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ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 258-264
 

Antimicrobial efficacy of medium chain fatty acids as root canal irrigants: An in vitro study


1 Department of Paedodontics and Preventive Dentistry, Kannur Dental College, Kannur, Kerala, India
2 Department of Conservative Dentistry and Endodontics, PSM College of Dental Science and Research, Thrissur, Kerala, India

Date of Web Publication30-Sep-2019

Correspondence Address:
Dr. Krishnapriya Devan
Department of Paedodontics and Preventive Dentistry, Kannur Dental College, Kannur, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_63_19

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   Abstract 


Background: Enterococcus faecalis and Candida albicans are the microbes that are most resistant to elimination by disinfecting agents and are the causative agents for reinfection of the root canal treated teeth. Medium chain fatty acids (MCFAs), which are the main components of coconut oil, are proven antimicrobial agents. Thus, the aim of this study was to evaluate their antimicrobial efficacy against E. faecalis and C. albicans. Methodology: Ninety extracted single-rooted mandibular premolar teeth were decoronated, biomechanically prepared, autoclaved, and divided into three groups (n = 30): Group A (inoculated with E. faecalis), Group B (inoculated with C. albicans), and Group C (control group). Each group was again subdivided into three groups (n = 10) and irrigated with lauric acid (LA), decanoic acid (DA), and octanoic acid, respectively, for 5 min. Paper point samples were taken from canal walls and transferred into Brain Heart Infusion broth and potato dextrose broth and placed in an incubator at 37°C. The appearance of tubidity was checked at 24, 48, 72, and 96 h using direct contact test. The data were then statistically analyzed using the analysis of variance and Tukey honestly significant difference post hoc tests. Results: Among the three MCFAs, LA showed the maximum inhibitory against E. faecalis at 24 h and the inhibitory activity decreased considerably at 48, 72, and 96 h. DA was the most effective against C. albicans with a maximum inhibition at 48 h. DA also showed significant substantivity at 72 and 96 h. Conclusion: Within the limitations of this study, it can be concluded that MCFAs show promising antimicrobial efficacy against E. faecalis and C. albicans.


Keywords: Candida albicans, Enterococcus faecalis, medium chain fatty acids, root canal irrigants


How to cite this article:
Devan K, Peedikayil FC, Chandru T P, Kottayi S, Dhanesh N, Suresh K R. Antimicrobial efficacy of medium chain fatty acids as root canal irrigants: An in vitro study. J Indian Soc Pedod Prev Dent 2019;37:258-64

How to cite this URL:
Devan K, Peedikayil FC, Chandru T P, Kottayi S, Dhanesh N, Suresh K R. Antimicrobial efficacy of medium chain fatty acids as root canal irrigants: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Oct 23];37:258-64. Available from: http://www.jisppd.com/text.asp?2019/37/3/258/268186





   Introduction Top


Microorganisms are ubiquitous in nature, and it is not surprising to see their involvement in pulpal diseases. The most frequently isolated microorganisms before root canal treatment include Gram-negative anaerobic rods, Gram-positive anaerobic cocci, Gram-positive anaerobic and facultative rods, Lactobacillus species, and Gram-positive facultative Streptococcus species.[1] The obligate anaerobes are rather easily eradicated during root canal treatment. On the other hand, facultative bacteria such as nonmutans Streptococci, Enterococci, and Lactobacilli, once established, are more likely to survive chemomechanical instrumentation and root canal medication.[2]Enterococcus faecalis has gained attention in the endodontic literature, as it can frequently be isolated from root canals in cases of failed root canal treatments.[3] In addition, fungi (especially Candida species) may also be found in root canals associated with therapy-resistant apical periodontitis.[4] Among the Candida species, Candida albicans is the most frequently isolated variety. They enter the root canal from the oral cavity in cases of pathologically open endodontium, improper isolation techniques, or when the cavity is not properly sealed during lengthy treatments.

