|Year : 2019 | Volume
| Issue : 1 | Page : 5-11
Antimicrobial activity of probiotics against endodontic pathogens:- A preliminary study
Aarti Ashok Bohora1, Sharad R Kokate2, Smita Khedkar3, Ashok Vankudre4
1 Department of Conservative Dentistry and Endodontics, SMBT Institute of Dental Science and Research, Dhamnagaon, Nasik, Maharashtra, India
2 Department of Conservative Dentistry and Endodontics, YMT Dental College and Hospital, Kharghar, Navi Mumbai, Maharashtra, India
3 Department of Microbiology, Bac-Test Laboratory, Nasik, Maharashtra, India
4 Department of Community Medicine, Dr. Vasantrao Pawar Medical College, Hospital and Research Centre, Nasik, Maharashtra, India
|Date of Web Publication||16-Aug-2019|
Dr. Aarti Ashok Bohora
472, Bohora Bhavan, Raviwar Peth, Nasik - 422 001, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: The purpose of this study was to evaluate the antibacterial effectiveness of probiotics lactobacilli group and Bifidobacterium against Enterococcus faecalis and Candida albicans in both planktonic stage and biofilm stage. Materials and Methods: Phase 1 of the study was conducted by agar well diffusion method. About 0.5 ml of test pathogen culture was inoculated on 20 ml of molten agar and allowed to solidify. 4–5 circular wells of diameter 8–10 mm were punched in each poured plates and 150 μl of diluted test samples were added to the wells. Phase 2 was deferred antagonism test, wherein purified culture of pathogen strain was streaked at right angle to the original producer growth and incubated at 37°C for 24 h. Zone of inhibition was measured for both the phases. Phase 3 biofilm stage evaluation was conducted by mixing 9 ml of 30% poloxamer 407 and De Man, Rogosa and Sharpe (MRS) broth in a test tube with 500 μl of either pathogen, together with 500 μl of test probiotic strains and incubated (37°C, 48 h), followed by serially diluting the mixture by 1 ml into 9 ml sterile saline till 108 dilutions for evaluation of colony-forming unit/ml counts. Controls were endodontic pathogens in 30% poloxamer with MRS broth and no probiotics. Results: Results were evaluated and statistically analysed using one-way analysis of variance and unpaired t-test. In the planktonic stage, probiotics showed inhibitory activity against endodontic pathogens with valid statistical significance (P < 0.05), while there was no activity by deferred antagonism method. In biofilm stage, all three probiotics showed growth reduction for E. faecalis, while lactobacilli group showed reduction in C. albicans colonies. Conclusion: This preliminary study suggested that probiotics are effective for preventing the growth of endodontic pathogens in vitro. Poloxamer could be utilised as an ideal delivery vehicle for probiotics.
Keywords: Bacteriotherapy, Bifidobacterium, lactobacilli, root canal system
|How to cite this article:|
Bohora AA, Kokate SR, Khedkar S, Vankudre A. Antimicrobial activity of probiotics against endodontic pathogens:- A preliminary study. Indian J Med Microbiol 2019;37:5-11
|How to cite this URL:|
Bohora AA, Kokate SR, Khedkar S, Vankudre A. Antimicrobial activity of probiotics against endodontic pathogens:- A preliminary study. Indian J Med Microbiol [serial online] 2019 [cited 2019 Dec 13];37:5-11. Available from: http://www.ijmm.org/text.asp?2019/37/1/5/264479
| ~ Introduction|| |
Endodontic treatment aims to heal apical periodontitis, but treatment success depends on the elimination of underlying infection and prevention of re-infection. Interestingly, Kakehashi et al. have reported that germ-free rats did not develop apical periodontitis despite mechanical exposure of their molar pulps to the oral cavity, but control specimens with conventional oral microflora developed significant periapical radiolucency.
Root canal flora is polymicrobial and predominantly comprises anaerobic species. The environment of root canals is particularly conducive to harbour anaerobic bacteria, which can ferment the available amino acids and peptides for their metabolic needs. Various microbial species interact during infection, producing microbial population shifts.,, Moreover, these microbial interactions are responsible for creating polymicrobial flora in endodontic habitats and for ecological regulation. Thus, there is a continued speculation in endodontics, as to whether we can actually eliminate these pathogenic microorganisms from infected root canals.
