Mycopathologia DOI 10.1007/s11046-012-9520-4 Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses Sara Carvalhinho • Ana Margarida Costa • Ana Cla´udia Coelho • Euge´nio Martins • Ana Sampaio Received: 24 June 2011 / Accepted: 2 January 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Forty Candida albicans strains isolated DD and the Etest was 100% for amphotericin and from patient’s mouth with fixed orthodontic appliances fluconazole. One isolate was resistant to econazole were analyzed to their susceptibilities to antifungal (2.5%) and the other to ketoconazole (2.5%). Econa- agents, mouth rinses and essential oils. Susceptibility zole and ketoconazole had the highest percentages of to fluconazole, econazole, miconazole and ketocona- susceptible dose dependent (SDD), 55 and 95%, zole, amphotericin B and nystatin was assessed by the respectively. Regarding to the susceptibility isolates disk diffusion (DD) method based on the Clinical and profile, seven phenotypes were detected, and the 3 Laboratory Standards Institute M44-A protocol, and more represented (90% of the isolates) of them were by Etest (fluconazole and amphotericin B). The SDD to one, two or three azoles. The study of mouth susceptibilities to mouth rinses and essential oils were rinses showed a high variability of efficacy against also determined by the DD technique. All isolates C. albicans. The results showed that the isolates tested were susceptible (S) to amphotericin B, nystatin susceptibility to essential oils differed (P \ 0.05). The and fluconazole. The overall concordance between the profile activity was: cinnamon [ laurel [ mint [ euca- lyptus [ rosemary [ lemon [ myrrh [ tangerine. The main finding was that the susceptibility to cinnamon and S. Carvalhinho Á A. M. Costa Á A. Sampaio laurel varied among the three more representative Department of Biology and Environment, University antifungal phenotypes (P \ 0.05). The susceptibility of Tra´s-os-Montes and Alto Douro (UTAD), PO Box 1013, 5001-911 Vila Real, Portugal of econazole-SDD isolates to cinnamon and lemon was higher than those of the econazole-S yeasts (P \ 0.05). S. Carvalhinho Á A. Sampaio (&) In contrast, econazole-SDD isolates were less affected Centre for the Research and Technology of Agro- by laurel than econazole-S counterparts (P \ 0.05). Environment and Biological Sciences (CITAB), Universidade de Tra´s-os-Montes e Alto Douro, PO Box 1013, 5001-801 Vila Real, Portugal Keywords Candida albicans Á Susceptibility Á e-mail: [email protected] Antifungal Á Essential oils Á Mouth rinses A. C. Coelho Department of Veterinary Sciences, Centre for Animal and Veterinary Science (CECAV), UTAD, Introduction PO Box 1013, 5001-911 Vila Real, Portugal The yeast Candida albicans is a commensal organism E. Martins Faculty of Dentistry, University of Porto, Rua Dr. Manuel frequently found in the oral cavity [1] that can cause Pereira da Silva, 4200-393 Oporto, Portugal opportunistic infections when some predisposing 123 Mycopathologia factors are present among the immunodeficiency, appliances who had attended a dental clinic, using the endocrine disorders, age extremes, radiotherapy, anti- medium CHROMagarTM Candida. The green colo- biotic therapies, transplants, malignant diseases and nies, presumptively identified as C. albicans, were the use of orthodontic appliances [2–5]. The most purified and cryo-preserved (-80°C). The identifica- common treatment is the use of antifungal agents, such tion of the isolates were confirmed based on pheno- as azoles (fluconazole, itraconazole, miconazole and typic features, such as their macro- and micro- ketoconazole) and polyenes (amphotericin B or nys- morphology, fermentation of D-glucose, assimilation tatin). The control of the infections caused by Candida of carbohydrates D-galactose, maltose, sucrose, cello- faces several problems, including the limited number biose, trehalose, raffinose, melezitose, soluble starch, of effective antifungal agents, their high toxicity and L-arabinose and D-glucosamine, formation of hyphae/ costs, the recurrence of the infection and, mainly, the pseudohyphae [18], chlamydospore production [19] increasing resistance to them [6, 7]. and germ tube formation [20]. In general, oral C. albicans isolates have high levels of susceptibilities to a range of antifungal agents [8], but Antifungal Susceptibility Tests some studies reported high levels of azoles resistance in C. albicans strains isolated from the throat and mouth Disk diffusion (DD) testing of amphotericin B, nysta- [9, 10]. The use of some mouth wash solutions to control tin, ketoconazole, econazole, fluconazole and mico- microbial mouth growth might represent a valid alter- nazole was performed as described by CLSI guidelines native to topical use of antifungal substances. In vitro (M44-A protocol [21]) and [22] except the use of studies provided evidence that chlorhexidine digluco- methylene blue at 0.5 lg/ml. Amphotericin B (10 lg), nate (CHX) was fungicidal [11, 12]. Additionally, nystatin (50 lg), ketoconazole (15 lg), econazole mouth rinses may contain alcohol or other compounds (10 lg), fluconazole (25 lg) and miconazole (10 lg) that could significantly affect their antimicrobial action were from Neo-sensitabsTM ROSCOÒ. Agar plates (90- [13]. Among those compounds, plant extracts and mm diameter) containing Mueller–Hinton (MH) agar essential oils are used. Due to their antibiotic properties, (Difco Laboratories) supplemented with 2% glucose essential oils use in the pharmaceutical and food were inoculated with yeast cells, previously suspended industry had been generalized, constituting an alterna- in a saline solution (0.85%) with the turbidity 0.5 in a tive to the use of antimicrobials [14]. Also, the growing McFarland scale. The plates were incubated at resistance of C. albicans to antifungal agents stimulated 36 ± 1°C for 24 h. After 24 h cultivation, the inhib- the research of new therapeutic alternatives, like the use itory diameter zone (dz) was measured [23]. The of essential oils [15–17]. interpretative criteria for fluconazole according to This study aims to test the susceptibility of C. CLSI guidelines [21] were: susceptible (S) C19 mm; albicans isolates from patients with orthodontic susceptible dose dependent (SDD) 18–15 mm and appliances, to (1) different antifungals (fluconazole, resistant (R) B14 mm. For the other antifungal agents, miconazole, econazole, ketoconazole, nystatin and we followed the manufacture’s interpretation: keto- amphotericin B); (2) mouth rinses and essential oils conazole (S C 28 mm; SDD 27–21 mm and R B 20 (lemon, eucalyptus, myrrh, cinnamon, laurel, mint, mm), econazole (S C 20 mm; SDD 19–12 mm and rosemary and tangerine) efficacies against the same R B 11 mm), miconazole (S C 20 mm; SDD 19–12 isolates; and (3) to search for possible relations among mm and R B 11 mm), amphotericin B (S C 15 mm; the isolates susceptibilities to antifungal drugs, essen- SDD 14–10 mm and R \ 10 mm) and nystatin tial oils and mouth rinses. (S C 15 mm; SDD 14–10 mm and R B no zone). The minimum inhibitory concentration (MIC) for fluconazole and amphotericin B was also determined Materials and Methods by the Etest method (ET), using the ET strips (AB BIODISK) with the concentration range from 0.002 to Origin of C. albicans Isolates 32 lg/ml for amphotericin B and 0.016–256 lg/ml for fluconazole. We used the MH agar to perform the test, Forty isolates of C. albicans used in this study were with 2% glucose. The inoculated suspension was obtained from 25 patients using fixed orthodontic treated as for DD testing. The interpretative MIC 123 Mycopathologia breakpoints were recommended by the manufacture: fruits peal, myrrh oil from the plant resin and fluconazole (S B 8 lg/ml and R C 64 lg/ml) and cinnamon essential oil from bark and leaves. For the amphotericin (S B 1 lg/ml and R C 4 lg/ml). susceptibility tests, 15 ll of each essential oil was put on blank disks (6-mm diameter), allowed to dry and Quality Control placed in a MH plate, previously inoculated with a 0.5 McFarland yeast suspension. The plates were incu- Quality control (QC) for DD and ET was performed by bated at 36 ± 1°C for 24–48 h, and dz (mm) read. using C. albicans ATCC 90028 and Candida parapsi- The seven commercial mouthwashes tested varied losis ATCC 22019 [22]. These species were included in their composition, namely in the main active in all runs, and the results were within published compounds (chlorhexidine digluconate-CHX, alcohol limits. or hexetidine-HEX) and their concentration (Table 1): five had CHX with (n = 2) or without alcohol Essential Oils and Mouthwashes Susceptibility (n = 3), one had HEX and the last only alcohol. They Tests also differ in the type of the excipients. For the susceptibility test, the same procedure was followed All essential oils were obtained from one standard for essential oils. Additionally to C. albicans isolates commercial supplier and derived from plants whose (n = 40), the two ATCC yeasts were also tested. extracts or active substances are frequently found in toothpastes. The plants belongs to 5 families: Laura- Data Analysis ceae, Cinnamomum zeylanicum Blume (cinnamon) and Laurus nobilis L. (laurel); Rutaceae, Citrus To test whether mouthwashes or essential oils affect limonum L. (lemon) and Citrus reticulata Blanco yeast growth (dz), we used the Wilcoxon matched pair (tangerine/mandarin); Lamiaceae, Mentha piperita L. test. The phenotypes and the susceptibility classifica- (mint) and Rosmarinus officinalis L. (rosemary); tion of the isolates to the antifungals were compared Burseraceae, Commiphora myrrha (Nees) Engl. against each essential oil and mouthwash, by the non- (myrrh); and Myrtaceae, Eucalyptus globulus Labill parametric Kruskal–Wallis, for multiple independent (blue gum eucalyptus). The rosemary, mint, laurel and groups, or Mann–Whitney’s, for two independent eucalyptus essential oils were obtained from the leaves groups, tests. All analyses were computed by STAT- of their plants, the lemon and tangerine oils from the ISTICA version 9.1.