Elimination of microorganisms from infected root canals is not an easy task. Numerous methods have been described to reduce the number of microorganisms in the root canal system, including the use of various instrumentation techniques, irrigation regimens, and intracanal medicaments.[5] Irrigation is an adjuvant to mechanical instrumentation in facilitating the removal of pulp tissue and/or microorganisms.[6] The effectiveness of irrigation depends on the working mechanism of the irrigant and the ability to bring the irrigant in contact with the microorganisms and tissue debris in the root canal.

A large number of substances have been used as root canal irrigants, including acids (citric and phosphoric), chelating agent (ethylene diamine tetraacetic acid [EDTA]), proteolytic enzymes, alkaline solutions (sodium hypochlorite, sodium hydroxide, urea, and potassium hydroxide), oxidative agents (hydrogen peroxide and gly-oxide), local anesthetic solutions, and normal saline.[7] However, the search for an ideal root canal irrigant continues with new studies focusing on natural derivatives.

The hydrolytic products of triglycerides and phospholipids, particularly the fatty acids, have antimicrobial activities. In addition to being natural compounds, they have the advantage of being both environmentally safe and generally harmless to the body in concentrations which kill pathogenic microbes. They are nonallergenic and are fully metabolized in the body. Fatty acids are abundant in nature and are an inexpensive source of antimicrobial products.[8]

Lauric acid (LA) or systematically, dodecanoic acid, is a saturated fatty acid with a 12-carbon atom chain. It is a white, powdery solid with a faint odor of baby oil or soap. Decanoic acid (DA) (capric acid) is a saturated fatty acid with a ten-carbon atom chain. Caprylic acid is the common name for the eight-carbon saturated fatty acid known by the systematic name octanoic acid (OA). Their main natural sources include coconut oil, laurel oil, and palm kernel oil.[9]

The literature on the effects of fatty acids dates as far back as the work of Clark reported in 1899.[10] In the subsequent years, antifungal and bactericidal properties of fatty acids have been extensively investigated. Early studies suggest that medium-chain fatty acids (MCFA) are bactericidal for Gram-positive bacteria, more so than Gram-negative bacteria, fungi, protozoa, and viruses.[11]

Thus, the aim of this study is to evaluate the antimicrobial efficacy of MCFA as root canal irrigants against E. faecalis and C. albicans.


   Methodology Top


The study was approved by the Review Board and Institutional Ethical Committee of Kannur Dental College, Anjarakandy. LA, DA and OA were purchased from Sigma Aldrich, Bengaluru, India [Figure 1]. One hundred percent ethanol was used to dissolve the fatty acids and was, therefore, taken as a control. The test organisms E. faecalis (ATCC 29212) and C. albicans (ATCC 10231) were obtained from Biogenix Research Centre, Thiruvananthapuram, India.
Figure 1: Medium chain fatty acids

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Ninety noncarious single-rooted mandibular premolar teeth with matured, closed apices, extracted for orthodontic treatment were selected. The external root surfaces were debrided with a curette and all teeth were placed in 0.5% NaOCl for 24 h for surface disinfection and stored in 0.9% sterile saline at room temperature till use.

Teeth were decoronated at the cementoenamel junction using the diamond disc and root lengths standardized to approximately 14 mm. Root canals were biomechanically prepared by apico coronal (passive step back) technique and instrumented 1 mm beyond the apical foramen up to size 50. Irrigation with sterile saline was done during preparation.

Each canal was then rinsed with 1 ml of 17% EDTA for 1 min followed by 3 ml of 5.25% NaOCl to facilitate removal of the smear layer. Following root canal preparation, the enlarged apical foramina was sealed with cyanoacrylate adhesive to prevent bacterial leakage. To make both handling and identification easier, the teeth were mounted vertically in dental stone blocks [Figure 2].
Figure 2: Decoronated teeth mounted on dental stone

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The media and tooth samples were sterilized by autoclaving at 121°C; 15l bps for 15 min. The sterilized tooth samples were kept in the media for 24 h for sterility check. After 24 h, the media was observed for any turbidity. After sterilization, the ninety teeth were divided into three groups: Group A, Group B, and Group C. Group A, Group B, and Group C, each containing thirty teeth.

