Lactobacilli Antagonize the Growth, Motility, and Adherence of Brachyspira pilosicoli: a Potential Intervention against Avian Intestinal Spirochetosis

Luke J Mappley; Monika A Tchórzewska; William A Cooley; Martin J Woodward; Roberto M La Ragione

doi:10.1128/AEM.00185-11

. 2011 Aug;77(15):5402–5411. doi: 10.1128/AEM.00185-11

Lactobacilli Antagonize the Growth, Motility, and Adherence of Brachyspira pilosicoli: a Potential Intervention against Avian Intestinal Spirochetosis ^{^▿}

Luke J Mappley ^1,^*, Monika A Tchórzewska ¹, William A Cooley ², Martin J Woodward ¹, Roberto M La Ragione ^1,³

PMCID: PMC3147436 PMID: 21666022

Abstract

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

INTRODUCTION

Avian intestinal spirochetosis (AIS) is recognized worldwide as an enteric disease that affects layer and broiler breeder chickens, leading to clinical enteritis and reduced performance (52). The disease results from the colonization of the ceca and colorectum by the fastidious anaerobic spirochete Brachyspira. Clinical symptoms of AIS include reduced egg production with delayed onset of laying, chronic diarrhea with fecal staining of eggs, weight loss, and increased flock morbidity rates (7, 49). Currently, Brachyspira alvinipulli, B. intermedia, and B. pilosicoli are considered pathogenic to poultry (20, 50, 51). However, other Brachyspira species have been isolated from poultry with decreased egg production (8). Although the mechanisms of pathogenesis are unclear, colonization of poultry, swine, and humans by B. pilosicoli is characterized by its ability to form end-on attachments to the intestinal epithelial surface and to invade the surface epithelium (18, 24, 25).

In the United Kingdom, the incidence of Brachyspira in commercial and free-range flocks has been estimated at 74% and 90%, respectively (8). AIS disease associated with Brachyspira infection is reported to be increasing, which may be attributed at least partially to the 2006 European Union (EU) ban on the use of antibiotics as growth promoters in livestock (9). In 2006, the annual cost of the disease to the United Kingdom laying industry was estimated at £14 million, and this figure continues to rise (7). Additionally, antibiotic resistance appears to be increasing among Brachyspira organisms, including an emerging resistance to the most commonly used antibiotic for AIS treatment, tiamulin. Resistance has been reported for porcine Brachyspira strains (27, 34, 42), and elevated MICs have been demonstrated for strains of poultry origin (21). The rise of endemic diseases since the ban and of antimicrobial resistance has increased interest in developing alternative intervention strategies; one such alternative therapy which is being researched extensively is the use of probiotics (12).

Probiotics, which include genera such as Bifidobacterium and Lactobacillus (12), are described as live microorganisms that confer health benefits on the host when administered in adequate quantities (17). Multiple mechanisms have been proposed for the competitive exclusion (CE) of pathogenic microorganisms by probiotics, and they include secretion of antimicrobial compounds, competition for essential nutrients, competition for host cell binding receptors, and immunomodulation of the gut mucosa (54). Probiotics have demonstrated promise, in vitro and in vivo, as CE agents against Escherichia coli, Salmonella, Clostridium, and Campylobacter infection in poultry (31, 32, 46, 53, 55) and have been shown to colonize the ceca (41), the host niche of many of these pathogens, including Brachyspira.

Recently, a patent application was published (46a) describing the use of Lactobacillus johnsonii D115 as a probiotic against Brachyspira species, based on its ability to inhibit B. pilosicoli and B. hyodysenteriae through the production of hydrogen peroxide and a proteinaceous antimicrobial compound. Lactobacillus rhamnosus and Lactobacillus farciminis strains have also been implicated in inhibiting the motility of Brachyspira by coaggregation with spirochetes and by eliciting a stress response (6). However, to date, no studies have investigated the adherence and invasion dynamics of avian B. pilosicoli with epithelial cells in relation to treatment with probiotics. Here we have employed motility, growth inhibition, adhesion, and invasion assays to investigate the in vitro antagonistic effects of Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2 on B. pilosicoli B2904.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

B. pilosicoli B2904 was originally isolated in the United Kingdom from the feces of a chicken exhibiting clinical signs of AIS. B. innocens B2960 was isolated in the United Kingdom from the feces of a healthy chicken. L. reuteri LM1 and L. salivarius LM2 were isolated from conventional commercial poultry feces proven free from Brachyspira by culture and multiplex PCR (M-PCR) (1). Escherichia coli DH5α-K-12 was obtained from Gibco. Stock cultures of lactobacilli and E. coli were maintained in heart infusion broth (HIB) plus 30% (vol/vol) glycerol (Oxoid), and Brachyspira stock cultures were maintained in fetal calf serum (FCS) (Sigma-Aldrich) plus 30% (vol/vol) Brachyspira enrichment broth (BEB) (43), at −80°C.

