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Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE Enhanced activity of drugs using natural phenolics against yeast strains of and Cryptococcus N.C.G.Faria1, J.H. Kim3, L.A.P. Gonc¸alves2, M. de L. Martins1, K.L. Chan3 and B.C. Campbell3

1 Instituto de Higiene e Medicina Tropical ⁄ CREM, Universidade Nova de Lisboa, Portugal 2 Instituto de Higiene e Medicina Tropical ⁄ CEAUL, Universidade Nova de Lisboa, Portugal 3 Plant Mycotoxin Research Unit, Western Regional Research Center, ARS-USDA, Albany, CA, USA

Keywords Abstract amphotericin B, phenolic, synergism, . Aims: Determine whether certain, natural phenolic compounds enhance activ- Correspondence ity of commercial antifungal drugs against yeast strains of Candida and Crypto- Bruce C. Campbell, Plant Mycotoxin Research coccus neoformans. Unit, Western Regional Research Center, Methods and Results: Twelve natural phenolics were examined for fungicidal USDA-ARS, 800 Buchanan Street, Albany, CA activity against nine reference strains of Candida and one of C. neoformans. Six 94710, USA. compounds were selected for synergistic enhancement of antifungal drugs, E-mail: [email protected] amphotericin B (AMB), fluconazole (FLU) and (ITR). Matrix assays 2010 ⁄ 1438: received 19 August 2010, revised of phenolic and drug combinations conducted against one reference strain, each, 14 February 2011 and accepted 15 February of and C. neoformans showed cinnamic and benzoic acids, thy- 2011 mol, and 2,3- and 2,5-dihydroxybenzaldehydes (-DBA) had synergistic interac- tions depending upon drug and yeast strain. 2,5-DBA was synergistic with doi:10.1111/j.1472-765X.2011.03032.x almost all drug and strain combinations. Thymol was synergistic with all drugs against Ca. albicans and with AMB in C. neoformans. Combinations of benzoic acid or thymol with ITR showed highest synergistic activity. Of 36 combinations of and drug tested, none were antagonistic. Conclusions: Relatively nontoxic natural products can synergistically enhance antifungal drug activity, in vitro. Significance and Impact of the Study: This is a proof-of-concept, having clini- cal implications. Natural chemosensitizing agents could lower dosages needed for effective chemotherapy of invasive mycoses. Further studies against clinical yeast strains and use of animal models are warranted.

included are the polyene drugs, namely amphotericin B Introduction (AMB) and , which complex with membrane Resistance to antifungal agents is widely recognized (Den- sterols resulting in cellular leakage and echinochandins, ning et al. 1997; Pfaller et al. 2006) requiring continuous which inhibit synthesis of cell wall b-(1,3)-glucans (Ste- development of new antifungal agents (Stevens et al. vens et al. 2004; VandenBussche and Van Loo 2010). 2004). and of continuously Treatment of cryptococcal has chiefly involved expanding global incidence are a result of increased combination antifungal therapy, more than one drug, immunosuppressive disorders, including AIDS and certain with AMB or , sometimes having severe side chemo- or radiotherapies (Sobel et al. 2004). effects (Johnson and Perfect 2007). Commonly prescribed drugs for the treatment of can- Thorough investigations of fungal resistance mecha- didiases include a variety of and triazole drugs nisms have involved antifungal agents and a wide that disrupt biosynthesis of , a fungal-specific number of Candida species (Pfaller et al. 2005). Extensive sterol of cellular membranes (Heimark et al. 2002). Also use of one antifungal drug, fluconazole (FLU), has