Microscopic examinations of serial sections of the roots of many teeth have demonstrated the prevalence of multiple accessory and lateral canals. These branches are never completely devoid of microorganisms and the best that can be achieved is a reduction in the biological load. Any clinical success achieved from endodontic treatments can probably be ascribed to the reduction in the number of microorganisms, removal of the most inflamed or necrotic tissue and a favourable systemic background.,
Conventional thinking is restricted by the belief that all microorganisms must be removed from the root canal system regardless of their pathogenicity or other characteristics. However, extensive research in the fields of microbiology and probiotics has led to the proposal that we should maintain a state of equilibrium within the human microbiome. The human microbiome is defined as the recognised, normal microbial component of all humans and animals, which is required for maintaining health., Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host (World Health Organisation)., In this way, normal bacterial flora of the oral cavity and dentition can be considered the 'oral human microbiome', and probiotics may be introduced into the root canals of teeth for improving the resilience of this microbiome.
Probiotics act by immune modulation; downregulation of inflammatory responses; production of antimicrobial substances, such as peroxides, organic acids and bacteriocins production of mucin; inhibition of epithelial invasion by inhibiting pathogens; mucosal adherence; stimulation of Immunoglobulin A and competition with other flora, including potential pathogens.,,,,, In the oral cavity, probiotics tend to create a biofilm that protects oral tissues from bacterial pathogens by filling a space that could serve as a future niche for pathogens., The concept of probiotics has already been implemented in dentistry for the prevention of dental caries and for the treatment of oral- and gut-associated halitosis, oral candidiasis and periodontal diseases.
Most species found in infected root canals are also present in periodontal pockets; thus, using probiotics in root canals may prove beneficial in endodontics., However, there has been little research into the use of probiotics for endodontic health, and probiotics have not been examined in terms of which particular strains are effective or what is the necessary colony-forming unit (CFU) count to eliminate pathogens. Hammad hasshown that Lactobacillus had no inhibitory effect against Enterococcus faecalis. Seifelnasrhave shown that commercially available probiotics were effective against endodontic pathogens although most products contained low numbers of viable organisms and were of little clinical benefit at the time of purchase. Even with better-characterised isolates, trial results can vary, reflecting the production and stability of products. There is no consensus across studies regarding the role of probiotics in endodontic therapy. Of note, these studies also did not evaluate the antimicrobial properties of normal flora using the deferred antagonism method.
Therefore, this study was performed to determine whether certain probiotics have therapeutic efficacy in the treatment of endodontic disease. Our primary hypothesis was that probiotics could eliminate or decrease the amount of E. faecalis and Candida albicans in planktonic and biofilm microbial stages when tested in an in vitro model.
| ~ Materials and Methods|| |
Study design and probiotic and pathogenic strain selection
This pilot study was approved by the Ethics Committee of our centre in Mumbai, India. To test our hypothesis, we designed an in vitro study adapted from that reported by Seifelnasr et al. (2014). The antimicrobial activity of probiotics was tested in the planktonic and biofilm stages.
Pure cultures of three probiotic strains, Lactobacillus plantarum (ATCC 8014), Lactobacillus rhamnosus (ATCC 7469) and Bifidobacterium bifidum (ATCC 11863) obtained from HiMedia, Mumbai, India, were selected on the basis of literature review, which showed their efficacies as probiotics. E. faecalis ( ATCC 29212) and C. albicans ( ATCC 10231) were selected as endodontic pathogens and were obtained from HiMedia, Mumbai, India.,,
Phase 1: Testing the efficacy of probiotics against Enterococcus faecalis and Candida albicans in planktonic stage using the agar cup-well method
L. plantarum and L. rhamnosus cultures (0.5 mL) of 0.1 optical density (OD) at 620 nm were inoculated into sterile molten agar (20 mL) cooled to 45°C ± 2°C. This suspension was thoroughly mixed and poured into a sterile, empty Petri dish More Details and allowed to solidify. Cell-free supernatant (CFS) and cell-free filtrate (CFF) were prepared by propagating pure isolates of Lactobacillus spp. in a 100-mL flask containing De Man, Rogosa and Sharpe (MRS) agar broth (pH 6.0) and incubated at 37°C for 72 h under microaerophilic conditions. The supernatant contained crude bacteriocin, and a cell-free solution was obtained by centrifuging the culture at 10,000 rpm for 20 min at 4°C. Thereafter, CFS was passed through a 22-μm membrane filter and evaluated for antimicrobial activity. CFS/CFF was used in three forms for testing: crude CFS, CFS adjusted to pH 6.0 (using 1N NaOH) and crude CFS diluted 1:2.
