Limosilactobacillus reuteri and Its Probiotic Potential against Cariogenic Bacteria

Abstract

Bacteriotherapy is a promising option in addressing dental caries, a persistent global public health challenge with multifactorial origin, including dysbiosis. Despite the exploration of various probiotics, outcomes remain inconclusive. Objective: This study aimed to assess the inhibitory potential of L. reuteri and other potential probiotics like S. salivarius and S. oralis on the growth, adhesion, colonization, and viability of major cariogenic pathogens, comparing their probiotic efficacy. Methods: An in vitro experimental study was conducted, encompassing direct competition assays in solid and liquid co-culture tests and the characterization of adhesion to dental enamel and cell viability by life or death assay. Results: L. reuteri exhibited the significant inhibition of S. sobrinus and S. mutans growth in both solid and liquid cultures, with statistically notable differences. Scanning electron microscopy and confocal microscopy demonstrated reduced cariogenic biofilm formation when combined with L. reuteri, corroborated by diminished bacterial viability and decreased dental enamel coverage. These findings underscore L. reuteri’s potential as an effective agent in caries prevention. Conclusion: The study suggests L. reuteri could serve as an effective probiotic in bacteriotherapy against dental caries. It displayed substantial inhibitory activity in vitro against cariogenic bacteria, impeding biofilm formation and adhesion, thereby impacting cell viability.

1. Introduction

Dental caries is a multifactorial disease characterized by the demineralization of dental tissues, influenced by various factors such as the composition and dysbiosis of the oral microbiome; diet; and behavioral, psychosocial, and environmental influences [1]. This condition represents a substantial challenge to public health globally, affecting both children and adults, and resulting in high costs for society [2].

Despite the availability of numerous preventive and curative treatments for dental caries, its prevalence remains high. The World Health Organization has issued evidence-based recommendations for the prevention and treatment of dental caries, including the use of pit and fissure sealants, topical and systemic fluorides, as well as minimally invasive or atraumatic restorative techniques [3]. While these strategies are effective, they do not directly address the underlying microbiological factors that trigger and perpetuate dental caries.

Recently, there has been growing interest in an alternative approach aimed at restoring the balance of the oral microbiome, bacteriotherapy. This strategy focuses on displacing the main pathogenic microorganisms, Streptococcus mutans and Streptococcus sobrinus, from dental ecological niches, and represents a promising alternative to combat oral diseases through the use of “beneficial” bacteria, considered probiotics [4].

Probiotics are live microorganisms that confer health benefits on the host when administered through different routes such as foods, supplements, or oral hygiene products [5]. They contribute to the formation of plaque and participate in maintaining the balance of the oral and even intestinal microbiome, leading to significant systemic repercussions [6].

The immunomodulatory, anti-inflammatory, and antibacterial properties of certain probiotics, particularly from the genera Streptococcus, Lactobacillus, and Bifidobacterium, have been studied [7,8]. Although the exact mechanisms of interaction between these bacteria remain unclear, proposed mechanisms include coaggregation; the inactivation of toxins and metabolites; competition with cariogenic bacteria for nutrients and adhesion sites; and the release of antimicrobial substances including organic acids, hydrogen peroxide, diacetyl, and bacteriocins [9,10].

One of the species of particular interest is Lactobacillus reuteri, recently reclassified as Limosilactobacillus reuteri [11]. This bacterium produces reuterin, a water-soluble bacteriocin with inhibitory effects on both Gram-positive and Gram-negative bacteria, which acts in an environment with a wide pH range, resisting proteolytic and lipolytic enzymes [12]. L. reuteri is an obligate heterofermentative resident in the gastrointestinal and urogenital tracts in humans and some animals, as well as in breast milk [13], and could be a promising probiotic candidate for bacteriotherapy.

Additionally, Streptococcus oralis and Streptococcus salivarius have been considered potential probiotics due to their low cariogenic potential and competitive ability against Streptococcus mutans for colonization of the niche [14,15]; however, there is still no conclusive information in this regard.

Based on the above considerations, the present in vitro experimental study aims to investigate the inhibitory capacity of L. reuteri and other potential probiotics such as S. salivarius and S. oralis on the growth, adhesion, colonization, and viability of the main cariogenic pathobionts and to compare their probiotic capabilities.

