Microbiome is the community of microbial residents in human body and oral microbiome is the microorganisms resided in the oral cavity. It is known that oral microbiome is the second largest microbial habitat in humans after the gut and oral bacteria are the main components of this oral microbiota. ¹ It is a home for more than 700 bacterial species because oral cavity can provide a favorable habitat for the growth of microorganisms. ^{1 , 2} Bacteria could have a stable environment to survive in oral cavity with the constant average of temperature is 37°C and steady pH of 6.5-7. ¹

Generally oral microbiome consists of a core microbiome and a variable microbiome. The core microbiome is community of predominant species that are present in various parts of the body under healthy condition. Meanwhile, a variable microbiome is a community of microorganisms species that varies and is exclusive between individuals because it has evolved in response to unique lifestyle and genotypic determinants. ¹

The fetus inside the uterus is usually sterile. The baby comes in contact with the microflora for the first-time during delivery, specifically with the uterine microflora and the mother’s vaginal microflora. At birth, the baby then also comes into contact with atmospheric microorganisms. Normally, the newborn’s oral cavity is sterile and regularly inoculated with microorganisms from the first feeding onward, and the process of acquisition of resident oral microflora begins. ¹ Previous studies suggest that there were significant changes in the composition of the microbiome over time in humans, and this was caused by many factors. One of the influencing factors is the decrease in salivary flow rate and oral pH as an individual gets older. ³

Oral microbiomes usually reside in the oral cavity as biofilms. Maintaining the homeostasis of this biofilm is important to protect the oral cavity. ¹ The physiology and ecology of the microbiota are closely related to the host, this has an impact on the promotion of health or the development of disease. Since the oral cavity is the main gateway to the body, it can also lead to the spread of infection to other parts of the body causing systemic disease. ⁴ It is currently recognized that unresolved inflammation promotes conversion to a dysbiotic community, and host-related intrinsic factors and defense mechanisms may play a major role in disease pathogenesis. ^{2 , 5} Antibiotics are commonly used in dentistry to treat oral diseases, not infrequently they are also used as prophylactic treatment. ⁶ On the other hand, excessive use of antibiotics can increase the risk of developing antimicrobial resistance. ⁷ Unfortunately, the current condition of antimicrobial resistance has been declared by the WHO (World Health Organization) as a global public health emergency and is known as a silent pandemic. ⁸

Probiotics are living and viable microorganisms which in adequate quantities will provide benefits to the host. ⁹ Probiotics can improve oral flora through a bacteriotherapy mechanism which is increasing the composition of harmless microflora to maintain or restore beneficial oral flora and modulating the oral microbiota to prevent pathogen colonization. ¹⁰ In addition to bacteriotherapy mechanisms, probiotics can also prevent or treat oral microbiota dysbiosis through stimulation of the host’s immune system and produce molecules or substances with antimicrobial effects. ^{9 , 11 , 12} These concept provides an alternative therapy to fight infection with fewer side effects and is safer than antibiotics. ¹⁰

Over the past few years, probiotic therapy has been used as an adjunct treatment in clinical dentistry. ¹³ For oral or dental purposes, probiotics need to survive in the oral ecosystem. ¹⁴ In terms of oral health, Limosilactobacillus reuteri has been extensively researched and is considered one of the most studied probiotics. ¹⁵ Limosilactobacillus reuteri is known for its ability to control oral microbiota through reuterin, a wide-spectrum antimicrobial agent. ¹⁶ However, some studies discovered that the Limosilactobacilluss reuteri probiotic should not be recommended as an adjunct treatment for oral infection as there were no microbiota alterations between the test and control groups. ^{11 , 17 , 18} The purpose of this study is to provide a comprehensive overview of the existing literature on the antimicrobial effect of Limosilactobacillus reuteri on oral bacteria. This review aims to synthesize and map out prior research in order to identify knowledge gaps and research needs related to this topic. By examining the breadth and depth of available literature, this review will evaluate the quality and quantity of existing research on the subject, and inform future research and practice. Ultimately, this scoping review aims to provide a foundation for future research and practice in this important area of alternative antimicrobial drug research.

