Comparative evaluation of antimicrobial toothpastes on periodontal bacteria in orthodontic patients: A randomized controlled study

Abstract

OBJECTIVE:

To evaluate the effects of Lactobacillus paracasei probiotic toothpaste, cetylpyridinium chloride (CPC) toothpaste, and amyloglucosidase–glucose oxidase toothpaste on the levels of Porphyromonas. gingivalis, Aggregatibacter actinomycetemcomitans, and plaque index in individuals undergoing fixed orthodontic treatment.

MATERIALS AND METHODS:

A double-blind randomized controlled clinical trial was conducted using purposive sampling. Participants were randomly assigned to use one of the toothpaste types. Saliva samples were collected at baseline and one month after using the toothpaste. Bacterial levels were quantified using quantitative polymerase chain reaction, and plaque accumulation was assessed using the Orthodontic Plaque Index.

RESULTS:

All groups showed a reduction of P. gingivalis and A. actinomycetemcomitans following the intervention; however, no significant changes were observed in the plaque index. Statistical analysis using two-way repeated measures analysis of variance with sphericity assumed revealed no significant differences between the groups (p < 0.05).

CONCLUSION:

Toothpastes containing L. paracasei, CPC, and amyloglucosidase–glucose oxidase enzyme show potential for reducing periodontal pathogens, suggesting a preventive benefit against periodontal complications in patients with fixed orthodontic appliances.

Introduction

Malocclusion is a common condition with potential impacts on patients’ quality of life, psychosocial well-being, and self-confidence.[1] In Indonesia, approximately 80% of the population experiences some form of malocclusion, making it a significant public oral health issue.[2] The increasing public awareness of dental and facial aesthetics has led to a rising demand for orthodontic treatment.[3] Recent studies indicate a rising prevalence of adult patients seeking orthodontic care, with estimates suggesting that adults now represent 20%–30% of all orthodontic patients in many countries.[4]

Fixed orthodontic appliances, although effective in correcting malocclusion, create plaque-retentive areas that complicate oral hygiene. This can result in the accumulation of dental biofilm, which shifts the oral microbial balance and promotes colonization by pathogenic species.[5] Clinical signs of periodontal changes, including increased gingival inflammation, bleeding on probing, and periodontal pocketing, are often observed in patients wearing fixed appliances.[6,7]

Two major periodontal pathogens of concern in orthodontic patients are Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.[8] These organisms are capable of adhering to both tooth surfaces and oral mucosa, contributing to periodontal tissue destruction.[9,10] Conventional plaque control methods, such as mechanical brushing, may not be sufficient, highlighting the need for adjunctive antimicrobial strategies.[5,11]

Various active agents in toothpaste, such as Lactobacillus paracasei probiotics, cetylpyridinium chloride (CPC), and amyloglucosidase–glucose oxidase enzyme, have shown promising antimicrobial activity in previous studies.[12,13,14] However, most studies have focused on their effects against cariogenic bacteria rather than periodontal pathogens. Therefore, further investigation is warranted to explore the efficacy of these formulations in reducing P. gingivalis, A. actinomycetemcomitans, and plaque index in patients undergoing fixed orthodontic treatment.

Materials and Methods

This randomized double-blind clinical trial was conducted on orthodontic patients with fixed appliances. Ethical approval for this study (876A/S2/KEPK/FKG/11/2024) was provided by the Research Ethics Committee of the Faculty of Dentistry, Universitas Trisakti, on November 11, 2024. After informed consent was obtained, subjects were screened based on inclusion criteria through anamnesis, intraoral clinical examination, and assessment using the index of orthodontic treatment need (IOTN) and Gingival Index (GI). Participants with the Dental Health Component of IOTN scores ≤3 and GI scores between 0 and 2.0 were purposively selected. The exclusion criteria in this study were established to minimize potential confounding factors that could influence the outcomes. Participants were excluded if they had a history of probiotic consumption within the preceding three months or were undergoing pharmacological treatment that could interfere with salivary secretion. Individuals receiving systemic or topical antimicrobial therapy were also not considered eligible. In addition, subjects who reported habitual smoking or presented with systemic diseases were excluded from participation, also patients who had undergone professional oral hygiene procedures during the observation period were not included in the study.