2. Materials and Methods

This in vitro experimental study was conducted in the laboratory of the Master’s Degree in Dental Sciences Program at UASLP and was approved by the Research Ethics Committee of the Dental Faculty at UASLP (CONBIOÉTICA-24-CEI-001-20190213), assigned the code CEI-FE-019-021.

All assays were performed using the following bacterial strains: Streptococcus mutans ATCC 35665, Streptococcus sobrinus ATCC 6715, Streptococcus salivarius NCTC 8618, and Streptococcus oralis NCTC 11427. Lactobacillus reuteri was isolated from a commercially available food supplement (Lactipan Baby, Italmex Pharma, Coyoacán, Mexico). The identity of all strains was verified through quantitative PCR assays using primers previously reported in the literature [16,17,18].

These species were cultured in brain–heart infusion (BHI) and incubated at 37 °C for 24 h. The concentrations were adjusted to 0.5 on the McFarland scale, corresponding to 1.5 × 10⁸ bacteria/mL, for their use in different assays.

2.1. Direct Competition Assays on Solid Culture Medium

Competition assays on solid culture medium were conducted following the modified Kirby–Bauer method [19]. Inoculations were performed on BHI agar plates to generate a monolayer of different cariogenic species through serial dilutions (1:10, 1:100). Effector strains were inoculated by applying 20 μL of each probiotic bacterial species, while sterile water served as the negative control and 5.25% sodium hypochlorite served as the positive control. The plates were incubated under anaerobic conditions for 24 and 48 h (BD GasPakTM EZ System, Sparks, MD, USA). After this time period, inhibition zones were measured using a digital caliper, and the results were reported in millimeters.

2.2. Competition Assays in Liquid Culture Medium

For competition assays in a liquid culture medium, the strains were pre-inoculated in BHI, considering S. sobrinus as the cariogenic strain. In 96-well microplates, 100 µL of the cariogenic strain culture was dispensed, followed by the addition of 100 µL of the corresponding probiotic strain culture: S. salivarius, S. oralis, or L. reuteri. As controls, the cariogenic strain culture served as the positive control, while sterile BHI medium acted as the negative control. Incubation was performed at 37 °C for 24 h. After this period, DNA extraction from each well content was carried out, and real-time qPCR assays were conducted with a TaqMan probe (StepOneTM system, ThermoFisher, Waltham, MA, USA) (Applied Biosystems, Waltham, MA, USA) [20], constructing a standard curve through serial dilutions of the corresponding DNA plasmid. Results were expressed as the number of copies of S. sobrinus per milliliter of bacterial culture.

2.3. Analysis of Bacterial Life or Death with Confocal Microscopy

This analysis evaluated the bacterial viability in each group. Dental enamel disks were placed in a bacterial culture plate, the formed biofilms were washed, and live/dead fluorescent staining (Molecular Probes, LIVE/DEAD1 Yeast Viability Kit, Eugene, OR, USA) was added. After 30 min in darkness at 30 °C, they were observed using an argon ion LASER with a LASER scanning confocal microscope (TCS SPE, Leica DMI 4000B, Wetzlar, Germany). Images were processed for visualization using specialized software LAS X Office 1.4.6 (LAS AF lite, Leica Microsystems, Wetzlar, Germany), performing qualitative/quantitative analysis where green staining indicated live bacteria and red staining indicated dead bacteria, obtaining the percentage based on arbitrary fluorescence units.

2.4. Bacterial Colonization Assays on Dental Enamel

To investigate the competition for dental enamel colonization between cariogenic species (S. mutans, S. sobrinus) and L. reuteri, 6 mm cuts were made in extracted healthy molars. These cuts underwent ultrasonic treatment with 17% EDTA and 5.25% sodium hypochlorite to eliminate organic and inorganic tissue and were subsequently autoclaved.

A mature mixed biofilm formed on the enamel pieces; our first group formed by carigenic bacteria (S. mutans, S. sobrinus) was called the cariogenic group. The second group contained the cariogenic group + L. reuteri in combination, while the third group contained only L. reuteri. Obtained after 21 days, with BHI culture medium changes every 48 h, maintaining incubation at 37 °C, these assays started from the same concentration of bacteria corresponding to 1.5 × 10⁸ bacteria/mL. Therefore, cariogenic bacteria were in a 1:1 ratio, as was L. reuteri, so as not to favor any particular species. Gram staining was performed every 24 h to ensure the purity of the microorganisms.

