuteri Lr1, auto-aggregation and co-aggregation with C. difficile C6

Hydrophobicity, auto-aggregation and co-aggregation were determined according to the methods described by Botes et al. (2008a). L. reuteri Lr1 was grown in MRS broth for 18 h at 37 °C, the cells harvested (10,000 g, 10 min, 4 °C) and washed twice with quarter-strength Ringer’s solution (1.5 g NaCl, 0.02 g KCl, 0.03 g CaCl₂ and 0.03 g NaHCO₃). The optical density (OD) of the cell suspension was recorded at 580 nm (reading 1). An equal volume of n-hexadecane was added to the cell suspension, mixed for 2 min and left at 26 °C for 30 min to separate into two phases. One ml of the top phase was carefully extracted and the OD reading recorded at 580 nm (reading 2). Hydrophobicity was expressed as a percentage value, calculated by using the equation of Doyle and Rosenberg (1995)

$$ \%\mathrm{Hydrophobicity}=\left[\left({\mathrm{OD}}_{580\mathrm{nm}}\mathrm{reading}1-{\mathrm{OD}}_{580\mathrm{nm}}\mathrm{reading}2\right)/{\mathrm{OD}}_{580\mathrm{nm}}\mathrm{reading}1\right]\times 100. $$

Auto-aggregation of L. reuteri Lr1 cells was determined by washing the cells in quarter-strength Ringer’s solution, as described before. Cells were resuspended in sterile saline and the cell density adjusted to 0.3 (at 660 nm). One ml of the cell suspension was transferred to a sterile 2 ml plastic cuvette and centrifuged for 2 min at 2,000 g, 18 °C. Cell density in the supernatant was recorded immediately after centrifugation (OD₆₆₀0) and 60 min later (OD₆₆₀60). The percentage of cells that aggregated was calculated using the equation of Malik et al. (2003):

$$ \%\mathrm{Auto}-\mathrm{aggregation}=\left[\left({\mathrm{OD}}_{660}0-{\mathrm{OD}}_{660}60\right)/{\mathrm{OD}}_{660}0\right]\times 100. $$

Co-aggregation of L. reuteri Lr 1 and C. difficile C6 was determined as follows. Strain Lr1 was cultured in MRS broth for 18 h at 37 °C and strain C6 in DRCM broth for 24 to 48 h at 37 °C under anaerobic conditions as described before. Cells of both strains were harvested (10,000 g, 10 min, 18 °C), washed with sterile saline and equal volumes of the two cell suspensions mixed. The cell density of the supernatant was recorded (OD₆₆₀Tot) to represent the initial OD. Cells were then harvested (2,000 g, 2 min, 18 °C) and the OD of the cell-free supernatant determined (OD₆₆₀S). The percentage co-aggregation was calculated using the equation of Malik et al. (2003):

$$ \%\mathrm{Co}-\mathrm{aggregation}=\left[\left({\mathrm{OD}}_{660}\mathrm{Tot}-{\mathrm{OD}}_{660}\mathrm{S}\right)/{\mathrm{OD}}_{660}\mathrm{Tot}\right]\times 100. $$

Adhesion of L. reuteri Lr1 to buccal epithelial cells

Epithelial cells collected by swabs from the inside of the cheeks of healthy horses were suspended in Minimal Essential Medium (MEM) Earle’s Base (Highveld Biological PTY LTD, Kelvin, Johannesburg, SA) and were used to seed the wells of an eight-chamber slide (Nalge Nunc International, Rochester, NY, USA). Each well received 200 μl epithelial cells (approximately 1 × 10³ epithelial cells ml⁻¹). Lactobacillus reuteri Lr1 cells were prepared by suspending 200 μl of an 18 h-old culture (OD_600nm = 1.5) into 1 ml sterile distilled water. The cells were harvested (10,000 g, 5 min, 18 °C) and resuspended in 1 ml sterile PBS. To this suspension, 1.5 ml SYTO 9 (3.34 mM stock solution) and 1.5 ml propidium iodide (20 mM stock solution; LIVE/DEAD BacLight bacterial viability kit, L34856) were added and the cells incubated for 15 min at 25 °C. The epithelium-seeded wells were each inoculated with 150 μl (1 × 10⁴ CFU ml⁻¹) of the labelled bacterial suspension and incubated for 2 h at 37 °C. At specific time points, bacterial cells that did not adhere to the epithelial cells were carefully withdrawn with a micropipette, serially diluted and plated onto MRS agar (Biolab). Colonies were counted after 24 h of incubation at 37 °C. The percentage cells that adhered to the epithelial cells were calculated as follows: % Adhesion = [(CFU ml⁻¹ ₁₂₀ – CFU ml⁻¹ ₀) / CFU ml⁻¹ ₁₂₀] × 100. CFU ml⁻¹ ₀ refers to the initial number of bacteria and CFU ml⁻¹ ₁₂₀ to the number of bacteria that adhered to the epithelial cells after 2 h. The experiment was repeated with non-viable buccal epithelial cells, i.e., cells collected from swabs that were suspended in MEM Earle’s Base and left at 25 °C for three days.

