INTRODUCTION

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Lactobacilli comprise a large percentage of the indigenous flora of the gastrointestinal tract. It has been well documented that lactobacilli exert various beneficial effects on the host, which has led to their classification as a probiotic organism. Although specific health-related claims are generally not made, probiotic bacteria have been shown to possess the ability to inhibit various intestinal pathogens (5, 7), provide a barrier effect, and also modulate the immune function of the host (6, 10, 14), as well as to have a variety of other effects.

Luminal antigens gain access to the mucosal lymphoid tissues via the Peyer's patches in the small intestine. The delivery of vaccines directly to this site could enhance the probability that the host will encounter the immunizing antigen. Although the currently used vaccines are effective, they make use of attenuated pathogenic bacteria such as mycobacteria, salmonellae, and clostridia, many of which have been shown to associate with the Peyer's patches. Lactic acid bacteria are organisms that are generally regarded as safe and are being evaluated for use as a live-vector antigen delivery system (12).

It has been shown that lactobacilli associate with the gastrointestinal tract in a number of ways. Both specific proteinaceous (4, 13) and carbohydrate (8) structures are involved in the adhesion of lactobacilli to various regions within the gastrointestinal tract. The interaction of gram-negative pathogenic bacteria with M cells has been extensively studied (3, 9, 11), and more recently, the interaction of gram-positive bacteria with the surface of M cells has been examined (2). The study described here aims to determine whether lactobacilli associate with the Peyer's patches, in preference to nonlymphoid intestinal tissue, and to examine this adhesion both in vitro and in vivo.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The Lactobacillus strains used in the study (Table 1) were obtained from the Cooperative Research Centre (CRC) for Food Industry Innovation Culture Collection at the University of New South Wales and were maintained as glycerol stocks stored at 70°C. Primary cultures for each experiment were grown from the glycerol stocks by inoculation (1%) into Mann Rogosa Sharpe (MRS) broth (Difco) and incubation for 18 h anaerobically (Don Whitley Scientific Mark 3 Anaerobic Chamber). The bacterial cultures were centrifuged at 3,000 × g for 10 min, and the pellets were washed twice in 0.1 M phosphate-buffered saline (PBS; pH 7.2). The pellets were adjusted to give an optical density at ₆₀₀ of 0.5 for use in the in vitro adhesion assay. Bacteria used in the in vivo adhesion assay were concentrated by resuspension in a smaller volume of PBS to yield an optical density of 1.2, which corresponded to approximately 10⁹ CFU ml¹, as determined by serial dilution and plating of the suspension on MRS agar. For the radiolabeling of the bacteria used in the in vitro adhesion assay, the medium was supplemented with [methy-l,1',2'-³H]thymidine (124 Ci mmol¹) to give a final concentration of 10 µCi ml¹.

Preparation of tissue pieces. Peyer's patch and nonlymphoid intestinal tissue samples were taken from 6-week-old specific-pathogen-free female BALB/c mice which had been obtained from CULAS, Little Bay, Australia. The tissue pieces were washed so that they were visibly clear of debris and were placed into wells of a 24-well tissue culture plate (Nunc) with the villi facing upwards and with six pieces per well. The tissue pieces obtained were roughly 2 mm². The tissue pieces were kept on ice for no more than 2 h prior to use.

In vitro adhesion. The in vitro adhesion assay was conducted as described by Henriksson and Conway (8). Briefly, the radioactively labeled Lactobacillus cells were incubated with the tissue pieces (n = 6 per Lactobacillus suspension) at 37°C for 20 min with constant gentle agitation with an orbital shaker. After the incubation, the tissue pieces were washed three times in 1 ml of PBS with gentle agitation at room temperature for 5 min per wash. The tissue pieces were weighed and then digested with perchloric acid (150 µl) and hydrogen peroxide (300 µl) at 70°C for 12 h in glass scintillation vials. Scintillation fluid (9 ml) was added to each vial, and the radioactivity of the samples was measured after 10 min with a Packard Tricarb 2100TR liquid scintillation counter. Statistical calculations were carried out by the Student t test.

Scanning electron microscopy. The adhesion assay described above was also conducted with nonradiolabeled lactobacilli. Tissue pieces were prepared for examination by scanning electron microscopy instead of being weighed and digested. Briefly, following the adhesion assay tissue pieces were fixed in glutaraldehyde (3% in PBS) and were dehydrated with a graded ethanol series and 100% dry acetone. The tissue pieces were dried with the E3100 Jumbo Series II Critical Point Drier apparatus (Polaron, Watford, United Kingdom). The tissue pieces were gold coated with a Polaron sputter coater, according to the manufacturer's instructions. The mounted sections were examined with a scanning electron microscope (S360; Cambridge Instrument Co., Cambridge, United Kingdom). Fifty randomly selected fields from more than six tissue pieces from at least six individual mice were examined at ×3,000 magnification.