Lactobacilli were cultured in de Mann-Rogosa-Sharpe (MRS) broth in an anaerobic jar (94% H₂ and 6% CO₂) in a GasPak Plus system (BBL) at 37°C for 16 h. L. reuteri LM1 tested positive for hydrogen peroxide production by a previously described method (36). E. coli cells were cultured aerobically in Luria-Bertani (LB) broth for 16 h at 37°C, with gentle agitation (225 rpm). Brachyspira organisms were cultured on fastidious anaerobe blood agar (FABA) or in BEB in an anaerobic cabinet (10% H₂ and 10% CO₂ in N₂) (Don Whitley Scientific) at 37°C for 3 to 5 days.

Heat-inactivated lactobacilli were prepared by heating aliquots of viable bacterial suspensions for 20 min at 80°C. Spent Lactobacillus growth medium was obtained by centrifuging Lactobacillus broth cultures (10⁹ CFU/ml) at 2,500 × g for 10 min at ambient temperature and then filtering the supernatant through a 0.2-μm filter (Sartorius Stedim) to yield the cell-free supernatant (CFS). The pH value of the CFS was adjusted using 10 M sodium hydroxide (Sigma-Aldrich).

Growth and inhibition assays.

Heat-inactivated lactobacilli (10⁶ CFU/ml) and their CFS (10% [vol/vol]), at an original (3.8) or adjusted pH value (4.5 or 7.2), were added to BEB inoculated with either B. pilosicoli B2904 or B. innocens B2960 (10⁶ CFU/ml) and incubated anaerobically at 37°C. Control broths were prepared with MRS (10% [vol/vol]) at pH 3.8, 4.5, and 7.2. Brachyspira organisms were enumerated at 24-h intervals over a 120-h period by using a Helber counting chamber (Hawksley) under dark-field microscopy (Olympus CX21; magnification, ×400). Additionally, 100 μl of each broth mixture was inoculated into wells of a microtiter plate (Iwaki) and incubated in a FLUOstar Optima instrument containing an anaerobic atmosphere at 37°C. Optical densities at 600 nm (OD₆₀₀ values) were taken every 2.77 h for 125 h. Assays were performed in triplicate on three separate occasions.

Agar motility inhibition assay.

Agar motility inhibition assays were performed using the spot test as previously described (6). Each Lactobacillus strain, either viable or heat inactivated and resuspended in 0.1 M phosphate-buffered saline (PBS) (1 × 10⁹ CFU/ml), was preincubated anaerobically with either a B. pilosicoli B2904 or B. innocens B2960 cell suspension in 0.1 M PBS (1 × 10⁹ CFU/ml) (1/1 [vol/vol]) for 4 or 24 h in a microcentrifuge tube (Eppendorf) at 37°C. Following preincubation, 5 μl of each mixed suspension was spotted in triplicate onto Brachyspira selective agar (43) and incubated anaerobically at 37°C for 8 days. The extents of motility and hemolysis were examined visually at 24-h intervals and compared to the growth of B. pilosicoli B2904 and B. innocens B2960 cell suspension controls. Following the monitoring period, growth from each assay was subcultured onto FABA to assess viability. Assays were performed in triplicate on three separate occasions.

Culture of mucus-secreting, colonic HT29-16E 3D cells.

HT29-16E cells were cultured in a three-dimensional (3D) cell model as previously described (22). HT29-16E bead stock cultures were stored in liquid nitrogen (−196°C). Cells were thawed at 37°C in a water bath, reconstituted in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% FCS, 1% nonessential amino acids (100×), 2 mM l-glutamine, and gentamicin (50 μg/ml) (Sigma-Aldrich) in a 250-ml tissue culture flask, and incubated in the presence of 5% CO₂ at 37°C until a confluent monolayer was present. Trypsinized cells were resuspended in GTSF-2 medium (33) to 2 × 10⁵ cells/ml, combined with 5 mg/ml Cytodex microcarrier beads (Cytodex 3; 133 to 215 μm) (Sigma-Aldrich) and 500 μl sterile glucose solution (1 g/liter), and then dispensed into a 50-ml rotating wall vessel (RWV) (Synthecon). The RWV was incubated in the presence of 5% CO₂ at 37°C for 21 to 23 days; to allow cell adherence to the beads, the culture medium was not changed for the first 48 h, but subsequently, 90% of the culture medium was changed every 24 h. Over 21 to 23 days, the rotation speed was gradually increased from 13.0 to 30.0 rpm to ensure that cell-bead aggregates remained in suspension.

Adhesion and invasion assays using HT29-16E 3D cells.

Human HT29-16E mucus-secreting colonic cells were selected for use in adhesion and invasion assays because this cell line is well established for studies of the interaction of enteroinvasive bacteria, such as Salmonella and E. coli, with the intestinal epithelium (28, 37) and has demonstrated the ability to differentiate (10, 26). Furthermore, preliminary studies (not described here) confirmed that B. pilosicoli B2904 adhered to and invaded this cell line.