In a separate experiment, L. reuteri Lr1 was grown in 10 ml MRS broth (Biolab) for 18 h, harvested (10,000 g, 5 min, 18 °C) and washed with 0.8 ml sterile PBS. The cells were suspended in 500 μl pepsin (5.0 mg ml⁻¹, Roche Diagnostics GmbH, Mannheim, Germany), incubated at 37 °C for 1 h, harvested (14,000 g, 10 min, 4 °C), washed in two volumes 0.8 ml sterile PBS, and resuspended in 1 ml. The experiment was repeated with pronase (5.0 mg ml⁻¹, Roche). Adhesion experiments on the pepsin and pronase treated cells were performed as described before.

Images of the bacteria were recorded using an Olympus Cell^R system, attached to an IX-81 inverted fluorescence microscope equipped with a F-view-II cooled CCD camera (Soft Imaging Systems), as described by Botha et al. (2012). Adherence of the bacteria to epithelial cells was expressed as a percentage value, calculated by using the equation % Adhesion = [(CFU ml_T ⁻¹) / (CFU ml₀ ⁻¹)] x 100 (Botha et al. 2012). CFU/ml₀ refers to the initial number of bacteria and CFU/ml_T to the number of bacteria that adhered to the epithelial cells after exposure for a specific time.

Competition between L. reuteri Lr1 and C. difficile C6 for adhesion to epithelial cells

To determine if L. reuteri Lr1 has the ability to compete with C. difficile C6 for adherence to epithelial cells, the adhesion experiment was repeated. In this case, wells seeded with buccal epithelial cells were inoculated with 100 μl (1 × 10⁶ CFU ml⁻¹) viable cells of L. reuteri Lr1 plus 100 μl (1 × 10⁶ CFU ml⁻¹) viable cells of C. difficile C6. After 2 h of incubation at 37 °C, bacteria that did not adhere to epithelial cells were carefully withdrawn, plated onto MRS agar and DRCM agar and incubated at 37 °C for 24 h. Adhesion to epithelial cells was calculated from the difference in the cell numbers recorded before and after exposure to epithelial cells, and it was expressed as a percentage.

Screening for virulence genes in L. reuteri Lr1

Strain Lr1 was cultured in MRS broth for 18 h. Genomic DNA was isolated using the ZR Fungal/Bacterial DNA kit (Zymo Research, CA, USA). Plasmid DNA was isolated using the Qiagen plasmid midi kit (Qiagen, Inc., Valencia, USA). Genomic and plasmid DNA were amplified with primers detecting genes encoding adhesion to collagen, production of aggregation substances, cytolysin and β hemolysin III, and resistance to vancomycin A, B, and C. The primers used and PCR conditions were as described by Botes et al. (2008b). Gelatinase activity was tested by streaking active growing cells onto MRS agar, supplemented with 3 % (w/v) gelatin (BDH Laboratory Supplies, Poole, England). After 24 h of incubation at 37 °C, the plates were stored at 4 °C for 5 h and examined for opaque zones surrounding the colonies.

Administration of L. reuteri Lr1

Lactobacillus reuteri Lr1 was grown in MRS broth, as described before, the cells were harvested (10,000 g, 5 min, 18 °C) and then resuspended in sterile 10 % (w/v) molasses to obtain a final cell concentration of 1 × 10⁹ CFU per 20 ml. Each horse received 20 ml of the cell suspension, daily for seven days. The dosage of approximately 1 × 10⁸ CFU per 50 kg body weight represents the original cell numbers of L. reuteri Lr1 that were isolated from a healthy 450 kg horse (Botha 2011).

