FEMS Yeast Research, 15, 2015, fov012 doi: 10.1093/femsyr/fov012 Advance Access Publication Date: 20 March 2015 Research Article
RESEARCH ARTICLE
Exogenous tyrosol inhibits planktonic cells and biofilms of Candida species and enhances their susceptibility to antifungals Rossana de A. Cordeiro1,∗ , Carlos E.C. Teixeira1 , Raimunda S.N. Brilhante1 , ´ Debora S.C.M. Castelo-Branco1 , Lucas P. Alencar1,2 , Jonathas S. de Oliveira1 , Andre´ J. Monteiro3 , Tereza J.P.G. Bandeira1 , Jose´ J.C. Sidrim1 , Jose´ Luciano Bezerra Moreira1 and Marcos F.G. Rocha1,2 1
Department of Pathology and Legal Medicine, College of Medicine, Post Graduate Program in Medical ´ Fortaleza, Ceara, ´ CEP Microbiology, Specialized Medical Mycology Center, Federal University of Ceara, 60430-270, Brazil, 2 College of Veterinary Medicine, Post Graduate Program in Veterinary Sciences, State ´ Fortaleza, Ceara, ´ CEP 60430-270, Brazil and 3 Department of Statistics and Applied University of Ceara, ´ Fortaleza, Ceara, ´ Brazil Mathematics, Federal University of Ceara, ∗ Corresponding author: Department of Pathology and Legal Medicine, College of Medicine, Post Graduate Program in Medical Microbiology, Specialized ´ Fortaleza, Ceara, ´ CEP 60430-270, Brazil. Tel: +55 85 33668594; E-mail: [email protected] Medical Mycology Center, Federal University of Ceara, One sentence summary: Exogenous tyrosol destroys Candida albicans and C. tropicalis biofilms. Editor: Richard Calderone
ABSTRACT Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth. Keywords: tyrosol; candida; biofilms; susceptibility; synergism
INTRODUCTION In humans, Candida spp. infections are the most frequent opportunistic mycoses in immunocompromised individuals (Nucci et al. 2010). Although Candida albicans is still the most recur-
rent pathogen isolated, non-C. albicans (NCA) species have been emerging as a common problem in several hospitals throughout the world (Miceli, D´ıaz and Lee 2011; Silva et al. 2012). Epidemiological studies have shown that C. tropicalis is the first or the second most frequently isolated NCA pathogen from candidemia
Received: 28 June 2014; Accepted: 13 March 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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and candiduria (Colombo et al. 2006; Negri et al. 2012). Candidiasis may also occur in animals as an opportunistic infection as a result of predisposing conditions, such as prior antibacterial and/or immunosuppressive therapy, malnutrition and poor ˜ 2010; Skoric et al. 2011). management (Rogers et al. 2009; Cabanes Superficial, mucocutaneous or systemic infections have been described in dogs, cats, swine, bovines, horses and poultry (Zlo˜ towski et al. 2006; Osorio et al. 2007; Cabanes 2010; Lamm et al. 2013). Candida albicans is also considered the most important pathogen in these cases, although the list of opportunistic NCA species has increased in recent years (Crawshaw, MacDonald and Duncan 2005; Gaudie, Wragg and Barber 2009; Lamm et al. 2013). As with C. albicans, resistance to antifungals, especially fluconazole (FLC), in NCA is considered an important issue among physicians (Brion, Uko and Goldman 2007; Garnacho-Montero et al. 2010) and has gained attention among veterinarians (Brito et al. 2009; Brilhante et al. 2011; Castelo-Branco et al. 2013). Candida spp. recovered from both human and animal sources are able to produce an array of virulence factors that modulate pathogenesis. Of these, biofilms are recognized as important elements, especially in chronic candidiasis and catheter-related infections (Ramage, Robertson and Williams 2014). Previous studies have already shown that farnesol—an autoregulatory molecule produced by C. albicans—prevents biofilm formation (Weber et al. 2008), blocks the morphological transition from yeast to hyphae (Ramage et al. 2002) and alters fungal gene expression (Cao et al. 2005; Davis-Hanna et al. 2008). Tyrosol [2-(4-hydroxyphenyl)] is another quorum-sensing molecule in C. albicans which is known to stimulate germ-tube formation and hyphal development in the early and intermediate stages of biofilm growth (Alem et al. 2006). According to Alem et al. (2006), addition of exogenous tyrosol (TYR) at 1 mM during the formation step of C. albicans biofilms had no significant effect on its growth. As antifungal resistance is a hallmark of biofilms (Taff et al. 2013; Ramage, Robertson and Williams 2014), different approaches have been tested to enhance the susceptibility of these ´ structures to antimycotic drugs (Cuellar-Cruz et al. 2012). In the present study, we evaluated the susceptibility of planktonic cells and biofilms of C. albicans and C. tropicalis from both human and animal sources to concentrations above the endogenous threshold of TYR. In addition, we investigated whether TYR could act synergistically with antifungals that target cellular ergosterol.
MATERIAL AND METHODS Fungal strains Antifungal-resistant strains of Candida spp. from healthy animals (C. albicans, n = 6; C. tropicalis, n = 4) and human cases of candidemia (C. albicans, n = 4; C. tropicalis, n = 6) were included in this study (Brito et al. 2009; Sidrim et al. 2010; Cordeiro et al. 2013). Concerning veterinary strains, yeasts were recovered from healthy dogs (C. albicans, n = 2; C. tropicalis, n = 2) and cockatiels (C. albicans, n = 4; C. tropicalis, n = 2). Previous studies have already shown the antifungal resistance of such isolates to amphotericin, azoles and caspofungin. Detailed information can be found in Brito et al. (2009) and Sidrim et al. (2010) for veterinary strains and Cordeiro et al. (2013) for human strains. The strains were identified by the following procedures: differential growth on chromogenic medium, micromorphology on corn meal agar supplemented with Tween 80, physiological tests based on carbohydrate and nitrogen assimilation and sugar fer-
mentation (De Hoog et al. 2000). Fungal identification was confirmed by PCR reaction according to Ahmad et al. (2012) and Vitek ´ 2 ID-YST system (BioMerieux). The strains belong to the culture collection of the Specialized Medical Mycology Center of the Federal University of Ceara´ (CEMM, UFC, Brazil), where they were stored in Sabouraud agar slants recovered with 50% glycerol at −80◦ C
Antifungal agents and TYR Stock solutions of amphotericin B (AMB), itraconazole (ITC) and FLC were prepared according to M27-A3 guidelines (CLSI 2008). TYR (Sigma Chemical Co., USA) was diluted in sterile distilled water, as suggested by the manufacturer. Serial 2-fold dilutions of each antifungal, as well as TYR, were prepared in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma Chemical Co., USA), buffered to pH 7.0 with 0.165 M MOPS (Sigma Chemical Co., USA).
Inoculum preparation for susceptibility testing Inocula of all tested isolates were prepared from cultures previously grown on potato dextrose agar for 24 h at 35o C. The colonies were suspended in 5 mL of sterile 0.9% saline and the turbidity was adjusted to 0.5 on the McFarland scale. Afterwards, the suspension was diluted to 1:100 and then to 1:20 with RPMI 1640 medium to obtain an inoculum of planktonic cells containing 0.5–2.5 × 103 cells mL−1 (CLSI 2008).
