Comparison of Antibacterial Effect of Probiotic Yogurt and Xylitol-Containing Chewing Gum in Geriatric Patients: A Randomized Controlled Clinical Trial.

OBJECTIVES: To evaluate the effect of probiotic bacteria in yogurt on Streptococcus mutans (MS) count, plaque adherence and salivary pH compared to xylitol-containing chewing gum in geriatric patients.
MATERIALS AND METHODS: Total number of 96 high caries risk geriatric patients were randomized into two equal groups (n=48). Group 1 (intervention group) received probiotic yogurt (Activia, Danone) once per day, and group 2 (control group) received xylitol chewing gum (Trident original) three times per day. The primary outcome was salivary Streptococcus mutans count and secondary outcomes were interdental plaque Streptococcus mutans count, salivary pH and bacterial adherence.
RESULTS: For Streptococcus mutans count in saliva and plaque, a statistically significant reduction in the level of MS over all the examined follow up periods of the study in probiotic yogurt group as well as xylitol gum group was found. An intergroup comparison for salivary MS count showed statistically significant difference between the two materials in a two week and a three month period of time and there was no statistically significant difference between both materials at one month time period. Salivary pH results showed statistically significant increase in pH in both groups along the follow-up periods. Bacterial adherence results showed statistically significant reduction in both groups.
CONCLUSIONS: Probiotic yogurt is an effective antibacterial agent against salivary and plaque bacteria in geriatric patients.

Introduction:

Dysbiosis of the biofilm, with change of the bacterial composition, occurs with stress factors (). Many factors can be classified as caries predictors: dietary changes, neglecting of oral hygiene, medical treatment or medicines that affect salivary flow and changes in host immune response are considered risk factors (). These factors lead to flourishing of cariogenic Streptococcus mutans, the most important bacterial species involved in the demineralization of tooth tissue and initiation of dental caries ().

In geriatric population, people's immune functions decline, the lack of awareness increases, and their ability for oral hygiene care decreases (). Antibacterial compounds such as xylitol have been suggested to reduce the activity of cariogenic bacteria (). The delivery of xylitol via chewing gum is considered as a convenient, effective and simple way for geriatric patients (). However, long term use of xylitol containing products causes adverse effects such as cramps, bloating, constipation, flatulence and loose stool or diarrhea ().

Probiotics are live microorganisms that provide benefits to oral health. They adhere to the oral mucosa and teeth surfaces and become part of the biofilm preventing the adhesion, colonization, and proliferation of other cariogenic bacteria inhibiting the formation of pathogenic plaque ()

Probiotics are found in different dairy products, including yoghurt, which are known as carriers of probiotics (). Therefore, the incorporation of dairy products that do not require a caregiver in their daily routine, can be used as a simple means of protection from caries in geriatric patients.

It was found beneficial to test the null hypothesis that probiotic yogurt has similar antibacterial effect in plaque and saliva, similar pH and similar effect on bacterial adherence as xylitol-containing chewing gum in geriatric patients.

Material and methods

Study setting

This randomized controlled clinical study was conducted at the Faculty of Dentistry, Cairo University, Egypt. The protocol of this study was registered in www.clinicaltrials.gov/ database, with unique identification number NCT03889015. All the procedures done in this study involve human participants, and the study has been approved by the Research Ethics Committee (CREC) of Faculty of Dentistry, Cairo University, and approval number 19-4-15.

This study was conducted for approximately 3 months and it was single blinded, parallel-arm, randomized clinical trial with 1:1 allocation ratio. The participants of the study were randomly assigned into two equal groups.

Sample size calculation

The sample size was calculated based on the previous study by Hanno et al. in 2011 in which the probability of patients without reduction in bacterial count (≥105) after 3 months for xylitol chewing gum was (0.471). The estimated probability of patients without reduction in bacterial count (≥105) after 3 months for probiotic yogurt was (0.171) with a difference of 30%. By adopting an alpha (α) level of 0.05 (5%), power (80%), the predicted sample size (n) was a total of 74 (37 patients per group) to be able to reject the null hypothesis that the success rates for case and controls are equal. The sample size was increased by (30%) to compensate for possible dropouts during follow-up to be total of (96) cases i.e. (48) for each group. The sample size calculation was performed using G*Power 3.1.9.2, Universität Kiel, Germany.

