The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Abstract

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.

Pathogen adhesion, Probiotic adhesion, Intestinal mucus, Adhesion inhibition

1 Introduction

Adhesion of pathogenic bacteria to mucosal surfaces is considered to be the first step of intestinal infections [1,2]. The adhesion of pathogens is mediated by bacterial adhesins, which recognise specific mucosal receptors. Inhibition of adhesion may prevent colonisation of the intestine by the pathogen and thereby prevent the infection. Adhesion may be inhibited by blocking the receptor with specific adhesin analogues or by steric hindrance. Some probiotic bacteria with beneficial health effects have been found to adhere to the intestinal mucosa. Therefore, adhesive probiotics could prevent the subsequent attachment of pathogens, referred to as competitive exclusion.

Some Lactobacillus strains, either the cells alone or in combination with their spent culture supernatant (SCS), have been shown to inhibit adhesion of pathogens. For example, specific Lactobacillus strains were found to inhibit the adhesion of Escherichia coli to porcine enterocytes [3]. The SCS of lactobacilli was reported to prevent E. coli attachment to porcine mucus [4,5]. Lactobacilli have also been shown to inhibit the adhesion of human uropathogens to uroepithelial cells in vitro [6]. Inhibition of pathogen adhesion by probiotics has also been reported using the Caco-2 cell line as a model for human intestinal epithelium [7,8]. In the intestine, epithelial cells are covered with a mucus layer protecting the epithelial cells from physical and chemical damage as well as from pathogenic bacteria. The mucus layer is most likely the first place of contact between the host and the pathogen. Human intestinal glycoproteins extracted from faeces and human ileostomy glycoproteins were recently used as a model for intestinal mucus to investigate the adhesion potential of probiotic bacteria [9–11]. In this study, we investigated whether intestinal glycoproteins isolated from faeces could provide an in vitro model for human intestinal mucus to study adhesion of pathogenic bacteria as well as the interactions between pathogenic and probiotic bacteria.

2 Materials and methods

2.1 Micro-organisms and growth conditions

SfaII-fimbriated Escherichia coli HB101(pAZZ50) [12], hereafter abbreviated as E. coli SfaII, was a gift from Prof. J. Hacker (University of Würzburg, Germany). Human and bovine enterotoxigenic E. coli H10407 and B44, respectively, were provided by Dr M. Saxelin (Valio Ltd, Finland). The bovine strain, E. coli B44, was used as a non-adhering control strain. Salmonella typhimurium (ATCC 14028) was obtained from the American Type Culture Collection (USA). S. enteritidis, isolated from a human patient, was obtained from the National Public Health Institute (Turku, Finland). Pathogenic bacteria were grown at 37°C for 18–20 h in Luria-Bertani broth containing 5 µl ml⁻¹ of methyl-1,2-[³H]-thymidine (113 Ci mmol⁻¹). In the case of E. coli SfaII, 50 µg ml⁻¹ ampicillin was added to the broth. After growth, the radiolabelled bacteria were harvested by centrifugation (1500×g, 7 min), washed with HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid)-Hanks' buffer (HH; 10 mmol l⁻¹ HEPES; pH 7.4) containing 0.1% (w/v) Na-azide and resuspended in HH. The optical density of bacterial suspensions was adjusted to 0.25±0.01 to give approximately 1–2×10⁸ colony forming units (cfu) ml⁻¹. Different bacterial dilutions were tested in adhesion assays.

Lactobacillus GG (L. rhamnosus GG; ATCC 53103), L. johnsonii LJ1 (isolated from a LC1® product from Nestlé), L. casei Shirota (isolated from a Yakult® product from Yakult Ltd.), L. rhamnosus (human isolate) and L. rhamnosus LC-705 were obtained from Dr. M. Saxelin (Valio Ltd, Finland). Lactobacillus strains were grown in de Man, Rogosa and Sharpe (MRS) broth at 37°C for 18–20 h. A 0.2% inoculum from stocks stored at −70°C in 40% glycerol was used. To label the bacteria, the radiolabel was added as described above. The bacteria were harvested by centrifugation (1500×g, 7 min) and the pellet was washed with HH. The bacteria were resuspended in HH. The optical density (OD) at 600 nm of each bacterial suspension was adjusted to 0.25±0.01 give approximately 1–2×10⁸ cfu ml⁻¹ with the exception of L. johnsonii LJ1 containing 1×10⁷ cfu ml⁻¹. The adhesion was tested with different dilutions of the suspension.