The sample size for the study was calculated using the following formula:

Zα represents the alpha standard deviation of 1.96 corresponding to a 95% confidence interval, while Zβ refers to the beta standard deviation of 0.84 with the same confidence level. The value S denotes the pooled standard deviation, and x1 – x2 indicates the minimum difference considered statistically significant. The symbol n represents the total number of samples required. The calculated sample size (n) was increased to eight samples per group. This study consisted of three treatment groups, resulting in a total of 24 research subjects included in the study.

From a total of 32 participants who were initially assessed in this study, with 24 participants meeting the inclusion criteria, 16.67% were male and 83.33% were female, with ages ranging from 18 to 23 years. Participants were assigned to the study groups using block randomization, and the order of these blocks was further randomized to ensure balanced and unpredictable allocation. The randomization sequence was prepared in advance by an independent third party. Allocation concealment was maintained using sealed opaque envelopes. Throughout the study, both participants and outcome assessors remained blinded to group assignments to preserve the methodological integrity of the double-blind design. The participants were then assigned to one of three intervention groups: (1) probiotic toothpaste containing L. paracasei, (2) toothpaste with CPC, or (3) toothpaste with amyloglucosidase–glucose oxidase enzymes.

In this double-blind clinical trial, blinding procedures were rigorously implemented to minimize performance and assessment bias. All toothpaste formulations were dispensed in identical, unlabeled tubes to prevent participants from recognizing the type of toothpaste they received. Consequently, participants were unaware of their group allocation throughout the study period. Similarly, the investigators responsible for distributing the products, monitoring adherence, and performing clinical evaluations were blinded to the allocation codes. No visual, textual, or sensory cues distinguished one formulation from another. The allocation codes were generated and securely held by an independent third party and were not disclosed to the research team until all data collection, data entry, and preliminary analyses had been completed. This approach ensured that both participants and outcome assessors remained fully blinded, thereby preserving the methodological rigor of the double-blind design. Each participant was also given an orthodontic toothbrush and instructed to brush twice daily using the Bass technique for one month.

Saliva samples were collected at baseline (T0) and after one month (T1). Saliva offers a noninvasive, rapid, and reproducible sampling method that reflects the overall microbial load and oral health status, including the presence of periodontal pathogens, such as P. gingivalis and A. actinomycetemcomitans. Participants were instructed to avoid food, drink, and physical activity one hour before collection. Stimulated saliva was collected via paraffin wax chewing and spitting into sterile tubes. Samples were stored at 2°C–8°C temporarily and later frozen at − 20°C to − 80°C.

DNA extraction from the saliva was performed using heat-shock and centrifugation protocols. Quantification of P. gingivalis and A. actinomycetemcomitans was conducted using quantitative real-time polymerase chain reaction (qPCR). A total of 10 μL of DNA extraction from saliva was mixed with 90 μL of nuclear free water (NFW). These two mixtures were diluted seven times and produced a concentration of 10⁰ μL or equivalent to 1 μL. Homogenization was carried out using a vortex. Every 2 μL of the dilution results was put into a 96-well plate (Nest Biotech, China). Then, mix 10 μL of SYBR green (Thermo Fisher Scientific, Massachusetts, USA), 6 μL of NFW, 1 μL each of the forward and reverse primers [Table 1][15] into the PCR mix and put into the qPCR plate wells that already contained the previous dilution. The qPCR plate wells were inserted into the qPCR machine at 95°C for 10 minutes for one initiation denaturation cycle, followed by 40 cycles of denaturation at 95°C for 15 seconds per cycle. The expression results of the samples using qPCR were then quantified relative DNA gene expression by calculating using the formula 2⁻ΔΔ^Ct. Plaque levels were assessed using the Orthodontic Plaque Index (OPI) at both T0 and T1.

