Since persistent bacteria in the root canal system is the main cause of root canal failure, there is an increasing demand to find a potent antibacterial agent against most endodontic pathogens with less toxic effects [1, 2]. Subsequently, this study was conducted to assess the antibacterial effect of Lactobacilli probiotics as a potential endodontic medication in comparison with Ca(OH)2 paste.
The study involved two stages; (stage one): determination of the antibacterial potential of PS in liquid and gel phase against E. faecalis then compare it with Ca(OH)2 paste. This was followed by (stage two): assessment of MIC of the PS that showed the maximum ZOI.
The first stage of the study revealed that the PS cocktail mix is the most potent PS and was more effective than Ca(OH)2. The PS cocktail mix showed the largest ZOI both in solution and gel forms while stage two showed that the MIC of PS cocktail solution and gel were the same without any significant difference between them. Subsequently, the broth microdilution method was done on the PS solution only to confirm the MIC which was found to be similar.
The null hypothesis was rejected as the antibacterial effect of the Lactobacilli cocktail mix of the three supernatants (gel and solution) was significantly higher than the Ca(OH)2 paste.
E. faecalis was selected as an endodontic pathogen example due to its resistance to the high pH of Ca(OH)2 [6, 9]. In addition, it is one of the most frequently isolated bacteria from failed root canal treatments as it has a substantial role in persistent infection and is frequently encountered in primary infections [7, 47].
Calcium hydroxide Ultra Cal XS (Ca(OH)2) is the routinely used medication in root canal treatments, commercially available in the market, but has a limited ability against E. faecalis infection [13, 48]. Therefore, it was selected as a historical control for comparison with the new antibacterial agent.
Recently, probiotics had a growing role in the dental field owing to their antibacterial and anti-inflammatory potential in controlling infectious and inflammatory diseases like gingivitis and periodontitis [17, 27, 28, 49]. Three strains of Lactobacilli probiotics were selected L. plantarum (LP), L. rhamnosus (LR) and L. acidophilus (LA) as being the most commonly used probiotics in dental research [34, 38, 49].
Poloxamer 407 was chosen as a vehicle material for development of PS gel since it was used as an in-situ gel containing antibiotics in treatment of periodontal disease, also used in toothpastes, contraceptive gels and burn dressing materials [43,44,45]. The thermo-reversible properties of poloxamer gel 30% make it an appropriate drug delivery vehicle as it exists in gel form at room temperature and liquid form at low temperature mainly 4 °C [33, 43].
Although the study started with a concentration of 200 mg/ml PS in the first stage, 300 mg/ml was used as a higher concentration to compare the effectiveness of a concentration higher than 200 mg/ml, but it was found not significant. Therefore, this study recommends using another higher concentration like 400 mg/ml or more and comparing its efficiency with the 200 mg/ml concentration.
Agar well diffusion and broth microdilution are valid antimicrobial susceptibility testing methods that are commonly used to detect new antimicrobial agents [50, 51]. One of the drawbacks of the agar diffusion assay is that its result depends on the diffusibility of the tested agents , thus it is recommended to use another assessment method like the direct exposure test (DET) and compare its results with the agar diffusion test as Estrela et al.  performed in their study.
All PS supernatants showed a variable potential of growth inhibition on E. faecalis with the superiority of the PS cocktail mix indicating a possible synergistic effect of the three strains. These findings were supported by Plaza-Diaz , who reviewed the main mechanism of action of probiotics which included prevention of biofilm formation by competitive exclusion of pathogens and production of bacteriocin and other antimicrobial substances.
The results agreed with Seifelnasr’s study , which showed that L.rhamnosus L. acidophilus, L. casei had an inhibitory action on E. faecalis and are the most common used probiotics in commercial cocktail. Furthermore, Bohora et al.  who used commercial probiotics cocktails (Ecobion and Darolac), stated that probiotics effectively prevented the growth of E. faecalis. Another study held by the same author using individual probiotics strains (L. rhamnosus (ATCC 7469, L. plantarum (ATCC 8014),) and Bifidobacterium bifidum (ATCC 11,863)) showed the same result . On the contrary, Hammad  used two strains of Lactobacillus, PTA 5289 and DSM 17,938 and found that probiotics had a non-significant effect on E. faecalis.
On comparing the (PS) cocktail mix (gel and solution) with 35%, Ultra Cal XS Ca(OH)2 paste against E. faecalis, Ca(OH)2 paste exhibited the least ZOI. The possible reason for this result could be due to the limited diffusion of Ca(OH)2 paste through blood agar because of its high viscosity, as the viscosity of the paste can affect the antibacterial activity .
However, Ultra Cal XS contains 3% propylene glycol and 2% methylcellulose [53, 54]. Propylene glycol is a viscous vehicle that reduces dispersion and diffusion due to its high molecular weight but maintains prolonged release and improves handling of the material [11, 55]. Methylcellulose is an aqueous stabilizing matrix that allows rapid dissociation of ions and improves the diffusibility and viscosity of the paste [11, 56].
Blanscet et al.  used the agar diffusion method to compare the antibacterial effect of Ca(OH)2 paste with different concentrations and vehicles on E. faecalis and found that the effect of 35% Ultra Cal XS Ca(OH)2 paste with aqueous methylcellulose was less than 60- 40% Ca(OH)2 but greater than 30% Vitapex (Neo-Dental Int, Federal Way, WA) with non-aqueous silicone vehicle.
The present study advocated that Lactobacilli PS could be used as an irrigant (liquid form) or an intracanal medication (gel form) as no significant differences were found between PS solution and gel. This study could be considered the baseline for using PS in endodontics as the minimum effective concentration of PS was determined, but further evaluations are needed in future studies.
Similar to the present study, El-Sayed et al.  and Kumari  et al. revealed that probiotics are potential natural irrigants. In future studies, it is recommended to compare the antibacterial efficacy of PS cocktail solution with the gold standard irrigant (sodium hypochlorite).
Moreover, Noushad et al.  reported that probiotics could be a promising intracanal medication that could inhibit E. faecalis more than and Ca(OH)2 but less than triple antibiotic paste.
Regarding limitations of the present study, the study was performed on E. faecalis in the Planktonic state; however, persistent bacteria are present in a biofilm form, which is harder to eradicate. Nevertheless, a tooth model is a more reliable method to assess antibacterial activity as intra-canal agents can be inactivated by dentin . Another limitation, the culture and antimicrobial testing methods (agar diffusion assay and broth microdilution) are highly subjective. Results should have been confirmed by more accurate techniques as time kill assay.
Benbelaïd et al.  evaluated the antimicrobial effect of essential oil against E. faecalis in both planktonic and biofilm states and determined biofilm eradication concentration (BEC). Accordingly, it is recommended to assess the BEC of the PS cocktail against E. faecalis and compare it with the MIC.
Future studies can evaluate PS cocktail gel with different vehicles to detect the ideal delivery vehicle of PS for clinical application. This study was the first to use a cocktail mix of three supernatants of PS in gel form and assess its antibacterial activity, besides determining the MIC of the PS against E. faecalis.