R E S E A R C H A R T I C L E
Salivary IgA response to probiotic bacteria and mutans streptococci
after the use of chewing gum containing
Lactobacillus reuteri
Dan Ericson1, Kristina Hamberg1, Gunilla Bratthall2, Gabriela Sinkiewicz-Enggren3 & Lennart Ljunggren3
1 Department of Cariology, Faculty of Odontology, Malm€o University, Malm€o, Sweden 2 Department of Periodontology, Faculty of Odontology, Malm€o University, Malm€o, Sweden
3 Department of Biomedical Science, Faculty of Health and Society, Malm€o University, Malm€o, Sweden
In this small-scale random controlled trial, the authors have tested the effect of the probiotic bacterium, Lactobacillus reuteri, instilled into chewing gum versus a placebo. They found that the probiotic intervention did indeed affect salivary IgA concentration thus indicating an effect on local immunity, and providing yet another effective approach to the delivery of probiotics.
Keywords
probiotics; salivary IgA; mutans streptococci; Lactobacillus reuteri.
Correspondence
Dan Ericson, Department of Cariology, Faculty of Odontology, Malm€o University, SE 205 06 Malm€o, Sweden.
Tel.: +46406658537 fax: +4640935359 e-mail: dan.ericson@mah.se
Received 26 March 2013; revised 18 May 2013; accepted 23 May 2013. Final version published online 2 July 2013.
doi:10.1111/2049-632X.12048 Editor: Peter Timms
Abstract
We investigated whether ingestion of probiotic bacteria could influence salivary IgA levels, specific anti-mutans streptococci IgA levels and specific antibodies towards the ingested probiotic bacterium. The study was a randomised, double-blind, placebo-controlled trial, where the test group (n= 11) received twice daily chewing of gum containing Lactobacillus reuteri (29 108CFU per dose) and the control group (n= 12) received placebo. Resting saliva was collected before and after 12 weeks of treatment and 4 weeks after end of treatment. Total salivary IgA concentrations were measured by ELISA. Specific IgA reactivity was determined using a whole-cell ELISA. Results were expressed as % IgA per protein in saliva. The level of total IgA% per protein increased significantly between pretreatment levels (13.5%) and follow-up treatment levels (14.4%) within the test group only (P< 0.05). No changes were seen in the control group during the trial. The level of probiotic-reactive antibodies decreased significantly between pre- and post-treatment samples (from 12.2% to 9.0%, P< 0.05) in the test group. Similarly, the level of specific mutans streptococci antibodies decreased significantly between pre- and post-treatment samples (P< 0.05) in the test group only (for Streptococcus mutans from 20.1% to 15.0%; for Streptococcus sobrinus from 7.4% to 5.3%). Ingestion of probiotic bacteria might influence the adaptive immune response of the host.
Introduction
Probiotic bacteria have created increased awareness within oral health care as a complementary way to prevent diseases caused by the oral microbial flora (Meurman & Stamatova, 2007; Teughels et al., 2007; Twetman & Stecksen-Blicks, 2008). Proposed mechanisms of action for beneficiary effects of intake of probiotics include that probiotic bacteria compete for space and nutrients as well as release of antagonistic substances with pathogens to establish a healthy and diverse flora (Meurman & Stamatova, 2007). The stimulating effect on the immune response by probiotic bacteria has also been suggested as another mechanism in some studies (Surono et al., 2011; Yan & Polk, 2011). Probiotics may also affect levels of inflammatory mediators in crevicular fluid (Twetman et al., 2009).
Several studies have demonstrated that lactobacilli-derived probiotic strains (i.e. Lactobacillus reuteri ATCC PTA 5289, L. reuteri ATCC 55730, Lactobacillus rhamnosus GG ATCC 53103 and L. rhamnosus LC 705) during short-term (10– 21 days) intake reduced the counts of mutans streptococci in saliva and plaque (Ahola et al., 2002; Nikawa et al., 2004; Caglar et al., 2008), but no effect could be seen with one strain (L. rhamnosus LB21; Lexner et al., 2010). Only one study using probiotic bacteria has demonstrated a clinical car-ies-preventive effect where N€ase et al. (2001) showed that drinking milk supplemented with L. rhamnosus GG for 7 months could be caries preventive among 3- to 4-year-old children.