Circular wells (diameter, 10 mm) were punched with a sterile steel cork borer in each of the plates. Precisely, 150 μL of the prepared lactobacilli group probiotic samples were added to these wells. The plates were incubated under aerobic conditions in an upright position at 37°C for 24 h. Post-incubation, the zone of inhibition (ZOI) was measured.
B. bifidum was cultured in glucose broth under anaerobic conditions at 37°C for 48 h. Culture density was adjusted to 2 McFarland units. Test pathogens were cultured in glucose broth at 37°C for 24 h. The cultures were vortexed to obtain a uniform suspension, and the density was finally adjusted to 1 McFarland unit. Then, 0.1 mL of E. faecalis culture was spread onto a blood agar plate and 0.1 mL of C. albicans culture was spread onto Mueller-Hinton agar with 2% glucose and blood. B. bifidum was propagated in 10-mL tubes containing glucose broth and incubated under anaerobic conditions at 37°C for 48 h. Cell-free solution was obtained by filtration through a 0.45-μ millipore membrane filter. CFF was evaluated for antimicrobial activity by the agar cup-plate method. Circular wells (diameter, 8 mm) were punched in each of the inoculated plates, and precisely, 150 μL of the diluted probiotic culture was added to them. The plates were incubated under anaerobic conditions in upright positions at 37°C for 24 h using a BD GasPak EZ system. Post-incubation, ZOI was measured.
Phase 2: Testing the efficacy of probiotics against Enterococcus faecalis and Candida albicans in planktonic stage using the deferred antagonism test
Lactobacilli group strains were standardised to 0.1 OD at 600 nm and inoculated in a 1-cm-wide streak across the surface of Tryptic soy broth (TSB) with 1.5% bacteriological agar, 2% yeast extract and 0.25% CaCO3(trypticase soy yeast extract calcium agar [TSYCa]) agar using a sterile cotton swab. The culture was incubated at 37°C for 24 h under microaerophilic conditions. Macroscopically visible growth was removed by scraping with the edge of a glass slide. The plate was then inverted over a circle of chloroform-soaked filter paper in the lid of a Petri dish. After 30 min, the plate was removed from the lid and exposed to air for 15 min, before being standardised overnight (18 h at 37°C) to 0.1 OD at 600 nm. The purified culture of the pathogenic strain was streaked at a right angle to the line of the original producer growth, again using a sterile cotton swab. All TSYCa plates with the probiotic and pathogen strains were aerobically incubated at 37°C for 24 h. All procedures were performed in triplicates. Bifidobacterium strain was standardised to 2 McFarland units and inoculated in a 5mm-wide diametric streak across the surface of blood agar using a sterile cotton swab and standardised overnight (18 h at 37°C) to 1 McFarland unit. Purified cultures of pathogenic strains were then streaked at right angles to the line of probiotic culture using a cotton swab and the inoculated plate was incubated under anaerobic condition using BD Anaerobic GasPak EZ system (37°C, 24 h).
In both phases of planktonic stage testing, the inhibitory activity of probiotics was considered significantly positive if ZOI produced by the probiotic strain against the pathogenic strain was at least 10 mm.
