|Year : 2019 | Volume
| Issue : 2 | Page : 50-55
Inhibitory effect of probiotic lactobacilli against Streptococcus mutans and Porphyromonas gingivalis biofilms
Armelia Sari Widyarman1, Endang Winiati Bachtiar2, Boy Muchlis Bachtiar2, Chaminda Jayampath Seneviratne3
1 Department of Microbiology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
2 Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia
3 National Dental Centre Singapore, SingHealth Academic Clinical Programme, Duke National University of Singapore, Singapore
|Date of Web Publication||18-Jun-2019|
Dr Armelia Sari Widyarman
Department of Microbiology, Faculty of Dentistry, Trisakti University, Jakarta
Source of Support: None, Conflict of Interest: None
Background: Lactobacillus reuteri and Lactobacillus casei have been proposed as probiotic bacteria that promote oral health. Objectives: The present study aimed to evaluate the in vitro effects of L. reuteri and L. casei on the biofilm formation of major oral pathogens, Streptococcus mutans and Porphyromonas gingivalis. Materials and Methods: L. casei strain Shirota and L. reuteri ATCC 55730 were isolated from the commercial products and cultured in de Man, Rogosa, and Sharpe broth. Polymerase chain reaction was used to confirm the identity of the species. S. mutans ATCC 25175 and P. gingivalis ATCC 33277 were cultured in brain–heart infusion broth and used for biofilm formation on 96-well microplate platform. The biofilms were treated with the probiotics and appropriate controls in a time-dependent experiment from 15 min to 24 h. The biofilm biomass was evaluated using crystal violet and safranin. Results: The statistical analysis showed a significant reduction in the S. mutans and P. gingivalis biofilms after treatment with the L. reuteri and L. casei probiotics at all incubation times (P < 0.05). Conclusion: The present study demonstrated the potential antibiofilm activity of L. casei strain Shirota and L. reuteri ATCC 55730 against S. mutans and P. gingivalis biofilms in vitro. The foregoing data have formed a basis for future clinical studies to evaluate the beneficial oral health effect of probiotic Lactobacilli strains.
Keywords: Biofilm, Lactobacilli, Lactobacillus casei, Lactobacillus reuteri, probiotics
|How to cite this article:|
Widyarman AS, Bachtiar EW, Bachtiar BM, Seneviratne CJ. Inhibitory effect of probiotic lactobacilli against Streptococcus mutans and Porphyromonas gingivalis biofilms. Sci Dent J 2019;3:50-5
|How to cite this URL:|
Widyarman AS, Bachtiar EW, Bachtiar BM, Seneviratne CJ. Inhibitory effect of probiotic lactobacilli against Streptococcus mutans and Porphyromonas gingivalis biofilms. Sci Dent J [serial online] 2019 [cited 2019 Sep 16];3:50-5. Available from: http://www.scidentj.com/text.asp?2019/3/2/50/260562
| Background|| |
Dental caries and periodontal disease are highly prevalent oral infectious diseases worldwide. The etiological agent for the foregoing diseases is pathogenic microorganisms residing in the dental plaque biofilm. Dental plaque biofilm is formed on tooth surfaces coated with a salivary pellicle. Oral biofilms are composed of diverse microorganisms, including both Gram-positive and Gram-negative bacteria. These biofilm bacteria are embedded in an extracellular polymeric substance making them more resistant to the antibiotics. Hence, researchers have attempted to alternative method to control the pathogenic transformation of dental plaque biofilms.
Streptococcus mutans is a Gram-positive facultative anaerobic bacterium that has been implicated as a primary cause of dental caries in humans. In the human oral cavity, S. mutans adheres to the tooth surfaces and becomes a part of the dental plaque biofilm. It produces acid by fermenting the dietary sucrose, which leads to demineralization of the tooth substance. Porphyromonas gingivalis, on the other hand, is a Gram-negative anaerobic bacterium associated with chronic adult periodontitis., P. gingivalis has been found in the periodontal pockets as a keystone pathogen associated with periodontal disease. Under favorable condition, P. gingivalis produces inflammatory molecules such as lipopolysaccharides that leads to periodontal tissue inflammation and eventually resulting in tooth loss.
