Effects of amine fluoride and probiotic mouthwash on levels of Porphyromonas gingivalis in orthodontic patients: A randomized controlled trial.

INTRODUCTION: Porphyromonas gingivalis is one among the major etiological agent in periodontal diseases and has been proved to cause gingival inflammation under orthodontic appliances. AIM: To assess the effect of amine fluoride and probiotic mouthwashes on levels of P. gingivalis during orthodontic treatment, using real time-polymerase chain reaction (RT-PCR).
MATERIALS AND METHODS: A randomised controlled trial was performed including 45 patients. There were three groups: Group A (control group), Group B (patients using Amflor mouthwash) and Group C (patients using probiotic mouthwash). During the treatment, all the 3 groups were advised to brush twice a day using Colgate tooth paste and Group B patients were advised to swish 15 ml fluoridated mouthwash (Amflor) and Group C advised to swish probiotic for 60 s before swallowing, once in the morning and once before sleep. After gently removing supragingival plaque, subgingival plaque samples were collected by inserting a sterile dental curette into the bottom of the gingival crevice around 31, 32, 41, and 42 at two different intervals: T1 at start and T2 after 6 months of fixed orthodontic treatment. Sub-gingival plaque samples collected were transferred to laboratory within 48 h for RT-PCR analysis in Tris-buffer solution, to maintain the integrity of bacterial DNA.
RESULTS: The levels of P. gingivalis were significantly decreased with probiotic mouth wash.
CONCLUSIONS: Probiotic mouth wash can be used as an adjunctive measure along with regular brushing to improve periodontal status during fixed orthodontic treatment.

INTRODUCTION

The ecosystem of oral cavity is very complex, inhabiting various microbial pathogens. Any external interference such as fixed orthodontic appliances could affect the equilibrium between components of microflora in this environment. Conditioning of enamel during bonding of brackets results in changes in the morphology and chemical nature of the oral cavity, increase in the quantity of bacteria within dental plaque and may change a healthy bacterial population to one that is able to cause disease.[1] Having an orthodontic appliance in oral cavity may hinder complete plaque removal and may also act as plaque retentive area. Thus, the appliances may complicate in achievement of an optimal oral hygiene. Ample evidence indicates Porphyromonas gingivalis as a putative periodontal pathogen in subgingival dental plaque and it is implicated as an indicator of periodontal disease.[2] Gingival changes and oral bacteria in plaque have been studied during orthodontic treatment in older children and young adults. Hence, orthodontic treatment further aggravates the periodontal destruction in patients with inappropriate plaque control.

Researches have been conducted to elucidate the need of controlling P. gingivalis in orthodontic patients using chemical plaque control such as mouthwash. Recently, probiotics have been introduced which are live micro-organisms and also benefits oral health by inhibiting the growth of harmful bacteria or by regulating mucosal immunity in the oral cavity.[3]

Aim

To assess the effect of amine fluoride and probiotic mouthwashes on the levels of P. gingivalis during orthodontic treatment, using real-time-polymerase chain reaction (RT-PCR).

MATERIALS AND METHODS

Patient selection

The study subjects included 45 patients who required orthodontic treatment, reporting to department of orthodontics and dentofacial orthopaedics, in the institution where study was conducted. Microbiological testing using RT-PCR was carried out in a Research Department at Bengaluru.

The inclusion criteria were – (1) Patients with age between 15 and 35 years and indicated for pre-adjusted edgewise appliance - 022 MBT prescription, (2) angle's malocclusion i, ii, iii, (3) no periodontal treatment within 6 months, (4) absence of grossly and deep carious lesions and (5) absence of any gross restoration. The exclusion criteria included (1) presence of acute gingival diseases, (2) severe periodontal diseases, (3) presence of any systemic disease, (4) use of any antibiotic or hormone during the study, (5) pregnant or lactating women, (6) presence of large prosthetic restorations, (7) patient on any other fluoride therapy and those who were allergic to milk or milk products.

The study was initiated with 45 patients. Thirty samples were finally assessed. Rest had to be excluded from study as 6 patients had to use antibiotics during the study period which would have given false positive results, 5 patients had poor patient compliance and 4 did not report on time.