In vivo adhesion. Specific-pathogen-free female BALB/c mice, as described above, were orally dosed with approximately 10⁹ lactobacilli by orogastric intubation. Each group contained six mice. At 2 h postdosing, the mice were killed by CO₂ asphyxiation and the Peyer's patches and control nonlymphoid intestinal tissue were examined by enumeration of the associated lactobacilli. This was performed by homogenizing the tissue with an Ultra Turrax homogenizer (Janke and Kunkel) and serially diluting and plating aliquots on Rogosa agar (Oxoid). The numbers of lactobacilli were enumerated by counting according to known colony morphologies. The results were analyzed by the Mann-Whitney rank sum test. Isolates were confirmed by protein profiling (n = 5), carbohydrate fermentation with an API 30 (BioMérieux) (n = 4), and immunodetection (n = 4) with Lactobacillus fermentum KLD-specific antiserum to positively identify L. fermentum isolates from indigenous organisms.

	RESULTS

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Examination of the in vitro association of Lactobacillus spp. with the FAE of the Peyer's patches by scanning electron microscopy. Initial screening of the strains of Lactobacillus which associate with the follicle-associated epithelium (FAE) of the Peyer's patches was performed by adhesion assays. Low levels of association were seen for most of the 16 different strains examined, with negligible Lactobacillus cells detected in most fields when the samples were examined by scanning electron microscopy (Table 2). It can been seen that L. fermentum KLD associated with the FAE in large numbers, while, in contrast, small numbers were observed on the nonlymphoid villous intestinal tissue pieces (Fig. 1). Of the five other strains of Lactobacillus seen to associate with the Peyer's patch tissue, a strong association with the nonlymphoid villous intestinal tissue was also evident. No correlation between the source of the lactobacilli and the pattern of Peyer's patch assocation was evident, with some strains isolated from all sources having affinity for the FAE. L. fermentum KLD and Lactobacillus delbruckii subsp. bulgaricus were selected from the initial screen for further quantification by scanning electron microscopy and using radiolabeled bacterial cells in the adhesion assay. L. delbruckii subsp. bulgaricus was chosen as a negative control strain because it associated with neither the Peyer's patches nor the nonlymphoid villous intestinal tissue.

View larger version (73K): [in this window] [in a new window]   FIG. 1.   Scanning electron micrograph of mouse tissue after incubation in L. fermentum KLD cell suspension. (a) Typical aggregates of L. fermentum KLD-like cells on the FAE of the Peyer's patch; (b) absence of cells on nonlymphoid villous intestine. Bars, 10 µm.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Recovery of viable Lactobacillus cells following a 20-min exposure of L. fermentum KLD and L. delbruckii subsp. bulgaricus with Peyer's patch and nonlymphoid intestinal tissue. More than 10 tissue pieces from at least six animals were examined. Results are expressed as the the numbers of CFU per milligram (wet weight) of tissue.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Association of bacterial cells with Peyer's patches (a) and nonlymphoid intestinal tissue (b) following a 20-min exposure to L. fermentum KLD and L. delbruckii subsp. bulgaricus and analysis by scanning electron microscopy. Results of the extent of bacterial coverage of the tissue surface when viewed by scanning electron microscopy at ×3,000 magnification are represented as a percentage. Fifty fields with tissue from six animals were examined per treatment.

Examination of the in vivo association of Lactobacillus spp. with the Peyer's patches. In order to determine whether the association of L. fermentum KLD with the Peyer's patches of mice in vitro was demonstrable in vivo, viable lactobacilli were isolated from small intestinal tissue following oral dosing (Fig. 4). L. fermentum KLD was recovered from both the Peyer's patches and the nonlymphoid villous intestinal regions in numbers significantly greater than those of L. delbruckii subsp. bulgaricus (P < 0.05), according to the Mann-Whitney rank sum test. As with the in vitro result, there were significant differences between the association of L. fermentum KLD with the Peyer's patches and the association of L. fermentum KLD with the villous intestine (P < 0.05). The association of L. delbruckii subsp. bulgaricus with both tissue types showed no significant difference, as there were generally no associated viable cells recovered from the tissue homogenates.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Association of L. fermentum and L. delbruckii subsp. bulgaricus with the Peyer's patches and nonlymphoid villous intestine 2 h following administration of an oral dose of 109 viable Lactobacillus cells. Results are expressed as numbers of CFU per milligram (wet weight) of tissue (n = 35 per treatment) as the mean value. Error bars indicate standard deviations.

