A Promising Probiotic Irrigant: An In Vitro Study

Abstract

AIM:

The present study aimed to investigate the inhibitory effect of Lactobacillus rhamnosus (B-445) as a probiotics irrigant on the growth of Enterococcus faecalis.

METHODS:

Forty-two extracted single human canal anterior teeth were prepared with rotary instrumentation and sterilised. Teeth were divided into 3 groups according to the type of irrigant, N = 14. Three experimental groups were inoculated with E. faecalis and cultured for 21 days before use; Group 1 was 2.5% NaOCl (positive control), Group 2 was saline (negative control), Group 3 was the experimental probiotic irrigant. Paper point sampling of the canals of each group was obtained before irrigation (S1), immediately after irrigation (S2) and after 24 hours (post irrigation samples) (S3) to determine remaining colony forming units for E. faecalis. Also, Colony counts for L. rhamnosus in Group 3 after immediate irrigation, as well as 24 hours post irrigation, was performed to determine the survival profile of these bacteria in infected root canal with E. faecalis.

RESULTS:

The NaOCl irrigant group had the lowest mean value of (log 10 CFU/mL) of E. faecalis after immediate irrigation and after 24 hrs post irrigation followed by the probiotic group, while the highest mean value was the saline group (P ≤ 0.001). The survival profile for L. rhamnosus in Group 3 after immediate irrigation and post-irrigation were slightly higher than for E. faecalis (P ≤ 0.001).

CONCLUSION:

Lactobacillus rhamnosus which revealed a potential inhibitory effect on the growth of Enterococcus faecalis, could be used as a new natural, safe probiotic irrigant agent.

Introduction

In Endodontics, success depends on chemomechanical removal of bacteria, bacterial endotoxin and debris from the root canal system (Sjögren et al., [1] 1997; Katebzadeh et al., [2] 2000). Enterococcus faecalis (E. faecalis) is an anaerobic, facultative microorganism that is highly resistant to conventional chemomechanical preparation and usually found in failing root canal cases [3]. E. faecalis is a part of the human flora and appears to be highly resistant to the medicaments used in treatment [4]. Also, E. faecalis can form a surface attached microbial community known as a biofilm. This allows it to be protected from host defences as well as systemic treatment.

Different methods of combatting E. faecalis were explored through the use of different irrigants [5]. Sodium hypochlorite has remained a popular root canal irrigant because of its ability to dissolve necrotic tissue and organic remnants and its antimicrobial activity. On the other hand, sodium hypochlorite has certain adverse effects such as ineffectiveness against some microorganisms when used at low concentration, corrosion of endodontic instruments and lack of differentiation between necrotic and vital tissues when in contact with apical and periapical tissues [6]. “Out of the box” treatments were evaluated for use against E. faecalis such as the use of passion fruit juice as an endodontic irrigant as well as the use of Photomedicine and laser surgery [7]. In endodontics because of the cytotoxic reactions of irrigants used and their inability to eliminate bacteria from dentinal tubules, the trend of recent medicine attends to use biologic medication.

Probiotics have been shown to promote health in the intestines and the oral cavity through the consumption of certain yoghurts and lozenges. The mechanisms of action of these probiotics include the productions of bacteriocin-like inhibitory substances and the altering of the local pH, competing for nutrients, forming physical barriers and stimulating the immune response [8]. The World Health Organization recognises probiotics as a significant avenue of health preservation if current antibiotics become useless due to the development of bacterial resistance. The purpose of this study was shifting the established paradigm of endodontic treatment from eliminating all bacteria from the canal system to focusing on the elimination of the problematic bacteria through introducing probiotics.

Therefore, this study aimed to explore the probiotics as new irrigant agent that would probably have an inhibitory effect against the growth of Enterococcus faecalis.