Antibiotic resistance is progressively increasing. This emphasizes the urgency of finding alternative antibacterial substances. ¹⁹ Many studies have tried to find alternative methods, other than antibiotics, to treat oral cavity diseases, one of which is through biofilm inhibitors. Quorum sensing (QS) research is one of the new frontiers in this regard. ²⁰ Quorum sensing is a chemical signaling process of microorganisms that aims to monitor and regulate their population density. This chemical signaling system allows bacteria to coordinate their behavior and enhance their survival skills to respond to environmental changes through the growth and synthesis of biofilm matrix, enabling them to increase their resistance to conventional therapies such as antibiotics and antiseptics. ^{20 , 21}

Generally, oral biofilms consist primarily of commensal species that are harmless to the host, but this balance may be altered by local or systemic causes, leading to a dysbiotic state or a condition characterized by increased number of pathogenic species. The involvement of quorum sensing mechanisms in facilitating biofilm formation helps bacteria perpetuate this dysbiotic state. ²⁰ On the other side, interest in probiotics has tremendously risen over the last 10 years, and several health claims have been made regarding probiotics’ antibacterial activity. ^{19 , 21} In digestive studies, probiotics have a good adhesion to the gut, effectively enhancing intestinal epithelial homeostasis and interfering with the quorum sensing that favors a dysbiotic state. ²² Since probiotics act on biofilm composition, this current review is important as a preliminary knowledge to identify the effect of Lactobacillus reuteri on pathogenic bacteria in the oral cavity. ¹⁵

Six studies in this review analyzed the antibacterial properties of Limosilactobacillus reuteri against Mutans streptococci in healthy humans. Despite variations in probiotic delivery mechanisms, intervention duration, and observation periods, these studies found an overall positive antibacterial effect. Additionally, among the six studies, four used Limosilactobacillus reuteri ATCC 55730, and two studies combined this strain with Limosilactobacillus reuteri ATCC PTA 5289. ^{23 – 27} Two study performed by Alamoudi et al. (2018) and Badri et al. (2020) also used Limosilactobacillus reuteri DSM 17938 and ATCC PTA 5289. ^{27 , 28} Nikawa et al. (2004) conducted a study with a 2-week intervention period where Limosilactobacillus reuteri was administered via yoghurt, and the count of Mutans streptococci was observed for 4 weeks. ²⁶ Five other studies observed the count of Mutans streptococci immediately after the intervention duration. ^{23 – 25 , 28} The results of these studies showed that Limosilactobacillus reuteri has an immediate and long-term effect of up to two weeks against Mutans streptococci. Related to this result, Caglar et al. (2009) conducted a study to investigate whether Limosilactobacillus reuteri ATCC 55730 survived in the oral cavity after the discontinuation of intervention. They found that Limosilactobacillus reuteri decreased gradually, where after 1 week, only 8% of bacteria were detected, and after 5 weeks, the bacteria were completely undetected. ²⁹

One limitation of the findings on Mutans streptococci presented in this scoping review is that the subjects included in the literature are all healthy individuals. While these studies provide valuable insights into the prevalence and distribution of Streptococcus mutans in healthy populations, they may not necessarily reflect the same patterns observed in individuals with other oral health conditions. In fact, other studies have shown that there are significant differences in the levels of Mutans streptococci in individuals with severe periodontitis compared to healthy individuals. ³⁰ Therefore, future research should aim to explore the distribution of Mutans streptococci in a more diverse range of populations, including those with varying levels of oral health conditions. This will help to better understand the role of Mutans streptococci in the development and progression of oral diseases and inform more targeted prevention and treatment strategies.