Table 1.

Primers of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans used in quantitative real-time polymerase chain reaction

Primer	Sequence (5’–3’)
P. gingivalis forward	TGC AAC TTG CCT TAC AGA GGG
P. gingivalis reverse	ACT CGT ATC GCC CGT TAT TC
A. actinomycetemcomitans forward	CTT ACC TAC TCT TGA CAT CCG AA
A. actinomycetemcomitans reverse	ATG CAG GAC CTG TCT CAA AGC

P. gingivalis=Porphyromonas gingivalis, A. actinomycetemcomitans=Aggregatibacter actinomycetemcomitans

The normality test on the data uses the Shapiro–Wilk test (n ≤ 50), if the p > 0.05 then the data are normally distributed. The homogeneity test uses Mauchly’s test of Sphericity. Next, a multivariate two-way repeated measures analysis of variance (ANOVA) test will be conducted with a p < 0.05 to see any significant differences and interactions between variables.

Results

A total of 32 individuals were examined in this study, of whom 24 fulfilled the inclusion criteria. With respect to gender, 16.67% were men and 83.33% were women, and the overall age range was 18 to 23 years. The initial assessment consisted of a clinical examination that included evaluation of malocclusion type, jaw relationship, IOTN, GI, and OPI. The most prevalent malocclusion type was Class I, observed in 54.17% of the subjects, while the most frequent jaw relationship was orthognathic, found in 70.83% of participants. The IOTN examination revealed that 41.67% of the subjects were classified in grade 1. All participants (100%) demonstrated mild gingivitis based on the GI and OPI score of 4, corresponding to the poor oral hygiene category.

Based on the type of toothpaste, the P. gingivalis count showed a change in 2⁻ΔΔ^Ct values before (T0) and one month after (T1) treatment. The L. paracasei probiotic toothpaste group showed an average decrease of 5.59 × 10⁶ before treatment to 5.03 × 10³ after one month using the toothpastes. The CPC toothpaste group showed an average decrease from 3.11 × 10³ to 4.79 × 10². The amyloglucosidase–glucose oxidase enzyme toothpaste group showed a greater average decrease from 1.19 × 10⁷ to 1.92 × 10³. The A. actinomycetemcomitans count also showed a change in 2⁻ΔΔ^Ct values before (T0) and one month after (T1) treatment in all three toothpaste groups. The group using L. paracasei probiotic toothpaste showed an average decrease of 9.24 before treatment to 1.31 after treatment. The CPC toothpaste group saw an average decrease from 2.89 to 0.65. The amyloglucosidase–glucose oxidase enzyme toothpaste group also showed a greater average decrease from 18.62 to 2.82 [Table 2].

Table 2.

Minimum, maximum, and average Ct values of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans bacteria before (T0) and one month after (T1) treatment based on the type of toothpaste group (2^−ΔΔCt)

Toothpaste groups	Treatment time	P. gingivalis			A. actinomycetemcomitans
		Minimum value	Maximum value	Average value	Minimum range	Maximum range	Average value
L. paracasei probiotic	T0	2.22	4.22×107	5.59×106	1.54	53.10	9.24
	T1	0.28	3.89×104	5.03×103	0.07	8.45	1.31
CPC	T0	6.82	2.11×104	3.11×103	0.84	9.88	2.89
	T1	1.26	2.56×106	4.79×102	0.02	2.55	0.65
Amyloglucosidase–glucose oxidase enzyme	T0	1.33	9.53×107	1.19×107	2.05	83.34	18.62
	T1	0.43	1.22×104	1.92×103	0.33	9.49	2.82

P. gingivalis=Porphyromonas gingivalis, A. actinomycetemcomitans=Aggregatibacter actinomycetemcomitans, L. paracasei=Lactobacillus paracasei, CPC=Cetylpyridinium chloride