Material and Methods

Forty-two freshly extracted single-rooted human teeth with fully formed apices were collected for this study. All teeth were examined under a stereomicroscope (Technical, Germany) to exclude teeth with any existing defects or cracks. Teeth were thoroughly washed under running water and immersed in 5.25% Sodium Hypochlorite (Clorox, Egyptian company for household products, Egypt) for 30 minutes to remove soft tissues on the root surface. Teeth were gently cleaned with ultrasonic scaler to remove any remaining soft tissues or calculus. The teeth were stored in saline solution until instrumentation. Before canal instrumentation, the crown of each tooth was sectioned at cementoenamel junction using a sectional disc (Toolouip, Germany) mounted in a low-speed handpiece (NSK, Japan) under water coolant. This was done for ease of manipulation and to standardise the tooth length at 15 mm.

A size 15 k-type file (Mani, Japan) was used to assure patency of the canal and the apical foramen. The working length was established by subtracting 1 mm from the length obtained when the file tip just appeared at the apical foramen. Cleaning and shaping were performed by using rotary Ni-Ti Protaper system that was driven by an endodontic handpiece of 1:16 gear reduction and powered by X-Smart electric motor (Speed 400 rpm and torque setting 5 Ncm, Dentsply, Japan). All canals were enlarged to the same file size F5. During instrumentation, irrigation was performed with 3 ml of 5.25% sodium hypochlorite after each file. After completion of the instrumentation, the smear layer was removed with 3 ml 17% ethylene-diamine-tetra-acetic acid solution (EDTA-Q.A-Dent, Egypt) by using a disposable plastic syringe. This was followed by irrigation with distilled water. Teeth were then autoclaved at 121°C for 30 minutes. After that, the apical foramen of each tooth was sealed by using Putty silicone for impression, and the root surface was sealed with varnish after which the samples were handled under strict aseptic measures.

Lactobacillus rhamnosus (L. rhamnosus B-445) was selected as an example of probiotic species and provided in lyophilised form by the Northern Regional Research Laboratory, Illinois, USA. Bacterial activation was carried out through inoculation of L. rhamnosus in De Man, Rogosa, Sharpe (MRS) broth medium (Oxide, Basingstoke, Uk) followed by anaerobic incubation (BBL Gas Pak, Becton Dickinson, Cockeysville, MD, USA) at 37°C for 48 hrs. The experimental probiotic irrigant was formulated through inoculation 5 ml of L. rhamnosus (2x10⁸ CFU/ml) in 10 ml of sterile distal water.

A Total of Forty-two samples were randomly assigned to three main groups according to the type of irrigant was applied (N = 14); Group1 was 2.5% NaOCl (positive control), Group2 was saline (negative control), Group3 was the experimental probiotic irrigant.

The three groups were infected by using a 24-hours pure culture suspension of Enterococcus faecalis (E. faecalis; ATCC 19434) in Brain heart infusion broth medium (BHI broth medium; 53286 Sigma-Aldrich, USA). In each previously sterilised samples 1 × 10⁻⁸ CFU/mL suspension of E. faecalis (determined by serial dilution and plating) was inoculated in root canals and incubated at 37°C and 95% humidity for 21 days. The BHI broth was removed from the canal by gentle aspiration and renewed every 48 hours.

At the end of 21 days infection with E. faecalis, sterile paper point pre-irrigation sample was taken from each specimen. The sterile paper points were placed in the canals to the apical foramen and were moved circumferentially along the walls of the canal. Each paper point was left for 1 minute in the canal to collect the pre-irrigation samples from the canals of all teeth. Paper points were placed in airtight sterile vials containing 5 ml of nutrient broth for transportation to the microbiological laboratory for culturing procedure. After that, each group was irrigated immediately with 5 mL of its specific irrigant, and all irrigants were delivered by using a side-vented 30-gauge needle (Max-I-Probe; Dentsply). The immediate paper point irrigation sample was obtained from each root canal of each tested groups. Again the experiment was repeated after 24 hours irrigation of root canals (post irrigation samples) and storage in an incubator at 37°C.