Samples were prepared for SEM analysis, washed with a 0.1 M phosphate-buffered solution, fixed with 2% glutaraldehyde and 1% Alcian Blue Stain 8GX (Sigma-Aldrich, St. Louis, MO, USA), and stored at 4 °C for 24 h. After fixation, they were washed; dehydrated in a series of anhydrous ethanol (Industrial Chemical Technology, Ltd, Howell, MI, USA) at 20%, 40%, 60%, 80%, 90%, 95%, and 100%; and critical point-dried (CPD 030 BAL-TEC GmbH, Schalksmühle, Germany). Samples were coated with a 20 nm gold–palladium mixture (Spi SUPPLIES, West Chester, PA, USA) [21].

The qualitative evaluation of the bacterial colonization of the cariogenic biofilm and L. reuteri on dental enamel was performed using a scanning electron microscope (JEOL JSM-6510LV, Tokyo, Japan) at two magnifications (1000× and 2000×)

For the quantitative interpretation of the results, thirty-two images (1000×) were analyzed for each group, using a system based on the percentage of microbial cells covering the surface, as previously reported in the literature. Four scores were assigned: 1 for the presence of bacteria covering <5% of the enamel surface, 2 for coverage from 5 to 33%, 3 for 34–66%, and 4 for 67–100% [22,23].

Two experienced SEM evaluators independently and blindly assessed the images. In the case of disagreement, the higher score was selected.

2.5. Statistical Analysis

The statistical analysis was performed utilizing GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA). The Shapiro–Wilk test was applied to assess the distribution of variables. The outcomes of direct competition assays in solid culture are expressed as mean and standard deviation, with comparisons conducted using ANOVA–Tukey tests. Concurrently, competition assays in liquid culture are represented as median values, with comparisons made using Kruskal–Wallis–Dunn tests.

The growth coverage on enamel surfaces is described qualitatively as frequency and percentage, assessed using the chi-square test. Additionally, a quantitative analysis was conducted, presenting the data as mean, standard deviation, range, and median, with comparisons made using the Kruskal–Wallis–Dunn test.

3. Results

In Figure 1, the results of direct competition assays in solid culture are depicted, showing a significant statistical difference (p = 0.0181). The mean of growth inhibition halos obtained in the S. sobrinus monolayer when adding L. reuteri as the effector strain was 7.8 mm, similar to that obtained with S. salivarius (p > 0.9999). However, S. oralis produced significantly smaller growth inhibition halos compared to the others (5.5 mm, p < 0.05). Regarding the assay where S. mutans was the monolayer, it was observed that both S. salivarius and S. oralis generated inhibition halos of approximately 5 mm, in contrast to L. reuteri, which reached values close to 7 mm (p = 0.0127). A statistically significant difference was found between S. oralis and L. reuteri (p = 0.00109). It is important to note that S. salivarius showed greater inhibition against S. sobrinus compared to S. mutans (p = 0.0008).

Regarding the direct competition assays in liquid culture medium, where the quantification of S. sobrinus copies was performed using real-time PCR, a result of 1.3 × 10⁸ copies of S. sobrinus per milliliter of culture medium was recorded after 24 h of incubation. This count significantly decreased when co-cultured with S. oralis and even more in the presence of S. salivarius (2.9 × 10⁷ and 2.2 × 10⁵, respectively, p < 0.05). Conversely, in the presence of L. reuteri, the counts were comparable to those obtained in the absence of this strain, recording 1.1 × 10⁷ copies per milliliter (p > 0.05) (Figure 2).

Regarding bacterial adhesion assays, 32 SEM images were analyzed. Figure 3 presents representative images with a 2000× magnification of dental enamel, illustrating the configuration of the cariogenic biofilm covering the surface (3a). This biofilm exhibited an abundance of clustered cells with a coccobacillary morphotype, unlike the surface covered with L. reuteri biofilm, where a lower quantity of bacteria with elongated rod-like morphological characteristics covering dental enamel was observed (3c). When combined, a reduced growth of the cariogenic biofilm was evident (3b).