No claim to US Government works 506 Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology N.C.G.Faria et al. Natural antifungal chemosensitizers resulted in widespread resistance (Spanakis et al. 2006) – PYCC 3097T, – PYCC associated with drug efflux through ABC plasma mem- 3341, Candida lusitaniae – PYCC 2705T. Cryptococcus: brane transporters (Holmes et al. 2008). Centraalbureau voor Schimmelcultures (CBS), Utrecht, There are frequent reports of cross-resistance to both The Netherlands – – CBS 132T. FLU and itraconazole (ITR), and other azole drugs for the treatment of candidiasis and other opportunistic Antifungal activities of natural products mycoses associated with AIDS chemotherapy (de Repent- igny et al. 2004; Charlier et al. 2006). High levels of resis- All reference strains of Candida and C. neoformans were tance to azole antifungal drugs have also been observed in used to determine antifungal activity of 12 natural prod- other conditions involving filamentous fungi (Mocroft ucts using microtitre plate assays. Natural products were et al. 2005). dissolved in DMSO and then added to stock synthetic ) The upsurge of fungal resistance to currently available glucose (SG) media (6Æ7mgml 1 yeast nitrogen base w ⁄ o agents, and stagnation in development of new agents, amino acids, 2% glucose) prior to distribution into compels the development of a supplementary strategy for microtitre plate wells. For each well, one containing only antifungal chemotherapy. Natural compounds are a SG and DMSO (control) and another one containing also potential source of antimycotic agents either in their nas- the natural product to be tested, an inoculum of cent form or as template structures for more effective c. 5 · 103 yeast cells (ATCC and PYCC) in SG liquid derivatives (Barrett 2002; Jacob and Walker 2005). medium was added to each well containing SG and natu- Recently, natural products were found to augment in vitro ral product to a final volume of 200 ll and final concen- ) activity of FLU against strains of resistant filamentous tration of natural product to 5 mmol l 1. Each test was fungi causing (Kim et al. 2008a, 2010). We carried out in triplicate and incubated at 30C. Yeast cell report here the potential for safe, natural compounds to multiplication was monitored at 48 and 72 h for Candida similarly improve effectiveness of selected azole and poly- strains and C. neoformans, respectively, by optical density ene antifungal drugs against the yeasts Candida albicans at 595 nm (OD595) (Zenyth 3100 multimode detector; and Cryptococcus neoformans. These natural compounds Anthos Labtec Instr., Salzburg, Austria). Inhibition was are common phenolics found in edible plants and are measured as percentage OD595 treated ⁄ OD595 control. considered to be innately safe for humans. This proof-of- concept, which chemosensitization by natural products Determination of inhibitory concentrations enhances antifungal drug activity, warrants further testing on clinical strains and in animal models. Inhibitory concentrations (IC) of antifungal drugs and natural compounds against the yeasts were determined using microdilution assays measured by optical density Materials and methods (Galagiani and Stevens 1978). All IC assays were performed in triplicate in microtitre plates and used an inoculum of Antifungal drugs and chemicals c. 5 · 103 cells in SG. The final volume including SG, cells All benzo analogues, antifungal drugs and culture media and test compound in each well was 200 ll. IC assays of were procured from Sigma-Aldrich, or Fluka (St Louis, natural products involved six serial, twofold dilutions using ) ) MO, USA): natural products: cinnamic, 2-hydroxycinnamic, SG, starting at 5 mmol l 1 and ending at 78Æ125 lmol l 1. 3-hydroxycinnamic, 4-hydroxycinnamic, benzoic and sali- IC determinations of antifungal drugs involved diluting cylic acids, thymol, vanillin, 3,4,5-trimethoxybenzaldehyde, FLU and ITR in DMSO, and AMB in water, and included veratraldehyde, 2,3-dihydroxybenzaldehyde (2,3-DBA) and 10 twofold serial dilutions from the starting concentration. 2,5-dihydroxybenzaldehyde (2,5-DBA). Antifungal drugs: The range of drug concentrations for calculating ICs AMB, FLU, ITR. Solvent for test compounds: dimethylsulf- was based on published results (Cuenca-Estrella et al. oxide (DMSO). 2002). For the calculation of ICs for AMB, concentrations ) ranged from 32 to 0Æ03125 mg l 1 for strains of Candida ) and 64 to 0Æ0625 mg l 1 for C. neoformans. For FLU, con- Yeast species and strains ) centrations ranged from 64 to 0Æ0625 mg l 1 for Candida ) Candida. American Type Culture Collection (ATCC), and 128 to 0Æ125 mg l 1 for C. neoformans. For ITR, con- ) Manassas, VA, USA – Candida albicans – ATCC 90028, centrations ranged from 4 to 0Æ00391 mg l 1 for Candida ) ATCC 10231; – ATCC 22019. Portu- and 8 to 0Æ00781 mg l 1 for C. neoformans. Control wells guese Yeast Culture Collection (PYCC), Universidade received only SG and DMSO and the respective yeast-strain Nova de Lisboa – Ca. albicans – PYCC 3436T, Ca. par- inoculum. Inhibition was monitored and calculated based T apsilosis – PYCC 2545, – PYCC 2418 , on OD595 of treated vs control.