Eligibility criteria

Subjects were assessed for eligibility to be included in the study. High caries risk geriatric patients (over 65 years old) with Streptococcus mutans count more than 105 CFU (by culturing on mitis salivarius bacitracin agar) were included in the study. Their cognitive ability had to be sufficient to understand consent procedures.

Subjects who were milk intolerant or habitual consumers of xylitol were excluded from the study. The geriatric patients who used antibacterial mouthwash or were under any antibiotic treatment were not included in the study.

Participants were recruited in the study to fulfil the eligibility criteria and screening continued until the target geriatric population (96 participants) was achieved. An Arabic informed consent supplied by the Research Ethics Committee (REC) at Faculty of Dentistry, Cairo University containing all the ethical aspects of the trial was signed by the eligible participants prior to the initiation of the study.

Randomization, Sequence generation and blinding

Simple randomization was done by generating numbers from 1:96 using Random Sequence Generator, Randomness and Integrity Services Ltd (www.randomization.com). Each generated random number represented assigning intervention and comparator in a random manner. The investigator and the microbiologist were blinded to the material assigned. However, the patients could not be blinded due to the difference between probiotic yogurt and chewing gum.

The materials used were Probiotic yogurt (Activia; Danone, Egypt) and xylitol Chewing Gum (Trident Original) presented in Table 1. For Intervention Group, participants had to eat 100 grams of the provided probiotic yogurt 10 minutes after dinner. They were advised not to brush their teeth until one-hour later3. For Comparator Group, the participants were instructed to chew the provided gum three times daily for five minutes after each meal ().

Table 1

Materials’ description, composition, lot number and manufacturers

Material	Product description	Composition	Lot #	Manufacturer
Activia	Probiotic containing yogurt (100 gm)	Skim milk, Ultrafiltered milk, Cream, Skim milk powder, Active probiotic culture (Bifidobacterium) and active bacterial cultures.	NA	Danone, Obour city, kaliobeya, Egypt. https://www.dannon.com
Trident Original	Xylitol chewing gum	Sorbitol, gum base, Xylitol, Glycerin, Natural and Artificial Flavor; less than 2% of: Acesulfame Potassium, aspartame, Butylated hydroxytoluene (to maintain freshness), Mannitol, Soy Lecithin and Sucralose. Phenylketonurics: contains phenylalanine	RK09419222V	Mondelez Global LLC, East Hanover, NJ 07936 USA. https://www.tridentgum.com

For both groups, the participants were instructed not to take other probiotic products or mouth rinse throughout the study period. All participants were asked to continue their routine toothbrushing and flossing habits during the study period.

Salivary and interdental plaque MS counts, salivary pH and bacterial adherence were evaluated at different assessment times T0: before taking either the intervention or the comparator, at T1: two weeks, at T2: one month and T3: three months.

For salivary Streptococcus mutans count, the subjects were instructed not to eat or drink anything for at least one hour before the collection of saliva sample on the day of saliva collection. The participants were asked to rinse their mouth with water before collection of saliva to avoid the contamination of food debris (). In order to control the circadian variations, the samples were collected between 10:00 am and 11:00 am.

The subjects were instructed to chew on a piece of paraffin wax for 5 minutes and the pooled saliva was expectorated into 15 ml sterile graduated falcon collection tube. Samples were mixed by vortexing for 30 seconds to disperse bacterial aggregates, and subsequently100 μL of each sample was diluted 1:10 with double-distilled water. After that, 10 μl aliquots of each diluted sample was transferred to the sterile mitis salivarius bacitracin agar. Inoculated culture media were incubated at 37°C in a candle extinction jar for 48 hours anaerobically in an atmosphere of 5-10% CO2 in a precision incubator.