The minimum inhibitory concentration (MIC) of the three MCFAs against E. faecalis and C. albicans were determined using two-fold serial dilution method.

Inoculation of organism

  • Group A (n = 30): The tooth samples were inoculated with a pure culture of E. faecalis (10 μl/tooth) and were kept for incubation at 37°C for 24 h. The 30 teeth were then divided into three subgroups (n = 10): A1, A2, and A3 irrigated with 2 ml of LA, DA, and OA each dissolved in 100% ethanol for 5 min, respectively [Figure 3]
  • Group B (n = 30): The tooth samples were inoculated with pure culture of C. albicans (10 μl/tooth) and were kept for incubation at 37°C for 24 h. The 30 teeth were then divided into three subgroups: B1, B2, and B3 irrigated with 2 ml of LA, DA and OA each dissolved in 100% ethanol for 5 min, respectively
  • Group C (n = 30): Divided into three subgroups: C1 negative control where root canals not contaminated with E. faecalis or C. albicans and not irrigated with any solutions. In C2 and C3 groups, root canals were contaminated with E. faecalis and C. albicans, respectively and irrigated with pure 100% ethanol for 5 min.


The canals were then irrigated with 1 ml sterile saline solution and a size 45 sterile paper point was selected to sample the microorganisms from the root canals. The paper points were left in the wet canal for 1 min and then transferred to the tubes containing 5 ml of the Brain Heart Infusion broth for E. faecalis and potato dextrose broth for C. albicans, respectively [Figure 4]. The tubes were then vortexed for 5 min and incubated at 37°C for 4 days.
Figure 3: Root canal irrigation of the teeth sample with respective irrigant test solution

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Figure 4: Paper points in Eppendorf tubes containing selected broth

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Direct contact test (DCT) was used to determine the optical density. It is based on the turbidimetric determination of bacterial growth in 96-well microtiter plates. Kinetics of outgrowth in each well was monitored at 630 nm at 37°C and recorded using a spectrophotometer (ELISA reader: Stat Fax 2100; Awareness Technology) after 24, 48, 72, and 96th h.

The data were analyzed using analysis of variance (ANOVA) to compare the significance of difference between means of more than two independent groups at 5% level of significance in the Statistical Package for the Social Sciences for Windows, SPSS 17 (IBM Corporation, Chicago, US). If P < 0.05, it can be concluded that there is a significant difference between the groups considered with regard to mean.


   Results Top


The MICs of the three MCFAs against E. faecalis and C. albicans are represented in [Table 1] and [Table 2].
Table 1: MIC values of test materials against Enterococcus faecalis

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Table 2: MIC values of test materials against Candida albicans

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[Table 3] shows the mean values and ANOVA for Group A, i.e; E. faecalis group, for 24, 48, 72, and 96 h. All the three materials showed inhibitory activity against E. faecalis., LA showed maximum inhibitory activity followed by OA and the least inhibitory activity was shown by DA at 24, 48, 72, and 96 h. Further, it can be noted that the maximum inhibitory activity was shown by all the materials at 24 h. The inhibitory activity decreased considerably at 48, 72, and 96 h. On comparing the three materials using ANOVA, statistically significant difference was found only at 24 h. There was no statistically significant difference observed among the materials studied at 48, 72, and 96 h.
Table 3: Mean and analysis of variance in Group A for 24, 48, 72 and 96 h

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[Table 4] shows the mean values and ANOVA for Group B, i.e.; C. albicans group, for 24, 48, 72, and 96 h. All the three materials showed inhibitory activity against C. albicans. DA showed maximum inhibitory activity followed by OA and the least inhibitory activity was shown by LA at 24, 48, 72, and 96 h. DA maintained considerable inhibitory activity for 48, 72, and 96 h. On comparing the three materials using ANOVA, statistically significant difference was found at 24, 48, and 72 h.
Table 4: Mean and analysis of variance in Group B for 24, 48, 72 and 96 h