No claim to US Government works Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology 507 Natural antifungal chemosensitizers N.C.G.Faria et al.

natural product, as follows: FICI = (lowest IC of natu- Matrix assays with drugs and natural products 90 x ral product in combination with antifungal drug resulting

To determine interactions of natural products and drug in ‡90% growth inhibition ⁄ IC90 of natural product, activities, a series of microtitre plate matrix assays were alone) + (lowest ICx of antifungal drug resulting in ‡90% performed. For these assays, reference strains Ca. albicans growth inhibition in combination with natural prod-

ATCC 90028, recommended as a standard for drug resis- uct ⁄ IC90 of antifungal drug, alone). Interactions between tance studies (Clinical and Laboratory Standards Institute natural products and antifungal drugs were defined as fol- T 2007), and C. neoformans CBS 132 , originally isolated lows: synergy = FICI90 £ 0Æ5; additive = 0Æ5 < FICI90 £ 1; from fermenting fruit juice (Gue´ho et al. 1993), were neutral = 1 < FICI90 £ 2; antagonistic = 2 < FICI90 (Isen- used. Neither of these strains had previously been exposed berg 1992). to AMB, FLU or ITR. Individual drug and natural prod- uct were combined in a matrix-like fashion, in triplicate, Results using combinations of control, IC25,IC50,IC75 and IC90 levels respective to each yeast strain, using microdilution Effects of natural products on reference strains protocols described. Of the 12 natural products tested, cinnamic, benzoic and salicylic acids, thymol, 2,3- and 2,5-DBAs (Group A) sig- Statistical methods nificantly inhibited growth ‡90% (P <0Æ05) of all strains Significant differences (P <0Æ05) in growth inhibition by of Candida and C. neoformans (Table 1). Although vanil- natural products were based on the nonparametric Fried- lin showed significant inhibitory activity against the Can- man test using spss (Chicago, IL, USA). IC levels corre- dida strains, it had only moderate antifungal activity sponding to 25, 50, 75 and 90% growth inhibition (i.e. against C. neoformans.

IC25,IC50,IC75 and IC90) were determined by generalized IC levels of Group A compounds varied against Candida linear model and probit procedures (Finney 1971) of the strains (Table 2). For example, based on IC50, fungicidal SPSS package. Interactions between natural compounds ranking, highest to lowest, was 2,3-DBA > salicylic > ben- and antifungal drugs were determined from drug ⁄ natural zoic > cinnamic acids > thymol > 2,5-DBA. Also, among product concentrations resulting in 90% growth inhibi- the Candida strains, there was a variation in sensitivities. tion to reduce errors in assigning interactions to ‘neutral’ Both Ca. albicans PYCC 3436T and Ca. lusitaniae PYCC (Meletiadis et al. 2005). Fractional Inhibitory Concentra- 2705T were significantly more susceptible to the natural tion Indices (FICI90) were calculated from the lowest IC compounds, in general, than the other Candida, whereas (i.e. IC25,IC50,IC75 or IC90) of antifungal drug showing Ca. krusei PYCC 3341and Ca. parapsilosis PYCC 2545 were ‡90% growth inhibition with corresponding lowest ICx of significantly less sensitive than other Candida.