Antifungal susceptibility of Candida planktonic cells Antifungal susceptibility to AMB, ITC and FLC of Candida strains was investigated by broth microdilution, according to the document M27-A3 (CLSI 2008). TYR susceptibility testing was performed as previously described (Cordeiro et al. 2013). To determine the susceptibility of planktonic cells, the final concentrations of each drug ranged from 0.03125 to 16 μg mL−1 for AMB and ITC and from 0.125 to 64 μg mL−1 for FLC. TYR was tested at concentrations ranging from 0.04 to 22 mM, which are approximately 2× to 1000× the concentration reached by endogenous TYR in C. albicans (Alem et al. 2006). Susceptibility testing for planktonic cells was performed in 96-well plates at 35o C for 48 h. All isolates were tested in duplicate. For AMB, the minimum inhibitory concentration (MIC) was defined as the lowest drug concentration capable of inhibiting 100% of fungal growth, while for the azole derivatives (ITC and FLC), MICs were defined as the lowest drug concentration capable of inhibiting 50% of fungal growth, when compared to the control well (CLSI 2008). TYR MIC was defined as 50% inhibition, similar to that of azole drugs, based on previous studies with farnesol (Cordeiro et al. 2013). Candidia parapsilosis ATCC 22019 was included as quality control for each test. Isolates with MICs >1.0, ≥1.0 and ≥8 μg mL−1 were considered resistant to AMB, ITC and FLC, respectively (CLSI 2008; Pfaller et al. 2010). Drug interaction was evaluated in duplicate through the microdilution assay as previously described (Chaturvedi et al. 2011). Positive growth controls were performed in RPMI medium without antimicrobials. TYR was combined with AMB, FLC or ITC. Combinations were formed with each drug at the following concentrations: from 0.0078 to 4.0 μg mL−1 for AMB; from 0.125 to 64 μg mL−1 for FLC, from 0.03125 to 16 μg mL−1 for ITC and from 0.005 to 5.4 mM (0.7–750 μg mL−1 ) for TYR. The MIC of each drug combination was defined as the lowest concentration that caused 50% inhibition of visible fungal growth for TYR plus
Cordeiro et al.
azoles and 100% inhibition for TYR plus AMB. Drug interactions were classified as synergistic, indifferent or antagonistic according to the fractional inhibitory concentration index (FICI). The interaction was defined as synergistic when FICI was ≤0.5, indifferent, when 0.5 < FICI < 4, and antagonistic when FICI > 4 (Odds 2003).
Biofilm formation For biofilm testing, inocula were prepared as described by Chatzimoschou et al. (2011), with some modifications. In brief, strains of C. albicans (n = 10) and C. tropicalis (n = 10) were grown in Sabouraud dextrose broth for 24 h at 30◦ C in a rotary shaker at 150 rpm. After this period, the cells were collected by centrifugation (3000 rpm, 10 min) and the pellet was washed twice with PBS. Suspensions were adjusted to 1 × 106 cells mL−1 in RPMI medium and then 100-μl inoculum aliquots were transferred to flat wells of 96-well polystyrene plates. The plates were incubated at 37◦ C for 48 h and the wells were washed three times with 0.05% Tween 20 in Tris-buffered solution to remove nonadhered cells.
Effect of exogenous TYR on growth of Candida biofilms The ability of TYR and antifungal combinations in inhibiting formation of C. albicans and C. tropicalis biofilms was evaluated as suggested by Yu et al. (2012). The following solutions were tested: AMB, FLC, ITC, TYR, AMB/TYR FLC/TYR and ITC/TYR. Antifungals were prepared at MIC, 10 × MIC and 50 × MIC concentrations, as previously determined for planktonic cells. Solutions formed by antifungals plus TYR were assayed at 10 × MIC and 50 × MIC concentrations previously established in synergism testing. Then biofilm formation was conducted as described in the previous section of this paper. After incubation at 37◦ C for 48 h, the supernatant was aspirated and biofilm metabolic activity was quantified by the 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2Htetrazolium hydroxide (XTT, Sigma, Germany) reduction assay. For each strain, 75 μL of XTT salt solution (1 mg mL−1 in PBS), 6 μL of menadione solution (1 mM in acetone, Sigma, Germany) and 50 μL of sterile PBS were added to each well. The microtiter plates were incubated at 36◦ C for 5 h. The metabolic activity of the yeast cells within the biofilm was evaluated by enzymatic reduction of XTT tetrazolium salt to XTT formazan, resulting in a colorimetric change, which was then measured at 492 nm. Microtiter wells containing RPMI without fungal cells were included as negative control. Experiments were performed in duplicate and repeated three times independently.
Effect of exogenous TYR on mature Candida biofilms To evaluate the inhibitory activity of TYR against mature C. albicans and C. tropicalis biofilms, inocula were prepared as described above. The following solutions were tested: AMB, FLC, ITC, TYR, AMB/TYR, FLC/TYR and ITC/TYR. Aliquots of 200 μL of each test solution at three different concentrations (MIC, 10 × MIC and 50 × MIC) were added to viable 48-h biofilms. Controls were grown in medium without antimicrobials. Measurement of Candida biofilms was obtained by the XTT assay, as described above. Experiments were performed in duplicate and repeated three times independently.
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Statistical analysis Considering that the MIC data presented low asymmetry, Student’s t-test for paired samples was used to compare differences in MIC values before and after combining TYR with antifungal drugs. Finally, in order to verify differences in optical density, to evaluate the effects of TYR and antifungals on biofilm production, Student’s t-test for paired samples was used. For all the analyses, a significance level lower than 5% indicated statistically significant findings (P < 0.05).
RESULTS Antifungal susceptibility of Candida planktonic cells All the tested strains showed resistance to at least one antifungal drug. Antifungal resistance to AMB was detected in C. albicans, (n = 1) and C. tropicalis (n = 4) strains. Simultaneous resistance to ITC and FLC was also seen (C. albicans n = 7; C. tropicalis n = 10). The MIC for TYR ranged from 2.5 to 5.0 mM for both species. Detailed description of MIC values is shown in Table 1, along with the results of the in vitro interaction between antifungals and TYR. TYR significantly reduced the MICs for all antifungal drugs tested (P < 0.01). Synergism was observed between AMB/TYR (11/20 strains), ITC/TYR (18/20 strains) and FLC/TYR (18/20 strains).
Effect of exogenous TYR on Candida biofilms Exogenous TYR alone was able to significantly reduce biofilm formation of both C. albicans and C. tropicalis only when tested at 50 × MIC (P < 0.001). At this concentration, TYR was able to decrease metabolic activity of growing biofilms at approximately 24 and 30% for C. albicans and C. tropicalis, respectively. Antifungal drugs alone were effective at both 10 × MIC and 50 × MIC (P < 0.001). Reduction in biofilm formation was more pronounced when TYR was combined with antifungal drugs, resulting in statistically significant reduction in biofilm metabolic activity starting at MIC (P < 0.001). Candida biofilm formation declined by 60% in metabolic activity after incubation with AMB/TYR, FLC/TYR or ITC/TYR at 50 × MIC. Details regarding inhibition of biofilm formation are shown in Table 2. Exogenous TYR at 50 × MIC was able to reduce mature biofilms of C. albicans and C. tropicalis at nearly 50% (P < 0.001). Antifungal drugs alone were effective against mature biofilm at both 10 × MIC and 50 × MIC (P < 0.001). Reduction of biofilm formation was more pronounced when TYR was combined with AMB, resulting in a significant reduction in biofilm metabolic activity starting at MIC (P < 0.0001). Mature biofilm exposed to AMB/TYR at 50 × MIC presented a 35% metabolic reduction. On the other hand, the combination of TYR with either azole antifungal at both 10 × MIC or 50 × MIC resulted in a significant increase in biofilm metabolism (Table 2).
DISCUSSION TYR (2-(4-hydroxyphenyl)ethanol)—a derivative of the amino acid tyrosine—was the second QS molecule described in C. albicans (Chen et al. 2004). Earlier studies have shown that the accumulation of TYR in a culture medium is directly related to the increase in fungal cell density (Chen and Fink 2006). Besides being a fungal quorum-sensing molecule, TYR is also a majority component of olive oils used in human diets. The antioxidative properties of such oils have been attributed to phenolic compounds, including TYR and hydroxytyrosol (Visioli et al. 2000).