Figure 3 also displays representative images of bacterial viability through confocal laser microscopy, demonstrating a higher number of live cells, stained in green, in images depicting the cariogenic biofilm formation (3d) and the L. reuteri biofilm (3f). In contrast, the image representing the combination of the cariogenic biofilm + L. reuteri showed a higher number of non-viable cells, stained in red (3e).

These findings are confirmed in the results of Figure 4, where bacterial viability is expressed as a percentage, obtained through the transformation of arbitrary fluorescence units. For the cariogenic group, a viability of 61.82% was obtained, comparable to the L. reuteri group with 63.63%. In contrast, the combination group of cariogenic biofilm and L. reuteri recorded a viability of 43.46%.

Regarding the enamel surface coverage in the 32 SEM-acquired images analyzed, a marked disparity in score distribution was observed among the evaluated groups, revealing statistically significant differences (p = 0.0025). Specifically, it was found that in both the cariogenic bacteria group and the L. reuteri group, approximately 60% of the examined areas exhibited bacterial coverage over the majority of the surface (67 to 100%), corresponding to a score of 4. In stark contrast, this condition was observed in 31.2% of the bacteria combination group. Conversely, a score of 1, reserved for images showing <5% coverage, corresponded to 9.3% in the cariogenic bacteria group, while no images from the L. reuteri group reached this classification. However, in the bacteria combination group, 34.3% of the images were assigned a score of 1.

Upon quantitative analysis of these scores across the different groups, a statistically significant difference was detected (p = 0.0031), as both the cariogenic bacteria group and the L. reuteri group yielded an average score exceeding 3, as opposed to the combined group which exhibited an average score of 2.5 (see

Table 1. Scores of surfaces covered by biofilms on dental enamel in the evaluated groups.

Resolution 1000×	Cariogenic Group	L. reuteri Group	Cariogenic + L. reuteri Group	p
	Frequency %
Score of 1	3 (9.3)	0 (0)	11 (34.3)	0.0025 *
Score of 2	4 (12.5)	2 (6.2)	4 (12.5)
Score of 3	6 (18.7)	12 (37.5)	7 (21.8)
Score of 4	19 (59.3)	18 (56.2)	10 (31.2)
	Mean + SD (Range) Median
	3.28 + 1.02(1–4)4	3.5 + 0.62(2–4)4	2.5 + 1.27(1–4)3	0.0031 **

Score of 1: clean enamel or isolated residual microbial cells covering less than 5% of enamel surface; score of 2: coverage from 5% to 33%; score of 3: coverage from 34% to 66%; score of 4: coverage from 67% to 100% of enamel surface. n = 32. * Kruskal–Wallis–Dunn’s test; ** chi-square.

).

4. Discussion

We recognize the importance of evaluating the impact of L. reuteri on biofilm formation, as this can generate dysbiosis, leading to oral diseases. It is crucial to understand the oral microbiome as a complex ecosystem composed of numerous bacterial species that interact with each other and maintain a balance, since any alteration in its composition could lead to the development of diseases such as dental caries [24].

Dental caries affects a large percentage of the world’s population and represents a major public health problem. Its etiology is multifactorial and is related to dysbiosis in the oral microbiome, which leads to the production of acids through bacterial metabolism, primarily by S. mutans, triggering the demineralization process of dental tissue [25].

Among the promising preventive strategies is bacteriotherapy, which involves the administration of probiotics, live microorganisms that, in adequate quantities, confer health benefits to the host, according to the definition of the World Health Organization (WHO) [9]. Several mechanisms of action of probiotics against cariogenic pathogens have been proposed, including the release of bacteriocins, antimicrobial effects through coaggregation, and competition with cariogenic bacteria for nutrients and adhesion sites [10].

Despite growing research in this field, the effectiveness of probiotics in preventing dental caries remains inconclusive. In this study, the inhibitory capacity of some candidate bacteria, especially L. reuteri, was evaluated against the main bacteria associated with dental caries, S. mutans and S. sobrinus, using various techniques. The results revealed that L. reuteri, as well as the other two bacteria tested, S. salivarius [26] and S. oralis [14], were capable of inhibiting the growth of these cariogenic bacteria to varying degrees.