The analysis then continued with the evaluation of the mean natural logarithm (NL) values of P. gingivalis at baseline (T0) and one month after treatment (T1) across the three toothpaste groups, as presented in Table 3. At baseline, the highest mean NL value was observed in the L. paracasei probiotic toothpaste group (7.35 ± 6.63), followed by the amyloglucosidase–glucose oxidase enzyme toothpaste group (6.84 ± 6.42), and the CPC toothpaste group (4.81 ± 2.81). The overall mean NL value of the three groups before treatment was 6.33 ± 5.44. After one month of treatment, a reduction in the mean NL values was observed in all groups. The L. paracasei probiotic toothpaste group demonstrated a mean NL value of 1.99 ± 4.37, the amyloglucosidase–glucose oxidase enzyme toothpaste group recorded 2.48 ± 4.10, and the CPC toothpaste group showed 2.93 ± 2.98. The combined mean NL value across all groups after treatment was 2.47 ± 3.71. The control of Ct values obtained from the laboratory procedure was 36.25 for P. gingivalis ATCC 33277 and 31.48 for A. actinomycetemcomitans ATCC 29522.

Table 3.

Analysis of the normal logarithm (NL) values of the average Porphyromonas gingivalis and mean values of Aggregatibacter actinomycetemcomitans mean values before (T0) and one month after (T1) treatment in the three toothpaste groups

Toothpaste groups	n	NL values of P. gingivalis		Mean values of A. actinomycetemcomitans
		T0	T1	T0	T1
L. paracasei probiotic	8	7.35±6.63	1.99±4.37	9.24±17.79	1.31±2.90
CPC	8	4.81±2.81	2.93±2.98	2.89±3.15	0.65±0.89
Amyloglucosidase–glucose oxidase enzyme	8	6.84±6.42	2.48±4.10	18.62±27.62	2.82±3.26
Total	24	6.33±5.44	2.47±3.71	10.25±19.37	1.59±2.63

NL=Natural logarithm, P. gingivalis=Porphyromonas gingivalis, A. actinomycetemcomitans=Aggregatibacter actinomycetemcomitans, L. paracasei=Lactobacillus paracasei, CPC=Cetylpyridinium chloride

The analysis of the mean values of A. actinomycetemcomitans was conducted at baseline (T0) and one month after treatment (T1) across the three toothpaste groups [Table 3]. At baseline, the L. paracasei probiotic toothpaste group demonstrated a mean value of 9.24 ± 17.79, the CPC toothpaste group recorded 2.89 ± 3.15, and the amyloglucosidase–glucose oxidase enzyme toothpaste group demonstrated the highest value at 18.62 ± 27.62. The overall mean value of the three groups before treatment was 10.25 ± 19.37. Following one month of treatment, a reduction in mean values was observed in all groups. The L. paracasei probiotic toothpaste group exhibited a mean value of 1.31 ± 2.90, the CPC toothpaste group recorded 0.65 ± 0.89, and the amyloglucosidase–glucose oxidase enzyme toothpaste group demonstrated 2.82 ± 3.26. The combined mean value across all groups after treatment was 1.59 ± 2.63 [Table 3].