For interdental plaque Streptococcus mutans count, samples were collected from four sites using separate dental floss threads for each site. These sites were: maxillary right molar – mesial surface, maxillary incisor – mesial surface, mandibular incisor – distal surface and mandibular left molar – distal surface. The plaque samples were then dispersed in a falcon tube containing saline. Samples were then prepared for culture in the same manner as salivary samples.

Salivary pH was assessed with the aid of a pH meter. The required minimum volume of saliva (2 ml) was placed in a sterile test tube so that the bulb of the measuring electrode could dip sufficiently into the saliva samples.

Measuring bacterial adherence was done by placing C-shaped cheek retractors to retract cheek, plaque disclosing tablets (Visuplac, Maquira, Brazil) were rubbed over the upper teeth to make dental plaque visible by staining the teeth areas that were covered by dental plaque. Standard photos were taken using digital camera mounted on a tripod. Digital image analysis used the Cool PHP Tools image color extract software by Kepler Gelotte (http://www.coolphptools.com/) to analyze plaque covering the upper six anterior teeth. Images were converted into pixels to calculate the percentage of the tooth covered with plaque divided by the total tooth area calculated by the software.

Statistical analysis

Statistical Analyses were performed using Medcalc software, version 19 for Windows (MedCalc Software Ltd, Ostend, Belgium). Data were explored for normality using the Kolmogorov-Smirnov test and the ShapiroWilk test. Continuous data were described using mean and standard deviation. An intergroup comparison between continuous data was performed using t-test, while intragroup comparison was performed using repeated measures ANOVA, and two-way ANOVA was used to test interaction of variables. A p-value less than, or equal to 0.05, was considered statistically significant, and all tests were two tailed.

Results

The intergroup comparison between both materials have shown non-significant differences within follow up periods; baseline and 4 weeks respectively (P = 0.9020, P = 0.0852), but there was a statistically significant difference at 2 weeks where probiotics showed higher level of reduction in MS count and 3 months where xylitol showed higher level of reduction in MS count than probiotics (P < 0.0001). Intragroup comparisons with xylitol chewing gum or probiotic yoghurt have shown statistically significant difference between different follow-up periods (P < 0.001). The results are presented in Table 2.

Table 2

Mean and standard deviation of Log CFU/ml of salivary bacterial count

InterventionFollow-up	Xylitol		Probiotic		P value
	Mean	SD	Mean	SD
Baseline	6.5909	0.07892	6.5889	0.07314	P = 0.9020 NS
2 weeks	6.4619	0.06707	6.2441	0.05879	P < 0.0001*
4 weeks	5.5235	0.05586	5.5505	0.09162	P = 0.0852 NS
3 months	4.2242	0.06763	4.4078	0.1023	P < 0.0001*
P value	‎‎<0.001‏‏		‎<0.001‏‏

The intergroup comparison between both materials has shown a statistically significant difference within follow up periods; 2 weeks (P = 0.0021) where probiotics showed greater reduction in plaque bacterial count, 4 weeks and 3 months respectively (, P < 0.0001 and P < 0.0001) where xylitol showed greater reduction in plaque bacterial count, but there was no statistically significant difference at baseline (P = 0.3297). The intragroup comparison within xylitol chewing gum or probiotic yoghurt groups has shown a statistically significant difference between different follow-up periods (P < 0.001). The results are presented in Table 3.

Table 3

Mean and standard deviation of Log CFU/ml of plaque bacterial count

InterventionFollow-up	Xylitol		Probiotic		P value
	Mean	SD	Mean	SD
Baseline	4.5711	0.08206	4.5864	0.07065	P = 0.3297 NS
2 weeks	4.2344	0.08567	4.1648	0.1263	P = 0.0021*
4 weeks	3.3677	0.06156	3.7700	0.1774	P < 0.0001*
3 months	2.8785	0.1161	3.2799	0.09644	P < 0.0001*
P value	‎<0.001‏‏		‎<0.001‏‏

The intergroup comparison between both materials has shown no statistically significant differences within follow up periods; baseline, 2 weeks, 4 weeks and 3 months respectively (P = 0.1709, P = 0.0700, P = 0.0811 and P = 0.0894). The intragroup comparisons within xylitol chewing gum or probiotic yoghurt groups have shown statistically significant differences between different follow-up periods (P < 0.001). The results are presented in Table 4.