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[Table 5] shows the mean values for Group C, i.e.; control group, for 24, 48, 72, and 96 h. There was no visible turbidity observed in the C1 group, but the DCT showed optical density of 0.0021, 0.0024, 0.0026 and 0.0028 at 24, 48, 72, and 96 h. In C2 (E. faecalis) and C3 (C. albicans) groups, the maximum inhibitory activity the maximum inhibitory activity by 100% ethanol was at 24 and 48 h.
Table 5: Mean and standard deviation of Group C

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   Discussion Top


E. faecalis is a facultative anaerobe that plays a major role in the etiology of persistent periradicular lesions after root canal treatment.[12] Studies show that fungi are present in about 10%–25% of endodontic infections, their significance being that they express a variety of virulence factors, particularly in association with bacteria, and are difficult to eradicate from the root canal environment.[13]E. faecalis and C. albicans were selected for the present study because they are the microbes that are most resistant to elimination by disinfecting agents and are causative agents for reinfection.

Therapeutically, extracted single-rooted mandibular premolars were selected in this study. Extracted human teeth were used to simulate the clinical condition and to test the efficiency of the test solutions within the root canal system.[14] Teeth were decoronated at the cementoenamel junction using diamond disc and root lengths finalized to approximately 14 mm. This was done to standardize the root lengths and make all the specimens uniform.

The teeth were biomechanically prepared and the apex enlarged to number 50 k file. This was done to ensure a better penetration of irrigation solutions to the apex of the root canal and to rule out the possibility that the lack of penetration into the apex of the canal is not the reason for a deficient antimicrobial activity.[15],[16] To prevent bacterial microleakage, the apical foramina of all the specimens were sealed with cyanoacrylate adhesive after the canal preparation.[15]

After the preparation of root canals, the teeth were autoclaved to ensure the sterilization of root canals and to make sure that there was no microorganism in the root canal other than the selected microorganisms. The sterilized tooth samples were kept in the media for 24 h for sterility check. After 24 h the media was observed for any turbidity.

The root canals were then artificially infected with the selected microorganisms E. faecalis and C. albicans and incubated for 48 h at 37°C to ensure the growth of the incubated organisms. After inoculation of the organisms, mechanical preparation was not done as the purpose of the study was to determine the antimicrobial efficacy of the irrigants following standard irrigation protocols.[15],[16]

The MIC of the materials to be tested was done using the broth microdilution method. Broth microdiluton test was chosen for the determination of MIC in this study because it is reproducible, easy to perform as channels are prepared, cost-effective, and saves reagents and space.

After determining the MIC, irrigation of the teeth sample were done using the test solutions. Each root canal was irrigated with 2 ml volume of the selected test solution and the irrigant was allowed to remain in the canal for 5 min to ensure that test solutions get sufficient time to interact with the inoculated microorganisms. Final irrigation was performed with 1 ml of sterile normal saline for all sample groups to demonstrate the substantivity of the test irrigants and to prevent the carryover of the antimicrobial solutions.[16]

Sterile paper points have been used to collect the samples in this study, as previous studies [17],[18],[19] have reported that the paper point cultures of the root canal detected bacteria more frequently than the sample collected by using files or reamers. Paper point samples were transferred to tubes containing Brain Heart Infusion broth for E. faecalis and potato dextrose broth for C. albicans as these media are selective for the test organisms and risk of false results due to the growth of potential bacterial contaminants that might have occurred during handling can be avoided.