Immune-stimulating effects of probiotic bacteria have been reviewed by Kelly et al. (2005), and it is documented that intake of probiotic lactobacilli will enhance production of
Pathogens and Disease (2013), 68, 82–87,© 2013 The Authors. 82
IgA (Corthesy et al., 2007). Relevant to the oral ecosystem, high salivary IgA titres against mutans streptococci antigens have been demonstrated to be caries protective in humans (Smith & Mattos-Graner, 2008) and recently also in a rat model (Shi et al., 2012). Of particular interest for immune regulation affecting the mucosal immunity and caries prevention, an increase in salivary IgA, but not serum IgA, has been demonstrated after 90 days ingestion of a probi-otic strain Enterococcus faecium among under-nourished children (Surono et al., 2011).
Therefore, the hypothesis in this study was that ingestion of a probiotic strain for prevention of oral diseases can have immunomodulating effects measurable in saliva. The aim of this study was to investigate whether the introduction of L. reuteri ATCC PTA 5289 through the use of probiotic chewing gum can elicit an increased immune response towards mutans streptococci as well as towards the ingested probiotic bacterium.
Materials and methods
Patients and study designThe study was a randomised, double-blind, placebo-controlled trial. Exclusion criteria were use of antibiotics, oral antiseptics or periodontal treatment 6 months prior to the trial. Detailed description of methods and results on clinical outcome, plaque formation, plaque and salivary bacteria from the current investigation has been reported by Sinkiewicz et al. (2010). The research protocol was approved by Ethics Committee at Lund University, Sweden. Twenty-four healthy volunteers aged 18 years or more randomised into test or control subjects. One subject in the test group was excluded due to the use of antibiotics during the trial period (Sinkiewicz et al., 2010). The antibody response in saliva towards mutans streptococci and towards the probiotic strain L. reuteri ATCC PTA 5289 was measured before and after chewing gum containing the probiotic strain.
Probiotic bacteria
Chewing gum active product containing L. reuteri (an equal mix of ATCC PTA 5289 and ATCC 55730, at a total of 29 108CFU per dose; BioGaia AB, Sweden) was chewed 10 min directly after dental hygiene procedures in the morning and in the evening. The placebo contained no bacteria, but was identical to the active chewing gum in terms of taste, shape, texture and composition.
Clinical trial outline
Patients were randomised in two groups; test group (n= 11) received active product and the control group (n= 12) received placebo. At baseline, resting saliva samples were collected (1 mL) from each participant in a tube (without any oral movements) and diluted 10 times by adding 8 mL 0.15 M NaCl and 1 mL glycerol, mixed and frozen to
80°C.
The study/placebo products were taken daily for 12 weeks after which the subjects were re-analysed as at baseline. After completion of the study, subjects were invited to return 4 weeks following the last intake of the product for saliva sampling to determine washout of L. reuteri ATCC PTA 5289.
Sampling was carried out systematically in the morning. Resting saliva samples were collected before and after 12 weeks of treatment and 4 weeks after end of treatment. At follow-up, saliva from one individual was not collected. Lactobacillus reuteri reuteri in saliva
The level of L. reuteri ATCC PTA 5289 in saliva was enumerated using Man–Rogosa–Sharpe (MRS) agar plates. To confirm the identity of L. reuteri ATCC PTA 5289, a PCR method described by Magnusson et al. (2003) was employed.
Salivary IgA and protein determinations
Salivary IgA concentrations were measured by enzyme-linked immunosorbent assay (ELISA) previously described in detail by Sonesson et al. (2011). The wells of MicroWellTM
plates (MaxiSorpTM
surface, Nunc-ImmunoTM
Plate; Thermo Fisher Scientific, Denmark) were coated overnight using 100lL alpha-chain-specific goat anti-hu-man IgA (Sigma I0884; Sigma Chemical Co., St. Louis, MO) in a coating buffer (0.05 M carbonate buffer, pH 9.6). Each well was washed three times with 200lL 0.05% PBS Tween 20 (PBST) and blocked with 100lL PBST contain-ing 1% BSA (Sigma Chemical Co.) for 1 h at 37°C. After washing, 100lL of standards (Colostrum standard; Sigma Chemical Co.) and diluted saliva samples in PBST was added and incubated for 1 h at 37°C.