Four of the six previously mentioned studies also analyzed salivary lactobacilli count as a dependent variable. All of the studies performed with healthy adults had the result that Limosilactobacillus reuteri has no significant antibacterial effect on salivary lactobacilli. ^{23 – 26} However, a study by Alamoudi et al. (2018) with healthy children as participants, discovered a positive antibacterial effect of Limosilactobacillus reuteri on salivary lactobacilli. ²⁷ This result may be influenced by the dental hygiene performed by the children using the provided fluoride toothpaste. It may also be due to the absence of salivary lactobacilli in newborns, and 40% of the 3-year-old population have it to varying degrees. Whereas salivary lactobacilli in adults depend on ecological conditions, such as pits and fissures or partially erupted wisdom teeth which provide an environment that supports its growth. ³¹

The studies performed on healthy subjects concerning Mutans streptococci and salivary lactobacilli count were based on the prior knowledge that both microbiomes are related to dental caries. One of the salivary lactobacilli species, Lactobacillus acidophilus, has characteristics that will increase significantly 2-3 months before dental caries appear. This phenomenon is called the “explosion of Lactobacillus”. However, the main oral acid production was not from lactobacilli, thereby making it only a secondary microbiome in dental caries. Lactobacillus cannot adhere to tooth surfaces on their own, as they need retention niches. On the other hand, Streptococcus mutans could produce extracellular polysaccharides to help them adhere to the tooth structure. Streptococcus mutans’ main feature is its acidophilic nature. This allows it to become the dominant bacteria in the oral cavity during acidic conditions. Moreover, the intracellular polysaccharides in Streptococcus mutans create energy reserves, so the level of acid produced, especially lactic acid, remains constant even when external sugar intake is low. Hence, Mutans streptococci and salivary lactobacilli are strongly associated with dental caries. ³²

Laleman et al. (2019), Tada et al. (2017, and Galofre et al. (2017) used peri-implantitis patients as their research subjects. These three studies analyzed the antibacterial effect of Limosilactobacillus reuteri DSM 17938 and ATCC PTA 5289 on Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans. Furthermore, Tada et al. (2019) and Galofre et al. (2017) also analyzed Treponema denticola and Tannerella forsythia, whereas only Galofre et al. (2017) analyzed Peptostreptococcus micros, Campylobacter rectus, and Eikenella corrodens. All of the patients received non-surgical mechanical debridement of the peri-implant sites. ^{18 , 33 , 34} Two of these studies found that there were no significant differences between the treated and control groups in microbiome counts after 24 weeks and 30 days of intervention with Limosilactobacillus reuteri lozenges. ^{18 , 34} However, the study conducted by Laleman et al. (2019) that used probiotic drops as the drug delivery mechanism found different results compared to the other two studies. The intervention period was 12 weeks and the observation time was the baseline, week 6, week 12, and week 24. This study also evaluated the microbiome counts in the following three sites: saliva, tongue, and subgingival. The count of Porphyromonas gingivalis was only reduced in the saliva of the treated group at the week 6 observation. The count of Aggregatibacter actinomycetemcomitans significantly decreased in the saliva and tongue at week 12. On week 24, the count of Aggregatibacter actinomycetemcomitans also decreased in the saliva. Next, the count of Prevotella intermedia in the tongue of the treated group showed a significant decrease in all of the observation periods, and a decrease was also found in the subgingival area on week 24. ³³ These varying results may be influenced by the drug delivery mechanisms and the sites evaluated. Nevertheless, we can conclude that the number of peri-implant microbiota evaluated at the subgingival site was generally not affected by Limosilactobacillus reuteri, whether administered by lozenge or drops. Thus, similar studies need to be conducted for the tongue area and saliva evaluation, and a larger number of samples needs to be used to obtain conclusive results.

Regarding peri-implant mucositis patients, four of the selected studies analyzed the antibacterial effect of Limosilactobacillus reuteri DSM 17938 and ATCC PTA 5289 on Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. ^{11 , 34 – 36} Three of the previous four studies also analyzed Prevotella intermedia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Campylobacter rectus. Two of these three studies analyzed Peptostreptococcus micros and Eikenella corrodens, whereas the other study analyzed Fillifactor alocis, Porphyromonas endodontis, and Parvimonas micra. ^{34 – 36} Overall, it can be concluded that Limosilactobacillus reuteri had no antibacterial effect on Tannerella forsythia and Treponema denticola in peri-implant mucositis patients as there was no positive result from the four studies despite differences in the intervention period, observation time, and drug delivery mechanisms. ^{11 , 34 – 36} Similar negative results from the three studies were also noted in Prevotella intermedia, Aggregatibacter actinomycetemcomitans, and Campylobacter rectus. Additionally, Eikenella corrodens was found to be not affected by Lactobacillus reuteri in two studies. ^{34 – 36}