Table 4

Mean and standard deviation of pH

InterventionFollow-up	Xylitol		Probiotic		P value
	Mean	SD	Mean	SD
Baseline	7.3250	0.2817	7.4083	0.3093	P = 0.1709 NS
2 weeks	7.3896	0.2707	7.4875	0.2523	P = 0.0700 NS
4 weeks	7.4313	0.2502	7.5188	0.2358	P = 0.0811 NS
3 months	7.4812	0.2312	7.5583	0.2082	P = 0.0894 NS
P value	‎<0.001‏‏		‎<0.001‏‏

The intergroup comparison between both materials has shown a statistically significant difference at 3 months (P < 0.0001), but there was no statistically significant differences at baseline (P = 0.0836). The intragroup comparison within xylitol chewing gum or probiotic yoghurt has shown statistically significant difference between different follow-up periods (P < 0.0001). The results are presented in Table 5.

Table 5

Mean and standard deviation of plaque adherence percentage

InterventionFollow-up	Xylitol		Probiotic		P value
	Mean	SD	Mean	SD
Baseline	56.1250	6.2284	58.4167	6.6070	P = 0.0836 NS
3 months	6.0833	1.6221	3.8333	1.7785	P < 0.0001*
P value	P < 0.0001		P < 0.0001

Discussion

Dental caries is a chronic biofilm mediated disease caused by dysbiosis that manifests when the cariogenic oral pathogens dominate over the healthy commensals (). Improvement of the oral flora is one of the effective strategies to prevent dental caries (). Biofilm-targeted strategies to restore ecological symbiosis can either be biofilm inhibitory or biofilm modulating ().

Inhibition of bacterial biofilm is achieved using antimicrobial agents that act against Streptococcus mutans such as xylitol. Xylitol, a sugar alcohol, has a unique inhibitory effect on glycolysis of MS where intracellular accumulation of xylitol-5-phosphate leads to reduced acid formation from glucose and a reduction in bacterial count in both plaque and saliva (). However, unabsorbed xylitol can be fermented in the colon by bacteria, resulting in the production of a considerable amount of hydrogen that causes laxation and diarrhea (). Therefore, the aim of the current study was to achieve a modality with suitable efficacy against MS but without these complications.

The biofilm modulating approach aims to restore and maintain oral homeostasis by enhancing the growth of healthy oral commensals, effectively attenuating the presence of pathogens, thereby maintaining a diverse symbiotic ecological microbiome (). Probiotics have been confirmed to co-aggregate with MS and inhibit their growth by colonizing and competing with such oral pathogens for adhesion sites ().

Preventive care in geriatric individuals must be simple and fast with techniques and materials adapted to their specific features. Since geriatric people lean towards a soft diet that is easier to chew, probiotic yogurt is ideal for them ().

Measuring Streptococcus mutans in saliva is one of the most popular methods to recognize subjects at risk of dental caries. All the groups in the current study had similar salivary count of Streptococcus mutans at baseline so that the changes seen after the administration of the test products could be attributed to the use of these products.

In the current study, the statistically significant reduction in the level of salivary MS count over all the examined follow up periods in xylitol gum group was due to xylitol inhibitory effect on glycolysis and subsequent intracellular accumulation of xylitol-5-phosphate leading to a dramatic reduction in MS count (, ).

The probiotic yogurt group showed statistically significant reduction in the level of salivary MS count over all the examined follow up periods of the study since probiotic bacteria produce antibacterial agents against oral pathogens such as hydrogen peroxide organic acids and bacteriocins (). They are also effective in regulating local and systemic immune systems. They compete with the pathogenic MS bacteria and impede their viability in saliva ().