Adhesion and invasion assays were performed essentially as described previously (14, 47). Briefly, B. pilosicoli and Lactobacillus inocula were prepared by centrifugation (2,447 × g, 10 min) of 5-day and 24-h broth cultures, respectively, after which the pellets were resuspended in tissue culture medium to yield 5 × 10⁷ CFU/ml. Following 21 to 23 days of incubation, cell aggregates were removed from the RWV, resuspended to yield 5 × 10⁵ cells/ml, and seeded into 1.5-ml microcentrifuge tubes (Eppendorf). Cells were inoculated by different experimental strategies (Table 1) with a total of 1 ml bacterial inoculum prepared as described above and then were incubated at 37°C in an anaerobic cabinet with gentle agitation.

Table 1.

Experimental strategies for HT29-16E 3D cell and avian cecal IVOC infection studies

Assay	Experimental strategy or step	Bacterial inoculum (CFU/ml) (incubation time)
Assay	Experimental strategy or step	HT29-16E 3D cells	Avian cecal IVOC^b
Protection assay^a	L. reuteri or L. salivarius preincubation	5 × 10⁷ (30 min)	10⁸ (30 min)
	B. pilosicoli inoculation	5 × 10⁷ (5 h)	10⁸ (2 h)
Competition assay	Simultaneous inoculation of B. pilosicoli with L. reuteri or L. salivarius	5 × 10⁷ (5 h)^c	10⁸ (2 h)^c
Displacement assay^a	B. pilosicoli inoculation	5 × 10⁷ (5 h)	10⁸ (2 h)
	L. reuteri or L. salivarius administered postincubation	5 × 10⁷ (30 min)	10⁸ (30 min)
CFS assay	B. pilosicoli inoculation with 10% L. reuteri or L. salivarius CFS	5 × 10⁷ (5 h)	10⁸ (2 h)
Controls
B. pilosicoli B2904	B. pilosicoli inoculation with medium in place of lactobacillus inoculation (containing 10% MRS for CFS assay control)	5 × 10⁷ (5 h)	10⁸ (2 h)
L. reuteri LM1/L. salivarius LM2	L. reuteri or L. salivarius inoculation with medium in place of B. pilosicoli inoculation	5 × 10⁷ (30 min in protection and displacement assays; 5 h in competition assay)	10⁸ (30 min in protection and displacement assays; 2 h in competition assay)
Uninfected	Complete medium only added (supplemented with 10% MRS, pH 5.8 or 3.8, for CFS assay control)	No bacterial inoculum added	No bacterial inoculum added

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Between delivery of different bacterial inocula, HT29-16E cells or IVOC tissues were washed twice with HBSS.

Both viable and heat-inactivated lactobacilli were administered under separate conditions for avian cecal IVOC assays.

The values refer to the concentrations and incubation times of both B. pilosicoli and the Lactobacillus spp.

To enumerate the B. pilosicoli organisms associated with infection, the cells were washed three times with Hanks' balanced salt solution (HBSS) (Sigma-Aldrich), and a homogenous cell suspension was achieved by gentle pipetting. To differentiate the intracellular (invaded) B. pilosicoli organisms, 100 μg/ml gentamicin solution (Sigma-Aldrich) was added to incomplete DMEM, delivered to each microcentrifuge tube, and incubated for another 2 h. Cells were subsequently washed three times with HBSS, and a homogenous cell suspension was prepared as described above. Cell suspensions were diluted serially (10⁰ to 10⁻⁷), plated onto Brachyspira selective agar, and incubated anaerobically for 3 to 5 days at 37°C. The number of adherent Brachyspira organisms was determined by subtracting the number of invaded from the number of associated Brachyspira organisms. All HT29-16E 3D cell experiments were conducted in triplicate on three separate occasions.

Avian cecal IVOC association assays.

Immediately prior to in vitro organ culture (IVOC) studies, 36 commercial 20-week-old ISA brown laying hens (confirmed free of Brachyspira by pooled fecal culture and M-PCR) were euthanized by cervical dislocation, and at postmortem examination, the ceca were sampled aseptically and stored in precooled complete RPMI 1640 medium containing 10% FCS, 0.25% lactalbumin hydrolysate, 75 mM mercaptoethanol, 0.2 μg/ml hydrocortisone (in chloroform-ethanol [1:1]), 0.1 μg/ml insulin, and 2 mM (each) l-glutamine and l-aspartate (Sigma-Aldrich) (19). Tissues were prepared as described previously (11). Briefly, tissues were washed in complete medium and trimmed aseptically to remove excess mesenteric adipose tissue. Tissue sections were immobilized in CellCrown inserts (Scaffdex) so that the mucosal side was immobilized between the insert and its base, providing a polarized IVOC system with a fixed surface area where the bacterial inoculum was limited to the mucosal side of the explant tissue. Immobilized tissues were placed into a 24-well plate (BD Biosciences) with the mucosal side facing upwards and then submerged in 500 μl complete medium.