Biofilm formation contributed to the increased antibiotic resistance of the bacterial cells. Therefore, effective therapeutic alternatives are required for the prevention and treatment of these oral infectious diseases. The use of probiotics has been suggested as a promising strategy for the biofilm-related diseases. In addition, some studies have provided evidence of the beneficial effect of probiotics on dental caries and periodontitis., Probiotics have been defined by the World Health Organization as living microorganisms that, when administered in a sufficient amount, it might be promoted health benefits in the host. In 1906, Tissier isolated the Bifidobacterium, which was subsequently used for the treatment of diarrhea. In 1907, Ellie Metchnikoff observed that Bulgarian people lived longer by consuming Bulgarian yogurt, which contained lactic acid bacteria. Recently, probiotics have been used successfully to control gastrointestinal diseases and reduce allergy symptoms. There are several strains that can be classified as probiotics, and they commonly belong to the Lactobacillus and Bifidobacterium genera.
Lactobacillus reuteri, a known probiotic bacteria, has been reported to produce a number of antibacterial compounds including reuterin, a broad-spectrum antimicrobial compound structurally related to 3-hydroxypropionaldehyde (3-HPA), as a result of glycerol fermentation. Reuterin has been shown to have inhibitory effects against pathogenic oral bacteria and also has the anti-inflammatory factors., According to Nikawa et al. 2004, L. reuteri in yogurt can decrease the oral carriage of S. mutans. For example, chewing gum containing L. reuteri ATCC 55730 resulted in the significant growth inhibition of S. mutans in saliva.
Lactobacillus casei is another probiotic bacteria that have an antagonist effect toward the pathogenic oral bacteria. The short-term daily consumption of L. casei strain Shirota in the children has shown potential cariostatic effects. There was also a significant reduction in P. gingivalis after the daily consumption of L. casei for 1 month., Hence, foregoing studies are suggestive of both L. reuteri and L. casei have the ability to inhibit pathogenic oral bacteria. However, the exact mechanism by which the inhibitory effect of these probiotics exerts on oral bacteria has not been explicitly shown to date. Taking this research gap, we hypothesized that these probiotic can inhibit the biofilm formation of oral pathogenic bacteria. Hence, the present study aimed to evaluate the activity of L. reuteri and L. casei against S. mutans and P. gingivalis biofilms in vitro.
| Materials and Methods|| |
Isolation and identification of Lactobacillus reuteri and Lactobacillus casei
The L. casei strain Shirota was isolated from Yakult® (Yakult Persada, Indonesia). In brief, 100 μl of the probiotic drink was spread on a de Man, Rogosa, and Sharpe (MRS) agar plate. The L. reuteri ATCC 55730 was isolated from BioGaia ProDentis® lozenges (Kalbe, Singapore). The tablet was crushed aseptically, suspended in phosphate-buffered saline, and inoculated on MRS agar. All the isolates were incubated at 37°C for 24 h, anaerobe conditions.
DNA extraction and polymerase chain reaction
The DNA extraction was performed using an Extract-N-Amp™ Tissue Kit (Sigma-Aldrich, Merck, Germany). For the preparation, 100 μl of the extraction solution was mixed with 25 μl of the tissue preparation solution. A 10 μl sample was pipetted into the solution and mixed by vortexing. The sample was incubated at room temperature for 10 min, then at 95°C for 3 min. For each sample, 100 μl of neutralization solution B was added. The extracts were stored at 4°C until used for the polymerase chain reaction (PCR).
The L. reuteri and L. casei specific 16S rDNA primer sequences, i.e., 5' ACC TGA TTG ACG ATG GAT CAC CAGT (forward); CCA CCT TCC TCC GGT TTG TCA 3' (reverse) and 5' TGG TCG GCA GAG CTG TTG TCG 3' (forward); and CCA CCT TCC TCC GGT TTG TCA 3' (reverse), respectively, were used for the PCR reaction. The PCR Master Mix contained the following: 10 μl of the DreamTaq (Thermo Scientific, USA), 2 μl of the forward primers, 2 μl of the reverse primers, 4 μl of water, and 2 μl of the DNA sample. The PCR was performed as follows: initial heating at 94°C for 2 min, followed by 35 cycles consisting of denaturation at 94°C for 20 s, annealing at 51°C for 40 s, extension at 68°C for 30 s, and a 7 min final extension step at 68°C. The amplicons were separated on a 1.5% agarose gel by electrophoresis.
Probiotic treatment on oral pathogenic biofilms
The L. reuteri ATCC 55730 and L. casei strain Shirota were cultured on MRS agar at 37°C for 72 h. Each suspension was centrifuged at 5000 ×g for 15 min, and diluted in MRS broth to achieve a concentration of 108 CFU/ml. Then, different concentrations of glycerol (62.5 mM, 125 mM, and 250 mM) were added to the L. reuteri suspension. The biofilm assays were performed as follows: S. mutans ATCC 25175 was cultured in the brain–heart infusion (BHI) broth and incubated in a CO2 enriched, 37°C atmosphere, while the P. gingivalis ATCC 33277 was cultured in BHI broth using the gas pack jar system. After 48 h, each suspension was diluted in saline to achieve a concentration of 107 CFU/ml. The suspensions were distributed into a 96-well microplate, which was incubated for 48 h to form the biofilms.
