Antifungal activity of synthetic antiseptics and natural compounds against Candida dubliniensis before and after in vitro fluconazole exposure

ABSTRACT

INTRODUCTION:

This study evaluated the susceptibilities of oral candidiasis-derived Candida albicans, fluconazole-resistant (FR) Candida dubliniensis, and fluconazole-susceptible (FS) C. dubliniensis to synthetic antiseptics [chlorhexidine gluconate (CHX), cetylpyridinium chloride (CPC), and triclosan (TRC)] and natural compounds (carvacrol, eugenol and thymol).

METHODS:

Susceptibility tests were performed based on the M27-A3 reference method. The fluconazole-resistant C. dubliniensis strains were obtained after prolonged in vitro exposure to increasing fluconazole concentrations. The geometric mean values for minimum inhibitory concentrations and minimum fungicidal concentrations were compared among the groups.

RESULTS:

Fluconazole-susceptible C. dubliniensis was more sensitive to CPC and TRC than FR C. dubliniensis and C. albicans were. However, eugenol and thymol were more active against FR C. dubliniensis. The fungicidal activities of CHX and TRC were similar for the three groups, and FR C. dubliniensis and C. albicans had similar sensitivities to CPC.

CONCLUSIONS:

The resistance of C. dubliniensis to fluconazole affects its sensitivity the synthetic antiseptics and natural compounds that were tested.

Keywords:
Antiseptics; Susceptibility; Candida dubliniensis

INTRODUCTION

Candidiasis is the most common fungal infection among immunocompromised patients. These infections frequently involve the oral cavity, as Candida spp. are commensal organisms, and may contaminate other lesions. Candida albicans is the most frequently occurring species, although other Candida species (e.g., Candida dubliniensis) are becoming more common. C. dubliniensis was recognized as a new species in 1995, when it was isolated from the oral cavity of patients with human immunodeficiency virus (HIV) infections and acquired immunodeficiency syndrome (AIDS)¹. Although C. dubliniensis shares many phenotypic characteristics with C. albicans, C. dubliniensis has a notable ability to acquire resistance to fluconazole²_.

In odontology, as well as during treatment of cancer using antineoplastic and/or radiotherapy, mouthwash use has become an established adjunct to antimicrobial treatment. These mouthwashes have also been formulated to contain various antiseptics, such as chlorhexidine gluconate (CHX), cetylpyridinium chloride (CPC), triclosan (TRC), thymol, and eugenol^3-6. These compounds have well-known antibacterial activities, although the susceptibility of fungi, especially Candida spp., remains unclear.

Among Candida spp., the development of antifungal resistance is an emergent phenomenon that can be confirmed using standardized susceptibility tests⁷. However, it remains unclear whether oral antiseptics can inhibit fluconazole-resistant (FR) Candida spp. Therefore, we compared the susceptibilities of C. albicans, fluconazole-susceptible (FS) C. dubliniensis, and FR C. dubliniensis to well-known antiseptics and several natural compounds (eugenol, carvacrol, and thymol).

METHODS

Microorganisms

The present study evaluated 20 Candida dubliniensis strains and 20 Candida albicans strains that were isolated from oropharyngeal candidiasis cases. Because the strains’ susceptibility to fluconazole was already known, these isolates were classified as the FS C. dubliniensis group and the C. albicans group. Based on the methods of Fekete-Forgács et al.⁸, a third group was created from the FS C. dubliniensis group by exposing the strains to increasing concentrations of fluconazole, and this group was named the FR C. dubliniensis group (n = 20). The three groups’ minimum inhibitory concentration (MIC) ranges were found to be 0.25-4µg/mL (FS C. dubliniensis), 0.25-64µg/mL (FR C. dubliniensis), and 0.25-16µg/mL (C. albicans).