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Table 1. MICs of antifungals (μg mL−1 ) and TYR (mM) and FICI for each drug combination against Candida planktonic cells. Strains
Origin
MIC (individual drugs) AMB ITC FLC TYR
AMB/TYR
MIC (combined drugs) ITC/TYR FLC/TYR
AMB/TYR
FICI index∗ ITC/TYR
FLC/TYR
C. albicans 05–05–005 01–05–006 01–05–004 02–01–070 01–03–069 01–03–037 01–01–027 03–02–033 01–03–068 01–02–081
H H H H V V V V V V
0.5 0.5 0.5 0.5 2.0 0.5 0.25 0.125 0.5 0.5
>16 4.0 2.0 4.0 2.0 >16 >16 >16 >16 >16
>64 2.0 2.0 4.0 >64 >64 >64 32 >64 >64
2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0
0.0625/1.25 0.125/0.3125 0.5/1.25 0.25/0.625 1.0/2.5 0.125/0.3125 0.125/1.25 0.125/0.3125 0.125/0.3125 0.0625/1.25
2.0/0.312 0.0625/0.156 0.0625/0.156 2.0/2.5 2.0/2.5 2.0/0.625 0.0625/0.156 2.0/0.625 2.0/0.0312 1.0/0.625
8.0/0.3125 0.125/0.039 1.0/1.25 1.0/1.25 0.0156/0.625 1.0/1.25 0.125/0.039 16/2.5 2.0/0.625 8.0/1.25
0.625 0.312 1.25 0.625 1.5 0.312 1.0 1.062 0.375 0.375
0.249 0.046 0.094 1.0 2.0 0.5 0.101 0.5 0.5 0.187
0.5 0.14 1.0 0.5 0.027 0.375 0.0175 1.0 0.281 0.375
H H H V H H H V V V
2.0 0.5 0.5 1.0 4.0 4.0 4.0 0.5 0.5 1.0
2.0 >16 >16 >16 >16 >16 >16 8.0 4.0 >16
8 >64 >64 >64 >64 >64 8 32 >64 >64
5.0 5.0 2.5 5.0 5.0 5.0 5.0 5.0 2.5 5.0
0.125/0.3125 0.25/0.625 0.125/0.3125 0.5/1.25 0.5/1.25 0.5/1.25 0.25/0.625 0.125/0.3125 0.25/0.625 0.125/0.3125
0.25/0.625 0.5/0.3125 2.0/0.3125 0.0625/0.156 0.125/0.3125 0.125/0.3125 2.0/0.625 0.125/0.3125 1.0/0.625 0.5/0.3125
1.0/1.25 16/1.25 8.0/0.3125 0.125/0.039 8.0/1.25 4.0/0.625 1.0/1.25 2.0/0.625 4.0/0.625 1.0/1.25
0.125 0.625 0.375 0.75 0.375 0.375 0.077 0.312 0.75 0.187
0.25 0.093 0.5 0.07 0.07 0.07 0.25 0.078 0.5 0.07
0.375 0.5 0.25 0.097 0.375 0.187 0.375 0.187 0.187 0.375
C. tropicalis 01–02–184 02–01–016 03–02–048 01–01–031 02–01–012 02–02–179 02–02–199 03–02–047 01–02–078 01–03–075
H: human; V: veterinary; AMB: amphotericin B; ITC: itraconazole; FLC: fluconazole; TYR: tyrosol. Resistance breakpoint: AMB>1 μg mL−1 , ITC ≥1 μg mL−1 and FLC ≥ 8 μg mL−1 . ∗ The interaction was defined as synergistic when FICI ≤ 0.5; indifferent when 0.5 < FICI < 4, and antagonistic when FICI>4.0.
In recent years, many studies have shown the inhibitory effect of fungal QSMs against bacteria (Brilhante et al. 2012) and fungi (Derengowski et al. 2009; Cordeiro et al. 2013). Until now, a total of five QSMs have been described in fungi (Alem et al. 2006; Chen and Fink 2006), but farnesol and TYR from Candida have attracted the most attention from researchers. Farnesol is the most studied fungal QSM and experimental evidence has shown that this compound can also induce cell death in Aspergillus (Dinamarco, Goldman and Goldman 2011), inhibit yeastto-hypha transition in C. albicans and impair biofilm formation in Candida spp. (Ramage et al. 2002). Even though the biological effects of TYR on fungal pathogens is not well understood, it is known to have the opposite activity of farnesol, i.e. stimulation of germ-tube formation and hence hyphal development (Alem et al. 2006). The results of the present study show that exogenous TYR caused a significant reduction of C. albicans and C. tropicalis planktonic growth at concentrations that ranged from 2.5 to 5.0 mM. According to Alem et al. (2006), TYR production by C. albicans can reach concentrations of approximately 11 μM in the supernatant of planktonic cell cultures, which is lower than the TYR MICs found in this study. Although C. albicans produces significantly more TYR when compared to C. tropicalis (Tumbarello et al. 2007), a similar susceptibility pattern to exogenous TYR was seen in both Candida species. In this study, synergistic interactions between TYR and azoles were detected against both C. albicans and C. tropicalis strains. TYR reverted the resistance phenotype to FLC of 4/7 C. albicans and 7/10 C. tropicalis and the resistance phenotype to ITC of 3/10 C. albicans and 7/10 C. tropicalis. Recently, Cordeiro et al. (2013) showed that the QSM farnesol was able to reduce MICs of AMB, azoles and caspofungin against Candida species, making them susceptible to these antifungals. Although far-
nesol and TYR are both autoregulatory molecules in Candida, they have different biological effects and probably different receptors in the fungal cell (Alem et al. 2006). We believe that TYR interferes with ergosterol biosynthesis, since the fungal cells became more susceptible to azoles after exposure to this compound. Several studies have shown that fungal QSMs have a dramatic impact on Candida biofilms (Alem et al. 2006; Albuquerque and Casadevall 2012). Exogenous farnesol, for example, added at a very early stage inhibits biofilm formation, blocks the yeasthyphae transition required for mature biofilms (Lindsay et al. 2012). Indeed, when C. albicans cells were treated with farnesol at 300 μM, thin biofilms formed predominantly of blastoconidia and pseudohyphae were seen (Ramage et al. 2012). In the present study, exogenous TYR added at time zero was able to significantly reduce biofilm formation, which was also observed when TYR was combined with antifungals, at all tested concentrations (MIC, 10 × MIC and 50 × MIC). Even though further studies should be conducted to elucidate the mechanisms involved in the effects of high concentrations of exogenous TYR on Candida biofilm growth, some hypotheses can be proposed, based on the dynamics of biofilm formation. It has been shown that farnesol, not TYR, is the QSM predominantly secreted by C. albicans planktonic cells and that exogenous TYR has no significant effect on early biofilm formation, despite accelerating hyphal growth (Alem et al. 2006). Thus, we believe that when Candida biofilms were exposed to high concentrations of exogenous TYR, the compound overcame the physiological effects of farnesol, then compromising biofilm formation. Antifungal-TYR combinations were even more effective against growing biofilms, when compared to each compound alone, likely due to an additive deleterious effect on biofilm growth.
TYR: tyrosol; AMB: amphotericin B; FLC: fluconazole: ITC: itraconazole; AMB/TYR: tyrosol plus amphotericin B; FLC/TYR: tyrosol plus fluconazole; ITC/TYR: tyrosol plus itraconazole. Different letters indicate statistically significant differences (P < 0.05). Numbers represent XTT absorbances (mean ± SEM) of Candida biofilms at 492 nm. Controls were performed in RPMI medium without inhibitory drugs.