The results of the intergroup comparison showed a statistically significant difference between two materials in a two week period of time where probiotics showed higher levels of reduction in the MS count. This could be due to the rapid antibacterial action of probiotics in saliva by co-aggregation with MS and fast elimination (). This was followed by statistically non-significant difference between both materials after one-month timeframe. This was due to increase in level of MS reduction in xylitol group between a two week time period and a four week period of time compared to probiotics. The results also showed a statistically significant difference between both groups where xylitol showed higher level of reduction in MS count than probiotics after three months. This could be due to long term action of xylitol in addition to the washing action of chewing gum which facilitates bacterial clearance from saliva ().

Regarding the count of plaque bacteria, statistically significant reduction in the level of MS between follow up periods in xylitol gum group was found owing to the ability of xylitol to disturb MS energy production and reduce the adhesion of these microorganisms to the teeth surface by diminishing the production of extracellular polysaccharides (, ).

In the current study, the statistically significant reduction in the level of plaque bacterial count between follow up periods in probiotic yogurt group could be explained by the local biofilm effect of probiotics on MS relying on bacterial coaggregation, bacteriocin and hydrogen peroxide production, competition for adhesion sites, and competition for nutrients with such pathogenic bacteria ().

The results of intergroup comparison showed statistically significant difference between the two materials in a two week time period where probiotics showed greater reduction in plaque bacterial count. This could be due to the rapid ability of probiotic in preventing the adherence of other bacteria to the tooth surface by modifying the protein composition of the salivary pellicle on tooth surface via binding to it, thus causing its degradation ().

However, the intergroup comparison showed statistically significant difference between the two materials in a four week period of time and a three month period of time where xylitol showed greater reduction in plaque bacterial count. This is attributed to the inability of microorganisms of dental plaque to ferment xylitol leading to inhibition of their growth as well as the ability of xylitol to produce intracellular vacuoles inside Streptococcus mutans causing degradation of their cell membrane (). Combining mechanical cleaning and salivary stimulation effect of chewing gum gives further benefits compared to the antibacterial effect alone.

The statistically significant increase in salivary pH in xylitol gum group along the follow-up periods could be attributed to the inability of MS to ferment xylitol, and therefore cannot produce acids that cause drop in the pH of the oral cavity. In addition, the action of stimulating salivary flow by chewing gum aids in increasing the action of saliva buffering systems ().

The statistically significant increase in salivary pH in probiotic yogurt group along the follow-up periods was due to immunomodulatory action of probiotics by increasing the number of secretory IgA (sIgA) producing cells found in saliva. These sIgA producing cells inhibit the function of the glucosyltransferase enzyme from Streptococcus mutans, and therefore diminishing its acid production ability preserving a homeostatic environment ().

In the current study, Xylitol gum led to statistically significant decrease in plaque adherence to tooth surface over a three month intervention period by inhibiting the glycolysis process in cariogenic bacteria by competing with the phosphofructokinase enzyme leading to reduction in insoluble polysaccharides which plays an important role in bacterial adherence ().

The statistically significant reduction in bacterial adherence in probiotic group after three months of consumption of probiotic yogurt was due to production of antioxidants by probiotic bacteria that utilize the free electrons required for mineralization of plaque, thus inhibiting plaque formation25. Probiotics also compete with bacteria for binding sites on host tissues causing disruption of plaque biofilm formation (, ).

The present study is the first study investigating the beneficial oral effect of using probiotics on geriatric people. For geriatric population, yogurt is a very simple and easy way to deliver an antibacterial agent. Therefore, according to the results of the current study, probiotic yogurt can be used as an alternative to xylitol in enhancing the oral condition and prevention from caries ().

Conclusions

Under the conditions used in this clinical trial, it could be concluded that probiotic yogurt is an effective antibacterial agent against salivary and plaque bacteria in geriatric patients and can be used as a caries prevention means.

This is the first study investigating the beneficial oral effect of using probiotics on geriatric patients. For geriatric population, yogurt is a very simple and easy way to deliver an antibacterial agent. Therefore, according to the results of the current study, probiotic yogurt can be used as an alternative to xylitol in enhancing the oral condition and prevention from caries.

More studies on geriatric population are required to investigate probiotic yogurt antibacterial effects, and long term follow up is required in future studies to identify the emergence of any bacterial resistance to probiotics.