Antimicrobial agents

The studied synthetic compounds were CHX, CPC, and TRC. The studied natural compounds were carvacrol, eugenol, and thymol. All compounds were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Antifungal susceptibility tests

The antimicrobial agents were diluted to create stock solutions and testing concentrations: CHX (10mg/mL; 0.4-250µg/mL), CPC (10mg/mL; 0.04-25µg/mL), TRC (10mg/mL; 0.04-25µg/mL), carvacrol (20mg/mL; 1.22-625µg/mL), eugenol (50mg/mL; 2.44-1,250µg/mL), and thymol (10mg/mL; 1.22-625µg/mL). CHX, CPC, and TRC were diluted in distilled water, and carvacrol, eugenol, and thymol were diluted in methanol. One hundred-microliter aliquots of the two-fold diluted compounds were dispensed into 96-well microtiter plates, and the compounds’ MICs were determined using the M27-A3 reference protocol (Clinical & Laboratory Standards Institute, 2008). The medium was Roswell Park Memorial Institute (RPMI) 1640 broth containing 2% dextrose buffered using 3-(N-morpholino) propanesulfonic acid. According to the M27-A2 protocol, the inocula were standardized by suspending the yeast (five colonies grown on Sabouraud dextrose agar) in saline solution (0.085%) and adjusting the turbidity. All tests were performed in triplicate, and each series included a positive control (diluted inoculum working solution) and a negative control (RPMI 1640 alone). The cell suspensions were diluted 1:50 using distilled water and 1:20 using RPMI 1640 medium. After adding the 100-µL cell suspension aliquots, the microdilution plate was incubated at 35°C for 48h. Yeast growth was monitored visually, and the MIC for each compound was defined as the lowest concentration required to arrest visible fungal growth at the end of the 48-h incubation. The minimal fungicidal concentrations (MFCs) were determined by subculturing 0.01mL from each well without visible growth during the MIC assay onto Sabouraud dextrose agar plates. The lowest concentration of the antimicrobial agents to prevent growth was defined as the MFC value.

Statistical analysis

The groups’ susceptibilities (MICs and MFCs) to each antiseptic compound were compared using the paired Wilcoxon test (FS C. dubliniensis vs. FR C. dubliniensis) and the unpaired Mann-Whitney test (C. albicans vs. FS C. dubliniensis and C. albicans vs. FR C. dubliniensis). P-values of <0.05 were considered statistically significant⁹.

RESULTS

The MICs and susceptibility profiles of C. albicans, FS C. dubliniensis, and FR C. dubliniensis are shown in Table 1. The CPC tests revealed that FS C. dubliniensis group was significantly more susceptible, compared to C. albicans (p < 0.001) or FR C. dubliniensis (p < 0.05). A similar susceptibility pattern was observed for TRC (FS C. dubliniensis vs. C. albicans, p < 0.05; FS C. dubliniensis vs. FR C. dubliniensis, p < 0.05). C. albicans was significantly less susceptible to CHX, compared to the FS C. dubliniensis and FR C. dubliniensis groups (p < 0.05). The susceptibility tests for TRC revealed that C. albicans was significantly less susceptible, compared to FS C. dubliniensis (p < 0.05). However, no differences were detected between the FR C. dubliniensis and C. albicans groups.

TABLE 1
Comparing the MICs of Candida albicans and fluconazole-susceptible and -resistant forms of Candida dubliniensis for synthetic and natural antiseptic compounds.