A written consent was obtained from all the participants before initiation of study. Randomisation was performed and patients were equally divided into three groups: (1) Group A - control group, patients undergoing therapy with regular cleansing aids, (2) Group B - patients using Amflor mouthwash, manufactured by group pharma, containining amine fluoride (Bis–[hydroxyethyl]-aminopropyl-N hydroxyethyl octadecylamine dihydrofluoride [Olaflur]) 1000 ppm) and (3) Group C - patients using probiotic mouthwash (1 g containing Lactobacillus reuteri [0.1 billion cfu], Lactobacillus rhamnosus [0.1 billion cfu], Bifidobacterium longum [0.06 billion cfu], Bifidobacterium bifidum [0.1 billion cfu], elemental zinc [10 mg], Fructo-oligosaccharides [20 mg], lactilol [10 mg]). Probiotic–Lactogut Kidz (Medispan ltd), in powder form was mixed in normal saline and used as a mouth wash according to the manufacturer's instructions.

All three groups were advised to brush twice daily using Colgate toothpaste and Group B patients were advised to swish 15 ml fluoridated mouth wash (Amflor) for 60 s before expectorating, once in the morning and once in the night after brushing. Group C patients were advised to swish 15 ml probiotic mouthwash for 60 s before swallowing, twice daily after brushing. No other oral hygiene aids were recommended and patients were instructed not to eat anything for ½ h after rinsing.

Study design

A randomised controlled trial was performed among 45 patients. There were three groups: group A (control group), Group B (patients using Amflor mouthwash) and Group C (patients using Probiotic mouthwash).

Method of sample collection

Supra gingival debris and plaque was removed before subgingival plaque sample collection. The area was isolated using cotton rolls. A sterile curette was inserted into the periodontal pocket and plaque samples from root surfaces were collected by a gentle pull stroke around 31, 32, 41, and 42 as these are usually the major plaque accumulation areas. Visible plaque can be assessed and plaque samples can be collected easily from this region. Sample was collected from a single site, where most of the plaque was present. Samples were collected using the same method at two different intervals for each patient: T1 (Before the start of orthodontic treatment) and T2 (6 months of fixed orthodontic treatment). The collected specimen were transferred to Ependroff tube containing TE Buffer (Tris–HCL, EDTA buffer) and sent to the laboratory within 24–48 h. Samples were stored at −70°C until processing in the laboratory (New Brunswick Scientific Ultra– Low Temperature Freezer).

P. gingivalis was quantitatively analysed in all the three groups using RT-PCR technique. Custom SYBR® Green assay reagents for P. gingivalis (Applied Biosystems, India) were used in this study.

The Primer sequence specific to P. gingivalis selected for the study was as follows: P. gingivalis forward– 3'-TGCAACTTGCCTTACAGAGGG-5' and P. gingivalis reverse– 5'-ACTCGTATCGCCCGTTATTC-3'. The primer sequence specific to 16 sRNA selected as control for the study was as follows: Forward Primer: 3'-TCCTACGGGAGGCAGCAGT-5' and Reverse Primer: 5'-GGACTACCAGGGTATCTAATCCTGTT-3'.

The conditions for RT-PCR were as follows: Holding stage at 95° C for 10 s followed by 40 cycles of shuttle heating at 95° C for 15 s and at 60° C for 1 min. The melt curve stage was at 95° C for 15 s, 60° C for 1 min and 95° C for 15 s. 16S RNA was used as an endogenous control (SYBR® Green assay reagents, Applied Biosystems, India). In a RT-PCR assay, presence of organism was detected by the construction of a dissociation curve/melting curve, produced in the range of 60°C to 95°C the curve is formed due to accumulation of a fluorescent signal. The cycle threshold is defined as the number of cycles required for the fluorescent signal to cross the threshold. Real-time assays undergo 40 cycles of amplification. If the sample does not reach this level even after 40 cycles, then it does not contain the organism and is called “undetermined.”

Statistical analysis

Paired t-test was used to compare the means of two variables within the group. Repeated measure ANOVA was used for descriptive analysis of all the groups.