In colony counting method, each pre-irrigation, immediate, post irrigation paper points samples were placed in airtight sterile vials containing 5 ml of saline and vortexed at the highest speed for 3 consecutive intervals of 15 seconds each. This was followed by serial dilutions 1:10, 1:100, 1:1000 and 1:10000. A volume of 50 µl was taken from each dilution using the automatic micropipette and was plated on BHI agar in triplicate. All plates were incubated at 37°C and 95% humidity for 24 hours, after which the colonies for E. faecalis on BHI agar plates were enumerated, and the most countable plates from pre-irrigation, immediate, post irrigation paper point’s samples of each group were selected. If any sample was too numerous to count, it was diluted, plated, and counted.

Colony counts for E. faecalis and L. rhamnosus in Group 3 after immediate irrigation as well as 24 hours post irrigation was performed to determine the survival profile of these bacteria. Colony count method was carried out as mentioned above through serial dilutions followed by a volume of 50 µl was taken from each dilution and plated on BHI agar in triplicate. Also, the duplicated paper point samples were plated on MRS agar in triplicate. The experiment was repeated after 24hrs post irrigation. All plates were incubated at 37°C and 95% humidity for 24 hours, after which both the colonies for E. faecalis on BHI agar plates and L. rhamnosus on MRS agar plates were enumerated and the most countable plates for each microorganism were selected.

Data presented as means and standard deviation (SD) values. One-way repeated measures ANOVA used to compare between the follow-up within each Irrigant solution. One Way ANOVA used to compare different irrigant solutions within each time.

For the Survival profile of microorganisms (Log10 CFU/mL); independent t-test used to compare between different type of bacteria, Dependent t-test used to compare between follow-up periods for each irrigant. The significance level was set at P ≤ 0.05 and was performed with IBM® SPSS® (SPSS Inc., IBM Corporation, NY, USA) Statistics Version 24 for Windows.

Results

Regarding the colony counting method, the results showed that there was a statistically significant difference in the mean (log-10 CFU/mL) of E. faecalis between the three irrigants groups immediately and after 24 hrs post irrigation (P ≤ 0.001). While there was no statistically significant difference in the mean (Log10 CFU/mL) of E. faecalis between tested groups pre-irrigation (P = 0.001).

The previous results revealed that the NaOCl irrigant group had the lowest mean value of (log-10 CFU/mL) of E. faecalis after immediate irrigation (0.00) and after 24 hrs post irrigation (2.20 ± 2.01) followed by the probiotic group with mean value (4.95 ± 0.11) for immediate irrigation and (4.40 ± 0.10) for post irrigation, while the highest mean value immediately and after 24 hrs post irrigation was the saline group (4.95 ± 0.11, 6.89 ± 0.32) respectively (Table 1, Figure 1).

Table 1.

Mean and SD for the Log10 CFU of (E. faecalis) for different irrigants

		Groups						p-value
		Saline		NaOCl		Probiotic
		Mean	SD	Mean	SD	Mean	SD
Log10 CFU (E. faecalis)	Pre-irrigation	7.04Ab	0.03	7.20Aa	0.07	7.09Ab	0.09	0.001*
	Immediately	4.95Ba	0.11	0.00Cc	0.00	4.40Bb	0.10	≤ 0.001*
	After 24 hours	6.89Aa	0.32	2.20Bc	2.01	4.04Bb	0.16	≤ 0.001*
p-value		≤ 0.001*		≤ 0.001*		≤ 0.001*

Means with same Capital Superscript within each Column are not significantly different at p=0.05; Means with same Small Superscript within each Row are not significantly different at p = 0.05; NS = Non-significant;

^*= Significant.

Figure 1.

Line chart showing the mean Log10 CFU (E. faecalis) for follow-up periods for each group

Concerning the survival profile for both bacterial growth E. faecalis and L. rhamnosus in Group 3, the results showed that the mean colony count (Log10 CFU/mL) for L. rhamnosus after immediate irrigation and post-irrigation (6.41 ± 0.06, 4.77 ± 0.44) was slightly higher than for E. faecalis (4.40 ± 0.10, 4.04 ± 0.16 respectively) (Table 2, Figure 2).

Table 2.