In direct competition tests in solid culture, L. reuteri, together with S. salivarius, were the most effective at inhibiting both S. mutans and S. sobrinus, compared to S. oralis, which presented lower inhibition zones. S. salivarius demonstrated a greater inhibitory capacity on S. sobrinus than on S. mutans. These findings are consistent with previous research using similar assays, where bacteria–bacteria contact was evaluated including various species of lactobacillus, especially L. reuteri. In these studies, higher values than those in the present report were obtained, but with consistent conclusions (10 to 23 mm and 7 respectively) [27,28].

There is evidence that L. reuteri expresses an inhibitor of sticky glycans [29] and both S. mutans and S. sobrinus are producers of insoluble and sticky glucans as biofilm material [30]. Furthermore, L. reuteri is a producer of reuterin, a bacteriocin that has antimicrobial properties against a wide range of microorganisms, both Gram-positive and Gram-negative. It is water-soluble and resistant to some proteolytic enzymes [31].

In the case of S. oralis, it has been reported that it is a potent producer of hydrogen peroxide through the pyruvate oxidase enzyme SpxB; however, it showed the lowest inhibitory capacity on both bacteria [32]. This finding is valuable since S. sobrinus has demonstrated great cariogenic potential compared to S. mutans, even in aspects such as adhesion capacity.

The liquid culture assays provide evidence of the inhibitory potential of the bacteria and their virulence factors, given that the contact was direct, in contrast to previous studies that have focused only on the effects of supernatants [27]. Depending on the strain studied, the inhibitory mechanisms include the release of fatty acids, hydrogen peroxide, competition for nutrients, and the production of bacteriocins [33].

It is relevant to mention that the tests were carried out by the absolute quantification of the number of copies per milliliter of S. sobrinus culture, representing the cariogenic strains, using the real-time qPCR technique. This method is a robust molecular test with great sensitivity and specificity, allowing precise quantification of the DNA of the microorganism of interest. In these trials, S. sobrinus was co-cultured with each of the three probiotic species.

The results indicated that although L. reuteri showed a tendency to inhibit the growth of S. sobrinus, reducing the counts by an order of magnitude compared to the control, a statistically significant difference was not observed, as in the case of S. oralis and S. salivarius. The aforementioned bacteria, although they have probiotic benefits, are also associated with infective endocarditis, with prevalences that vary depending on the species [34]. For this reason, our evaluation focused on the comparative effect, and we did not delve into its probiotic potential as much as we did with L. reuteri.

Importantly, the exact mechanism by which probiotics interfere with cariogenic pathogens is still not fully understood [10] but could be attributed to pH modulation and competition for nutritional resources [35].

It is also relevant to mention that this methodology has not been used in other studies and could be considered a suitable option to have a more precise view of the inhibitory capacity of the probiotics, due to its quantitative nature and its wide dynamic range of detection.

In order to obtain a more detailed understanding of the colonization, adhesion and coaggregation of the cariogenic/probiotic biofilm on the tooth enamel surface, qualitative adhesion assays were carried out, followed by observations under scanning electron microscopy. Different growth patterns were observed. It is important to highlight that microorganisms exhibit different morphologies: while cariogenic strains appear as cocci arranged in chains, probiotics (L. reuteri) adopt elongated shapes, initially being able to have a sporulated shape, but differentiating from each other.

In the micrographs showing the growth of the cariogenic biofilm, large agglomerations of bacteria stacked on practically the entire surface were observed, in contrast to the growth of the L. reuteri biofilm, characterized by small, homogeneous, separated, and barely overlapping groups, covering most of the area.

It is important to mention that both the individual biofilms and the co-culture started from the same concentration of bacteria to allow a precise comparison of colonization and adhesion patterns. In the micrographs showing the co-culture, it was possible to identify that, although there were colonies of agglomerated bacteria, they did not occupy large areas of the surface, evidencing notably less microbial growth. It is relevant to highlight that the cariogenic film in this study was multi-species in contrast to previous research focused on monospecies biofilms of S. mutans. It is confirmed that a composite biofilm tends to exhibit greater aggressiveness, thus evidencing the probiotic potential of L. reuteri. In similar studies in cariogenic biofilms of multiple species, this probiotic significantly decreased the loss of enamel minerals, the number of bacteria on the surface, and the cariogenic activity of the biofilm, showing superior anti-caries potential compared to other microorganisms [36].