For IVOC association assays, B. pilosicoli and both viable and heat-inactivated Lactobacillus inocula were prepared by centrifugation (2,447 × g, 10 min) of 5-day (B. pilosicoli) and 24-h (lactobacilli) broth cultures, after which the pellets were resuspended in complete medium to yield 10⁸ CFU/ml. Tissues were inoculated by a number of different experimental strategies (Table 1) with a total of 1 ml bacterial inoculum and then were incubated at 37°C in an anaerobic cabinet. Uninfected control tissues were used to confirm the absence of preexisting Brachyspira infection. Following infection, tissues were washed thoroughly using HBSS, homogenized in 0.1 M PBS, and serially diluted to facilitate enumeration. Dilutions (10⁰ to 10⁻⁷) were plated onto Brachyspira selective agar and incubated anaerobically for 3 to 5 days at 37°C to determine the numbers of associated Brachyspira cells. All IVOC experiments enumerating viable bacteria were repeated in quadruplicate on two separate occasions.

SEM and TEM.

Samples from 3D cell and IVOC studies were fixed in 3% (vol/vol) glutaraldehyde (Sigma-Aldrich) for at least 24 h prior to processing by the Electron Microscopy Unit of the Animal Health and Veterinary Laboratories Agency (AHVLA), Weybridge, United Kingdom. Scanning (SEM) and transmission (TEM) electron microscopy was carried out as previously described (30). Duplicate samples for each condition were examined blind.

Statistical analysis.

All results are presented as means and standard deviations of the means. A one-way analysis of variance (ANOVA) was performed with commercially available software (GraphPad Prism), using the Bonferroni test with a 95% confidence interval.

RESULTS

pH-dependent inhibition of B. pilosicoli growth by Lactobacillus CFS.

Lactobacillus CFS obtained from spent MRS was at pH 3.8, whereas MRS was at pH 5.8 prior to the growth of lactobacilli. The effects of 10% (vol/vol) L. reuteri LM1 and L. salivarius LM2 CFS at pH 3.8, 4.5, and 7.2 on B. pilosicoli B2904 growth were monitored using a FLUOstar Optima system to measure the OD (Fig. 1) and by bacterial cell counts using a Helber counting chamber. In comparison with MRS controls at the respective pH values, significant inhibition of growth of B. pilosicoli was observed with CFS from both L. reuteri (P < 0.05) and L. salivarius (P < 0.001) at pH 3.8 and with the CFS of L. reuteri only at pH 4.5 (P < 0.001). At pH 7.2, neither CFS had an effect on the growth of B. pilosicoli. Neither of the two heat-inactivated Lactobacillus strains had a significant impact on B. pilosicoli growth. Furthermore, the growth of B. innocens under each of the conditions was similar to that of B. pilosicoli.

Fig. 1. — Growth of *B. pilosicoli* B2904 in BEB supplemented with 10% *L. reuteri* LM1 (A) or *L. salivarius* LM2 (B) CFS (open shapes) adjusted to pH 3.8 (circles), 4.5 (squares), or 7.2 (triangles) or with heat-inactivated lactobacilli (diamonds), measured using OD₆₀₀ as a reporter of growth. Controls with pH-adjusted MRS (closed shapes) and with no additive to *B. pilosicoli* culture (x's) are also shown. *B. pilosicoli* cells were also enumerated using a Helber counting chamber: an OD₆₀₀ of 0.05 represents ∼1 × 10⁷ CFU/ml, and an OD₆₀₀ of 0.25 represents ∼3.5 × 10⁸ CFU/ml. Means ± standard errors for 9 repeats are presented. Significance, if any, is shown for differences between the final growth point readings for *B. pilosicoli* with *Lactobacillus* CFS and those for the respective control (+10% MRS). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Inhibitory effect of Lactobacillus whole cells on B. pilosicoli motility.

Agar motility inhibition assays were performed using the motile strain B. pilosicoli B2904 and viable and heat-inactivated lactobacilli; heat-inactivated lactobacilli were used to eliminate the potential effects of competition for nutrients and synthesis of antimicrobial substances. The motility of B. pilosicoli was inhibited by both viable and heat-inactivated L. reuteri and L. salivarius following 4-h and 24-h contact times, respectively. Also, viable lactobacilli inhibited hemolysis by B. pilosicoli in these tests. B. pilosicoli recovered by subculture from all assays displayed motility and hemolytic growth, indicating viability. The effects of each of the conditions on motility were similar for both B. pilosicoli and B. innocens.

Inhibitory effects of lactobacilli on B. pilosicoli adherence to and invasion of HT29-16E 3D cells.

HT29-16E 3D cells exhibited excellent cellular differentiation (brush borders, confluence across the cell surface, and tight junctions), giving multilayered cell aggregates (see Fig. 3) measuring up to 350 μm in diameter.