Porphyromonas gingivalis in peri-implant mucositis patients was found to be significantly affected by Limosilactobacillus reuteri application via probiotic lozenge for thirty days. However, this result was only positive on day-90 observation time while the study also performed observation on days 30 and 60. Nevertheless, as previously described, Porphyromonas gingivalis in the saliva of peri-implantitis patients was significantly affected by Limosilactobacillus reuteri in the week-6 of observation and there was no significant difference in the week 12 and week 24 samples. However, these results were considered inconclusive due to the small number of samples and require further research. ³⁴

Pena et al. (2018) conducted a study with no statistically significant different results between the treated and control group in all peri-implant microbiota after administering Limosilactobacillus reuteri tablets from day 15 until day 45 of the study. However, he also analyzed the difference between the bacteria count on the day of observation and at the beginning of the study in each group, then he compared those numbers between both groups. This method resulted in significantly reduced Peptostreptococcus micros in the treated group compared to the control group, as indicated by the difference in bacterial counts between day 15 and day 45, and between day 135 and baseline. Another positive result was also found in Fusobacterium nucleatum in bacterial count on day 135 minus the bacterial count on the baseline. ³⁶

One study conducted by Iniesta et al. (2012) provided evidence that the administration of Lactobacillus reuteri DSM 17938 and PTA 5289 probiotic tablets for 28 days effectively reduced the total anaerobic bacteria counts in saliva samples after 4 weeks and Prevotella intermedia counts after 8 weeks in gingivitis patients. This probiotic combination exhibited inhibitory effects against various periodontopathogens, such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, Capno, Eikenella corrodens, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans in saliva samples. However, despite the significant total microbiological changes observed, these reductions in the target species did not translate into any clinically significant outcomes, as the inter-group differences in the clinical variables were not significant. This lack of clinical efficacy could be attributed to either the sample population or the short evaluation period. On the other hand, a significant reduction was observed only in Porphyromonas gingivalis counts in the subgingival sample from baseline to 4 weeks. These findings suggest that using Lactobacillus reuteri DSM 17938 and PTA 5289 probiotic tablets as an adjunct therapy could be promising for managing gingivitis and its associated periodontal pathogens, particularly Porphyromonas gingivalis. ³⁷

Three articles in this scoping review examined the use of Limosilactobacillus reuteri DSM 17938 as a probiotic intervention in periodontitis patients. Of these three studies, one included diabetic patients with periodontitis, while the other two used healthy individuals with no systemic disease. The probiotic was administered in two studies as a tablet containing a combination of Limosilactobacillus reuteri DSM 17938 and ATCC PTA 5289, while the third study used only Limosilactobacillus reuteri DSM 17938 in the form of a suspension delivered through a blunt syringe to subgingival sites. ^{38 – 40} Interestingly, the study with diabetic patients showed a significant reduction in the number of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola after three weeks of probiotic tablet use, which was sustained after a three-month evaluation. ³⁸ On the other hand, in line with the previous result, the study conducted by Vivekananda with a split-mouth study design in India found that only the treatment modalities that included the Limosilactobacillus reuteri tablet, either alone or in combination with scaling root planing, were able to significantly reduce the counts of pathogens tested ( Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia). ³⁹ Lastly, the study conducted by El-bagoory et al. (2021) showed a significant reduction in the number of Porphyromonas gingivalis on the three-month and six-month evaluations after only four applications of the probiotic suspension delivered to subgingival sites, even though it was administered only at baseline, week 1, week 2, and week 4. ⁴⁰