Evaluation of P. gingivalis levels at different intervals in all the three groups:

One-way ANOVA was used to compare the levels of P. gingivalis from time intervals T1 to T2 in all the three groups. The mean difference was calculated between T1 and T2 and ANOVA was applied to compare the significance between all the three groups [Tables 1 and 2].

Table 1

Mean difference among three groups

	n	Mean difference	SD	Minimum	Maximum
Control	10	7.9000	2.31741	4.30	12.00
Probiotic	10	−1.5880	0.82135	−3.00	−0.57
Amflor	10	1.6930	3.20048	−5.02	6.43
Total	30	2.6683	4.58999	−5.02	12.00

n – Total number of study population; SD – Standard deviation

Table 2

One-way ANOVA to compare all groups

	Sum of squares	df	Mean square	F	Significance
Between groups	464.380	2	232.190	42.766	0.000*
Within groups	146.593	27	5.429
Total	610.973	29

*Highly significant, considered significant if P≤0.05. df – Degrees of freedom; F – F-distribution

Comparison of P. gingivalis levels at different intervals within Groups A, B and C:

Paired t-test was used to compare the levels of P. gingivalis levels from beginning of fixed orthodontic treatment (T1) to 6 months after fixed orthodontic treatment (T2).

Comparison of levels of P. gingivalis between Groups B and C:

T-test was used to compare levels of P. gingivalis between Group B and Group C [Tables 3 and 4].

Table 3

Description of levels of Porphyromonas gingivalis associated with Group B and C

Group	n	Mean±SD	SEM
Difference
Probiotic	10	−1.5880±0.82135	0.25973
Amflor	10	1.6930±3.20048	1.01208

n – Total number of study population; SD – Standard deviation; SEM – Standard error mean

Table 4

Independent t-test to compare levels of Porphyromonas gingivalis associated with Group B and C

	T-test for equality of means
	t	df	Significance (two-tailed)	Mean difference
Difference
Equal variances assumed	−3.140	18	NS	−3.28100

t – t-statistic; df – Degrees of freedom; NS – Statistically not significant

RESULTS

Group A showed mean values of P. gingivalis 5.8180 × 106 ± 2.05519 CFU/ml at T1 and 13.7180 × 106 ± 2.34148 CFU/ml at T2. For Group B the mean values of P. gingivalis are 5.5220 × 106 ± 3.28667 CFU/ml at T1, 7.2150 × 106 ± 3.80469CFU/ml at T2 and for Group C the mean values of P. gingivalis are 4.7170 × 106 ± 2.36815 CFU/ml at T1, 3.1290 × 106 ± 1.92080 CFU/ml at T2 [Tables 5 and 6].

Table 5

Relative quantity of Porphyromonas gingivalis (billion colony forming unit/ml) among groups at beginning of the fixed orthodontic treatment and 6 months after the fixed orthodontic treatment

	Group A (control)	Group B (amflor)	Group C (probiotic)
At T1	58.18	55.22	47.17
At T2	133.18	71.25	31.29

TI – At beginning of the fixed orthodontic treatment; T2 – 6 months after the fixed orthodontic treatment; CFU/ml – Colony forming unit/milliliter

Table 6

Descriptive analysis of all the three groups

	Group	Mean±SD
At T1	Control	5.8180±2.05519
	Probiotic	4.7170±2.36815
	Amflor	5.5220±3.28667
	Total	5.3523±2.57441
At T2	Control	13.7180±2.34148
	Probiotic	3.1290±1.92080
	Amflor	7.2150±3.80469
	Total	8.0207±5.19682

TI – At beginning of the fixed orthodontic treatment; T2 – 6 months after the fixed orthodontic treatment; SD – Standard deviation

A 1% significant difference was found within and between the groups [Tables 7 and 8]. Change in P. gingivalis count is highly significant when compared between Groups (P < 0.000).