After washing as above, 100lL alpha-chain-specific goat anti-human IgA conjugated with alkaline phosphatase in PBST (Sigma A 9669; Sigma Chemical Co.) was added and incubated for 1 h in 37°C. After washing, 200 lL per well of phosphatase substrate (Sigma P5869; Sigma Chemical Co.) in diethanolamine buffer (pH 9.8) was incubated for 30 min at 37°C. The colour reaction was stopped after 30 min by adding 3 M NaOH.
Absorbance was read in a microplate reader (ELx800TM;
BioTek Instruments, Inc., Winooski, VT) at 405 nm. Saliva samples in duplicates were plotted against a colostrum standard curve and multiplied with the dilution factor. Results are presented as mg 100 mL 1.
The total protein concentration in the saliva samples was determined using the Bio-Rad Laboratories protein assay (Richmond, CA). Levels of IgA were also expressed as percent per protein.
Specific IgA reactivity using a bacterial whole-cell ELISA
This method was developed for the one probiotic strain L. reuteri ATCC PTA 5289 that adhered well into the microtitre plates. Laboratory strains of Streptococcus
mutans NCTC 10449 and Streptococcus sobrinus B13 were cultured in Todd-Hewitt broth (5% CO2 in N2) at
37°C 18–24 h. Lactobacillus reuteri strain ATCC PTA 5289 was cultured in MRS broth for 18–24 h.
After harvesting and washing (9 3) in PBS, the optical density (OD) was set to 0.25 for mutans streptococci and for L. reuteri ATCC PTA 5289 to OD 0.50 in carbonate buffer (pH 9.6). ELISA plates (MaxiSorp, NUNC) were coated with 100lL of the bacteria in 0.05 M carbonate buffer (pH 9.6) and incubated at +4 °C overnight. After aspiration and washing with PBST9 3 (washing step), 100 lL of blocking agent, 1% BSA in PBST, was added and plates were incubated at 37°C for 1 h. After washing step as above, 100lL PBST (blank) and diluted saliva (1 : 10) were added and the plate was incubated 1 h at 37°C.
Washing step was performed as above, 100lL of goat anti-human alpha-chain IgA conjugated with alkaline phos-phatase 1 : 10 000 (Sigma A-9669) was added and incu-bated 1.5 h at 37°C. After washing step, 200 lL of substrate (Sigma P5869; Sigma Chemical Co.) and 1 mg mL 1in diethanolamine buffer were added, and after
15 min, the reaction was stopped by 50lL of 3 M NaOH and the absorption was read at 405 nm in a Bio-Tek plate reader. After subtracting blank values, the results were expressed as absorption per % IgA per protein (Abs/% IgA/protein9 103). The code for treatment and control samples was broken after the analyses were performed. Statistical methods
A comparison between salivary concentrations of antibodies in pretreatment samples, after treatment samples (12 weeks) and follow-up samples after 4 weeks of washout, was made using Student’s paired t-test and Wilcoxon rank sum test (within groups) and Student’s unpaired t-test and Wilcoxon signed-rank test (between groups). P-values< 5% were considered statistically significant.
Results
Relevant results reported previously from this investigation by Sinkiewicz et al. (2010) include that in saliva of nine of 11 test group individuals, the test strain L. reuteri ATCC PTA 5289 could be recovered after 12 weeks of chewing, but also in one of 12 individuals in the control group. After another 4 weeks without chewing, the strain could be recovered in saliva of one test individual only. At baseline, three of the 23 individuals harboured L. reuteri.
Protein concentrations
The total levels of protein were unchanged in test and control group throughout the trial (Fig. 1, Table 1).
Total levels of salivary IgA
The total levels of IgA did not change (Fig. 2, Table 1), but expressed as %IgA/protein, the levels increased significantly between pretreatment levels and
post-treat-ment levels within the test group only (P< 0.05; Fig. 3, Table 1).
Levels of specific salivary IgA
The level of specific antibodies did not change in the control group during the trial. Neither did the test and control group differ on any parameters. In the test group only, the level of L. reuteri ATCC PTA 5289-reactive antibodies decreased significantly between pre- and post-treatment samples (P< 0.05) and a difference remained between pretreatment and follow-up samples (P< 0.05). The follow-up samples increased with a trend towards pretreatment levels (Fig. 4, Table 1).