The effectiveness of Limosilactobacillus reuteri in treating specific periodontal pathogens in peri-implant mucositis and periodontitis patients can vary depending on the distinct microbial network shape present in these niches. Peri-implant biofilm is more homogeneous and similar to supragingival biofilm than subgingival biofilm, while subgingival biofilm in periodontitis patients contains a higher number of microorganisms. In addition to this, the host response and habitat structure surrounding implants and teeth can also affect the microbial community structure and the effectiveness of probiotics. ⁴¹ Furthermore, the rough surface of dental implants increases the rate of biofilm formation around the implant, and surface roughness contributes to increased plaque buildup. ⁴²

Based on our review, it was found that Limosilactobacillus reuteri is a probiotic bacterium that exhibits varying levels of antimicrobial activity against different pathogens. However, its effectiveness can be further enhanced by combining it with other clinical care and active ingredients such as natural compounds or prebiotics. For instance, Holm et al. (2022) showed that the antimicrobial effectiveness of Limosilactobacillus reuteri (ATCC PTA 5289) can be improved by combining it with glycerol against periodontal pathobionts and anaerobic commensals. This may be attributed to the increased production of reuterin by Limosilactobacillus reuteri from glycerol. ⁴³ Hence, combining Limosilactobacillus reuteri with other active ingredients such as natural compounds or prebiotics may be a useful strategy for improving its antimicrobial efficacy. However, further details on this strategy need to be explored, including relevant references and future research perspectives. It is worth mentioning that some newly introduced compounds have a significant influence on the oral environment. The use of paraprobiotics, lysates, and postbiotics can modify clinical and microbiological parameters in periodontal patients. ^{44 – 46} Therefore, these products should be considered in combination with Limosilactobacillus reuteri in future clinical trials.

In conducting a scoping review, it is important to consider the limitations of the study design in order to interpret the findings in a meaningful way. One limitation of the current scoping review is the potential for bias in quality assessment due to the inclusion of a non-randomized study with a high risk of bias. However, the review also included 15 other studies, all of which were randomized control trials. Of these, 9 studies were found to have a low risk of bias, while 6 studies had “some concerns” regarding risk of bias based on the Cochrane Risk-Of-Bias (RoB 2) Tool. Although the non-randomized study may have impacted the overall quality of the evidence included in the review, the findings of the randomized control trials provide important insights into the effectiveness of the interventions studied. Moreover, the inclusion of grey literature in the search strategy can help to enhance the comprehensiveness of the review. Overall, while limitations should be acknowledged and considered in the interpretation of the findings, the scoping review still provides valuable insights and can serve as a foundation for future research in the field.

Furthermore, in this scoping review, it was found that several studies included in the analysis received materials and funding from a sponsor. Additionally, one author from a study received a PhD grant from the sponsor, while another author received a lecturing fee. Nonetheless, the authors of these studies stated that the involvement of the sponsor did not have any impact on the results of their research. It is essential to acknowledge the presence of external funding sources and their potential influence on study outcomes, as transparency in reporting is crucial in scientific research. Despite the presence of sponsorship, the authors of the reviewed studies made it clear that the research conducted was not compromised, and their findings were not influenced by the sponsors. However, it is necessary to approach the results with caution, as the presence of external funding sources can still potentially affect the outcome of research studies.

Limosilactobacillus reuteri exhibits a strong antibacterial effect on Streptococcus mutans and has a less significant impact on Lactobacilli in healthy individuals. Additionally, Limosilactobacillus reuteri has antibacterial properties against Porphyromonas gingivalis in periodontitis and gingivitis patient although its effectiveness is not stable in patients with peri-implant infections. The study suggests that Lactobacillus reuteri does not have a noteworthy impact on Campylobacter rectus and Eikenella corrodens. However, the effectiveness of Limosilactobacillus reuteri varies concerning other bacteria such as Prevotella intermedia, Peptostreptococcus micros, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Fusobacterium nucleatum. Further research is essential to examine the antibacterial properties of Limosilactobacillus reuteri on the oral/dental microbiomes, taking into account variations in the strains used, intervention periods, subjects, and observation times. This research is crucial in advancing our understanding of Limosilactobacillus reuteri’s potential as a probiotic for maintaining oral/dental health.