Fig. 3. — SEM and TEM of HT29-16E 3D cells left uninfected (A), infected with *B. pilosicoli* B2904 (B to D), and following coincubation with *L. reuteri* LM1 (E and F). Uninfected control cell aggregates exhibited a prominent brush border that was confluent across the cell surface (i) and showed superiorly differentiated tight junctions (ii). When administered, *B. pilosicoli* was observed adhering at the epithelial cell surface (iii), and when lactobacilli were coadministered, adherent lactobacilli were observed (iv), in addition to their interactions and coaggregation with *B. pilosicoli* (v). Cells treated with *B. pilosicoli* only exhibited signs of blebbing (vi), loss of microvilli (vii), disintegrated cytoplasm with vacuolation (viii), chromatin condensation and fragmentation (ix), and cell sloughing (x). Overall minimal pathology was apparent in the presence of lactobacilli, and specifically, the integrity of the brush border was maintained (xi).

To determine whether viable L. reuteri LM1 or L. salivarius cells were able to reduce the adherence to and invasion of HT29-16E 3D cells by B. pilosicoli, protection, competition, and displacement assays were performed (Table 1). The adherence and invasion of B. pilosicoli were reduced significantly by both organisms in protection and competition assays (P < 0.001) but not in displacement assays (Fig. 2A, B, and C). Specifically, L. reuteri reduced invasion by B. pilosicoli 13.6-fold in protection assays and 30.0-fold in competition assays. L. reuteri was associated with significantly greater reductions of adherence and invasion by B. pilosicoli than those induced by L. salivarius (P < 0.05). The assays described above were performed with CFS rather than lactobacilli (Table 1) to determine whether the inhibitory effects on the adherence and invasion of B. pilosicoli were a result of compounds secreted by the lactobacilli or a result of the bacteria themselves. No significant reduction of B. pilosicoli adherence was observed with Lactobacillus CFS (Fig. 2D).

Fig. 2. — Effects of probiotic treatment on *B. pilosicoli* B2904 adherence to (white bars) and invasion of (hatched bars) HT29-16E cells grown in a 3D cell culture model. *L. reuteri* LM1 and *L. salivarius* LM2 were used in protection (A), competition (B), and displacement (C) assays, and their CFS were used at 10% in CFS studies (D). *B. pilosicoli*-only controls are shown, where tissue culture medium was added in place of lactobacilli and 10% MRS (pH 5.8 and 3.8) was added as a control in CFS assays. *E. coli* K-12 was used as a negative control for invasion (black bars). The values presented are means with standard errors for 9 repeats. Significance is shown in cases where adhered or invaded *B. pilosicoli* cell numbers differed significantly between probiotic treatment and the no-probiotic control and, for the CFS studies, in cases where CFS differed significantly from the MRS (pH 3.8) control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Electron microscopic analysis of HT29-16E 3D cells following adhesion and invasion assays.

Samples from competition assays were examined by SEM and TEM (Fig. 3). Qualitatively, larger numbers of adherent B. pilosicoli cells were observed in the absence of lactobacilli, with dense populations invading at tight junctions (Fig. 3), supporting the data derived from bacteriological counts. Of particular note were direct interactions between B. pilosicoli and both L. reuteri LM1 (Fig. 3E and F) and L. salivarius LM2.

EM of cell aggregates that were infected with Brachyspira alone indicated apoptosis (blebbing, loss of microvilli, disintegrated cytoplasm with vacuolation, chromatin condensation and fragmentation, and cell sloughing) (Fig. 3C and D). With coadministration of L. reuteri LM1 (Fig. 3E and F) or L. salivarius LM2, end-on attachment of B. pilosicoli was observed less frequently, with apparent interactions between the two bacterial species and colocalization at the cell surface. Minimal pathology was apparent in the presence of lactobacilli, and the integrity of the brush border was maintained (Fig. 3E and F).

Inhibitory effect of lactobacilli on association of B. pilosicoli with avian cecal IVOC tissues.

Avian cecal IVOC association assays were performed to determine whether L. reuteri LM1 or L. salivarius LM2, either viable or heat inactivated, was able to reduce the association of B. pilosicoli with IVOC tissues. This model generated reproducible bacterial association values and was used alongside the data generated in 3D cell assays to compare and validate the findings of each method. As found in the HT29-16E cell assays, viable L. reuteri and L. salivarius significantly reduced the association of B. pilosicoli with cecal IVOC tissue in protection and competition assays (Fig. 4A and B) (P < 0.001). Heat-inactivated lactobacilli shared a similar effect (P < 0.01), with the level of reduction in association observed in protection and competition assays decreased up to 6.5-fold. A greater reduction of B. pilosicoli association resulted when viable or heat-inactivated lactobacilli were administered in competition, as previously noted in the 3D cell assays, and L. reuteri reduced the association of B. pilosicoli to a greater degree than did L. salivarius. In displacement assays, neither of the Lactobacillus strains displayed any significant ability to reduce B. pilosicoli association (Fig. 4C).