The level of S. mutans 10449-reactive antibodies decreased significantly between pre- and post-treatment samples (P< 0.05, Student′s paired t-test only) in the test group only, and the follow-up samples increased with a trend towards pretreatment levels (Fig. 5, Table 1).
The level of S. sobrinus B13-reactive antibodies, decreased significantly between pre- and post-treatment samples (P< 0.01) only in the test group, and a difference remained through the washout period (P< 0.01). The follow-up samples tended to increase towards pretreatment levels (Fig. 6, Table 1).
Discussion
The main result from this investigation was that the total level of salivary IgA (abs/%IgA/protein) showed a significant increase within the test group, after ingestion and docu-mented colonisation (9 of 11 subjects) of the probiotic bacteria. In contrast to this total IgA increase, the reactivity of specific IgA towards the ingested probiotic strain (L. reuteri ATCC PTA 5289) as well as against S. mutans and S. sobrinus decreased in the test group and the levels tended to return to pretreatment values after a 4-week washout period. The probiotic strain could then not be detected in 11 of 12 subjects. Almost no differences between test and control group could be seen, except a barely significant (P= 0.0488) difference for reactivity towards S. sobrinus B13 (Table 1). Differences were, however,
Protein concentration mg per 100 mL
NS 150 100 50 0 Pre Post Control Test
Follow-up Pre Post Follow-up
Fig. 1 Total protein concentration. Bars indicate standard deviation. NS, no significant differences.
consistently seen longitudinally within the test group only. Therefore, the conclusive interpretation must be that inges-tion of probiotic bacteria has an effect on salivary IgA concentration, but not to an extent that reveals a difference between test and control groups.
The large variation among subjects and their relatively low number might explain lack of significant effect between
groups. The salivary IgA levels are highly dependent on salivary secretion rate (Ericson et al., 1982). So are salivary protein levels (Wu et al., 2008). To diminish the effect of fluctuation of salivary IgA concentrations related to differ-ences in flow rate among the subjects, the level of total and
Table 1 Summary of changes over time by variable and treatment group
Variable
Experimental groups
Control Test
Post–Pre Follow-up–Pre Follow-up–Post Post–Pre Follow-up–Pre Follow-up–Post
Protein (mg 100 mL 1) 14.4 (31.5)* 13.4 (40.7) 1.0 (26.7) 8.3 (33.3) 1.7 (20.7) 8.7 (46.6) Total IgA (mg 100 mL 1) 3.9 (8.5) 1.0 (3.0) 2.9 (6.6) 1.1 (3.4) 1.9 (4.5) 1.4 (6.6) %IgA/protein 0.1 (3.8) 1.1 (4.6) 0.9 (4.8) 1.8 (3.6) 1.8 (2.5)†‡ 0.6 (2.8) L. reuteri, Abs/%IgA/protein (91000) 0.7 (3.7) 1.3 (6.4) 0.6 (4.6) 3.2 (3.4)†‡ 2.5 (3.0)†‡ 0.8 (3.8) 10449, Abs/% IgA/protein (91000) 0.7 (4.9) 1.1 (8.8) 1.8 (9.0) 5.1 (7.2)†‡ 3.0 (4.1)† 1.5 (5.8) B13, Abs/%IgA/protein (9 1000) 0.7 (2.8) 0.3 (3.4)§ 0.4 (2.2) 2.1 (1.6)†‡ 1.8 (1.5)†‡§ 0.2 (1.4) *SD. †
Statistically significant (Student’s paired t-test, P< 0.05).
‡Statistically significant (Wilcoxon’s signed-rank test, P< 0.05) within test group.
§Statistically significant (Wilcoxon’s rank sum test, P< 0.05) between control and test group. Bold numbers indicate statistical significant values in the test group.
Total IgA mg per 100 mL
NS 25.0 20.0 15.0 10.0 5.0 0.0 Pre Post Control Test
Follow-up Pre Post Follow-up
Fig. 2 Total salivary IgA concentrations. Bars indicate standard deviation. NS, no significant differences.
% IgA/protein * 25.0 20.0 15.0 10.0 5.0 0.0 Pre Post Control Test
Follow-up Pre Post Follow-up
Fig. 3 Total salivary IgA concentrations expressed as % IgA per protein. Bars indicate standard deviation.*P < 0.05.