) Table 1 Growth inhibitory activity (%) of natural products (5 mmol l 1) on reference strains of Candida and Cryptococcus neoformans. Com- pounds having ‡90% growth inhibition (P <0Æ05) in all strains are in bold (Group A)

Candida Candida Candida Candida Candida Candida albicans parapsilosis glabrata tropicalis krusei lusitaniae C. neoformans

ATCC ATCC PYCC ATCC PYCC PYCC PYCC PYCC PYCC Natural product 90028 10231 3436T 22019 2545 2418T 3097T 3341 2705T CBS 132T

Cinnamic acid 99Æ899Æ999Æ099Æ999Æ399Æ899Æ999Æ999Æ996Æ1 2-Hydroxycinnamic acid 50Æ411Æ765Æ419Æ557Æ117Æ127Æ132Æ329Æ278Æ0 3-Hydroxycinnamic acid 63Æ244Æ980Æ532Æ141Æ68Æ739Æ34 19Æ040Æ881Æ0 4-Hydroxycinnamic acid 65Æ515Æ479Æ030Æ27Æ78Æ521Æ23 5Æ924Æ060Æ4 Veratraldehyde 32Æ016Æ287Æ732Æ554Æ010Æ215Æ034Æ318Æ188Æ7 Vanillin 96Æ397Æ898Æ898Æ097Æ890Æ598Æ790Æ297Æ979Æ3 Benzoic acid 99Æ799Æ999Æ999Æ23 98Æ899Æ699Æ899Æ599Æ895Æ8 3,4,5-Trimethoxybenzaldehyde 42Æ28Æ676Æ349Æ738Æ820Æ335Æ716Æ634Æ376Æ0 99Æ699Æ999Æ999Æ898Æ199Æ999Æ899Æ999Æ895Æ4 Thymol 99Æ999Æ999Æ999Æ699Æ799Æ999Æ899Æ999Æ895Æ23 2,5-DBA 96Æ396Æ093Æ290Æ091Æ493Æ895Æ993Æ695Æ993Æ5 2,3-DBA 99Æ499Æ799Æ398Æ897Æ399Æ999Æ499Æ799Æ595Æ1

DBA, dihydroxybenzaldehyde.

No claim to US Government works 508 Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology N.C.G.Faria et al. Natural antifungal chemosensitizers

Table 2 Inhibitory concentrations (IC; mmol l)1) of Group A natural products (see Table 1) at 25, 50, 75 and 90 levels against reference strains of Candida and Cryptococcus neoformans

Candida Candida Candida Candida Candida Candida albicans parapsilosis glabrata tropicalis krusei lusitaniae C. neoformans

ATCC ATCC PYCC ATCC PYCC PYCC PYCC PYCC PYCC CBS 90028 10231 3436T 22019 2545 2418T 3097T 3341 2705T 132T

Cinnamic acid

IC25 0Æ37 0Æ53 0Æ24 0Æ05 0Æ08 0Æ04 0Æ08 0Æ16 0Æ05 0Æ06

IC50 0Æ64 0Æ74 0Æ45 0Æ12 0Æ18 0Æ09 0Æ16 0Æ29 0Æ10 0Æ11

IC75 1Æ12 1Æ03 0Æ86 0Æ28 0Æ41 0Æ23 0Æ31 0Æ52 0Æ19 0Æ23

IC90 1Æ86 1Æ38 1Æ53 0Æ62 0Æ88 0Æ55 0Æ57 0Æ88 0Æ33 0Æ44 Ave. 1Æ00 0Æ92 0Æ768 0Æ27 0Æ38 0Æ23 0Æ28 0Æ46 0Æ17 0Æ21 Benzoic acid

IC25 0Æ08 0Æ43 0Æ04 0Æ03 0Æ04 0Æ01 0Æ23 0Æ24 0Æ02 0Æ13

IC50 0Æ19 0Æ73 0Æ10 0Æ09 0Æ13 0Æ05 0Æ48 0Æ71 0Æ06 0Æ26

IC75 0Æ48 1Æ24 0Æ25 0Æ29 0Æ36 0Æ223 1Æ01 2Æ13 0Æ14 0Æ51

IC90 1Æ12 2Æ00 0Æ58 0Æ85 0Æ92 0Æ94 1Æ96 7Æ74 0Æ31 0Æ94 Ave. 0Æ47 1Æ10 0Æ24 0Æ32 0Æ36 0Æ31 0Æ92 2Æ70 0Æ13 0Æ46 Salicylic acid