Phase 3: Biofilm stage testing of an intracanal delivery vehicle for the probiotics
Poloxamer F 127 (407) was dissolved in cold MRS broth at a concentration of 30% using a magnetic stirrer for 10–15 min until a homogenous mixture was obtained. Poloxamer was sterilised and placed in the refrigerator at 4°C until testing. Control testing was performed as follows: E. faecalis and C. albicans stocks were prepared in TSB to an absorbance of 0.25 OD; L. plantarum, L. rhamnosus and B. bifidum stocks were prepared in TSB to an absorbance of 0.3–0.45 OD. All stocks were adjusted to given confluence at 600 nm using an ultraviolet spectrophotometer. Poloxamer (9 mL) was placed in a test tube with 500 μL of pathogenic cultures and vortexed at 4°C in a refrigerated environment to allow a homogenous mixture. This mixture was then aerobically incubated (48 h at 37°C) before serial dilutions of the pathogenic biofilm samples were prepared and plated on brain–heart infusion (BHI) agar plates to count CFU of microorganisms. Serial dilutions were made by serially adding 1 mL of Poloxamer to 9.0 mL of sterile saline to a dilution of 108. Plating was done by adding 1 mL of the dilutions onto BHI agar plates, followed by aerobic incubation at 37°C for 72 h. Then, CFUs were counted.
Testing of the probiotic/pathogenic organism and poloxamer mixtures was performed. A mixture of 9 mL poloxamer, 500 μL test probiotic strain and either 500 μL E. faecalis or500 μL C. albicans was prepared [Figure 1]. After 48 h of incubation, serial dilutions of the pathogenic biofilm samples were plated on BHI agar plates to count in all test groups compared against controls according to dilution factors and actual numbers of probiotics and pathogenic organisms per group.
|Figure 1: Poloxamer 407 with pathogenic organisms and test probiotics mixed together|
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Statistical analysis was done using SPSS software 16.0 (IBM software and solutions New York, USA) and analysis of variance and independent t-tests were used to evaluate antimicrobial activity of probiotics against E. faecalis and C. albicans. Statistical significance was set at P < 0.05.
| ~ Results|| |
Phase I: Agar cup/well method
When tested against E. faecalis, L. plantarum, L. rhamnosus and B. bifidum showed antimicrobial activity for crude CFS, with mean ZOIs of 19.7 mm, 19.4 mm and 18.2 mm, respectively, and for 1:2 diluted CFS, with mean ZOIs of 11.2 mm, 11.8 mm and no zone, respectively [Figure 2]a and [Figure 2]b. When tested against C. albicans, lactobacilli and Bifidobacterium groups showed antimicrobial activity for crude CFS, with mean ZOIs of 19 mm and 18.3 mm, respectively, but not for 1:2 diluted CFS and crude CFS adjusted to pH 6.0 [Figure 2]a, [Figure 2]b and [Figure 3]a, [Figure 3]b. ZOIs for L. plantarum, L. rhamnosus and B. bifidum were statistically significant against E. faecalis (P = 0.0001) [Table 1] and [Table 2] and against C. albicans (P = 0.023) [Table 3] and [Table 4].
|Figure 2: (a) Agar cup/well diffusion method of Lactobacilli plantarum and Lactobacilli rhamnosus against Enterococcus faecalis.|
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|Figure 3: (a) Agar cup/well diffusion method of Lactobacilli plantarum and Lactobacilli rhamnosus against Candida albicans(b) Agar cup/well diffusion method of Bifidobacterium bifidum against Candida albicans|
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|Table 1: Distribution of zones of inhibition by probiotics against Enterococcus faecalis|
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|Table 2: Analysis of variance for zones of inhibition by probiotics against Enterococcus faecalis|
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|Table 3: Distribution of zones of inhibition by probiotics against Candida albicans|
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|Table 4: Analysis of variance for zones of inhibition by probiotics against Candida albicans|
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Phase II: Deferred antagonism method
No antimicrobial activity was observed for any of the probiotics strains against either of the endodontic pathogens.