According to a previous report, it is necessary for probiotics to adhere to the dental tissue together with the cariogenic microbiome to establish a competition effect that plays a cariostatic role, which is a fundamental requirement for the effective confrontation of cariogenic bacteria [4].

In a quantitative analysis, the extent of the surface covered by biofilms was evaluated. It was observed that both the cariogenic group and the L. reuteri group, in the majority of the specimens evaluated, presented level 4, which corresponds to more than two-thirds of the surface covered by bacteria. In contrast, in the cariogenic/L. reuteri group, 30% presented that level of coverage, while 30% showed less than 5% of the surface with bacteria. These results confirm what was observed in the qualitative analysis, indicating that L. reuteri generated an inhibitory change in colonization, competition, adhesion, and coaggregation.

As mentioned, L. reuteri can inhibit the synthesis of insoluble glucans by S. mutans and S. sobrinus through the expression of a substance that inhibits the glucosyltransferase D (gtfD) genes, which participate in the formation of soluble glucans, thus altering the adhesion of these bacteria [28]. This could be due to changes in the structure and quantity of the exopolysaccharide matrix formed by co-culture, as observed in studies on 24 h biofilms, where a lower number of bacteria was recorded. It is relevant to highlight that this modification in the biofilm could be related to the ability of certain Lactobacillus strains to generate hydrogen peroxide, a potentially toxic substance for organisms that lack hydrogen peroxide-eliminating enzymes, such as S. mutans [27]. These findings coincide with previous research that has studied strains of L. reuteri, demonstrating a significant inhibition in the proliferation and formation of biofilms of S. mutans, possibly due to the ability of lactic acid-producing bacteria to generate organic acids and thus inhibit the accumulation of cariogenic biofilm [12].

Certain Lactobacillus strains generate hydrogen peroxide, a potentially toxic substance for organisms that lack hydrogen peroxide-eliminating enzymes, such as S. mutans [25]. These findings coincide with previous research that has studied strains of L. reuteri, demonstrating significant inhibition in the proliferation and formation of biofilms of S. mutans, possibly due to the ability of lactic acid-producing bacteria to generate organic acids and thus inhibit the accumulation of cariogenic biofilm [12].

Because bacterial cell viability was not evaluated in any of the previous assays, a cell viability analysis was carried out using a fluorescence life or death assay. This analysis allowed us to determine the percentage of viable cells in each sample. The results revealed that in both the cariogenic and L. reuteri biofilms separately, the majority of cells remained viable. In contrast, in the combination, most of the cells had lost viability. This finding highlights the importance of cell functionality in addition to the total count since the production of acids as a final product of bacterial metabolism is the main etiological factor of dental caries. These results were consistent with previous findings that also showed a predominance of nonviable cells in the combined group. This variation could be attributable to previously described phenomena such as the inhibition of cariogenic microbial biofilm, adhesion, competitive colonization, and coaggregation with pathogens to inhibit cariogenic microorganisms.

As a limitation, our study only shows a part of the characterization of this probiotic potential; it does not allow us to reflect the impact of a more complex and dynamic oral microbiome, host responses, oral hygiene behaviors, or diets on cariogenicity. Due to this, we decided, in future studies, to carry out in vitro tests with dental plaque samples from pediatric patients with caries and without caries, which will be a working model where we evaluate the effect of the complex microbiome and the role of probiotics for their effect in the oral cavity.

5. Conclusions

The findings of this study suggest that L. reuteri could be a highly effective probiotic in bacteriotherapy for the prevention of dental caries. This microorganism demonstrated significant inhibitory capacity in vitro against cariogenic bacteria responsible for the initiation of dental caries. Additionally, it effectively inhibited the formation and adhesion of cariogenic biofilms, impacting the cell viability of these pathogens. These results indicate that L. reuteri could play a crucial role in maintaining a balanced oral microbiome, representing a valuable preventive resource against dental caries.

However, it is important to note that this study was carried out in a controlled in vitro environment, with these assays being very useful to complement the characterization and detection of possible probiotic candidates. Therefore, additional clinical studies will be necessary to validate these findings and determine the long-term effectiveness of probiotics in preventing dental caries in humans.