L. reuteri Abs/% IgA/protein (x 1000)
* * 25.0 20.0 15.0 10.0 5.0 0.0 Pre Post Control Test
Follow-up Pre Post Follow-up
Fig. 4 Specific antibodies towards Lactobacillus reuteri ATCC PTA 5289 expressed as absorption per % IgA per protein 9 1000. Bars indicate standard deviation.*P < 0.05.
10449 Abs/% IgA/protein (x 1000) * 25.0 20.0 15.0 10.0 5.0 0.0 Pre Post Control Test
Follow-up Pre Post Follow-up
Fig. 5 Specific antibodies towards Streptococcus mutans 10449 expressed as absorption per% IgA per protein 9 1000. Bars indicate standard deviation.*P < 0.05.
specific antibodies was expressed in relation to total salivary protein level.
The results of this study indicate that ingestion of probiotic bacteria induces an increase in total IgA in saliva. This might be interpreted as an immunostimulatory effect of probiotic bacteria, which has been suggested (Kelly et al., 2005; Corthesy et al., 2007). However, such an effect has been shown only among under-nourished children (Surono et al., 2011). Such increase in salivary IgA among under-nour-ished, but not in normal weight children, could be an effect not relating to a specific immunostimulation by the probiotic bacteria per se, but rather an effect of normalisation of the immune system along with better nourishment status among the children, similar to what was seen among malnourished mice (Maldonado Galdeano et al., 2011).
A similar lack of effect of probiotics on salivary IgA levels was reported among athletes (Cox et al., 2010). The use of probiotics to stimulate a specific response to a cholera vaccine (Paineau et al., 2008) actually resulted in an increase in the salivary IgA titre against the vaccine, but the total IgA was not measured in the study. A recent review (Maidens et al., 2013) indicates at most a modest stimula-tory effect of probiotics on the IgA response, when used concomitant with vaccines. Alas, the mechanisms by which probiotics influence the immune response are poorly understood. It is, however, suggested that the adjuvant effects might be mediated by components of the cell wall– like lipoteichoic acid and polysaccharide–peptidoglycan complexes (de Vrese et al., 2005). In the current study, the total IgA levels increased while ingesting probiotic bacteria and tended to decrease to baseline values after washout, indicating an immunostimulatory effect.
This study infers that the specific immune response to all target organisms seemed to decrease. The decrease in L. reuteri ATCC PTA 5289-specific IgA antibodies in col-lected saliva among the test subjects during the treatment phase might be attributed to antibody absorption onto bacteria in saliva during the collection procedure. Thus, the numerous (a dose 108 bacteria twice daily) probiotic bacteria present in saliva could act as specific immunosor-bents covered with specific antibodies that eventually were spun down, during centrifugation of saliva samples. The
antibodies would then be present in lower concentration in the supernatant that was analysed. Given a certain cross-reactivity with the tested mutans streptococcal strains (i.e. due common surface antigens), an adsorption onto L. reuteri ATCC PTA 5289 in saliva could also explain the decrease in the salivary-specific immune response towards the mutans streptococci. One would, however, anticipate that the measured specific IgA reactivity then would return quickly to pretreatment levels once the L. reuteri ATCC PTA 5289 strains disappeared from the oral cavity. This was not seen, but merely suggested by a trend of the specific IgA response to return to baseline values (Fig. 4). It is also conceivable that the probiotic strains induced a cross-tolerance towards the mutans streptococci (Edelman & Kasper, 2008) indicated by that the specific IgA responses to all strains followed the same trend (Figs 4–6).
The presence of anti-L. reuteri ATCC PTA 5289 IgA in saliva in baseline saliva samples in both test and control group could be due to presence of cross-reactive antibodies or to previous exposure to the test bacteria as 3 of 23 individuals harboured L. reuteri from start. A possible cross-reactivity with other common oral bacteria is plausible, as the reactivity towards S. mutans and S. sobrinus followed the pattern of that against the probiotic strain L. reuteri ATCC PTA 5289.
In conclusion, the results from this article indicate that ingestion of probiotic bacteria might influence the immune response of the host. Further investigations are necessary to verify and understand the mechanisms on the effect of probiotics bacteria on salivary immunology.
Acknowledgements
We thank Jayanthi Stjernsw€ard for language revision and Mikael Astr€om for statistical consultation.
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