Table 7

Intra group comparison

Source	Type III sum of squares	df	Mean square	F	Significance
Intercept	2682.557	1	2682.557	222.694	0.000*
Gpp	344.674	2	172.337	14.307	0.000*
Error	325.241	27	12.046

*Highly significant, considered significant if P≤0.05. Gpp – Differences between groups; df – Degrees of freedom; F – F-distribution

Table 8

Inter group comparison

Source	Type III sum of squares	df	Mean square	F	Significance
Change	106.800	1	106.800	39.342	0.000*
Change × Gpp	232.190	2	116.095	42.766	0.000*
Error (change)	73.296	27	2.715

*Highly significant, considered significant if P≤0.05. Gpp – Differences between groups; df – degrees of freedom; F – F-distribution

There was statistically significant change in levels of P. gingivalis from beginning of fixed orthodontic treatment (T1) to 6 months after fixed orthodontic treatment (T2) as shown in Table 6. Figure 1 and Figure 2 shows this significant changes within three study groups.

Figure 1

Line diagram indicating increase in Porphyromonas gingivalis levels in all the three groups from beginning to 6 months of fixed orthodontic treatment. Gpp – Differences between groups

Figure 2

Comparison of Porphyromonas gingivalis levels at different intervals in all three groups. TI – At beginning of the fixed orthodontic treatment; T2 – Six months after the fixed orthodontic treatment

Change in P. gingivalis count is highly significant at significance level of 1% within Group A and Group C (P < 0.000). This states that within Groups A and C there was significant increase or decrease in P. gingivalis count at two different intervals (T1 and T2) respectively.

In Group A, there was significant increase in P. gingivalis count at two different intervals T1 and T2 [Table 9]. In Group B, There was insignificant increase (p– 0.129) in P. gingivalis count at two different intervals (T1 and T2) respectively [Table 10]. In Group C, At 1% significance level there was a decrease in P. gingivalis count at two different intervals (T1 and T2) respectively [Table 11].

Table 9

Comparison of Porphyromonas gingivalis level at different intervals within Group A

	Mean	n	SD	SEM	df	Significance (two-tailed)
Pair 1
T1	5.8180	10	2.05519	0.64991	9	0.000*
T2	13.7180	10	2.34148	0.74044

*Highly significant, considered significant if P≤0.05. TI – At beginning of the fixed orthodontic treatment; T2 – 6 months after the fixed orthodontic treatment; n – Total number of study population; SD – Standard deviation; SEM – Standard error mean; df – Degrees of freedom

Table 10

Comparison of Porphyromonas gingivalis levels at different intervals within group

	Mean	n	SD	SEM	df	Significant (two-tailed)
Pair 1
T1	5.5220	10	3.28667	1.03934	9	NS
T2	7.2150	10	3.80469	1.20315

BTI – At beginning of the fixed orthodontic treatment; T2 – 6 months after the fixed orthodontic treatment; n – Total number of study population; SD – Standard deviation; SEM – Standard error mean; df – Degrees of freedom; NS – Statistically not significant

Table 11

Comparison of Porphyromonas gingivalis levels at different intervals within group

	Mean	n	SD	SEM	df	Significance (two-tailed)
Pair 1
T1	4.7170	10	2.36815	0.74887	9	0.000*
T2	3.1290	10	1.92080	0.60741

*Highly significant, considered significant if P≤0.05. CTI – At beginning of the fixed orthodontic treatment; T2 – 6 months after the fixed orthodontic treatment; n – Total number of study population; SD – Standard deviation; SEM – Standard error mean; df – Degrees of freedom

P. gingivalis levels was found to be insignificantly higher in Group B at T1 (P > 0.921) and T2 (P > 0.058). This suggested that there was increased level of P. gingivalis in association with amflor mouthwash than probiotic mouthwash [Tables 3 and 4, Figure 3]. At T1P. gingivalis level difference was insignificant between the groups and this suggests that randomization of subjects has been achieved. There was statistically significant difference between the two groups at a significance level of 5%.

Figure 3

Comparison of levels of Porphyromonas gingivalis between Amflor mouthwash and probiotic mouthwash. TI – At beginning of the fixed orthodontic treatment; T2 – Six months after the fixed orthodontic treatment

DISCUSSION

Intraoral environmental change like bonding of a fixed orthodontic appliance leads to an increase in the bacterial growth within dental plaque and may change a healthy bacterial population to one that is able to cause disease.[4] Many studies have focused on the interactions of cariogenic bacteria, such as Streptococcus mutans during fixed orthodontic treatment.[5678] However, only a few have looked at this interaction with periodontopathogens.