The average NL values for the P. gingivalis groups and mean values for the A. actinomycetemcomitans groups were then tested using Mauchly’s test of sphericity. The Mauchly’s test yielded a value of 1, indicating that the requirement for homogeneity of covariance for the two-way repeated measures ANOVA was fully met for those two groups. Overall, there was a significant difference between the P. gingivalis groups before (T0) and one month after (T1) treatment. This is evident in the average NL T0 value of P. gingivalis of 6.33 ± 5.44, which decreased to 2.47 ± 3.71 at T1. The results of the assumed sphericity test for treatment time [Table 4] showed a p value of 0.021 (p <0.05), which means that there was a significant difference between the A. actinomycetemcomitans groups before (T0) and one month after (T1) treatment. This can be seen in the average T0 value of A. actinomycetemcomitans of 10.25 ± 19.37, which decreased in the average T1 value to 1.59 ± 2.63. To assess the differences among the three toothpaste groups, the assumed sphericity test was applied to evaluate the interaction between time and treatment group [Table 4]. The analysis yielded a p value of 0.367 (p > 0.05), indicating no statistically significant difference. A decrease in the mean value of P. gingivalis was observed from baseline (T0) to one month after treatment (T1) across all three toothpaste groups, namely, L. paracasei probiotic toothpaste, CPC toothpaste, and amyloglucosidase–glucose oxidase enzyme toothpaste. Similarly, for A. actinomycetemcomitans, the assumed sphericity test produced a p value of 0.298 (p > 0.05), demonstrating no significant difference between the three groups. Although reductions in bacterial counts were evident in each group, the extent of decrease did not differ significantly, suggesting that all three toothpastes produced relatively comparable outcomes in reducing A. actinomycetemcomitans.

Table 4.

Results of the two-way repeated analysis of variance test with sphericity assumed on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans before (T0) and one month after (T1) treatment in the three toothpaste groups

Assumed sphericity test variable	P. gingivalis		A. actinomycetemcomitans
	Mean square	p	Mean square	p
Treatment time	1.79×102	<0.05	8.99×102	<0.05
Treatment time * treatment group	12.79	0.367	1.85×102	0.298

P. gingivalis=Porphyromonas gingivalis, A. actinomycetemcomitans=Aggregatibacter actinomycetemcomitans, p<0.05

The results of the OPI assessment. At baseline (T0), the mean OPI score in all three toothpaste groups, namely, L. paracasei probiotic toothpaste, CPC toothpaste, and amyloglucosidase–glucose oxidase enzyme toothpaste, was 4. Similarly, at one month after treatment (T1), the mean OPI score remained unchanged at 4 across all groups.

Discussion

Patients undergoing treatment with fixed orthodontic appliances frequently encounter difficulties in maintaining optimal oral hygiene, as the components of the appliances may hinder effective cleaning. Consequently, these patients are at increased risk of periodontal tissue damage due to plaque accumulation and bacterial colonization.[7] The primary determinant of oral health maintenance is effective plaque control, which includes toothbrushing, interdental cleaning, and the use of mouth rinses.[5,11] Beyond mechanical methods of plaque removal, the selection of toothpaste also plays an essential role in plaque control, aiming to reduce bacterial load within the oral cavity.[15]

Adolescents are an appropriate population for studying periodontal pathogens, such as A. actinomycetemcomitans and P. gingivalis, because they commonly undergo fixed orthodontic treatment, which promotes plaque retention and bacterial colonization due to appliance components that hinder cleaning.[8] Poor oral hygiene compliance in this age group further facilitates the proliferation of pathogenic bacteria associated with early periodontal changes. Studies have reported that A. actinomycetemcomitans and P. gingivalis are frequently detected in adolescents with gingival inflammation or early attachment loss during orthodontic treatment.[16] The prevalence of aggressive or early-onset periodontitis linked to these pathogens among adolescents ranges between 0.3% and 5.9%, emphasizing their importance as a high-risk group for periodontal research.[17]

In this study, saliva was employed as the diagnostic medium owing to its ease, rapidity, and noninvasive nature of collection. Saliva provides valuable insight into the oral environment, including bacterial load and the severity of periodontal disease.[18] Stimulated saliva was chosen because the mechanical action of chewing paraffin wax facilitates the release of bacteria from the gingival sulcus, thereby enhancing the detection of periodontal pathogens.[19] However, while gingival crevicular fluid (GCF) offers higher site specificity for sampling bacteria and mediators directly from the periodontal pocket, it has drawbacks. GCF collection is technically demanding, requires multiple site‐specific samples, prone to contamination with saliva, blood or plaque, and often involves low fluid volume and extensive laboratory processing.[20] Consequently, although GCF may provide more direct information about local periodontal microbiology, for larger scale screening or monitoring purposes saliva remains a more practical and efficient alternative.[20,21]