Each probiotic bacterial suspensions were tested against the S. mutans, P. gingivalis, and mixed-species (both oral pathogenic bacteria) biofilms. The probiotic suspension was added into the biofilm-containing wells, and the biofilm-inhibitiory effect was observed in a time-dependent experiment after 15 min and 3, 6, and 24 h. Crystal violet (0.5% w/v) and safranin (0.5% w/v) were used to stain the biofilm mass of the S. mutans and P. gingivalis, respectively. Next, the microplates were washed using saline. Then, 200 μl of ethanol (90%) was added to extract the remaining stain from the biofilm.
The biofilm mass was quantified by measuring the optical density (OD) at 655 nm using a microplate spectrophotometer. The biofilm-containing wells without the addition of probiotic bacteria were used as negative controls. The measurement of biofilm reduction in percentage was calculated from the blank, control, and treated absorbance values on the plate as follows:
Here, B denotes the average absorbance per well for blank wells (no biofilm, no treatment), C denotes the average absorbance per well for negative control wells (biofilm, no treatment), and T denotes the average absorbance per well in treated wells (biofilm, treatment). The assays were performed in triplicate. As a blank control, wells were filled with sterile broth.
The obtained results were statistically analyzed using one-way analysis of variance test to reveal significant differences of biofilm reduction between the experimental groups. P < 0.05 was considered statistically significant. Shapiro–Wilk test was used to test for normality, and Levene's test was used to test for homogeneity of variance previously. Statistical calculations were performed with SPSS Statistics for Windows software Version 20 (IBM, Armonk, NY, USA).
| Results|| |
Molecular identification of Lactobacillus reuteri and Lactobacillus casei
The results of the PCR assays were 720 bp for the L. casei and 1100 bp for the L. reuteri [Figure 1]. These results were confirmed the identity of both probiotic bacteria.
|Figure 1: Electrophoresis results (left to right): Lactobacillus casei Marker; Lactobacillus reuteri p)|
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Biofilm assays of Lactobacillus reuteri against Streptococcus mutans and Porphyromonas gingivalis
The biofilm ODs after incubation with L. reuteri and glycerol were decreased when compared to the negative control. The statistical analysis showed the significant reductions of S. mutans and P. gingivalis biofilms upon treatment with probiotic suspensions compared to the negative controls (P < 0.05). The most effective concentration for inhibiting the S. mutans biofilm was L. reuteri supplemented with 125 mM of glycerol at the 3-h incubation time with 73.05% of biofilm reduction [Figure 2]. For the P. gingivalis biofilm, the optimum inhibition occurred with 77.97% of biofilm reduction, when the L. reuteri were supplemented with the 250 mM glycerol concentration at the 15 min incubation time [Figure 3].
|Figure 2: Reduction of Streptococcus mutans biofilm (percentage) after probiotic treatment with Lactobacillus reuteri and Lactobacillus reuteri (108 CFU/ml) supplemented with glycerol at different concentrations (62.5 mM, 125 mM, and 250 mM) after 15 min, 3 h, 6 h, and 24 h of incubation|
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|Figure 3: Reduction of Porphyromonas gingivalis biofilm (percentage) after probiotic treatment with Lactobacillus reuteri and Lactobacillus reuteri (108 CFU/ml) supplemented with glycerol at different concentrations (62.5 mM, 125 mM, and 250 mM) after 15 min, 3 h, 6 h, and 24 h of incubation|
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Biofilm assays of Lactobacillus casei against Streptococcus mutans and Porphyromonas gingivalis
The result also showed there were significant reductions in the S. mutans and P. gingivalis biofilms (P < 0.05) in the incubation times after treated with L. casei. The most effective incubation time for inhibiting the S. mutans biofilm was L. casei at the 3 h period with 62.18% of biofilm reduction [Figure 4], and for the P. gingivalis biofilm, the best inhibition occurred with the L. casei at the 6 h incubation time with 46.88% of biofilm reduction [Figure 5].
|Figure 4: Reduction of Streptococcus mutans biofilm (percentage) after probiotic treatment with Lactobacillus casei (108 CFU/ml) after 15 min, 3 h, 6 h, and 24 h of incubation|
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|Figure 5: Reduction of Porphyromonas gingivalis biofilm (percentage) after probiotic treatment with Lactobacillus casei (108 CFU/ml) after 15 min, 3 h, 6 h, and 24 h of incubation|
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| Discussion|| |
In this study, we assessed the antibacterial effects of L. reuteri and L. casei probiotics against S. mutans and P. gingivalis biofilms, the major pathogens associated with dental caries and periodontitis, respectively. The L. casei and L. reuteri were successfully isolated from Yakult® and BioGaia Prodentis® lozenges, respectively. Both species were identified by using PCR with species-specific 16S rDNA primers. The probiotics bacteria used in the study showed a considerable inhibitory effect on S. mutans and P. gingivalis monospecies and mixed-species biofilms.