This study focused on the levels of P. gingivalis during fixed orthodontic treatment. Many periodontopathic bacterias have been implicated in periodontal diseases during fixed orthodontic treatment but P. gingivalis belongs to “red complex” as described by Socransky which is more associated with active periodontal destruction and bleeding on probing.[9]

RT-PCR assay was performed to identify and quantify P. gingivalis in subgingival plaque samples as it is an anaerobic bacteria. PCR was preferred over bacterial culture due to difficult isolation of small numbers of the organism on a nonselective medium and under large bacterial cell background.

Similar studies were conducted by Liu et al.[2] and Kim et al.,[10] but at intervals of 0, 3 and 6 months of appliance placement, and showed that there was a statistically significant increase of subgingival plaque from third to sixth month and statistically insignificant increase from initiation to third month. Liu et al.[2] also evaluated the samples even 3 months after the appliance removal and showed that quantity of subgingival P. gingivalis were increased at the end of orthodontic treatment and even after removal of appliance. So in this study, sample collection was done at the beginning and after 6 months of orthodontic therapy as significant levels of sub gingival plaque are demonstrable at 6 months in previously conducted researches.

The study compared the Amflor mouthwash with the probiotic, so probiotic was also supplied in the form of liquid mouth rinse. Commercially available probiora contains Streptococcus species but the probiotic used in the study contained Lactobacillus species which is more effective against P. gingivalis as shown by Kõll-Klais et al.[11] As per manufacturer's instructions, patients using Amflor mouthwash were advised to expectorate after swishing due to systemic side effects of fluoride but the patients using probiotic mouthwash were advised to swish and swallow twice a day.

Various studies [12131415] have shown the effect of mouthrinse and probiotic on streptococcus mutans present in supragingival plaque but due to placement of bands and brackets during fixed orthodontic treatment there is more alteration of subgingival plaque and colonization of anaerobic bacteria which are responsible for gingivitis, chronic periodontal conditions and halitosis etc., So, in this study sub-gingival plaque around lower anteriors was assessed to determine the amounts of P. gingivalis in oral cavity.

The results of the study proved that daily rinsing of Amflor mouth wash for 6 months slightly increased the levels of P. gingivalis which was statistically insignificant [Table 5] whereas a previous study by Qgaard et al.[16] showed significantly less plaque collection and gingivitis on the upper anterior teeth after using the amine fluoride (AmF) or Stannous Fluoride (SnF2) toothpaste products during the entire period of treatment. This could be due to different study design as in previous study supragingival plaque was assessed. The daily rinsing of probiotic mouth wash for 6 months decreased the levels of P. gingivalis levels in plaque samples which was in accordance with previous studies of bifidobacteria and lactobacilli derived probiotics.[17] This, was also in accordance with previous finding by Hillman [15] which showed low subgingival plaque levels of P. gingivalis with probiora mouthwash.

There was two fold increase in control group and slight increase in Amflor group showing restrictive effect of Amflor mouth wash in arresting the growth of P. gingivalis. The restriction can be attributed to reduction in levels of plaque PH by the amine fluoride present in the Amflor mouth wash and also reduction of anaerobic glycolysis carried by plaque bacteria.[16] There was significant decrease in the levels of P. gingivalis levels in probiotic group which can be due to the formation of a biofilm which prevents the adhesion of pathogens thereby, the competitive inhibition of pathogenic bacteria and the antimicrobial agents produced by the bacteria. This also suggested that Amflor mouthwash could not be able to penetrate subgingival plaque due to topical effect and probiotic could able to penetrate and inhibited the growth of bacteria.

No other opposing effects were registered with the probiotic use but brown staining was reported with Amflor mouthwash. The results of this human study suggested that probiotic mouthwash may be a safe product to be added as an effective routine oral hygiene regimen and use of both mouthwashes depends on patient selection.

CONCLUSIONS

Significant increase in levels of P. gingivalis can be expected during orthodontic treatment, but with the use of probiotic mouth wash, there is significant decrease in levels of P. gingivalis. Hence probiotic mouth wash can be used as an adjunctive measure along with regular brushing to improve periodontal status during fixed orthodontic treatment

Amflor mouthwash can effectively be used for white spot lesions and initial carious lesions than gingival and periodontal problems.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.