100.0a (0.590 ± 0.03) 100.0a (0.540 ± 0.03) 100.0a (0.442 ± 0.03) 100.0a (0.425 ± 0.03) Control
53.02 ± 2.20c 65.83 ± 3.56b 74.55 ± 3.53b 64.71 ± 4.16b 63.48 ± 3.88b 120.7 ± 2.38d 120.4 ± 2.9d 110.7 ± 2.85a 75.82 ± 4.19b 80.9 ± 2.99b 78.37 ± 4.29b 80.79 ± 3.45b 123.0 ± 3.37d 120.5 ± 3.52d 102.2 ± 2.89a 93.68 ± 2.79a 96.69 ± 2.6a 97.98 ± 1.95a 85.1 ± 3.68a 100.4 ± 1.94a 101.9 ± 1.2a 46.86 ± 5.33b 68.45 ± 4.38b 67.74 ± 3.94a 80.31 ± 3.18a 63.63 ± 2.97b 128.4 ± 3.08c 126.8 ± 4.42c 112.2 ± 6.06a 92.67 ± 6.08a 74.55 ± 1.84a 81.57 ± 3.36a 80.68 ± 1.89b 127.1± 6.21c 120.6 ± 2.07c 71.99 ± 4.36b 68.62 ± 1.91b 64.10 ± 2.53b 73.00 ± 2.83b 37.29 ± 2.36c 42.23 ± 3.10c 39.34 ± 3.11c 97.32 ± 4.96a 71.89 ± 3.74b 85.91 ± 1.59b 83.64 ± 3.64b 63.71 ± 3.40b 68.09 ± 3.93b 71.22 ± 2.88b 84.9 ± 4.95a 75.43 ± 1.91b 78.36 ± 2.00b 83.95 ± 4.10b 66.39 ± 2.41b 61.43 ± 2.99b 57.07 ± 3.26b TYR AMB FLC ITC AMB/TYR FLC/TYR ITC/TYR
103.9 ± 2.98a 101.8 ± 1.35a 99.41 ± 4.92a 94.58 ± 1.06a 91.52 ± 1.02a 87.82 ± 3.49a 82.14 ± 1.73a
76.76 ± 4.90b 67.28 ± 2.75b 71.32 ± 2.60b 74.11 ± 2.51b 31.05 ± 2.71c 44.98 ±2.51c 30.42 ± 2.79c
101.0 ± 3.04a 103.1 ± 4.20a 99.43 ± 0.90a 108.0 ± 7.75a 89.0 ± 1.77a 86.89 ± 1.42a 95.47 ± 5.83b
102.8 ± 2.62a 88.34 ± 2.63a 97.15 ± 1.14a 97.92 ± 1.84a 84.48 ± 1.70a 97.71 ± 2.16a 99.75 ± 1.12a
MIC 50 × MIC 50 × MIC MIC
50 × MIC
MIC
MIC
C. albicans 10 × MIC C. tropicalis 10 × MIC C. albicans 10 × MIC
Growing biofilm (%) Drugs
Table 2. Metabolic activity of Candida biofilms grown in RPMI medium supplemented with TYR or antifungals alone or in combination.
Mature biofilm (%)
C. tropicalis 10 × MIC
50 × MIC
Cordeiro et al.
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In the present study, it was shown that azoles-TYR combinations were not able to inhibit mature biofilms even at 50 × MIC concentration. We believe that the concentrations of TYR in these experiments—which ranged from 0.039 to 62.5 mM—were not able to reduce the metabolic activity in mature biofilms. On the other hand, concentrations as low as 0.6 mM of TYR were able to enhance the antifungal activity of AMB against mature biofilms. Although Alem et al. (2006) did not detect any inhibitory effect of TYR at 1 mM against mature biofilms, the results presented here show that TYR at this concentration is able to act synergistically with AMB, reducing biofilm viability by approximately 16%. Combinations formed by synergistic agents have been tested successfully against in vivo Candida biofilms. In a seminal study conducted by Uppuluri et al. (2008), it was confirmed that combinations formed by FK506 and FLC caused complete inhibition of C. albicans biofilms in a rat venous catheter biofilm model. More recently, Bink et al. (2012) demonstrated that the nonsteroidal anti-inflammatory diclofenac potentiates the antifungal activity of caspofungin against in vivo C. albicans biofilms. Li et al. (2012) described the in vivo inhibitory effect on the biofilm of C. albicans by the liverwort-derived riccardin D and FLC. Taken together, these results support that combination therapy is a good strategy to combat Candida biofilms infections. Further study with an in vivo model should be conducted to confirm the anti-biofilm potential of combinations formed by TYR and AMB. The results of the present study show that exogenous TYR alone or combined with antifungals inhibits planktonic cells and Candida biofilm growth. Mature biofilms were also inhibited by TYR alone or combined with AMB, but TYR plus azoles increased the biofilm activity by a dose-dependent mechanism. Because of the clinical importance of Candida infections and due to the paucity of drugs able to inhibit fungal biofilms, we believe that the results presented here might be of interest for the development of new antifungal strategies.
FUNDING This study was supported by CNPq, Brazil (302574/2009-3; PROTAX 562296/2010-7; 504189/2012-3; 307606/2013-9) and CAPES; Brazil (PNPD 2103/2009, AE1-0052-000630100/11). Conflict of interest. None declared.
REFERENCES Ahmad S, Khan Z, Asadzadeh M, et al. Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis. BMC Infect Dis 2012;12:230. Albuquerque P, Casadevall A. Quorum sensing in fungi: a review. Med Mycol 2012;50:337–45. Alem MAS, Oteef MDY, Flowers TH, et al. Production of tyrosol by Candida albicans biofilms and its role in Quorum Sensing and biofilm development. Eukaryot Cell 2006;5:1770–9. Bink A, Kuchar´ıkova´ S, Neirinck B, et al. The nonsteroidal antiinflammatory drug diclofenac potentiates the in vivo activity of caspofungin against Candida albicans biofilms. J Infect Dis 2012;1:1790–7. Brilhante RSN, Paiva MAN, Sampaio CMS, et al. Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp. FEMS Microbiol Ecol 2011;76:268–77.