Fig. 4. — Effect of probiotic treatment on association of *B. pilosicoli* B2904 with avian cecal IVOC tissues. Viable (white bars) and heat-inactivated (hatched bars) *L. reuteri* LM1 and *L. salivarius* LM2 were used in protection (A), competition (B), and displacement (C) assays, and their CFS were used at 10% in CFS studies (D). *B. pilosicoli*-only controls (gray bars) are shown, where tissue culture medium was added in place of lactobacilli and 10% MRS (pH 5.8 and 3.8) was added as a control in CFS assays. The values presented are means with standard errors for 8 repeats. Significance is shown in cases where associated *B. pilosicoli* cell numbers differed significantly between probiotic treatment and the no-probiotic control and, for the CFS studies, where CFS differed significantly from the MRS (pH 3.8) control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CFS assays were also performed with the IVOC model (Table 1) to assess the effects of secreted compounds from the lactobacilli on B. pilosicoli association. The L. salivarius CFS exerted no effect on B. pilosicoli association; however, the association was decreased significantly in the presence of the CFS from L. reuteri (P < 0.001), although numerically, the difference was small (Fig. 4D).

Samples from competition assays were processed by SEM (Fig. 5). In a qualitative analysis, adherent B. pilosicoli cells were observed in greater abundance in the absence of L. reuteri or L. salivarius coadministration (Fig. 5A), confirming bacteriological counts. Furthermore, direct interactions between both Lactobacillus species and B. pilosicoli were apparent.

DISCUSSION

Growth inhibition studies indicated that the CFS of L. reuteri LM1 and L. salivarius LM2 suppressed the growth of B. pilosicoli B2904 in a pH-dependent manner. L. salivarius CFS elicited a greater suppressive effect on the growth of B. pilosicoli at pH 3.8 than did L. reuteri CFS, although the L. reuteri CFS also induced significant suppression of B. pilosicoli growth at pH 4.5 (Fig. 1). The suppression induced by CFS at pH 3.8 was significantly greater than that by MRS at the same pH, suggesting that the suppressive effect was not attributed solely to acidity. A possible explanation may be the strain-dependent production of a pH-dependent active compound(s), such as hydrogen peroxide, and/or other antimicrobial compounds, such as reuterin or bacteriocins (29). Lactobacillus CFS has been shown to induce a stress response in Brachyspira, often with a lethal effect attributable to lactic acid (6). Heat-inactivated lactobacilli had no effect on B. pilosicoli growth, supporting the role of secreted compounds in inhibiting growth. Since heat-inactivated lactobacilli and CFS had similar effects on both B. innocens and B. pilosicoli growth, it appears that CFS has a universal effect on both pathogenic and nonpathogenic Brachyspira growth.

Incubation of B. pilosicoli B2904 or B. innocens B2960 with both Lactobacillus strains, whether viable or heat inactivated, resulted in a loss of motility. The similarity of results for the assays with viable and heat-inactivated lactobacilli suggests that this is a passive process and is not physiological. Since the motility of B. pilosicoli and B. innocens was inhibited under similar conditions, the factor which results in this inhibition most likely does not relate to the pathogenicity of Brachyspira. Our EM observations showed coaggregation between the two bacterial species (Fig. 3C and D and Fig. 5B), supporting similar observations described previously (6). Coaggregation may prove detrimental to Brachyspira by rendering it incapable of escaping the eliminating effect of mucus, for which motility and chemotaxis are considered key virulence features (35, 38). Interestingly, the motility of B. pilosicoli and B. innocens was inhibited after 4 h of incubation with L. reuteri but only after 24 h of incubation with L. salivarius. The biochemical basis of adherence and the avidity of binding (3, 44, 45) is worthy of further investigation, as this may identify lectins for further development and exploitation.

Prior to adhesion and invasion studies, preliminary studies (not described here) confirmed the adhesive and invasive properties of B. pilosicoli B2904 with HT29-16E cells; this tropism may be attributed to the mucus-secreting properties of this cell line, since B. pilosicoli previously exhibited a chemoattraction to mucin (39). A 3D cell model which maintains a differentiated 3D architecture of the parental tissue, creating a more physiologically relevant platform, was adopted for adhesion and invasion assays. Findings from 3D cell assays encouraged the continuation of studies using IVOC cecal tissue explants from laying hens, a novel and possibly more physiologically relevant in vitro platform with which to study the potential use of probiotics to protect against AIS. In IVOC studies, tissues were maintained as physiologically active tissues, with their natural architecture and mucin layers; SEM showed that the tissues remained well preserved throughout the study (Fig. 5). However, the nature of the IVOC study did not allow separate enumeration of adherent and invaded B. pilosicoli cells, so the total association was assessed. L. reuteri LM1 and L. salivarius LM2 significantly reduced the adherence and invasion of B. pilosicoli with HT29-16E cells and with the mucosal surface of avian cecal IVOC tissues in protection and competition assays (Fig. 2A and B and 4A and B). This is most probably explained by coaggregation between the lactobacilli and B. pilosicoli (Fig. 3E and F and 5B). High levels of exopolysaccharide (EPS) production have been associated with the coaggregative properties of lactobacilli with enteric pathogens such as E. coli (3). EPSs are produced by other probiotic members of the normal gut microflora, including bifidobacteria (44), and thus may provide additional protection against Brachyspira infection. Surface proteins such as (co)aggregation-promoting factor (Cpf) have also been implicated in the coaggregative phenotype of lactobacilli with pathogens (45). Whether there was any physiological, pH-dependent impact on adherence and invasion is unclear. However, this is less likely to have occurred than passive coaggregation, because there was no evidence of pH-associated cellular tissue damage of the HT29-16E cells or the mucosal surface of avian cecal IVOC tissues.