The broth containing the samples were then incubated at 37°C for 4 days, and the turbidity was checked by using the DCT for 24, 48, 72, and 96 h, respectively. The DCT was chosen as it is a quantitative assay which allows water-insoluble materials to be tested. DCT relies on direct and close contact between the test microorganism and the tested material. It is virtually independent of the diffusion properties of both the tested material and the media. In addition, it is reproducible, quantitative, and not affected by the size of the inoculum.[20]

Ten teeth were not contaminated with either E. faecalis or C. albicans and used as negative control. This was done to ensure that sterility was maintained throughout the procedure and contamination did not occur at any stage of the experimental procedure.[16]

In the present study, all the three MCFAs showed inhibitory activity against E. faecalis at 24, 48, 72, and 96 h. The inhibitory action of fatty acids may be due to their surfactant activity and their ability to cause cellular lysis by disrupting cell membranes.[21] Of the three MCFAs, LA showed the highest inhibitory activity and the maximum inhibitory activity was seen at 24 h. Studies by Hess et al.[22] and Hinton and Ingram [23] reported inhibitory action of LA on E. faecalis biofilm formation. Similarly, Ja-Hyung and Young-Wook,[24] reported that LA exhibit significantly high antimicrobial activity by inhibiting microbial survival and biofilm growth against Streptococcus mutans. Padgett et al.[25] reported that high level of LA addition (8%) significantly lower the film water permeability. The inhibitory activity of LA decreased substantially at 48, 72, and 96 h. Compared to DA and OA, LA exhibited better antimicrobial activity may be because of the difference in the carbon chain length.[26]

Literature search revealed very few studies comparing the antimicrobial efficacy of all the three MCFAs on E. faecalis. Furthermore, the substantivity of the material could not be compared due to paucity of similar studies.

All the three MCFAs showed inhibitory activity against C. albicans at 24, 48, 72, and 96 h in this study. The maximum inhibitory activity was shown by DA, which is a ten carbon MCFA, and it retained considerable antimicrobial substantivity at 48 h, 72 h, and 96 h. Following DA, OA exhibited inhibitory activity against C. albicans and the least inhibitory activity was shown by LA. This result was similar to the results obtained by Huang et al.,[26] Bergsson et al.,[27] Hayama et al.,[28] Takahashi et al.,[29] and Jadhav et al.[30]

Murzyn et al.[31] reported that DA can inhibit filamentous growth, adhesion, and biofilm formation by C. albicans. Jadhav et al.[30] reported that DA and OAs inhibited C. albicans planktonic growth, morphogenesis, adhesion, and biofilm growth in vitro. In addition, there was cell cycle arrest at S and G2/M phases. They also reported that DA and OA inhibited biofilm development and eradicated mature biofilms as observed by light microscopy and scanning electron microscopy. According to Jadhav et al.[30] DA was more effective than OA which is similar to the findings in this study. This could be because OA was less hemolytic compared to DA.

Contradictory to the findings in this study, Tsukahara [32] reported OA as the most powerful fungistat against C. albicans among the normal saturated fatty acids with even-numbered carbon atoms. This disparity could be due to the difference in concentration of the fatty acids tested and the medium used for their dissolution in the two studies. The extended effect of capric acid cannot be compared as the literature search failed to reveal studies checking the antimicrobial activity of MCFAs against C. albicans for a prolonged duration.

In the present study, some amount of optical density was observed in the negative control group which could be due to the leaching of mineral contents from the tooth sample. To rule out false results due to this, percentage of inhibition was calculated wherein the optical density of control was subtracted from the optical density of the test sample.

The fatty acid solutions were prepared in ethanol stock. Some amount of inhibitory activity was also shown by ethanol in the present study. Thus, the inhibitory activity of the MCFAs could be enhanced with ethanol. This is similar to the findings of Huang et al.[26]

In the present study, the test materials were not compared with any of the routinely used standard root canal irrigants. Hence, how much effective MCFAs are as root canal irrigants in comparison to other standard irrigants cannot be determined, which is a drawback of our study. It should also be kept in mind that in the oral cavity bacteria grow in complex biofilms. The biofilm itself has different properties, chemically and physically, while the community of organisms behaves differently to isolated species.[33],[34] Hence, the results obtained here can vary in a biofilm environment. Extrapolation to the clinical environment requires careful interpretation of the findings of in vitro studies.