	DISCUSSION

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Most of the current knowledge of the adhesion to Peyer's patches is based on the results of studies with gram-negative organisms and pathogens (3, 9, 11), and consequently, this interaction is comparatively well understood. The study described here sought to identify a strain of Lactobacillus that associated directly with the surfaces of the Peyer's patches. The importance of this phenomenon is reflected in the need for improved vectors for foreign antigens in vaccines against enteric diseases, such that mucosal protection can be provided, as well as the need for the exclusion of pathogens such as Escherichia coli from one of the important areas of invasion (1).

The selection of L. fermentum KLD was based on its high degree of association with the FAE of the Peyer's patch and its poor association with the nonlymphoid villous intestine (Table 2), since this would allow the ingested Lactobacillus cells to target the Peyer's patches and not be spread over the entire villous surface. Five other strains also associated with the Peyer's patches but also showed a high degree of association with the nonlymphoid villous intestine (Table 2). L. fermentum 104S was more adhesive to the Peyer's patches than the nonlymphoid intestinal tissue, which reflects results of previous studies which have shown that this strain adheres better to nonsecreting squamous tissue than to secreting gastric epithelium (8). The Peyer's patches are generally considered to be nonsecreting regions of the gastrointestinal epithelium due to the decreased numbers of goblet cells. Hence, the mechanism of adhesion to the squamous nonsecreting regions of the gastrointestinal tract and to the intestinal mucosa could be important when considering adhesion to the Peyer's patches, as they contain columnar cells but no overlying mucus.

The association of L. fermentum KLD with the surface of the Peyer's patches has been shown by an in vitro adhesion assay, as well as by in vivo recovery from the Peyer's patches following orogastric dosing of the strain. Although in most fields of view 30 to 100% of the field was covered by L. fermentum KLD (Fig. 1), this association does not necessarily imply a direct association with the M cells within these regions. Although the in vitro scanning electron microscope analysis reveals that L. fermentum KLD shows a preference for the FAE of the Peyer's patch tissue (Fig. 3), low levels of association were observed in some domes of the Peyer's patches, with some fields showing small numbers of associated bacteria or noncharacteristic adhesion patterns (data not shown). This variation in binding has been reported for other organisms. Salmonella enterica serovar Typhimurium SL 1344 shows variation in binding to M cells (3) and differential binding to domes. The nonuniform bacterial adhesion to the domes suggests that there are M-cell subtypes present in the domes of the Peyer's patches.

The large degree of association of L. fermentum KLD with the Peyer's patches was particularly evident compared with that for a nonassociating strain of L. delbruckii subsp. bulgaricus. This strain showed levels of association which were significantly lower than the level of association of L. fermentum KLD with the small intestinal tissue used in this study (Fig. 2). Bacterial cells were rarely seen in scanning electron micrographs of tissue incubated with L. delbruckii subsp. bulgaricus (Fig. 3). The larger numbers of organisms associated with the tissue following direct recovery from the tissue may be due to a less rigorous washing procedure compared to that used for scanning electron microscope analysis. L. fermentum KLD was highly autoaggregative in most of the fields examined (Fig. 2). This suggests not only that there is an association of the lactobacilli with the Peyer's patch surface but also that there are interactions between the bacterial cells. This clumping may be beneficial, as it could further enhance binding to the Peyer's patches and may account for the large numbers of L. fermentum KLD associated with the Peyer's patch surface.

Following administration of an orogastric dose either L. fermentum KLD or L. delbruckii subsp. bulgaricus to mice, it was observed that L. fermentum KLD associated with tissue in significantly larger numbers than L. delbruckii subsp. bulgaricus when measured by direct enumeration of viable cells from the tissue surface (Fig. 4). In this study, the tissue was sampled 2 h after orogastric intubation. It has previously been shown that the transit time through the entire gastrointestinal tract of mice is approximately 3.5 h (Xin Wang, personal communication). It is therefore assumed that 2 h after incubation, the Lactobacillus cells would have reached the terminal ileum. The same trend was observed for the association with the nonlymphoid villous intestine. As observed in the in vitro adhesion assay, large numbers of L. fermentum KLD were recovered from the nonlymphoid villous intestine sections (Fig. 2). However, the association with the Peyer's patches was statistically more significant (P < 0.05) than the association with the nonlymphoid villous intestine, suggesting that L. fermentum KLD does preferentially associate with the Peyer's patches in vivo.

The results of this investigation indicate that L. fermentum KLD is able to associate directly with Peyer's patches in mice, both in vitro and in vivo. The tissue association of L. fermentum KLD was determined by comparison with that of a nonassociating strain of Lactobacillus. The association is further supported by the persistence of this organism within the gastrointestinal tract for greater than 10 h and its high survival rate in this system (L. Plant, unpublished observation). This study has provided novel evidence that lactobacilli associate with the Peyer's patches in the murine intestine.