Antiseptics	Groups*	MIC range	GM	Comparisons	P-values
	(n = 20)	(µg/mL)	(µg/mL)
Cetylpyridinium chloride	A	0.78-6.25	1.56	A × B	<0.05
	B	1.56-6.25	3.44	A × C	<0.001
	C	3.12-6.25	4.26	B × C	ns
Chlorhexidine gluconate	A	0.97-7.8	3.63	A × B	ns
	B	1.95-15.6	3.51	A × C	<0.05
	C	1.95-7.8	5.14	B × C	<0.05
Triclosan	A	0.78-25	10.5	A × B	<0.05
	B	6.25-25	16.8	A × C	<0.05
	C	6.25-25	17.1	B × C	ns
Carvacrol	A	78.1-625	206.4	A × B	<0.01
	B	78.1-312.5	148.7	A × C	ns
	C	78.2-312.5	213.6	B × C	<0.05
Eugenol	A	312.5-625	603.7	A × B	<0.05
	B	156.5-625	371.6	A × C	ns
	C	156.2-625	583.1	B × C	<0.001
Thymol	A	39.06-625	272	A × B	ns
	B	156.2-625	286	A × C	ns
	C	78.2-625	301.9	B × C	ns

• MICs: minimum inhibitory concentration; GM: geometric mean; ns: not significant. *A: fluconazole-susceptible Candida dubliniensis; B: fluconazole-resistant Candida dubliniensis; C: Candida albicans.

The FR C. dubliniensis group was more susceptible to carvacrol than the FS C. dubliniensis group (p < 0.01) and the C. albicans group (p < 0.05). The FR C. dubliniensis group was less susceptible to eugenol than the FS C. dubliniensis group (p < 0.05) and the C. albicans group (p < 0.001). The FS C. dubliniensis and C. albicans groups had similar susceptibilities to eugenol. No significant differences were observed among the three groups’ susceptibilities to thymol.

The MFC values are shown in Table 2. Significantly differences in susceptibility were observed for CPC (FS C. dubliniensis was more susceptible than C. albicans; p < 0.001), carvacrol (FR C. dubliniensis was more susceptible than FS C. dubliniensis; p < 0.05), and eugenol (FR C. dubliniensis was more susceptible than FS C. dubliniensis; p < 0.01). All other tests did not reveal any significant differences among the groups.

TABLE 2
Comparing the MFCs of Candida albicans and fluconazole-susceptible and -resistant forms of Candida dubliniensis for synthetic and natural antiseptic compounds.

Antiseptics	Groups*	MFC range	GM	Comparisons	P-values
	(n = 20)	(µg/mL)	(µg/mL)
Cetylpyridinium chloride	A	0.78-50	2.43	A × B	ns
	B	1.56-25	5.38	A × C	< 0.001
	C	3.12-25	7.69	B × C	ns
Chlorhexidine gluconate	A	0.97-500	7.3	A × B	ns
	B	1.95-250	7.06	A × C	ns
	C	3.9-31.25	6.56	B × C	ns
Triclosan	A	1.56-50	17.7	A × B	ns
	B	6.25-50	18.5	A × C	ns
	C	6.25-50	18.9	B × C	ns
Carvacrol	A	156-625	301.8	A × B	< 0.05
	B	156-625	256.6	A × C	ns
	C	156-312	262.8	B × C	ns
Eugenol	A	625-2,500	915.1	A × B	< 0.01
	B	312-2,500	625	A × C	ns
	C	625-1,250	769.5	B × C	ns
Thymol	A	312-625	420.6	A × B	ns
	B	312-625	388.1	A × C	ns
	C	78.12-625	359	B × C	ns

• MFC: minimum fungicidal concentration; GM: geometric mean; ns: not significant. *A: fluconazole-susceptible Candida dubliniensis. B: fluconazole-resistant Candida dubliniensis . C: Candida albicans.

DISCUSSION

The present study evaluated Candida albicans because it is the most studied yeast-like fungi that can be responsible for oral candidiasis in immunocompromised patients. We also considered C. dubliniensis because it can acquire resistance to fluconazole and its susceptibility to antiseptics remains largely unknown.

Fluconazole exposure may alter resistance to other antifungal agents. Thus, we created a group of FR C. dubliniensis using prolonged in vitro exposure to this triazole, in order to evaluate the effect of this resistance on susceptibility to other oral antiseptics. However, it is important to recognize that this form of induced resistance may be difficult from naturally occurring resistance in patients with oral candidiasis.