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Dinamarco TM, Goldman MHS, Goldman GH. Farnesol-induced cell death in the filamentous fungus Aspergillus nidulans. Biochem Soc Trans 2011;39:1544–8. Garnacho-Montero J, D´ıaz-Mart´ın A, Garc´ıa-Cabrera E, et al. Risk factors for fluconazole-resistant candidemia. Antimicrob Agents Chemother 2010;54:3149–54. Gaudie CM, Wragg PN, Barber AM. Outbreak of disease due to Candida krusei in a small dairy herd in the UK. Vet Rec 2009;165:535–7. Lamm CG, Grune SC, Estrada MM, et al. Granulomatous rhinitis due to Candida parapsilosis in a cat. J Vet Diagn Invest 2013;25:596–8. Li Y, Ma Y, Zhang L, et al. In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D. PLoS One 2012;7:e35543. Lindsay AK, Deveau A, Piispanen AE, et al. Farnesol and cyclic AMP signaling effects on the hypha-to-yeast transition in Candida albicans. Eukaryot Cell 2012;11:1219–25. Miceli MH, D´ıaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect Dis 2011;11:142–51. Negri M, Silva S, Henriques M, et al. Insights into Candida tropicalis nosocomial infections and virulence factors. Eur J Clin Microbiol Infect Dis 2012;31:1399–412. Neppelenbroek KH, Seo´ RS, Urban VM, et al. Identification of Candida species in the clinical laboratory: a review of conventional, commercial, and molecular techniques. Oral Dis 2014;20:329–44. ´ AM, et al. Epidemiology of opNucci M, Queiroz-Telles F, Tobon portunistic fungal infections in Latin America. Clin Infec Dis 2010;51:561–70. Odds FC. Synergy, antagonism, and what the chequerboard puts between them. Antimicrob Chemother 2003;52:1. Osorio C, Fletcher O, Dykstra MJ, et al. Comb candidiasis affecting roosters in a broiler breeder flock. Avian Dis 2007;51: 618–22. Pfaller MA, Andes D, Diekema DJ, et al. Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Update 2010;13:180–95. Ramage G, Rajendran R, Sherry L, et al. Fungal biofilm resistance. Int J Microbiol 2012;2012:1–12. Ramage G, Robertson SN, Williams C. Strength in numbers: antifungal strategies against fungal biofilms. Int J Antimicrob Agents 2014;43:114–20. Ramage G, Saville SP, Wickes BL, et al. Inhibition of Candida albicans biofilm formation by farnesol, a quorum-sensing molecule. Appl Environ Microb 2002;68:5459–63. Rogers CL, Gibson C, Mitchell SL, et al. Disseminated candidiasis secondary to fungal and bacterial peritonitis in a young dog. J Vet Emerg Crit Care 2009;19:193–8. Sidrim JJC, Castelo-Branco DSCM, Brilhante RSN, et al. Candida species isolated from the gastrointestinal tract of cockatiels (Nymphicus hollandicus): In vitro antifungal susceptibility profile and phospholipase activity. Vet Microbiol 2010;145: 324–8. Silva S, Negri M, Henriques M, et al. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36:288–305. Skoric M, Fictum P, Slana I, et al. A case of systemic mycosis in a Hovawart dog to Candida albicans. Vet Med 2011;56:260–4.
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Weber K, Sohr R, Schulz B, et al. Secretion of E,E-farnesol and biofilm formation in eight different Candida species. Antimicrob Agents Chemother 2008;52:1859– 61. Yu LH, Wei X, Ma M, et al. Possible inhibitory molecular mechanism of farnesol on the development of fluconazole resistance in Candida albicans biofilm. Antimicrob Agents Chemother 2012;56:770–5. Zlotowski P, Rozza DB, Pescador CA, et al. Muco-cutaneous candidiasis in two pigs with post weaning multisystemic wasting syndrome. Vet J 2006;3:566–9.
RESEARCH ARTICLE
Exogenous tyrosol inhibits planktonic cells and biofilms of Candida species and enhances their susceptibility to antifungals Rossana de A. Cordeiro1,∗ , Carlos E.C. Teixeira1 , Raimunda S.N. Brilhante1 , ´ Debora S.C.M. Castelo-Branco1 , Lucas P. Alencar1,2 , Jonathas S. de Oliveira1 , Andre´ J. Monteiro3 , Tereza J.P.G. Bandeira1 , Jose´ J.C. Sidrim1 , Jose´ Luciano Bezerra Moreira1 and Marcos F.G. Rocha1,2 1
Department of Pathology and Legal Medicine, College of Medicine, Post Graduate Program in Medical ´ Fortaleza, Ceara, ´ CEP Microbiology, Specialized Medical Mycology Center, Federal University of Ceara, 60430-270, Brazil, 2 College of Veterinary Medicine, Post Graduate Program in Veterinary Sciences, State ´ Fortaleza, Ceara, ´ CEP 60430-270, Brazil and 3 Department of Statistics and Applied University of Ceara, ´ Fortaleza, Ceara, ´ Brazil Mathematics, Federal University of Ceara, ∗ Corresponding author: Department of Pathology and Legal Medicine, College of Medicine, Post Graduate Program in Medical Microbiology, Specialized ´ Fortaleza, Ceara, ´ CEP 60430-270, Brazil. Tel: +55 85 33668594; E-mail: [email protected] Medical Mycology Center, Federal University of Ceara, One sentence summary: Exogenous tyrosol destroys Candida albicans and C. tropicalis biofilms. Editor: Richard Calderone
ABSTRACT Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth. Keywords: tyrosol; candida; biofilms; susceptibility; synergism
INTRODUCTION In humans, Candida spp. infections are the most frequent opportunistic mycoses in immunocompromised individuals (Nucci et al. 2010). Although Candida albicans is still the most recur-
rent pathogen isolated, non-C. albicans (NCA) species have been emerging as a common problem in several hospitals throughout the world (Miceli, D´ıaz and Lee 2011; Silva et al. 2012). Epidemiological studies have shown that C. tropicalis is the first or the second most frequently isolated NCA pathogen from candidemia
Received: 28 June 2014; Accepted: 13 March 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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and candiduria (Colombo et al. 2006; Negri et al. 2012). Candidiasis may also occur in animals as an opportunistic infection as a result of predisposing conditions, such as prior antibacterial and/or immunosuppressive therapy, malnutrition and poor ˜ 2010; Skoric et al. 2011). management (Rogers et al. 2009; Cabanes Superficial, mucocutaneous or systemic infections have been described in dogs, cats, swine, bovines, horses and poultry (Zlo˜ towski et al. 2006; Osorio et al. 2007; Cabanes 2010; Lamm et al. 2013). Candida albicans is also considered the most important pathogen in these cases, although the list of opportunistic NCA species has increased in recent years (Crawshaw, MacDonald and Duncan 2005; Gaudie, Wragg and Barber 2009; Lamm et al. 2013). As with C. albicans, resistance to antifungals, especially fluconazole (FLC), in NCA is considered an important issue among physicians (Brion, Uko and Goldman 2007; Garnacho-Montero et al. 2010) and has gained attention among veterinarians (Brito et al. 2009; Brilhante et al. 2011; Castelo-Branco et al. 2013). Candida spp. recovered from both human and animal sources are able to produce an array of virulence factors that modulate pathogenesis. Of these, biofilms are recognized as important elements, especially in chronic candidiasis and catheter-related infections (Ramage, Robertson and Williams 2014). Previous studies have already shown that farnesol—an autoregulatory molecule produced by C. albicans—prevents biofilm formation (Weber et al. 2008), blocks the morphological transition from yeast to hyphae (Ramage et al. 2002) and alters fungal gene expression (Cao et al. 2005; Davis-Hanna et al. 2008). Tyrosol [2-(4-hydroxyphenyl)] is another quorum-sensing molecule in C. albicans which is known to stimulate germ-tube formation and hyphal development in the early and intermediate stages of biofilm growth (Alem et al. 2006). According to Alem et al. (2006), addition of exogenous tyrosol (TYR) at 1 mM during the formation step of C. albicans biofilms had no significant effect on its growth. As antifungal resistance is a hallmark of biofilms (Taff et al. 2013; Ramage, Robertson and Williams 2014), different approaches have been tested to enhance the susceptibility of these ´ structures to antimycotic drugs (Cuellar-Cruz et al. 2012). In the present study, we evaluated the susceptibility of planktonic cells and biofilms of C. albicans and C. tropicalis from both human and animal sources to concentrations above the endogenous threshold of TYR. In addition, we investigated whether TYR could act synergistically with antifungals that target cellular ergosterol.