Phase III: Biofilm stage evaluation
L. plantarum, L. rhamnosus and B. bifidum, when tested against the biofilm morphological stage of E. faecalis, showed counts of 4.85 × 107, 7.44 × 107 and 5.9 × 108 CFU/mL, respectively, corresponding to growth reductions of 93.78%, 90.60% and 24.35%, respectively [Figure 4]a. Similarly, L. plantarum and L. rhamnosus showed notable antimicrobial activities against the biofilm morphological stage of C. albicans, with colony reductions to 1.35 × 107 and 3.689 × 106 CFU/mL, respectively, corresponding to growth reductions of42.06% and 83.79%, respectively [Figure 4]b. However, B. bifidum showed no antimicrobial activity against C. albicans [Figure 4]b.
|Figure 4: (a) Poloxamer test of probiotics against Enterococcus faecalis.(b) Poloxamer test of probiotics against Candida albicans|
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| ~ Discussion|| |
The elimination of bacteria from the root canals has continued to be the priority of endodontic therapy despite evidence that some organisms may be beneficial. Indeed, there has only been limited research on the potential role of probiotics. The most commonly used probiotic strains belong to the Lactobacillus and Bifidobacterium genera., We evaluated the potential of a novel approach to introduce bacteriotherapy using these genera in endodontics.
Primary infections of necrotic pulp tissue typically comprise mixed flora, often dominated by anaerobic Gram-negative bacteria. In persistent infections, however, anaerobic Gram-positive bacteria, such as E. faecalis and C. albicans, tend to be predominant. The biphasic nature of C. albicans allows it to be the universal co-aggregate in biofilms and makes it the most frequently isolated fungus from root-filled teeth with apical periodontitis. E. faecalis appears to be highly resistant to calcium hydroxide therapy because of its proton-pump activity, which enables it to survive in isolation and form a biofilm.
The present in vitro study involved elements focusing on discovery (Phases 1 and 2) and application (Phase 3). To avoid bias, pure culture strains of probiotics and endodontic pathogens were used and tested in both planktonic and biofilm stages. Phases 1 and 2 involved testing with the agar cup-well and deferred antagonism methods, respectively. The agar cup-well method is the most practical method for antimicrobial susceptibility testing and is a standard in most laboratories. In the preliminary in vitro study, both probiotic groups (lactobacilli and Bifidobacterium) exhibited antimicrobial activity against E. faecalis and C. albicans in their planktonic stages. The antimicrobial activity of probiotics was determined again by deferred antagonism test in phase two of the study. The deferred antagonism method has been used to evaluate the antimicrobial properties of normal flora in the nasopharynx. It uses a bacteriocin-like inhibitory substance to identify the bacterial products that have inhibitory effects. However, under the conditions of this study, probiotics had no effect on endodontic pathogens by deferred antagonism test.
Phase 3 was the application phase of the study, in which we assessed the potential efficacy of a novel delivery vehicle for probiotics into the root canal system. This method used 30% poloxamer 407 (Pluronic F-127) mixed with an MRS broth containing probiotics. Poloxamers are non-ionic, biocompatible polyethylene oxides and polypropylene oxides copolymers used in several pharmaceutical formulations, including surfactants, emulsifying agents, solubilising agents, dispersing agents and in vivo absorbance enhancers. These functional excipients benefit from having inverse thermosensitivity, being soluble in aqueous solutions at low temperatures (mainly 4°C), but forming a gel at higher temperatures.,, These properties make them ideal delivery vehicles for intra-canal treatments between clinical appointments. Notably, colony counts (CFU/mL) in this study revealed significant growth restrictions for E. faecalis and C. albicans using the tested probiotics.
This preliminary in vitro study demonstrated potential benefits of a paradigm shift from a focus on the need for complete elimination of pathogens to a focus on the restoration of the normal microbial ecology. Probiotics can counteract the growth of endodontic pathogens within infected root canals and can provide a more conducive environment for beneficial bacteria to grow and restore pulpal health. A new two-step therapeutic protocol can be envisioned. At the first visit, this might include cleaning, shaping, irrigation and activation to decrease the microbial load and remove organic tissue. This would be followed by introducing the poloxamer-based probiotic mixture in the root canal and leaving it for a period of approximately 1 week. At the second visit, further disinfection would be followed by obturation. If organisms persist after that process, the presence of probiotic flora in the root canals might produce environments that are more conducive to favourable endodontic outcomes. Moreover, we believe that probiotics could be formulated with a poloxamer for use as a root canal sealer. Moving forward, we contend that bacteriotherapy in endodontics should focus on introducing beneficial bacteria inside the root canal space and on eliminating only pathogenic flora rather than striving for the unachievable goal of a sterile canal.
| ~ Conclusion|| |
Probiotic therapy represents a potential antimicrobial treatment option that should be developed further. This pilot study demonstrates that probiotics offer potential benefits for root canal therapy and that further in vitro and in vivo studies are warranted to determine the full potential of bacteriotherapy in endodontics.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Haapasalo M, Udnæs T, Endal U. Persistent, recurrent, and acquired infection of the root canal system post-treatment. Endod Top 2003;6:29-56.
Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340-9.
Bohora A, Kokate S. Concept of probiotics in endodontics. Int J Adv Res 2016;4:1137-42.
Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol 1994;78:522-530.
Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med 2004;15:348-81.
Seltzer S, Bender IB. Cognitive dissonance in endodontics 1965. J Endod 2003;29:714-9.
Dominguez-Bello MG, Blaser MJ. Do you have a probiotic in your future? Microbes Infect 2008;10:1072-6.
Gueimonde M, Collado MC. Metagenomics and probiotics. Clin Microbiol Infect 2012;18 Suppl 4:32-4.
Elavarasu S, Jayapalan P, Murugan T. Bugs that debugs: Probiotics. J Pharm Bioallied Sci 2012;4:S319-22.
Grover HS, Luthra S. Probiotics the nano soldiers of oral health. J Indian Acad Clin Med 2011;13:48-54.
Harini PM, Anegundi RT. Efficacy of a probiotic and chlorhexidine mouth rinses: A short-term clinical study. J Indian Soc Pedod Prev Dent 2010;28:179-82.
] [Full text]
Meurman JH. Probiotics: Do they have a role in oral medicine and dentistry? Eur J Oral Sci 2005;113:188-96.
Reddy JJ, Sampathkumar N, Aradhya S. Probiotics in dentistry: Review of the current status. Rev Clín Pesqui Odontol Curitiba 2010;6:261-7.
Reddy S, Swapna L, Ramesh T, Singh R, Vijayalaxmi N, Lavanya R. Bacteria in oral health probiotics and prebiotics a review. Int J Biol Med Res 2011;2:1226-33.
Dhawan S, Dhawan R. Role of probiotics on oral health a randomized, double blind, placebo-controlled microbiological study. J Res Pract Dent 2013;20:1-9.
Haukioja A. Probiotics and oral health. Eur J Dent 2010;4:348-55.
Hammad K. Probiotic use in Endodontic Therapy (Master's Thesis). Chicago, US: University of Illinois; 2013.
Seifelnasr K. A Preliminary Study Evaluating Potential Probiotic use in Endodontics (Master's Thesis). West Virginia, US: West Virginia University; 2014.
Haukioja A, Yli-Knuuttila H, Loimaranta V, Kari K, Ouwehand AC, Meurman JH, et al.
Oral adhesion and survival of probiotic and other lactobacilli and bifidobacteria in vitro
. Oral Microbiol Immunol 2006;21:326-32.
Roberfroid M. Prebiotics: The concept revisited. J Nutr 2007;137:830S-7S.
Mejía JB, Arrieta AA. Reduction of viable Enterococcus faecalis
in human radicular dentin treated with 1% cetrimide and conventional intracanal medicaments. Dent Traumatol 2016;32:321-7.
Siqueira JF Jr., Rôças IN. The oral microbiota in health and disease: An overview of molecular findings. Methods Mol Biol 2017;1537:127-38.
Jayahari NK, Niranjan NT, Kanaparthy A. The efficacy of passion fruit juice as an endodontic irrigant compared with sodium hypochlorite solution: An in vitro
study. J Investig Clin Dent 2014;5:154-60.
Walls T, Power D, Tagg J. Bacteriocin-like inhibitory substance (BLIS) production by the normal flora of the nasopharynx: Potential to protect against otitis media? J Med Microbiol 2003;52:829-33.
Cafaggi S, Russo E, Caviglioli G, Parodi B, Stefani R, Sillo G, et al.
Poloxamer 407 as a solubilising agent for tolfenamic acid and as a base for a gel formulation. Eur J Pharm Sci 2008;35:19-29.
Patel HR, Patel RP, Patel MM. Poloxamers: A pharmaceutical excipients with therapeutic behaviors. Int J Pharm Tech Res 2009;1:299-303.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]