2.2 Human intestinal mucus

Human intestinal glycoproteins were isolated from faeces of healthy adults (n=10) by extraction and dual ethanol precipitation [9,10]. Equal amounts of lyophilised mucus from each individual were pooled to make a stock suspension of 10 mg ml⁻¹ in HH. Any particulate material was removed by centrifuging the suspension. The stock suspension was stored at −20°C. For adherence assays, mucus was diluted (0.5 mg ml⁻¹) in HH and 100 µl of the suspension was immobilised in polystyrene microtitre plate wells (Maxisorp; Nunc, Denmark) by overnight incubation at 4°C. Excess mucus was removed by washing twice with 250 µl of HH.

2.3 In vitro adherence assay

Radioactively labelled bacteria (100 µl) were added to the wells coated with intestinal mucus and incubated at 37°C for 1.5 h. The wells were washed three times with 200 µl of HH to remove unattached bacteria. The bacteria bound to intestinal mucus were released and lysed with 1% SDS-0.1 M NaOH by incubation at 60°C for 1 h. The radioactivity of the lysed suspension was measured by liquid scintillation. The adhesion ratio (%) was calculated by comparing the radioactivity of the bacteria added (triplicate 100-µl samples) to the radioactivity of the bound bacteria.

2.4 Competitive exclusion of S. typhimurium and E. coli SfaII

Probiotic bacteria without radiolabel (OD 0.25±0.01; 100 µl) were added to the wells coated with intestinal mucus and incubated at 37°C for 1 h. To remove unbound bacteria, the wells were washed three times with 200 µl of HH. Radiolabelled pathogens were diluted 1:9 to avoid saturation of the substratum and 100 µl of the suspension was added to the wells. The wells were incubated at 37°C for 1.5 h. The assay was performed as described above.

2.5 Coaggregation between S. typhimurium and Lactobacillus GG

To investigate possible interaction between S. typhimurium and Lactobacillus GG, the coaggregation ability between the two bacteria was studied according to the method of Spencer and Chesson [3] with the exception that the test was performed in HH since this buffer was also used in the adhesion assay. The bacterial suspensions were incubated for 4 h at 37°C and the OD at 660 nm of the suspensions (both strains alone and together) was measured. In addition, the suspension of S. typhimurium and Lactobacillus GG was Gram-stained and observed by light microscopy.

2.6 Statistical analysis

Student's t-test was used to determine the significant difference (P<0.05) between the control and the test strain. The results shown are the average of at least three independent experiments performed in triplicate.

3 Results

3.1 Adherence of strains to human intestinal mucus

E. coli SfaII expressed relatively high adhesion to mucus glycoproteins with approximately 13% of the applied bacteria adhering (Table 1). The adhesion of the other tested pathogens was not significantly different from the adhesion of the non-adhesive control strain (adhesion; 0.94%). SfaII-fimbriated E. coli and type 1 fimbriated S. typhimurium were chosen for the competitive exclusion studies.

Table 1

Adherence of bacterial strains to human intestinal mucus glycoproteins

Bacterial strain	Adhesion (%) (mean±S.D.)
E. coli SfaII	13±2.2
E. coli H10407	0.54±0.31
S. enteritidis	0.47±0.01
S. typhimurium (ATCC 14028)	1.4±1.0
E. coli B44 (non-adhesive control)	0.94±0.17
L. casei Shirota	0.25±0.02
L. johnsonii LJ1	25±5.9
Lactobacillus GG	39±2.6
L. rhamnosus LC-705	0.43±0.21
L. rhamnosus (human isolate)	24±2.2

Mean±S.D. of individual experiments (n=2 for S. enteritidis and E. coli H 10407, n=6 for the rest of the strains).

Significantly higher than the non-adhesive control (P<0.05).

Significantly lower than the non-adhesive control (P<0.05).

Among the probiotic strains, Lactobacillus GG, L. johnsonii LJ1 and L. rhamnosus (human isolate) expressed high adhesion, but L. rhamnosus LC-705 and L. casei Shirota adhered poorly.