   Conclusion Top


From the findings of this study, it can be concluded that the MCFAs exhibit antimicrobial activity against E. faecalis and C. albicans. Even though MCFAs show promising effect as root canal irrigants, future studies should be conducted with larger sample size to investigate the effectiveness of these acids in comparison to standard root canal irrigating solutions. Future studies can also be directed on checking the antimicrobial effect of different combinations of MCFAs in a biofilm model. Finally, in vivo studies have to be conducted before the MCFAs can be introduced as root canal irrigants clinically.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Kumar J, Sharma R, Sharma M, Prabhavathi V, Paul J, Chowdary CD. Presence of Candida albicans in root canals of teeth with apical periodontitis and evaluation of their possible role in failure of endodontic treatment. J Int Oral Health 2015;7:42-5.  Back to cited text no. 13
    
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Radwan IN, Randa B, Hend AN, Camilia G. Evaluation of antimicrobial efficacy of four medicinal plants extracts used as root canal irrigant on Enterococcus faecalis: An in vitro study. Int Dent Med J Adv Res 2015;1:1-8.  Back to cited text no. 14
    
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Vijaykumar S, GunaShekhar M, Himagiri S.In vitro effectiveness of different endodontic irrigants on the reduction of Enterococcus faecalis in root canals. J Clin Exp Dent 2010;2:169-72.  Back to cited text no. 15
    
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Agrawal V, Rao MR, Dhingra K, Gopal VR, Mohapatra A, Mohapatra A. An in vitro comparison of antimicrobial effcacy of three root canal irrigants-bioPure MTAD, 2% chlorhexidine gluconate and 5.25% sodium hypochlorite as a final rinse against E. Faecalis. J Contemp Dent Pract 2013;14:842-7.  Back to cited text no. 16
    
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Siqueira JF Jr., Rôças IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite. J Endod 2000;26:331-4.  Back to cited text no. 17
    
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Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M. Bacterial reduction with nickel-titanium rotary instrumentation. J Endod 1998;24:763-7.  Back to cited text no. 18
    
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Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24:472-6.  Back to cited text no. 19
    
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Hess DJ, Henry-Stanley MJ, Wells CL. The natural surfactant glycerol monolaurate significantly reduces development of Staphylococcus aureus and Enterococcus faecalis biofilms. Surg Infect (Larchmt) 2015;16:538-42.  Back to cited text no. 22
    
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Ja-Hyung L, Young-Wook J. Antimicrobial effect of a lauric acid on Streptococcus mutans biofilm. Ann Int Med Dent Res 2016;2:21.  Back to cited text no. 24
    
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Huang CB, Alimova Y, Myers TM, Ebersole JL. Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms. Arch Oral Biol 2011;56:650-4.  Back to cited text no. 26
    
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Bergsson G, Arnfinnsson J, Steingrímsson O, Thormar H.In vitro killing of Candida albicans by fatty acids and monoglycerides. Antimicrob Agents Chemother 2001;45:3209-12.  Back to cited text no. 27
    
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Hayama K, Takahashi M, Yui S, Abe S. Inhibitory effects of several saturated fatty acids and their related fatty alcohols on the growth of Candida albicans. Drug Discov Ther 2015;9:386-90.  Back to cited text no. 28
    
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Takahashi M, Inoue S, Hayama K, Ninomiya K, Abe S. Inhibition of Candida mycelia growth by a medium chain fatty acids, capric acid in vitro and its therapeutic efficacy in murine oral candidiasis. Med Mycol J 2012;53:255-61.  Back to cited text no. 29
    
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Jadhav A, Mortale S, Halbandge S, Jangid P, Patil R, Gade W, et al. The dietary food components capric acid and caprylic acid inhibit virulence factors in Candida albicans through multitargeting. J Med Food 2017;20:1083-90.  Back to cited text no. 30
    
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33.
Barry AL, editor. Agar diffusion test. In: The Antimicrobial Susceptibility Test: Principles and Practices. Philadelphia, PA: Lea & Febiger; 1976. p. 163-213.  Back to cited text no. 33
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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