In general, the FS C. dubliniensis group was significantly more sensitive to the studied compounds’ fungistatic activity, compared to the other groups. Furthermore, the MICs in the FR C. dubliniensis group were similar to those in the C. albicans group. However, measurement of the compounds’ fungicidal activities did not reveal any significant differences in the groups’ susceptibilities to CHX and TRC. Furthermore, the FS C. dubliniensis group was more sensitive to CPC, compared to the other groups, and the FR C. dubliniensis group was less sensitive to CHX. Although this reduced susceptibility was not evident in the MFC tests, we believe that it may be a sign of emerging resistance.

Chlorhexidine gluconate is a biguanide compound that is commonly found in toothpastes, hand soaps, and mouthwashes. In addition, it can be used as adjunct antifungal therapy for candidiasis, as it induces coagulation of nucleoproteins, inhibits budding, and causes changes in the cell wall that lead to cytoplasmic component escape and yeast death⁵. Similar to our findings, Shresta et al.⁵ found that C. tropicalis was less susceptible, compared to C. albicans, albeit using different methods. Fathilah et al.¹⁰ have also reported elevated MICs for Candida tropicalis (75µg/mL) and Candida krusei (150µg/mL), which were much higher than the GM MICs from the present study (3.63-5.14µg/mL). These findings highlight the differences in the susceptibilities of Candida spp. to CHX. Thurnmond et al.¹¹ have also reported variations in the MICs of C. albicans after daily CHX exposure, with an increase in the MIC range from 5-10 during week 1 to 2.5-20µg/mL during week 8. These findings are consistent with reports regarding varying degrees of stomatitis that are related to reducing the numbers and occurrences of oral Candida spp., oral candidiasis, and Candida-related morbidity and mortality¹².

Cetylpyridinium chloride is a cationic quaternary ammonium compound that is widely used in mouthwashes to prevent or treat candidiasis and bacterial infections¹³. CPC alters the surface tension of the cell wall structure, which may lead to cell wall leakage. Based on the GM MIC values, we found that CPC provided greater activity (1.56-4.26µg/mL), compared to the results of Fathilah et al.¹⁰, who found MICs of 66 µg/mL for C. tropicalis and 33µg/mL for C. krusei. Edling et al.¹⁴ have reported that two strains of FR C. albicans have reduced CPC susceptibility, which suggests that mouthwashes with CPC might select for resistant strains. Our results did not confirm this possibility, because the FR C. dubliniensis and C. albicans groups had similar susceptibilities, although we did not test C. tropicalis and C. krusei.

Our results also revealed that the FR C. dubliniensis and C. albicans groups had similar sensitivities to TRC, although the FS C. dubliniensis group was more sensitive than the C. albicans and FR C. dubliniensis groups. In contrast, Jones et al.¹⁵ reviewed the activity of TRC against fungi and reported MICs that ranged from 1.63µg/mL for Epidermophyton floccosum to 5,000µg/mL for Blastomyces dermatitidis; C. tropicalis ATCC 750 was inhibited by 2,500µg/mL of TRC. Furthermore, Yu et al.¹⁶ studied the combination of TRC and fluconazole against FR C. albicans, and reported MICs of 32-64µg/mL. When fluconazole and TRC were combined, the MICs of TRC decreased to 4-8µg/mL¹⁶, which suggested that fluconazole resistance affected the susceptibility to TRC. However, we did not detect this phenomenon in the present study.