Isolation of L. reuteri Lr1 and Clostridium spp from faeces

Faecal samples were collected from each horse seven days before administration with L. reuteri Lr1, and on each successive day for the following 17 days of the experiment. Serial dilutions of the samples were prepared in sterile saline and plated onto MRS agar, DRCM agar, and Clostridium difficile agar base CM0601 supplemented with the selective reagent SR0096 (Oxoid). MRS agar plates were incubated as described before. DRCM and CM0601 plates were incubated at 37 °C for six days in anaerobic flasks with gas-generating envelopes (Oxoid). All colonies that developed on DRCM plates were counted. On CM0601 plates, grey-white colonies, resembling the morphology of C. difficile, were counted. Colonies randomly selected from DRCM and CM0601 plates were Gram stained and tested for catalase activity by dropping 5 % (vol/vol) H₂O₂ onto the colonies. Colonies on MRS plates were overlaid with 1 × 10⁶ CFU of 24 h-old cells of C. difficile C6, suspended in soft (0.8 %, w/v, agar) DRCM medium, and incubated for a further 24 h in anaerobic flasks with gas-generating envelopes. Colonies surrounded by a halo, indicating that growth of C. difficile C6 was repressed (Botha 2011), were regarded as L. reuteri Lr1 and counted. The identity of the organism was confirmed by isolating the DNA from a few randomly selected colonies and performing 16S rRNA sequencing. DNA was isolated using the ZR Fungal/Bacterial DNA kit (Zymo Research, CA, USA) and amplified with primers 8 F (CACGGATCCAGACTTTGATYMTGGCTCAG) and 1512R (GTGAAGCTTACGGYTAGCTTGTTACGACTT), according to Felske et al. (1997). Reaction mixtures were as described by Endo and Okada (2005). PCR amplification was as described by Walter et al. (2001). Sequencing of the 16S rRNA gene was done using an automatic sequencer (ABI Genetic Analyzer 3130X1, Applied Biosystems, SA) and BigDye Terminator chemistry (Biosystems, Warrington, England). Sequences were subjected to Blast analysis on the GenBank.

Blood analysis

Blood was drawn from the jugular vein of each of the horses before dosage with L. reuteri Lr1, daily during the seven days of administration and thereafter for ten consecutive days. One blood sample was kept in EDTA, one sample was kept without an anticoagulant (SST tubes), and another sample stored in the presence of sodium fluoride. The SST tubes were centrifuged and the serum stored at −80 °C. Samples with EDTA were used to determine full blood counts and white blood cell (WBC) counts, using the Celldyne 3700CS Haematology Analyser with Veterinary Software Package. Sodium fluoride tubes were used for glucose and lactate analysis. Stored serum was used to determine aspartate aminotransferase (AST), glucose, cholesterol, lactate, and urea levels. The tests were conducted by Pathcare Pathologists, Stellenbosch, South Africa.

Statistical analysis

Statistical analyses of the results obtained from the blood samples were done using two-way analysis of variance (ANOVA). Variability between groups was noted when P ≤ 0.05 and the F-value was more than the F-crit value.

Results

Resistance of L. reuteri Lr1 to pH and bile

Cell numbers of L. reuteri Lr1 declined from 2 × 10⁸ CFU ml⁻¹ in MRS broth (control tube) to 8 × 10⁶ CFU ml⁻¹, after 2 h of exposure to pH 2.0 with HCL. No viable cells were recorded after 2 h in MRS broth adjusted to pH 2.0 with DL-lactic acid. Cell numbers recorded after 2 h of exposure in MRS broth, adjusted to pH 2.5, 3.0, 4.0, and 5.0 with HCl, ranged between 1 × 10⁶ and 1 × 10⁷ CFU ml⁻¹. Lower cell numbers (1 × 10³ to 1 × 10⁷ CFU ml⁻¹) were recorded after 2 h of exposure in MRS broth that was adjusted with DL-lactic acid ranging from pH 2.5 to 5.0.

Cell numbers of L. reuteri Lr1 decreased from 2 × 10⁸ CFU ml⁻¹ in MRS broth pH6.4 (control tube) to 1 × 10⁶ CFU ml⁻¹ after 2 h in the same medium with added 0.5 %, 1.0 %, or 1.5 % (w/v) bile. The same decline in cell numbers was recorded after 2 h of exposure to 1.0 % and 1.5 % (w/v) bile.

Hydrophobicity, auto-aggregation and co-aggregation

Lactobacillus reuteri Lr1 revealed no hydrophobic properties (no hydrophobicity), but the cells did aggregate with each other (22 % auto-aggregation). A higher percentage of the cells (47 %) aggregated with C. difficile C6.