DNA-based detection methods, such as qPCR, are widely used to estimate bacterial load because they offer high sensitivity, specificity, and the ability to identify target species even at low concentrations.[22] Although these techniques cannot distinguish between live and dead bacteria, they provide a reliable measure of total bacterial presence and are less affected by sample handling or bacterial viability compared to culture-based methods.[23] Additionally, many oral pathogens, including P. gingivalis and A. actinomycetemcomitans, are fastidious and difficult to culture, making DNA quantification a practical and efficient alternative for evaluating microbial changes in clinical studies.[24]

Toothpaste is available in several forms, such as paste, gel, powder, and liquid. It generally contains two types of ingredients, like non-active and active components. Non-active ingredients do not have therapeutic effects but determine the toothpaste’s physical properties, including texture, taste, consistency, and appearance, and usually consist of water, abrasives, humectants, binders, flavors, surfactants, preservatives, and colorants.[25,26] Active ingredients, however, provide therapeutic benefits, such as preventing cavities, reducing plaque, controlling sensitivity, eliminating bad breath, and offering antimicrobial effects. These include enzymes, CPC, and probiotics.[25]

The findings demonstrated significant reductions in P. gingivalis and A. actinomycetemcomitans counts following the use of L. paracasei probiotic toothpaste, CPC toothpaste, and amyloglucosidase–glucose oxidase enzyme toothpaste among patients with fixed orthodontic appliances. This suggests that all three toothpaste formulations exhibit antibacterial effects.[12,13,14] However, no statistically significant differences were observed in the degree of bacterial reduction among the three groups, which may be attributed to the distinct mechanisms of action of the active ingredients in each toothpaste in inhibiting bacterial growth.

Probiotics are defined as microorganisms that confer health benefits to the host when consumed in adequate amounts. Over the past decade, a growing body of research has highlighted their therapeutic and preventive potential in maintaining oral health. Probiotics are known to modulate both specific and nonspecific immune responses, enhance epithelial barrier function, produce antimicrobial substances, and inhibit the adhesion of pathogenic bacteria within the oral cavity.[27] Among the antimicrobial substances produced by probiotics are bacteriocins and organic acids. Organic acids, particularly acetic acid and lactic acid, play a central role in the inhibitory activity of probiotics against pathogenic species. These acids are able to penetrate bacterial cell membranes, thereby acidifying the intracellular environment, which ultimately leads to bacterial death, especially in Gram-negative organisms.[28]

Chuang et al. reported that oral administration of L. paracasei GMNL-33 exhibited anticariogenic properties by significantly reducing Streptococcus mutans levels in the oral cavity.[29] Similarly, Lee et al. demonstrated in a clinical study that L. paracasei GMNL-143-based probiotic toothpaste possesses the ability to co-aggregate with oral pathogens and inhibit their adhesion to gingival tissues.[30] The antibacterial effect of L. paracasei is more pronounced under acidic conditions compared with neutral pH environments. This enhanced activity in acidic conditions occurs because peptides are attracted to the phosphate groups of lipopolysaccharide molecules, initiating pore formation in the bacterial membrane. Such changes in membrane permeability led to structural disruption and compromise membrane integrity, ultimately resulting in bacterial cell lysis.[31] These findings are consistent with the present study, in which L. paracasei-containing probiotic toothpaste was shown to effectively reduce bacterial counts in the oral cavity.