Carvacrol, eugenol, and thymol are natural compounds that are contained in the main fractions of essential oils from Origanum vulgare, Syzygium aromaticum, and Thymus vulgaris, respectively. All three compounds are terpenoids and have antimicrobial activities against a wide range of pathogens, including Candida spp.¹⁷_. In contrast to our findings with the synthetic compounds, we found that the FR C. dubliniensis group was significantly more susceptible to carvacrol than the FS C. dubliniensis and C. albicans groups. The susceptibility of C. dubliniensis to carvacrol is poorly understood, and only a small number of isolates have been reported⁴. In the present study, the carvacrol MICs for C. albicans (78.2-312.5µg/mL) were higher than the 0.16µg/mL values for C. albicans and C. dubliniensis that were reported by Vale-Silva et al.¹⁸. Those authors also reported that the MFC for carvacrol was similar to the MIC¹⁸, while we found that the MFCs were generally higher than the MICs.

Similar to the results for carvacrol, the MIC and MFC values for eugenol were higher in the FS C. dubliniensis group than in the FR C. dubliniensis group. Conflicting results have been reported by Ahmad et al.¹⁹, who noted that FR strains had higher sensitivity to eugenol than the standard or clinical strains did. In addition to its use as an antiseptic agent, eugenol is applied topically to dental cavities, used as a component of dental protectives, and combined with zinc oxide to form zinc oxide eugenol, which has restorative and prosthodontic applications in dentistry²⁰.

The fungistatic and fungicidal activities of thymol were similar in the three groups. Guo et al.²¹ have also studied the activity of thymol against FS and FR C. albicans, although our results (based on the MIC ranges) were higher than their results. Thymol causes protein denaturation and damage to cellular membranes, which results in the leakage of intracellular components⁵. As suggested by Ahmad et al.¹⁹, the antifungal activities of carvacrol, eugenol, and thymol against FR and FS C. dubliniensis highlight the possibility that these compounds could expand the existing class of useful antifungal agents. Thus, these compounds might be used in pharmaceutical products, such as the antiseptic ingredients for mouthwashes.

In conclusion, our results indicate that FS C. dubliniensis were more sensitive to antiseptics than FR C. dubliniensis and C. albicans, which highlights the possibility that acquired resistance to fluconazole may alter antiseptic susceptibility. Interestingly, we did not observe this cross-resistance for the natural compounds (carvacrol, eugenol, and thymol). As stated by Fraise²², changes in the cell wall may also contribute to cross-resistance between biocides and antibiotics, which most likely involves reduced permeability. Thus, researchers must be alert for changes in the susceptibility of yeasts to antiseptics, given the increasing number of antimycotics that may target the cell wall.

Acknowledgments

We thank the institutions that provided technical support for the development and implementation of this study.