Adhesion to buccal epithelial cells

Lactobacillus reuteri Lr1, harvested from MRS broth (Biolab) and suspended in PBS, did not adhere to viable buccal epithelial cells (Fig. 1). However, approximately 10 % of the cells adhered to non-viable buccal epithelial cells after 20 min (image B, Fig. 2). No further increase in adherence was recorded during the following 100 min. Treatment of L. reuteri Lr1 with pepsin did not increase adherence to non-viable epithelial cells (not shown). However, 25 % bacteria adhered to viable cells pretreated with pepsin after 20 min of exposure (image B and E, Fig. 3). Treatment of L. reuteri Lr1 with pronase did not facilitate adhesion to non-viable (not shown) or viable (Fig. 4) epithelial cells.

Fig. 1

Fluorescent microscopy images showing the interaction between L. reuteri Lr1 and viable buccal epithelial cells. a: Z-stack view at time zero, immediately after contact. b: Z-stack view recorded 20 min later. c: Fence view recorded after 20 min of contact

Fig. 2

Fluorescent microscopy images showing the interaction between L. reuteri Lr1 and non-viable buccal epithelial cells. a: Z-stack view at time zero, immediately after contact. b: Z-stack view recorded 20 min later. c: Fence view recorded after 20 min of contact

Fig. 3

Fluorescent microscopy images showing the interaction between L. reuteri Lr1 and viable buccal epithelial cells after strain Lr1 was treated with pepsin. a–c: Z-stack view at time zero and after 20 min, 60 min and 120 min, respectively. e–g: Fence view recorded after 20 min, 60 min and 120 min, respectively

Fig. 4

Fluorescent microscopy images showing the interaction between L. reuteri Lr1 and viable buccal epithelial cells after strain Lr1 was treated with pronase. a–d: Z-stack view at time zero and after 20 min, 60 min and 120 min, respectively. e–g: Fence view recorded after 20 min, 60 min and 120 min, respectively

Competition between L. reuteri Lr1 and C. difficile C6 for adherence to buccal epithelial cells

Clostridium difficile C6 adhered strongly (60 %) to buccal epithelial cells (Fig. 5). However, when combined with L. reuteri Lr1 in the same cell suspension, strain Lr1 competed with C. difficile C6 for adhesion to epithelial cells. After 20 min of exposure, only 3 % of C. difficile C6 cells adhered to the epithelial cells (images B and E, Fig. 5). The increase in adherence of L. reuteri Lr1 to the epithelial cells was difficult to determine as some bacterial cells stained less than others, but was estimated to be between 30 % and 40 % within the first 20 min (Fig. 5). Based on cell counts, the number of L. reuteri Lr1 and C. difficile C6 cells that adhered to epithelial cells did not change with further incubation.

Fig. 5

Fluorescent microscopy images showing the competition between L. reuteri Lr1 and C. difficile C6 for adhesion to viable buccal epithelial cells. a–d: Z-stack view at time zero and after 20 min, 60 min and 120 min, respectively. e–g: Fence view recorded after 20 min, 60 min and 120 min, respectively

Virulence of L. reuteri Lr1

Genes encoding adhesion to collagen, aggregation substance (AS), cytolysin, non-cytolysin (β hemolysin III), and vancomycin A, B, C1, C2, and C3 were not amplified. No gelatinase activity was recorded.

Isolation of L. reuteri Lr1 and Clostridium spp. from faeces

No viable cells of L. reuteri Lr1 were detected in faeces during the seven days before dosage started. Cell numbers of L. reuteri Lr1 in faeces increased from 1 × 10⁴ CFU g⁻¹ faeces after the first day of dosage to 1 × 10⁶ CFU g⁻¹ faeces after seven days of administration, but declined to 1 × 10² CFU g⁻¹ at day 9 (two days after the last dosage). No viable cells of L. reuteri Lr1 were detected thereafter. Colonies surrounded with growth inhibition zones were identified as L. reuteri by matching sequences in GenBank (97 % sequence homology). Cell numbers of clostridia declined from 1 × 10² CFU to 10 CFU g⁻¹ faeces during dosage with L. reuteri Lr1. Two days after the last dosage, clostridia cell numbers increased to 1 × 10² CFU g⁻¹ faeces.