When coadministered, viable lactobacilli were distributed evenly across the cell surface of the HT29-16E 3D cells (Fig. 3E and F) and the mucosal surface of the avian cecal IVOC tissue (Fig. 5), potentially occupying specific receptor sites and thus limiting the number of adherent Brachyspira cells by niche competition. Larger numbers of lactobacilli would have been present in competition assays than in protection assays, where the washes following the 30-min pretreatment would have removed nonadhered lactobacilli; hence, this may explain the more profound reduction of the adherence and invasion of B. pilosicoli in competition assays, as more lactobacilli were available to interact with Brachyspira. L. reuteri induced a greater reduction of B. pilosicoli adherence and invasion than did L. salivarius, regardless of delivery. This trait may be attributed to an ability of L. reuteri to compete for a wider range of receptor binding sites, produce additional antimicrobial compounds, or coaggregate more efficiently.

The inability of either Lactobacillus strain to reduce the adherence or invasion of B. pilosicoli in displacement assays (Fig. 2C and 4C) may have been due to the absence of lactobacilli to interact with B. pilosicoli during the 5-h incubation and the inability of the Lactobacillus posttreatment to reverse adherence and invasion. These results suggest that lactobacilli must be present prior to or with Brachyspira in order to interact with the spirochete and prevent association with epithelial cells. These data suggest that probiotic treatment may have little effect in birds that are already colonized with B. pilosicoli.

The inability of Lactobacillus CFS to reduce the adherence and invasion of B. pilosicoli in the 3D cell model (Fig. 2D) further supports the notion that direct interactions with lactobacilli are crucial in reducing B. pilosicoli association. In spite of this, CFS studies with the IVOC model revealed a small but statistically significant ability of L. reuteri CFS, but not L. salivarius CFS, to reduce the association of B. pilosicoli with the tissues. The effect of L. reuteri CFS on the association of B. pilosicoli may be due to the production and release of one or more metabolic by-products capable of limiting B. pilosicoli association or of a bioactive component(s) which may block adhesion molecules on B. pilosicoli or host cells. Interestingly, the CFS of Lactobacillus delbrueckii subsp. bulgaricus inhibits the cytotoxic effects of Clostridium difficile and its adhesion to Caco-2 cells, and these effects are attributable to the production of bioactive compounds that inhibit the toxin, its receptors, or bacterial adhesion molecules (4).

Further supporting the concept of passive coaggregation between the lactobacilli and Brachyspira was the ability of heat-inactivated lactobacilli to elicit a significant reduction in Brachyspira association in protection and competition assays with the IVOC model (Fig. 4A and B). However, heat-inactivated lactobacilli reduced B. pilosicoli association to a lesser degree than did viable lactobacilli; this may have been due to the increasing numbers of viable lactobacilli throughout the assays or, perhaps, to the active production and secretion of inhibitory compounds. A probable mechanism by which lactobacilli induce a significant reduction in B. pilosicoli association when delivered prior to or with Brachyspira is by passive coaggregation between the different species, which inhibits Brachyspira motility, hence trapping the spirochete and mitigating its ability to adhere to and invade host cells.

In 3D cell assays, HT29-16E cells that had been infected with B. pilosicoli showed signs of membrane blebbing (Fig. 4C and D), which is indicative of apoptosis due to physical or chemical stresses (13, 16). B. pilosicoli may induce bleb formation via a type 3 secretion system (T3SS)-dependent invasion mechanism, as has been observed with Pseudomonas aeruginosa (2), or it may adopt a mechanism similar to that of Bacteroides fragilis, which produces an enterotoxin that acts on the cytoskeleton (15). Genes encoding components of a T3SS and putative cytotoxin genes have been reported for Brachyspira (5, 56). Effacement of microvilli was observed in addition to shrunken cytoplasm, intracellular vacuolation, and cell sloughing, which are indicative of apoptosis; these findings are consistent with histopathological studies of tissues from infected birds (23, 48). Moreover, chromatin condensation was noted, which has been observed in infected avian tissues and human colorectal cell (Caco-2) monolayers (40), and genes encoding ankyrin proteins, which bind host cell chromatin, have been identified in Brachyspira (5). Further supporting the ability of lactobacilli to intervene in Brachyspira infection in vitro was the apparent protection against this cellular pathology that they conferred in competition assays (Fig. 3E and F).

The effects of lactobacilli on the growth, motility, and host cell association of B. pilosicoli encourage in vivo studies to assess the efficacy of these strains to protect against AIS. The rapid growth and robust nature of lactobacilli compared with the slow-growing, fastidious Brachyspira cells make these species ideal probiotic candidates for intervention against Brachyspira infection by niche competition. Our results indicate that acidification will inhibit B. pilosicoli; however, this may be detrimental to the host, and therefore a key effector in control may be the passive coaggregation we observed. Supplementing the diet of poultry with coaggregative lactobacilli may therefore be a useful control strategy for AIS.