3.2 Competitive exclusion of S. typhimurium and E. coli SfaII by probiotic strains

The adhesion of S. typhimurium and E. coli SfaII to intestinal mucus was assigned to 100% and adhesion to intestinal mucus that was incubated with a probiotic strain prior to pathogen adhesion was compared to this control (Table 2). Lactobacillus GG, L. rhamnosus LC-705 and L. rhamnosus (human isolate) reduced the adhesion of E. coli SfaII to 90–91%, but only the reduction by Lactobacillus GG and L. rhamnosus (human isolate) was significant (P<0.05).

Table 2

Adherence of E. coli SfaII and S. typhimurium (ATCC 14028) to human intestinal mucus glycoproteins pre-treated with probiotic strain

Probiotic strain	Adhesion (%) (mean±S.D.)
	E. coli SfaII	S. typhimurium
Control (buffer only)	100	100
L. casei Shirota	97±24	83±18
L. johnsonii LJ1	96±16	77±13
L. rhamnosus LC-705	90±15	96±31
Lactobacillus GG	90±7.6	190±45
L. rhamnosus (human isolate)	91±7.1	332±116

Mean±S.D. of individual experiments (n=6).

Significantly lower than the control (P<0.05).

Significantly higher than the control (P<0.001).

The adherence of S. typhimurium was reduced to 77% by L. johnsonii LJ1 and to 83% by L. casei Shirota (P<0.05). Lactobacillus GG and L. rhamnosus (human isolate) were found to significantly increase the adhesion of S. typhimurium to 190% and 332%, respectively. When Lactobacillus GG was further diluted (1 to 100) for the pre-treatment, the adhesion was not increased compared to the control (data not shown).

3.3 Coaggregation between S. typhimurium and Lactobacillus GG

No coaggregation between S. typhimurium and Lactobacillus GG was measured after the incubation. Coaggregation was neither observed after Gram staining, although a number of S. typhimurium cells seemed to be attached to Lactobacillus GG cells.

4 Discussion

In this study the potential of some probiotic strains to adhere and to affect the adhesion of pathogens to human intestinal glycoproteins was investigated. Lactobacillus GG, L. johnsonii LJ1 and L. casei Shirota have documented probiotic effects on human health [13]. Lactobacillus GG and L. johnsonii LJ1 have been previously reported to adhere to Caco-2 intestinal tissue culture cells [8,14,15], to intestinal mucus extracted from faeces [9] and to human ileostomy glycoproteins [11]. The strains expressed high adhesion also in the present study, as did L. rhamnosus (human isolate) which was previously shown to adhere both to Caco-2 cells [15] and to ileostomy glycoproteins [11]. Strain LC-705 was known to adhere to Caco-2 cells [15], but it adhered poorly to human ileostomy glycoproteins [11] as well as to intestinal mucus used in the current study. L. casei Shirota adhered poorly, previously it was also shown to adhere poorly both to Caco-2 cells [15] and to ileostomy glycoproteins [11].

E. coli B44 has been used as a non-adhering control strain in studies with human intestinal cells from ileostomy lavage [16], with the Caco-2 cell line [14,15] and with ileostomy glycoproteins [11]. The strain adhered poorly also in the present study. Surprisingly, S. typhimurium expressing mannose-sensitive type 1 fimbriae [17] did not adhere significantly better than the non-adhesive control strain suggesting that the mannose-containing receptors are not present or not available in great numbers in the current mucus model. In our previous study [18], the same Salmonella strain adhered to Caco-2 cells (approximately 7% of the applied bacteria adhered). The adhesion to Caco-2 cells was partly inhibited by pre-treating the bacteria with mannose [18] indicating that the type 1 fimbriae were expressed in Luria-Bertani broth used also in the present study. Craven and Williams [19] reported the adhesion of S. typhimurium to chicken caecal mucus, but the level of adhesion was not shown making the comparison of the adhesion ability of Salmonella between the studies impossible. Also E. coli H10407 (CFA/I fimbriae) and S. enteritidis adhered poorly, although E. coli H10407 has been shown to adhere to human intestinal cells [16] and to the Caco-2 cell line [14,15]. Ouwehand and Conway [5] reported that E. coli expressing CFA/I, II or IV also adhered poorly to human ileostomy glycoproteins. Therefore, it may be possible that the type 1 fimbriae and CFA/I fimbriae, known to mediate adhesion to epithelial cells, do not mediate the adhesion to human mucus.