MATERIAL AND METHODS Fungal strains Antifungal-resistant strains of Candida spp. from healthy animals (C. albicans, n = 6; C. tropicalis, n = 4) and human cases of candidemia (C. albicans, n = 4; C. tropicalis, n = 6) were included in this study (Brito et al. 2009; Sidrim et al. 2010; Cordeiro et al. 2013). Concerning veterinary strains, yeasts were recovered from healthy dogs (C. albicans, n = 2; C. tropicalis, n = 2) and cockatiels (C. albicans, n = 4; C. tropicalis, n = 2). Previous studies have already shown the antifungal resistance of such isolates to amphotericin, azoles and caspofungin. Detailed information can be found in Brito et al. (2009) and Sidrim et al. (2010) for veterinary strains and Cordeiro et al. (2013) for human strains. The strains were identified by the following procedures: differential growth on chromogenic medium, micromorphology on corn meal agar supplemented with Tween 80, physiological tests based on carbohydrate and nitrogen assimilation and sugar fer-
mentation (De Hoog et al. 2000). Fungal identification was confirmed by PCR reaction according to Ahmad et al. (2012) and Vitek ´ 2 ID-YST system (BioMerieux). The strains belong to the culture collection of the Specialized Medical Mycology Center of the Federal University of Ceara´ (CEMM, UFC, Brazil), where they were stored in Sabouraud agar slants recovered with 50% glycerol at −80◦ C
Antifungal agents and TYR Stock solutions of amphotericin B (AMB), itraconazole (ITC) and FLC were prepared according to M27-A3 guidelines (CLSI 2008). TYR (Sigma Chemical Co., USA) was diluted in sterile distilled water, as suggested by the manufacturer. Serial 2-fold dilutions of each antifungal, as well as TYR, were prepared in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma Chemical Co., USA), buffered to pH 7.0 with 0.165 M MOPS (Sigma Chemical Co., USA).
Inoculum preparation for susceptibility testing Inocula of all tested isolates were prepared from cultures previously grown on potato dextrose agar for 24 h at 35o C. The colonies were suspended in 5 mL of sterile 0.9% saline and the turbidity was adjusted to 0.5 on the McFarland scale. Afterwards, the suspension was diluted to 1:100 and then to 1:20 with RPMI 1640 medium to obtain an inoculum of planktonic cells containing 0.5–2.5 × 103 cells mL−1 (CLSI 2008).
Antifungal susceptibility of Candida planktonic cells Antifungal susceptibility to AMB, ITC and FLC of Candida strains was investigated by broth microdilution, according to the document M27-A3 (CLSI 2008). TYR susceptibility testing was performed as previously described (Cordeiro et al. 2013). To determine the susceptibility of planktonic cells, the final concentrations of each drug ranged from 0.03125 to 16 μg mL−1 for AMB and ITC and from 0.125 to 64 μg mL−1 for FLC. TYR was tested at concentrations ranging from 0.04 to 22 mM, which are approximately 2× to 1000× the concentration reached by endogenous TYR in C. albicans (Alem et al. 2006). Susceptibility testing for planktonic cells was performed in 96-well plates at 35o C for 48 h. All isolates were tested in duplicate. For AMB, the minimum inhibitory concentration (MIC) was defined as the lowest drug concentration capable of inhibiting 100% of fungal growth, while for the azole derivatives (ITC and FLC), MICs were defined as the lowest drug concentration capable of inhibiting 50% of fungal growth, when compared to the control well (CLSI 2008). TYR MIC was defined as 50% inhibition, similar to that of azole drugs, based on previous studies with farnesol (Cordeiro et al. 2013). Candidia parapsilosis ATCC 22019 was included as quality control for each test. Isolates with MICs >1.0, ≥1.0 and ≥8 μg mL−1 were considered resistant to AMB, ITC and FLC, respectively (CLSI 2008; Pfaller et al. 2010). Drug interaction was evaluated in duplicate through the microdilution assay as previously described (Chaturvedi et al. 2011). Positive growth controls were performed in RPMI medium without antimicrobials. TYR was combined with AMB, FLC or ITC. Combinations were formed with each drug at the following concentrations: from 0.0078 to 4.0 μg mL−1 for AMB; from 0.125 to 64 μg mL−1 for FLC, from 0.03125 to 16 μg mL−1 for ITC and from 0.005 to 5.4 mM (0.7–750 μg mL−1 ) for TYR. The MIC of each drug combination was defined as the lowest concentration that caused 50% inhibition of visible fungal growth for TYR plus
Cordeiro et al.
azoles and 100% inhibition for TYR plus AMB. Drug interactions were classified as synergistic, indifferent or antagonistic according to the fractional inhibitory concentration index (FICI). The interaction was defined as synergistic when FICI was ≤0.5, indifferent, when 0.5 < FICI < 4, and antagonistic when FICI > 4 (Odds 2003).
Biofilm formation For biofilm testing, inocula were prepared as described by Chatzimoschou et al. (2011), with some modifications. In brief, strains of C. albicans (n = 10) and C. tropicalis (n = 10) were grown in Sabouraud dextrose broth for 24 h at 30◦ C in a rotary shaker at 150 rpm. After this period, the cells were collected by centrifugation (3000 rpm, 10 min) and the pellet was washed twice with PBS. Suspensions were adjusted to 1 × 106 cells mL−1 in RPMI medium and then 100-μl inoculum aliquots were transferred to flat wells of 96-well polystyrene plates. The plates were incubated at 37◦ C for 48 h and the wells were washed three times with 0.05% Tween 20 in Tris-buffered solution to remove nonadhered cells.
Effect of exogenous TYR on growth of Candida biofilms The ability of TYR and antifungal combinations in inhibiting formation of C. albicans and C. tropicalis biofilms was evaluated as suggested by Yu et al. (2012). The following solutions were tested: AMB, FLC, ITC, TYR, AMB/TYR FLC/TYR and ITC/TYR. Antifungals were prepared at MIC, 10 × MIC and 50 × MIC concentrations, as previously determined for planktonic cells. Solutions formed by antifungals plus TYR were assayed at 10 × MIC and 50 × MIC concentrations previously established in synergism testing. Then biofilm formation was conducted as described in the previous section of this paper. After incubation at 37◦ C for 48 h, the supernatant was aspirated and biofilm metabolic activity was quantified by the 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2Htetrazolium hydroxide (XTT, Sigma, Germany) reduction assay. For each strain, 75 μL of XTT salt solution (1 mg mL−1 in PBS), 6 μL of menadione solution (1 mM in acetone, Sigma, Germany) and 50 μL of sterile PBS were added to each well. The microtiter plates were incubated at 36◦ C for 5 h. The metabolic activity of the yeast cells within the biofilm was evaluated by enzymatic reduction of XTT tetrazolium salt to XTT formazan, resulting in a colorimetric change, which was then measured at 492 nm. Microtiter wells containing RPMI without fungal cells were included as negative control. Experiments were performed in duplicate and repeated three times independently.
Effect of exogenous TYR on mature Candida biofilms To evaluate the inhibitory activity of TYR against mature C. albicans and C. tropicalis biofilms, inocula were prepared as described above. The following solutions were tested: AMB, FLC, ITC, TYR, AMB/TYR, FLC/TYR and ITC/TYR. Aliquots of 200 μL of each test solution at three different concentrations (MIC, 10 × MIC and 50 × MIC) were added to viable 48-h biofilms. Controls were grown in medium without antimicrobials. Measurement of Candida biofilms was obtained by the XTT assay, as described above. Experiments were performed in duplicate and repeated three times independently.
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Statistical analysis Considering that the MIC data presented low asymmetry, Student’s t-test for paired samples was used to compare differences in MIC values before and after combining TYR with antifungal drugs. Finally, in order to verify differences in optical density, to evaluate the effects of TYR and antifungals on biofilm production, Student’s t-test for paired samples was used. For all the analyses, a significance level lower than 5% indicated statistically significant findings (P < 0.05).