CPC, another active ingredient found in certain toothpaste formulations, is a quaternary ammonium compound with well-established antimicrobial properties. Following use, CPC remains distributed within the oral cavity due to its surfactant chains and cationic charges, which enable sustained absorption onto oral surfaces.[32,33] Structurally, CPC contains hydrophilic and hydrophobic groups. The positively charged hydrophilic groups promote electrostatic binding to the negatively charged surfaces of pathogenic bacteria, while the hydrophobic groups interact with bacterial membranes, facilitating integration into the cytoplasmic membrane. These dual interactions lead to disruption of membrane integrity, impairment of cellular metabolism, cytoplasmic leakage, and eventual bacterial death. In addition, CPC reduces microbial adhesion to oral surfaces, thereby limiting colonization.[32] These mechanisms are consistent with the findings of Vasconcelos et al., who demonstrated that CPC-containing toothpaste significantly reduced bacterial counts in the oral cavity through decreased plaque accumulation and gingival inflammation.[13]

Toothpaste formulations containing the enzymes amyloglucosidase and glucose oxidase are reported to exert antimicrobial effects. The amyloglucosidase enzyme inhibits bacterial proliferation by converting D-glucose into D-glucono-1,5-lactone, thereby reducing the availability of bacterial nutrients in the oral cavity. Meanwhile, glucose oxidase activates the salivary immune defense system, specifically the lactoperoxidase (LPO) pathway, by generating hydrogen peroxide. This hydrogen peroxide interacts with catalase to produce oxygen, reducing the prevalence of anaerobic bacteria. Furthermore, hydrogen peroxide activates the LPO system to generate hypothiocyanite, a compound with antibacterial activity against P. gingivalis.[34,35] The findings of this study indicate that toothpaste containing amyloglucosidase and glucose oxidase produced greater reductions in both P. gingivalis and A. actinomycetemcomitans compared to the other tested toothpastes. This outcome is consistent with the choice of saliva as a diagnostic tool, as the enzymatic mechanisms are directly linked to salivary immune activity.

As a member of the “red complex,” P. gingivalis exhibits strong virulence through its capacity to aggregate with other bacterial species, facilitating colonization during later stages of biofilm development and rendering it difficult to eliminate.[9] Likewise, A. actinomycetemcomitans produces a wide range of virulence factors to ensure survival within the oral cavity.[36] Both species contribute to robust biofilm formation, aided by antimicrobial-resistant fimbriae and extracellular polysaccharides that hinder immune cell penetration and phagocytosis. These properties allow both pathogens to induce periodontal tissue damage.[31] The present study demonstrates a reduction in the levels of P. gingivalis and A. actinomycetemcomitans, which may help mitigate the risk of periodontal complications in patients with fixed orthodontic appliances.

Several studies have demonstrated a strong association between the presence of P. gingivalis and A. actinomycetemcomitans in saliva, the gingival sulcus, and dental biofilm. These bacteria are recognized as key periodontal pathogens and have been shown to colonize multiple oral niches simultaneously. A qPCR study by Reddahi et al. found significantly higher levels of P. gingivalis and A. actinomycetemcomitans in both whole saliva and subgingival plaque from periodontitis patients compared to healthy controls. Moreover, they report a strong positive correlation between A. actinomycetemcomitans and P. gingivalis in the diseased subgingival sites and in saliva.[37] Saliva often serves as a reservoir that reflects the microbial composition of subgingival and supragingival biofilms, including the presence of P. gingivalis and A. actinomycetemcomitans. Their detection in saliva correlates with their colonization in periodontal pockets and dental biofilm, because these pathogens disseminate through oral fluids and are shed from biofilm communities on tooth surfaces. Furthermore, previous research has demonstrated that salivary levels of these bacteria are significantly associated with periodontal inflammation, pocket depth, and microbial loads within the gingival sulcus, supporting the relevance of saliva as a diagnostic medium for monitoring periodontal pathogens.[19,20,37] Taken together, the evidence supports that the presence of P. gingivalis and A. actinomycetemcomitans in saliva corresponds to their presence and activity within the gingival sulcus and dental biofilm.