In contrast to Salmonella and other E. coli strains, the S-fimbriated E. coli strain adhered significantly better to intestinal mucus than the non-adhesive control strain. S-fimbriated E. coli is associated with newborn meningitis [20], but the gastrointestinal tract and the oropharynx seem to be the reservoir for E. coli with the translocation ability [21]. Therefore, the inhibition of adhesion in the intestine could prevent the translocation and subsequently the infection. Ouwehand et al. [22] used the same E. coli SfaII strain, and showed that the strain was able to produce the SfaII fimbriae recognising sialyl galactosides [23] under similar culture conditions as used in the present study. The level of adhesion of the S-fimbriated E. coli reported here was similar to the adherence to human ileostomy glycoproteins reported in previous studies [11,22]. However, the same number of bacteria used with ileostomy glycoproteins saturated the substratum in the present study suggesting that fewer receptors were available. Therefore, it is important to optimise the number of bacteria used in in vitro studies.

Strongly adhesive Lactobacillus GG and L. rhamnosus (human isolate) and low-adhering L. rhamnosus LC-705 were able to slightly reduce the S-fimbriated E. coli adhesion. Similarly, the adhesion of S. typhimurium was partially inhibited by the adhesive L. johnsonii LJ1 and low-adhering L. casei Shirota. The results indicate that the inhibition was not related to the adhesion potential of the Lactobacillus strain. Craven and Williams [19] reported that the pre-treatment of immobilised chicken caecal mucus with Lactobacillus cells or with the SCSs inhibited the binding of S. typhimurium. The adhesion of S. typhimurium was reduced to 60% by one L. salivarius and one L. delbrueckii ssp. delbrueckii strain. Similar to the present study, not all tested isolates inhibited the Salmonella adhesion. In the present study SCS and inhibitory components excreted by the bacteria were not involved in the inhibition. Therefore, the detected inhibition is due to the adhesion of whole probiotic bacterial cells or products released during the incubation. The adhesion of the pathogens was inhibited by 10–23% and it is difficult to estimate the biological significance of the inhibition. However, the infective doses of pathogens in general are relatively low and therefore even partial inhibition may be important in preventing infection.

S. typhimurium adhesion was significantly (P<0.001) increased by Lactobacillus GG and L. rhamnosus (human isolate). Interestingly, only the two strongly adhesive L. rhamnosus strains (Lactobacillus GG and L. rhamnosus (human isolate)) increased the adhesion although all three L. rhamnosus strains (including the poorly adhering L. rhamnosus LC-705) were able to reduce E. coli SfaII adhesion. Reid et al. [24] observed that type 1 fimbriated E. coli coaggregated with L. casei spp. rhamnosus GR-1 and suggested the potential of lactobacilli to prevent pathogenic adhesion in the urinary tract by coaggregation. Also intestinal lactobacilli have been shown to coaggregate with pathogens, for example lactobacilli of porcine origin coaggregated with a porcine pathogen (E. coli K88) [3], indicating that similar prevention could be obtained in the intestine. Coaggregation was not observed between S. typhimurium and Lactobacillus GG in the present study. However, microscopic observations demonstrated that some of the S. typhimurium and Lactobacillus GG cells were attached to each other although the binding was not strong enough to aggregate the bacteria. Further studies are needed to clarify the mechanisms of the interactions between S. typhimurium and adhering probiotics, and the biological importance of the observed increase in adhesion needs to be investigated.

In conclusion, the tested bacteria showed a strain-dependent adhesion to intestinal glycoproteins. The probiotics had different effects on the subsequent adhesion of S-fimbriated E. coli and type 1 fimbriae expressing S. typhimurium. This may imply that the mechanisms for probiotic action depend on the probiotic and the pathogenic strain. In addition, intestinal glycoproteins were shown to provide a new model of human origin to investigate the interactions between pathogens and probiotics at mucosal surfaces.

Acknowledgements

Mrs. Satu Tölkkö is thanked for skilful technical assistance. This work was supported by the Academy of Finland.