RESULTS Antifungal susceptibility of Candida planktonic cells All the tested strains showed resistance to at least one antifungal drug. Antifungal resistance to AMB was detected in C. albicans, (n = 1) and C. tropicalis (n = 4) strains. Simultaneous resistance to ITC and FLC was also seen (C. albicans n = 7; C. tropicalis n = 10). The MIC for TYR ranged from 2.5 to 5.0 mM for both species. Detailed description of MIC values is shown in Table 1, along with the results of the in vitro interaction between antifungals and TYR. TYR significantly reduced the MICs for all antifungal drugs tested (P < 0.01). Synergism was observed between AMB/TYR (11/20 strains), ITC/TYR (18/20 strains) and FLC/TYR (18/20 strains).
Effect of exogenous TYR on Candida biofilms Exogenous TYR alone was able to significantly reduce biofilm formation of both C. albicans and C. tropicalis only when tested at 50 × MIC (P < 0.001). At this concentration, TYR was able to decrease metabolic activity of growing biofilms at approximately 24 and 30% for C. albicans and C. tropicalis, respectively. Antifungal drugs alone were effective at both 10 × MIC and 50 × MIC (P < 0.001). Reduction in biofilm formation was more pronounced when TYR was combined with antifungal drugs, resulting in statistically significant reduction in biofilm metabolic activity starting at MIC (P < 0.001). Candida biofilm formation declined by 60% in metabolic activity after incubation with AMB/TYR, FLC/TYR or ITC/TYR at 50 × MIC. Details regarding inhibition of biofilm formation are shown in Table 2. Exogenous TYR at 50 × MIC was able to reduce mature biofilms of C. albicans and C. tropicalis at nearly 50% (P < 0.001). Antifungal drugs alone were effective against mature biofilm at both 10 × MIC and 50 × MIC (P < 0.001). Reduction of biofilm formation was more pronounced when TYR was combined with AMB, resulting in a significant reduction in biofilm metabolic activity starting at MIC (P < 0.0001). Mature biofilm exposed to AMB/TYR at 50 × MIC presented a 35% metabolic reduction. On the other hand, the combination of TYR with either azole antifungal at both 10 × MIC or 50 × MIC resulted in a significant increase in biofilm metabolism (Table 2).
DISCUSSION TYR (2-(4-hydroxyphenyl)ethanol)—a derivative of the amino acid tyrosine—was the second QS molecule described in C. albicans (Chen et al. 2004). Earlier studies have shown that the accumulation of TYR in a culture medium is directly related to the increase in fungal cell density (Chen and Fink 2006). Besides being a fungal quorum-sensing molecule, TYR is also a majority component of olive oils used in human diets. The antioxidative properties of such oils have been attributed to phenolic compounds, including TYR and hydroxytyrosol (Visioli et al. 2000).
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Table 1. MICs of antifungals (μg mL−1 ) and TYR (mM) and FICI for each drug combination against Candida planktonic cells. Strains
Origin
MIC (individual drugs) AMB ITC FLC TYR
AMB/TYR
MIC (combined drugs) ITC/TYR FLC/TYR
AMB/TYR
FICI index∗ ITC/TYR
FLC/TYR
C. albicans 05–05–005 01–05–006 01–05–004 02–01–070 01–03–069 01–03–037 01–01–027 03–02–033 01–03–068 01–02–081
H H H H V V V V V V
0.5 0.5 0.5 0.5 2.0 0.5 0.25 0.125 0.5 0.5
>16 4.0 2.0 4.0 2.0 >16 >16 >16 >16 >16
>64 2.0 2.0 4.0 >64 >64 >64 32 >64 >64
2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0
0.0625/1.25 0.125/0.3125 0.5/1.25 0.25/0.625 1.0/2.5 0.125/0.3125 0.125/1.25 0.125/0.3125 0.125/0.3125 0.0625/1.25
2.0/0.312 0.0625/0.156 0.0625/0.156 2.0/2.5 2.0/2.5 2.0/0.625 0.0625/0.156 2.0/0.625 2.0/0.0312 1.0/0.625
8.0/0.3125 0.125/0.039 1.0/1.25 1.0/1.25 0.0156/0.625 1.0/1.25 0.125/0.039 16/2.5 2.0/0.625 8.0/1.25
0.625 0.312 1.25 0.625 1.5 0.312 1.0 1.062 0.375 0.375
0.249 0.046 0.094 1.0 2.0 0.5 0.101 0.5 0.5 0.187
0.5 0.14 1.0 0.5 0.027 0.375 0.0175 1.0 0.281 0.375
H H H V H H H V V V
2.0 0.5 0.5 1.0 4.0 4.0 4.0 0.5 0.5 1.0
2.0 >16 >16 >16 >16 >16 >16 8.0 4.0 >16
8 >64 >64 >64 >64 >64 8 32 >64 >64
5.0 5.0 2.5 5.0 5.0 5.0 5.0 5.0 2.5 5.0
0.125/0.3125 0.25/0.625 0.125/0.3125 0.5/1.25 0.5/1.25 0.5/1.25 0.25/0.625 0.125/0.3125 0.25/0.625 0.125/0.3125
0.25/0.625 0.5/0.3125 2.0/0.3125 0.0625/0.156 0.125/0.3125 0.125/0.3125 2.0/0.625 0.125/0.3125 1.0/0.625 0.5/0.3125
1.0/1.25 16/1.25 8.0/0.3125 0.125/0.039 8.0/1.25 4.0/0.625 1.0/1.25 2.0/0.625 4.0/0.625 1.0/1.25
0.125 0.625 0.375 0.75 0.375 0.375 0.077 0.312 0.75 0.187
0.25 0.093 0.5 0.07 0.07 0.07 0.25 0.078 0.5 0.07
0.375 0.5 0.25 0.097 0.375 0.187 0.375 0.187 0.187 0.375
C. tropicalis 01–02–184 02–01–016 03–02–048 01–01–031 02–01–012 02–02–179 02–02–199 03–02–047 01–02–078 01–03–075
H: human; V: veterinary; AMB: amphotericin B; ITC: itraconazole; FLC: fluconazole; TYR: tyrosol. Resistance breakpoint: AMB>1 μg mL−1 , ITC ≥1 μg mL−1 and FLC ≥ 8 μg mL−1 . ∗ The interaction was defined as synergistic when FICI ≤ 0.5; indifferent when 0.5 < FICI < 4, and antagonistic when FICI>4.0.