Our results are in agreement with the previous studies that have investigated the use of probiotic bacteria for various oral diseases, including dental caries, periodontitis, halitosis, oral Candida colonization, oral mucositis, and xerostomia., The commonly used probiotic bacteria include Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, L. reuteri, L. casei, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis, Streptococcus thermophilus, and Streptococcus salivarius. Of them, Lactobacillus spp. has been the most popular probiotic bacteria. The previous study showed that Lactobacillus spp. isolated from a subject without caries have the capacity to inhibit S. mutans, suggesting an anticaries potential. We have previously demonstrated that consumption of probiotic yogurt containing Bifidobacterium lactis reduce S. mutans in the saliva quantitatively from subjects with fixed orthodontic appliances. Further study showed that L. reuteri ATCC 55730-containing probiotic lozenges consumption reduces the number of S. mutans serotype c, Streptococcus sobrinus, P. gingivalis, and Aggregatibacter actinomycetemcomitans in saliva subjects during fixed orthodontic appliances.
Probiotic bacteria may exert effective inhibition of pathogens by several mechanisms. They can secrete antimicrobial substances, such as organic acids, hydrogen peroxide, and bacteriocins. They can also modify the pH and/or the oxidation-reduction potential of the surrounding environment., The stimulation of nonspecific immunity and modulation of the humoral and cellular immune responses are additional properties of probiotics. However, the effects of probiotic bacteria are both strain-specific and individual-specific.
Reuterin is one of the antimicrobial compounds secreted by probiotic bacteria. Reuterin is an equilibrium mixture of the hydrated, dehydrated, and dimeric forms of 3-HPA. L. reuteri secretes high levels of reuterin in the presence of excess amounts of glycerol. The antipathogenic activity of reuterin has been assessed against Gram-positive and Gram-negative bacteria, fungi, and protozoa. Previous research has been suggested that reuterin induces oxidative stress in cells, inhibiting bacterial growth by modifying the thiol groups in proteins and small molecules.,,
L. reuteri ATCC 55730 produced a relatively high amount of reuterin, even as a planktonic substance (31.89 mmol/10 cells), when compared to the other strains. According to the results of the present study, the optimal concentration of glycerol is 250 mM for the production of reuterin. Overall, the most effective incubation time to inhibit the S. mutans or P. gingivalis biofilms seemed to be 3 h [Figure 2] and [Figure 3]. Afterward, there was a dimension of the effectiveness of probiotic. In the mixed-species biofilms, the most effective incubation time was only 15 min and the effect decreased with time. Hence, it seems that oral pathogenic bacteria may overcome the effect of probiotic with time. Hence, more research is warranted to find a strategy to increase the effective time of the probiotic on these biofilms.
Another probiotic strain L. casei used in the present study significantly decreased the S. mutans biofilm and P. gingivalis on monospecies biofilm. Competitive inhibition may be one reason for this inhibition. The previous study reported L.casei might reduce S. mutans-induced dental caries in gnotobiotic rats. Its ability to attach to the enamel enables it to colonize the oral cavity and competes with S. mutans., Moreover, previous study isolated biosurfactants and amphipathic compounds from L. casei, which contributed to its antiadhesive and antibiofilm abilities against oral Staphylococcus aureus. However, L. casei was not effective against P. gingivalis biofilms. Unlike S. mutans, which uses water-insoluble glucans to adhere to tooth surfaces, P. gingivalis has fimbriae to attach to and penetrate the periodontal tissues., As Gram-negative bacteria, P. gingivalis releases an outer membrane vesicle that contains adhesins, toxins, hydrolytic enzymes, and lipopolysaccharides as antigens. These substances enhance the survival of P. gingivalis. Hence, P. gingivalis may have a mechanism to overcome the inhibitory effect of L. casei.
| Conclusion|| |
The present study demonstrated considerable antibiofilm activity of probiotic bacteria L. reuteri and L. casei against S. mutans and P. gingivalis biofilms. However, it should be noted that the inhibitory effect is time- and dose-dependent. Hence, more research is warranted to find optimal probiotic strategy to enhance probiotic effect on these oral pathogens.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]