Blood analyses

The blood biochemical profiles (average of four horses) are listed in Table 1. No statistically significant variations (P > 0.05) in red and white blood cell numbers, neutrophils, lymphocytes, monocytes, eosinophils, basophils, platelet counts, and aspartate aminotransferase levels were recorded before, during, and after administration with L. reuteri Lr1. No statistically significant changes in red blood, haemoglobin, and haemocrit numbers were recorded before and during dosage. However, the red blood cell, haemoglobin, and haemocrit levels were significantly (P ≤ 0.05) lower after dosage with L. reuteri Lr1 compared to before dosage. Hemoglobin levels remained constant during dosage with L. reuteri Lr1 but decreased significantly after dosage. Cholesterol and glucose levels were mildly elevated for one day during dosage (not shown), but decreased significantly thereafter to levels similar to that recorded before dosage. Lactate levels were slightly higher for one day during dosage (not shown), but decreased thereafter to levels similar to that recorded before dosage, or slightly lower. Urea levels decreased significantly during dosage with L. reuteri Lr1.

Table 1 Statistical analysis of blood biochemical profiles before, during, and after L. reuteri Lr1 administration

Discussion

In the anaerobic fundic section of the equine stomach, carbohydrates are fermented to lactic acid, mostly by Lactobacillus and Streptococcus spp. (de Fombelle et al. 2003), and the pH decreases to approximately 2.6 (Frape 2010). L. reuteri has been isolated from epithelial cells of the oesophageal section of the stomach (Yuki et al. 2000). Lactobacillus reuteri Lr1 survived 2 h in the presence of HCl, suggesting that cells may enter the duodenum in a viable state.

Bile secreted in the first section of the duodenum suppresses the growth of many microorganisms (Colville and Bassert 2008). However, de Fombelle and co-workers (2003) reported high bacterial cell numbers (10⁹ CFU ml⁻¹ digesta) in the duodenum of horses. Lactobacillus reuteri Lr1 survived bile concentrations as high as 1.5 % (w/v, in vitro). It is, thus, safe to assume that a large percentage of cells in the duodenum would remain viable and enter the lower GIT. The increase in cell numbers of L. reuteri Lr1, from 1 × 10⁴ CFU g⁻¹ faeces on day 1 to 1 × 10⁶ CFU g⁻¹ faeces on day 7, indicates that the strain survived passing through the GIT.

One of the key properties of a probiotic is the ability to compete with pathogens for binding to epithelial cells or mucus (Schillinger et al. 2005). Adherence is facilitated by the presence of surface located aggregation substances, such as carbohydrates, cell-surface proteins, haemagglutins, lipoteichoic acids, and S-layer proteins (reviewed by Dicks and Botes 2010). The level at which cells aggregate (co-aggregation) is usually an indication of adhesion properties. Almost half of the cells (47 %) of Lactobacillus reuteri Lr1 aggregated with C. difficile C6 and competed for binding to buccal epithelial cells, but only to non-viable cells (Fig. 2). Adherence to viable epithelial cells was only recorded after the bacterial cells were treated with pepsin (Fig. 3). Pepsin cleaves peptide bonds between hydrophobic and aromatic amino acids such as phenylalanine, tryptophan, and tyrosine (Dunn 2001) and would, thus, denature specific proteins on the surface of L. reuteri Lr1. By doing this, other receptors on the bacterial cell surface, including proteins not denatured by pepsin, may be exposed. Adhesion to non-viable epithelial cells may be facilitated by structures different from that required for adhesion to viable epithelial cells.

Treatment of L. reuteri Lr1 with pronase did not facilitate adhesion to dead or viable epithelial cells (Fig. 4). Pronase is a combination of serine-type proteases, Zn²⁺ endopeptidases, Zn²⁺-leucine aminopeptidases, and Zn²⁺ carboxypeptidase and denatures many different proteins (Sweeney and Walker 1993). This supports our hypothesis that cell surface proteins are involved in the adhesion of L. reuteri Lr1 to epithelial cells. The mucus binding protein (Mub) and mucus adhesion promoting protein (MapA) of Lactobacillus reuteri have been described in detail (Satoh et al. 2000; Roos and Jonsson 2002). Wang and coworkers (2008) identified a protein on the surface of a strain of L. reuteri that facilitated adhesion to porcine mucin and human enterocyte-like HT-29 cells.