The bacterial increase observed in patients with fixed appliances is attributable to the additional niches created by the orthodontic elements. Clinically, the number of oral bacteria has been shown to triple within the first six months following appliance placement.[38] Furthermore, plaque control becomes increasingly difficult in cases of dental misalignment. In this study, no significant changes were observed in plaque index scores before and after the use of probiotic L. paracasei, CPC, or amyloglucosidase–glucose oxidase toothpastes. This finding reflects the persistent cycle of plaque formation, as bacterial communities consistently recolonize tooth surfaces. Plaque development begins with pellicle formation initiated by Streptococcus sanguinis, followed by the coaggregation of pathogenic species such as P. gingivalis, A. actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia.[39,40]

Mechanical plaque removal through toothbrushing eliminates only part of the biofilm, as microbial colonization can lead to dysbiosis. P. gingivalis plays a central role in this process, functioning as a “keystone pathogen” that manipulates host immune responses and disrupts homeostasis within the oral microbiome. Even at low concentrations, P. gingivalis can interact with other microorganisms to promote colonization.[41,42] Consequently, reductions in bacterial counts observed in this study could occur despite relatively unchanged plaque index values. This is explained by the complex biofilm composition of dental plaque, which consists not only of microbial cells but also of extracellular polysaccharides, proteins, and structural molecules that stabilize the biofilm matrix.[40,43]

Additionally, the design and placement of orthodontic appliances contribute significantly to bacterial accumulation and plaque formation. Archwire ligatures serve as additional sites for bacterial colonization, and brackets positioned near the cervical margin can increase the risk of gingivitis.[44,45] The bracket material itself also plays a role: In this study, stainless steel appliances were used, which exhibit higher surface tension and are therefore more prone to plaque retention.[43]

Plaque retention varies among individuals due to differences in plaque formation patterns, oral hygiene practices, and dietary habits.[46] The effectiveness of toothbrushing as a plaque control method is highly dependent on patient compliance, as brushing is a complex and technique-sensitive process. Short-term use of toothpaste has been shown to exert only minimal influence on mechanical plaque removal.[47] Brushing technique plays a critical role in maintaining oral health, particularly for patients with fixed orthodontic appliances, who often experience challenges in adequately cleaning around appliance components. A common error is positioning the toothbrush too coronally, which results in neglect of the cervical region of the teeth and consequently increases plaque accumulation, predisposing patients to gingivitis.[33]

Plaque index was chosen instead of pocket depth or bleeding index because the presence of orthodontic brackets can make periodontal probing difficult and lead to measurement bias. The brackets and archwires hinder probe access and compromise the accuracy of assessing pocket depth and bleeding on probing.[48] Therefore, the plaque index provides a more practical and reliable parameter for evaluating oral hygiene during orthodontic treatment.[49] In addition, the plaque index reflects supragingival plaque accumulation, which is particularly relevant for orthodontic patients who are more prone to plaque retention due to appliance design.[49,50]

Toothbrush selection is also an important factor. The use of orthodontic toothbrushes characterized by a concave bristle arrangement and smaller brush head has been recommended, as these features allow for better adaptation to tooth surfaces and enhance cleaning efficacy around brackets, archwires, and interdental areas.[51] In addition, electric toothbrushes may serve as an effective alternative, as their vibratory action facilitates the removal of both supragingival and subgingival plaque. Professional dental cleaning at each follow-up appointment is likewise essential for patients undergoing fixed orthodontic treatment to further support oral hygiene maintenance.[38]

Conclusions

The use of probiotic toothpaste containing L. paracasei, CPC toothpaste, and enzymatic toothpaste containing amyloglucosidase–glucose oxidase was found to reduce the levels of P. gingivalis and A. actinomycetemcomitans but had no effect on the plaque index in patients with fixed orthodontic appliances. There was no significant difference in the reduction of these bacteria among the three types of toothpaste. Therefore, it can be concluded that all three formulations have similar potential in preventing plaque formation and periodontal disease in patients undergoing fixed orthodontic treatment. Further research is expected to include other bacteria than P. gingivalis and A. actinomycetemcomitans that cause periodontal disease and also longer periods of toothpaste use to provide more comprehensive results.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study received leading faculty research grant from Universitas Trisakti.