REFERENCES

• ¹
  Sullivan DJ, Haynes KA, Bennett DE, Coleman DC. Candida dubliniensis sp. nov. phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology. 1995;141(7):1507-21.
• ²
  Moran GP, Sullivan DJ, Henman MC, McCreary CE, Harrington BJ, Shanley DB, et al. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV infected subjects and generation of stable fluconazole-resistant derivatives in vitro Antimicrob Agents Chemother. 1997;41(3):617-23.
• ³
  Aroonrerk N, Dhanesuan N. Candida inhibitory effects of six commercial mouthwashes. Ann Microbiol. 2007,57(3):449-52.
• ⁴
  Marcos-Arias C, Eraso E, Madariaga L, Quindós G. In vitro activities of natural products against oral Candida isolates from denture wearers. BMC Complement Altern Med. 2011;11:119. doi: http://bmccomplementalternmed.biomedcentral.com/articles/10.1186/1472-6882-11-119
  » http://bmccomplementalternmed.biomedcentral.com/articles/10.1186/1472-6882-11-119
• ⁵
  Shresta A, Rimal J, Rao A, Sequeira PS, Doshi D, Bhat GK. In vitro antifungal effect of mouth rinses containing chlorhexidine and thymol. J Dent Sci. 2011;6(1):1-5.
• ⁶
  Giuliana G, Pizzo G, Milici ME, Musotto GC, Giangreco R. In vitro antifungal properties of mouthrinses containing antimicrobial agents. J Periodontol. 1997;68(8):729-33.
• ⁷
  Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing Yeasts; Approved Standard M27-A3, Third edition. Wayne: CLSI. 2008a. 25p.
• ⁸
  Fekete-Forgács K, Gyüre L, Lenkey B. Changes of virulence factors accompanying the phenomenon of induced fluconazole resistance in Candida albicans Mycoses. 2000;43(7-8):273-9.
• ⁹
  Olsen CH. Review of the use of statistics in infection and immunity. Infect Immun. 2003;71(12):6689-92.
• ¹⁰
  Fathilah AR, Himratul-Aznita WH, Fatheen ARN, Suriani KR. The antifungal properties of chlorhexidine digluconate and cetylpyrinidinium chloride on oral Candida J Dent. 2012;40(7):609-15.
• ¹¹
  Thurmond JM, Brown AT, Sims RE, Ferretti GA, Raybould TP, Lillich TT, et al. Oral Candida albicans in bone marrow transplant patients given chlorhexidine rinses: occurrence and susceptibilities to the agent. Oral Surg Oral Med Oral Pathol. 1991;72(3):291-5.
• ¹²
  McGaw WT, Belch A. Oral complications of acute leukemia: prophylactic impact of a chlorhexidine mouth rinse regimen. Oral Surg Oral Med Oral Pathol . 1985,60(3):275-280.
• ¹³
  McDonnell G, Russell D. Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev. 1999;12(1):147-79.
• ¹⁴
  Edling MP, Smith WL, Edlind TD. Effects of cetylpiridinium chloride resistance and treatment on fluconazole activity versus Candida albicans Antimicrob Agents Chemother . 2005;49(2):843-5.
• ¹⁵
  Jones RD, Jampani HB, Newman JL, Lee AS. Triclosan: a review of effectiveness and safety in health care settings. Am J Infect Control. 2000;28(2):184-96.
• ¹⁶
  Yu L, Ling G, Deng X, Jin J, Jin Q, Guo N. In vitro interaction between fluconazole and triclosan against clinical isolates of fluconazole-resistant Candida albicans determined by different methods. Antimicrob Agents Chemother . 2011;55(7):3609-12.
• ¹⁷
  Pozzatti P, Scheid LA, Spader TB, Atayde ML, Santurio JM, Alves SH. In vitro activity of essential oils extracted from plants used as spices against fluconazole-resistant and fluconazole-susceptible Candida spp. Can J Microbiol. 2008;54(11):950-6.
• ¹⁸
  Vale-Silva L, Gonçalves MJ, Cavaleiro C, Salgueiro L, Pinto E. Antifungal activity of the essential oil of Thymus x viciosoi against Candida, Cryptococcus, Aspergillus and dermatophyte species. Planta Med. 2010;76(9):882-8.
• ¹⁹
  Ahmad A, Khan A, Khan LA, Manzoor N. In vitro synergy of eugenol and methyleugenol with fluconazole against Candida isolates. J Med Microbiol. 2010;59(10):1178-84.
• ²⁰
  Jadhav BK, Khandelwal KR, Ketkar AR, Pisal SS. Formulation and evaluation of mucoadhesive tablets containing eugenol for treatment of periodontal diseases. Drug Dev Ind Pharm. 2004;30(2):195-203.
• ²¹
  Guo N, Liu J, Wu X, Bi X, Meng R, Wang X, et al. Antifungal activity of thymol against clinical isolates of fluconazole-sensitive and -resistant Candida albicans J Med Microbiol . 2009;58(8):1074-9.
• ²²
  Fraise AP. Biocide abuse and antimicrobial resistance-a cause for concern? J Antimicrob Chemother. 2002;49(1):11-2.

• Financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Grant. Proc. 304168/2012-2)

Publication Dates

• Publication in this collection
  Jan-Feb 2017

History

• Received
  02 Nov 2016
• Accepted
  07 Feb 2017