The aggregation of L. reuteri Lr1 to C. difficile C6 suggested that the bacteria may compete for binding to epithelial cells. Sixty percent of C. difficile C6 cells adhered to epithelial cells in vitro (Fig. 5). In the presence of L. reuteri Lr1, only 3 % of C. difficile C6 cells adhered to the epithelial cells, suggesting that C. difficile C6 was outcompeted by L. reuteri Lr1. This may be the case in vivo, but will have to be confirmed with further experiments.

The decline in cell numbers of clostridia in the faeces (from 1 × 10² CFU g⁻¹ to 10 CFU g⁻¹) during administration with L. reuteri Lr1 indicates that the strain may be used to control Clostridium infections in the GIT. The decline in Clostridium cell numbers was, however, only observed during administration with L. reuteri Lr1, suggesting that the probiotic will have to be administered on a daily basis.

In humans and animals probiotics play a role in the stimulation of specific-immune and non-specific immune responses (Parvez et al. 2006). Probiotics may increase cytokine levels and natural killer cell activity, activate macrophages, change systemic T cell balances, and increase immunoglobulin levels (Kopp-Hoolihan 2001; Parvez et al. 2006). In addition, probiotic bacteria promote the proper functioning of the epithelium barrier (Kopp-Hoolihan 2001). Neutrophil and white blood cell numbers remained stable during and after dosage with L. reuteri Lr1, which suggests that the innate immunity of the horses were not altered and that L. reuteri Lr1 did not cause inflammation. Eosinophil levels were also consistent, suggesting that strain Lr1 did not elicit an allergy response. The lower red blood cell numbers, and lower hemoglobin and hemocrit levels recorded during and after dosage with L. reuteri Lr1 is most probably due to longer stabling hours and less exercise. Horses that are routinely exercised have higher red blood cell numbers (Kopp-Hoolihan 2001).

The general decrease recorded in blood glucose levels was expected. Blood glucose levels in exercised horses are normally higher than in resting horses (Tateo et al. 2008). However, blood glucose levels have to be carefully monitored. High glucose levels may cause colic (Frape 2010). It would be interesting to monitor changes in the glucose levels of racing horses that receive L. reuteri Lr1.

The decrease in cholesterol in horses that received L. reuteri Lr1 correlated with results obtained by Strompová et al. (2006). However, unlike our findings, the authors reported an increase in cholesterol levels for the first six days during treatment. Probiotics administered to calves decreased cholesterol levels for as long as three weeks during administration (Frizzo et al. 2010).

The decrease in lactate levels is characteristic of resting horses (Zobba et al. 2011). High blood lactate levels may lead to acidosis, a phenomenon associated with irritated or damaged mucosa (Al Jassim et al. 2005). Acidosis often leads to laminitis, which is symptomatic of toxins secreted from the GIT into the blood stream (Biddle et al. 2013) or colic (Pagan 1998), but this is usually associated with blood glucose concentrations in excess of 200 mg dl⁻¹ (Thrall et al. 2004).

Aspartate aminotransferase levels remained stable and corresponded with results reported by Strompová et al. (2006). Aspartate aminotransferase levels are usually high in horses with acute colic (Patton et al. 2009) or when stabled for too long (Tateo et al. 2008).

Urea levels decreased gradually during and after dosage with L. reuteri Lr1. Urea is secreted into the ileum and transported to the caecum where it is hydrolysed by bacteria to ammonia (Dicks et al. 2014). Excessive levels of ammonia can lead to ammonia toxicity (Frape 2010).

None of the virulence genes screened for were detected in L. reuteri Lr1, suggesting the strain is a safe probiotic. Yuyama and co-workers (2004) administered L. equi, L. crispatus, L. johnsonii, L. reuteri and L. salivarius to horses. The authors reported better growth and increased resistance against diarrhoea.

The inability of L. reuteri Lr1 to adhere to viable epithelial cells and variation in adherence recorded after treatment with proteolytic enzymes suggest that the strain does not form a strong association with the GIT. This hypothesis is supported by an increase in viable cell numbers of L. reuteri Lr1 in faeces during administration and an absence of viable cells three days after the last dosage. Strain Lr1 did, however, prevent the adherence of C. difficile to epithelial cells (in vitro) and decreased the number of viable clostridia isolated from faeces tenfold when administered to horses. L. reuteri Lr1 may, thus, be used to control C. difficile cell numbers in the GIT. The demand for equine probiotics is on the increase, especially with the white paper published by the Equine Research Coordination Group in May 2013 (http://www.aaep.org/images/files/AntibioticResistancePaper(Final).doc) that warns against the abuse of antibiotics and the developing of antibiotic-resistant bacteria.