In recent years, many studies have shown the inhibitory effect of fungal QSMs against bacteria (Brilhante et al. 2012) and fungi (Derengowski et al. 2009; Cordeiro et al. 2013). Until now, a total of five QSMs have been described in fungi (Alem et al. 2006; Chen and Fink 2006), but farnesol and TYR from Candida have attracted the most attention from researchers. Farnesol is the most studied fungal QSM and experimental evidence has shown that this compound can also induce cell death in Aspergillus (Dinamarco, Goldman and Goldman 2011), inhibit yeastto-hypha transition in C. albicans and impair biofilm formation in Candida spp. (Ramage et al. 2002). Even though the biological effects of TYR on fungal pathogens is not well understood, it is known to have the opposite activity of farnesol, i.e. stimulation of germ-tube formation and hence hyphal development (Alem et al. 2006). The results of the present study show that exogenous TYR caused a significant reduction of C. albicans and C. tropicalis planktonic growth at concentrations that ranged from 2.5 to 5.0 mM. According to Alem et al. (2006), TYR production by C. albicans can reach concentrations of approximately 11 μM in the supernatant of planktonic cell cultures, which is lower than the TYR MICs found in this study. Although C. albicans produces significantly more TYR when compared to C. tropicalis (Tumbarello et al. 2007), a similar susceptibility pattern to exogenous TYR was seen in both Candida species. In this study, synergistic interactions between TYR and azoles were detected against both C. albicans and C. tropicalis strains. TYR reverted the resistance phenotype to FLC of 4/7 C. albicans and 7/10 C. tropicalis and the resistance phenotype to ITC of 3/10 C. albicans and 7/10 C. tropicalis. Recently, Cordeiro et al. (2013) showed that the QSM farnesol was able to reduce MICs of AMB, azoles and caspofungin against Candida species, making them susceptible to these antifungals. Although far-
nesol and TYR are both autoregulatory molecules in Candida, they have different biological effects and probably different receptors in the fungal cell (Alem et al. 2006). We believe that TYR interferes with ergosterol biosynthesis, since the fungal cells became more susceptible to azoles after exposure to this compound. Several studies have shown that fungal QSMs have a dramatic impact on Candida biofilms (Alem et al. 2006; Albuquerque and Casadevall 2012). Exogenous farnesol, for example, added at a very early stage inhibits biofilm formation, blocks the yeasthyphae transition required for mature biofilms (Lindsay et al. 2012). Indeed, when C. albicans cells were treated with farnesol at 300 μM, thin biofilms formed predominantly of blastoconidia and pseudohyphae were seen (Ramage et al. 2012). In the present study, exogenous TYR added at time zero was able to significantly reduce biofilm formation, which was also observed when TYR was combined with antifungals, at all tested concentrations (MIC, 10 × MIC and 50 × MIC). Even though further studies should be conducted to elucidate the mechanisms involved in the effects of high concentrations of exogenous TYR on Candida biofilm growth, some hypotheses can be proposed, based on the dynamics of biofilm formation. It has been shown that farnesol, not TYR, is the QSM predominantly secreted by C. albicans planktonic cells and that exogenous TYR has no significant effect on early biofilm formation, despite accelerating hyphal growth (Alem et al. 2006). Thus, we believe that when Candida biofilms were exposed to high concentrations of exogenous TYR, the compound overcame the physiological effects of farnesol, then compromising biofilm formation. Antifungal-TYR combinations were even more effective against growing biofilms, when compared to each compound alone, likely due to an additive deleterious effect on biofilm growth.
TYR: tyrosol; AMB: amphotericin B; FLC: fluconazole: ITC: itraconazole; AMB/TYR: tyrosol plus amphotericin B; FLC/TYR: tyrosol plus fluconazole; ITC/TYR: tyrosol plus itraconazole. Different letters indicate statistically significant differences (P < 0.05). Numbers represent XTT absorbances (mean ± SEM) of Candida biofilms at 492 nm. Controls were performed in RPMI medium without inhibitory drugs.
100.0a (0.590 ± 0.03) 100.0a (0.540 ± 0.03) 100.0a (0.442 ± 0.03) 100.0a (0.425 ± 0.03) Control
53.02 ± 2.20c 65.83 ± 3.56b 74.55 ± 3.53b 64.71 ± 4.16b 63.48 ± 3.88b 120.7 ± 2.38d 120.4 ± 2.9d 110.7 ± 2.85a 75.82 ± 4.19b 80.9 ± 2.99b 78.37 ± 4.29b 80.79 ± 3.45b 123.0 ± 3.37d 120.5 ± 3.52d 102.2 ± 2.89a 93.68 ± 2.79a 96.69 ± 2.6a 97.98 ± 1.95a 85.1 ± 3.68a 100.4 ± 1.94a 101.9 ± 1.2a 46.86 ± 5.33b 68.45 ± 4.38b 67.74 ± 3.94a 80.31 ± 3.18a 63.63 ± 2.97b 128.4 ± 3.08c 126.8 ± 4.42c 112.2 ± 6.06a 92.67 ± 6.08a 74.55 ± 1.84a 81.57 ± 3.36a 80.68 ± 1.89b 127.1± 6.21c 120.6 ± 2.07c 71.99 ± 4.36b 68.62 ± 1.91b 64.10 ± 2.53b 73.00 ± 2.83b 37.29 ± 2.36c 42.23 ± 3.10c 39.34 ± 3.11c 97.32 ± 4.96a 71.89 ± 3.74b 85.91 ± 1.59b 83.64 ± 3.64b 63.71 ± 3.40b 68.09 ± 3.93b 71.22 ± 2.88b 84.9 ± 4.95a 75.43 ± 1.91b 78.36 ± 2.00b 83.95 ± 4.10b 66.39 ± 2.41b 61.43 ± 2.99b 57.07 ± 3.26b TYR AMB FLC ITC AMB/TYR FLC/TYR ITC/TYR
103.9 ± 2.98a 101.8 ± 1.35a 99.41 ± 4.92a 94.58 ± 1.06a 91.52 ± 1.02a 87.82 ± 3.49a 82.14 ± 1.73a
76.76 ± 4.90b 67.28 ± 2.75b 71.32 ± 2.60b 74.11 ± 2.51b 31.05 ± 2.71c 44.98 ±2.51c 30.42 ± 2.79c
101.0 ± 3.04a 103.1 ± 4.20a 99.43 ± 0.90a 108.0 ± 7.75a 89.0 ± 1.77a 86.89 ± 1.42a 95.47 ± 5.83b
102.8 ± 2.62a 88.34 ± 2.63a 97.15 ± 1.14a 97.92 ± 1.84a 84.48 ± 1.70a 97.71 ± 2.16a 99.75 ± 1.12a
MIC 50 × MIC 50 × MIC MIC
50 × MIC
MIC
MIC
C. albicans 10 × MIC C. tropicalis 10 × MIC C. albicans 10 × MIC
Growing biofilm (%) Drugs
Table 2. Metabolic activity of Candida biofilms grown in RPMI medium supplemented with TYR or antifungals alone or in combination.
Mature biofilm (%)
C. tropicalis 10 × MIC
50 × MIC
Cordeiro et al.
5
In the present study, it was shown that azoles-TYR combinations were not able to inhibit mature biofilms even at 50 × MIC concentration. We believe that the concentrations of TYR in these experiments—which ranged from 0.039 to 62.5 mM—were not able to reduce the metabolic activity in mature biofilms. On the other hand, concentrations as low as 0.6 mM of TYR were able to enhance the antifungal activity of AMB against mature biofilms. Although Alem et al. (2006) did not detect any inhibitory effect of TYR at 1 mM against mature biofilms, the results presented here show that TYR at this concentration is able to act synergistically with AMB, reducing biofilm viability by approximately 16%. Combinations formed by synergistic agents have been tested successfully against in vivo Candida biofilms. In a seminal study conducted by Uppuluri et al. (2008), it was confirmed that combinations formed by FK506 and FLC caused complete inhibition of C. albicans biofilms in a rat venous catheter biofilm model. More recently, Bink et al. (2012) demonstrated that the nonsteroidal anti-inflammatory diclofenac potentiates the antifungal activity of caspofungin against in vivo C. albicans biofilms. Li et al. (2012) described the in vivo inhibitory effect on the biofilm of C. albicans by the liverwort-derived riccardin D and FLC. Taken together, these results support that combination therapy is a good strategy to combat Candida biofilms infections. Further study with an in vivo model should be conducted to confirm the anti-biofilm potential of combinations formed by TYR and AMB. The results of the present study show that exogenous TYR alone or combined with antifungals inhibits planktonic cells and Candida biofilm growth. Mature biofilms were also inhibited by TYR alone or combined with AMB, but TYR plus azoles increased the biofilm activity by a dose-dependent mechanism. Because of the clinical importance of Candida infections and due to the paucity of drugs able to inhibit fungal biofilms, we believe that the results presented here might be of interest for the development of new antifungal strategies.
FUNDING This study was supported by CNPq, Brazil (302574/2009-3; PROTAX 562296/2010-7; 504189/2012-3; 307606/2013-9) and CAPES; Brazil (PNPD 2103/2009, AE1-0052-000630100/11). Conflict of interest. None declared.
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