Mean and SD for the Survival profile of microorganisms (Log10 CFU/mL)

		Bacterial				p-value
		E. faecalis		Lactobacillus Rhamnosus (L.rhamnosus)
		Mean	SD	Mean	SD
Survival profile of microorganisms (Log10 CFU/mL)	Immediately	4.40	0.10	6.41	0.06	≤ 0.001*
	After 24 hours	4.04	0.16	4.77	0.44	≤ 0.001*
p-value		≤ 0.001*		≤ 0.001*

NS = Non-significant;

^*= Significant.

Figure 2.

Histogram showing the mean Survival profile of microorganisms (Log10 CFU/mL)

Discussion

As Enterococcus faecalis (E. faecalis) became more and more commonly isolated in failing root canal cases, it became the focus of interest in the medical and dental communities. Hence, E. faecalis was selected in our due to its ability to form a surface attached microbial community known as a biofilm. This allows it to be protected from host defences as well as systemic treatment [9], [10]. Additionally, It is the most common bacterial species found in persistent infections due to its relatively small cell diameter [11], [12]. The present study was designed to prepare a standardised tooth length of 15 mm, and the root canals were enlarged to F5 Protaper at the working length as this could attribute to create an adequate environment for bacterial growth.

Based on previous studies, the counting of CFUs for bacterial growth that was expressed in log CFU/mL could be used in the present study to evaluate the effects of the tested irrigants on E. faecalis growth [13], [14], [15], [16]. Several endodontic irrigants have been used for decontamination, but the most common was sodium hypochlorite (NaOCL) for its antimicrobial activity [17], [18], [19] and its ability of dissolution of organic tissues [20], [21]. However, due to its high toxicity [22], new irrigants were introduced. Therefore, this study was trying to explore a potential new way of treating endodontic infections which would probably be as effective or more and at the same time less irritating to the tissues than sodium hypochlorite. The use of probiotics has not yet been evaluated for use against E. faecalis. So, the present study was proposed to test known probiotics as an experimental irrigant in the presence of the established post-endodontic treatment disease bacteria E. faecalis and observe what the effect of the probiotics would have against E. faecalis.

Regarding colony counting method, the results showed that E. faecaliscolonies statistically significant decrease from (7.09 log CFU/mL) pre-irrigation to (4.04 log CFU/mL) after 24 hrs post irrigation with probiotic irrigant. The latter result was supported by [23] who documented that killing of pathogens through probiotic could be due to one of the mechanisms of probiotic action such as; the production of Bacteriocin-like inhibitory substance (BLIS) and acids/peroxides along with altering the local environments pH. However, (BLIS) is used to describe bacterial products that have inhibitory effects.

For the results that concerning the survival profile for both bacterial growth E. faecalis and L. rhamnosus in Group 3, it revealed that the mean colony count for L. rhamnosus after immediate irrigation and post-irrigation was statistically significantly higher than for E.faecalis (P ≤ 0.001). A more sophisticated method of observing the effects of the probiotic agents against E. faecalis could be conducted. The method of “Deferred Antagonism” which was demonstrated by [24] could explain the latter result that been used to evaluate the antibacterial properties of the normal flora of the nasopharynx. This deferred antagonism test which previously mentioned could be used to evaluate our hypothesis presented in a more controlled fashion as probiotic might compete for E. faecalis on nutrition and adhesion site which contribute to decreasing the survival profile of E. faecalis as found in the latter result of the present study. E. faecalis appears to be highly resistant to the medicaments used in the treatment and is one of the few microorganisms shown in vitro to be resistant to calcium hydroxide, due to its proton pump [4]. It is also able to survive as a single organism without the support of other bacteria. The fact that E. faecalis is not normally present or is present in very low numbers in untreated root canal cases implies that it can enter the canal, survive the antibacterial treatment and then persist after obturation [25]. Therefore, this deferred antagonism test which previously mentioned could be used to evaluate our hypothesis presented in a more controlled fashion as probiotic might compete for E. faecalis on nutrition and adhesion site which contribute to decreasing the survival profile of E. faecalis as found in the latter result of the present study.

In conclusion, within the limitation of this study; Lactobacillus rhamnosus which revealed a potential inhibitory effect on the growth of Enterococcus faecalis, could be used as a new natural, safe probiotic irrigant agent. Further studies using other known probiotics should be considered.