ACKNOWLEDGMENTS

We acknowledge receipt of funding for this study from the British Egg Marketing Board Research and Education Trust.

We acknowledge Laura Searle for technical assistance during tissue culture studies, the Cell and Tissue Culture Unit of AHVLA (Weybridge, United Kingdom) for preparing the HT29-16E cells, the Animal Services Unit of AHVLA (Weybridge, United Kingdom) for assistance with animal husbandry, and Andrew Steventon and David Welchman (AHVLA, Winchester, United Kingdom) for providing the B. pilosicoli B2904 strain used in these studies.

Footnotes

^▿

Published ahead of print on 5 June 2011.

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[B1] 1. Abdelrahman W. H. A., AbuOun M., Stärk K. D. C., Wieland B., La Ragione R. M. 2009. Abstr. 5th Int. Conf. Colonic Spirochaetal Infect. Anim. Hum., Leon, Spain, 8 to 10 June 2009, p. 63–64 [Google Scholar]

[B2] 2. Angus A. A., et al. 2008. Pseudomonas aeruginosa induces membrane blebs in epithelial cells, which are utilized as a niche for intracellular replication and motility. Infect. Immun. 76:1992–2001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Aslim B., Onal D., Beyatli Y. 2007. Factors influencing autoaggregation and aggregation of Lactobacillus delbrueckii subsp. bulgaricus isolated from handmade yogurt. J. Food Prot. 70:223–227 [DOI] [PubMed] [Google Scholar]

[B4] 4. Banerjee P., Merkel G. J., Bhunia A. K. 2009. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells. Gut Pathog. 1:8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Bellgard M. I., et al. 2009. Genome sequence of the pathogenic intestinal spirochete Brachyspira hyodysenteriae reveals adaptations to its lifestyle in the porcine large intestine. PLoS One 4:e4641. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Bernardeau M., Gueguen M., Smith D. G., Corona-Barrera E., Vernoux J. P. 2009. In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli. Vet. Microbiol. 138:184–190 [DOI] [PubMed] [Google Scholar]

[B7] 7. Burch D. G., Harding C., Alvarez R., Valks M. 2006. Treatment of a field case of avian intestinal spirochaetosis caused by Brachyspira pilosicoli with tiamulin. Avian Pathol. 35:211–216 [DOI] [PubMed] [Google Scholar]

[B8] 8. Burch D. G. S. 2010. Egg production drops from Brachyspira. World Poult. 26:24–25 [Google Scholar]

[B9] 9. Castanon J. I. 2007. History of the use of antibiotic as growth promoters in European poultry feeds. Poult. Sci. 86:2466–2471 [DOI] [PubMed] [Google Scholar]

[B10] 10. Cohen E., Ophir I., Shaul Y. B. 1999. Induced differentiation in HT29, a human colon adenocarcinoma cell line. J. Cell Sci. 112:2657–2666 [DOI] [PubMed] [Google Scholar]

[B11] 11. Collins J. W., et al. 2010. Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella enterica serovar Typhimurium SL1344. Appl. Environ. Microbiol. 76:6645–6657 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Collins J. W., La Ragione R. M., Woodward M. J., Searle L. E. J. 2009. Application of prebiotics and probiotics in livestock, p. 1123–1192In Charalampopoulos D., Rastall R. A. (ed.), Prebiotics and probiotics science and technology. Springer, New York, NY [Google Scholar]

[B13] 13. Cunningham C. C. 1995. Actin polymerization and intracellular solvent flow in cell surface blebbing. J. Cell Biol. 129:1589–1599 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Dibb-Fuller M. P., Allen-Vercoe E., Thorns C. J., Woodward M. J. 1999. Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis. Microbiology 145:1023–1031 [DOI] [PubMed] [Google Scholar]

[B15] 15. Donelli G., Fabbri A., Fiorentini C. 1996. Bacteroides fragilis enterotoxin induces cytoskeletal changes and surface blebbing in HT-29 cells. Infect. Immun. 64:113–119 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Fackler O. T., Grosse R. 2008. Cell motility through plasma membrane blebbing. J. Cell Biol. 181:879–884 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. FAO/WHO 2001. Health and nutritional properties of probiotics of food including powder milk with live lactic acid bacteria. Report of a joint FAO/WHO expert consultation. WHO, Geneva, Switzerland [Google Scholar]

[B18] 18. Feberwee A., et al. 2008. Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both. J. Clin. Microbiol. 46:593–600 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Lactobacilli Antagonize the Growth, Motility, and Adherence of Brachyspira pilosicoli: a Potential Intervention against Avian Intestinal Spirochetosis ^{^▿}

Luke J Mappley

Monika A Tchórzewska

William A Cooley

Martin J Woodward

Roberto M La Ragione

Abstract

INTRODUCTION

MATERIALS AND METHODS