IC25 0Æ14 0Æ43 0Æ01 0Æ05 0Æ09 0Æ07 0Æ24 0Æ11 0Æ04 0Æ14

IC50 0Æ26 0Æ76 0Æ05 0Æ17 0Æ25 0Æ20 0Æ45 0Æ20 0Æ10 0Æ35

IC75 0Æ46 1Æ34 0Æ19 0Æ61 0Æ68 0Æ62 0Æ84 0Æ36 0Æ25 0Æ90

IC90 0Æ78 2Æ23 0Æ70 1Æ96 1Æ66 1Æ72 1Æ47 0Æ61 0Æ58 2Æ12 Ave. 0Æ41 1Æ19 0Æ24 0Æ70 0Æ67 0Æ65 0Æ75 0Æ32 0Æ24 0Æ88 Thymol

IC25 0Æ60 0Æ25 0Æ07 0Æ06 0Æ24 0Æ14 0Æ14 0Æ14 0Æ05 0Æ06

IC50 1Æ10 0Æ51 0Æ19 0Æ34 0Æ56 0Æ54 0Æ52 0Æ48 0Æ18 0Æ16

IC75 2Æ02 1Æ02 0Æ49 1Æ97 1Æ28 2Æ06 1Æ95 1Æ72 0Æ66 0Æ46

IC90 3Æ49 1Æ92 1Æ13 5Æ00* 2Æ72 5Æ00* 5Æ00* 5Æ00* 2Æ08 1Æ18 Ave. 1Æ80 0Æ92 0Æ47 1Æ84 1Æ20 1Æ97 1Æ90 1Æ86 0Æ74 0Æ46 2,5-DBA

IC25 0Æ71 0Æ35 0Æ02 0Æ21 0Æ13 0Æ35 0Æ99 0Æ20 0Æ14 0Æ02

IC50 1Æ13 0Æ59 0Æ06 0Æ88 0Æ68 1Æ16 2Æ50 0Æ82 0Æ35 0Æ08

IC75 1Æ79 1Æ00 0Æ24 3Æ65 3Æ45 3Æ79 5Æ00 3Æ41 0Æ88 0Æ26

IC90 2Æ72 1Æ59 0Æ81 5Æ00* 5Æ00* 5Æ00* 5Æ00* 5Æ00* 2Æ04 0Æ76 Ave. 1Æ59 0Æ88 0Æ28 2Æ43 4Æ26 2Æ57 3Æ38 2Æ35 0Æ85 0Æ28 2,3-DBA

IC25 0Æ03 0Æ03 <0Æ01 0Æ03 0Æ05 <0Æ01 0Æ03 0Æ03 0Æ03 0Æ01

IC50 0Æ04 0Æ05 0Æ01 0Æ08 0Æ09 <0Æ01 0Æ06 0Æ08 0Æ16 0Æ03

IC75 0Æ07 0Æ07 0Æ02 0Æ18 0Æ18 0Æ01 0Æ10 0Æ14 0Æ78 0Æ12

IC90 0Æ10 0Æ10 0Æ06 0Æ37 0Æ38 0Æ03 0Æ17 0Æ61 3Æ17 0Æ38 Ave. 0Æ058 0Æ06 0Æ02 0Æ16 0Æ18 0Æ01 0Æ09 0Æ21 1Æ04 0Æ13 Average: all compounds 1Æ94 1Æ92 0Æ40 1Æ60 4Æ20 1Æ40 2Æ21 6Æ32 0Æ59 0Æ40

*5 mmol l)1 (Table 1). DBA, dihydroxybenzaldehyde.

ICs of natural products also varied against C. neofor- Antifungal drug activities mans CBS 132T (Table 2). Overall, the C. neoformans ) strain had an average IC level (25–90) of 0Æ40 mmol l 1, IC levels for 25, 50, 75 and 90% growth inhibition of AMB, similar to the most sensitive Candida strain, PYCC 3436T FLU and ITR for Ca. albicans ATCC 90028 were c. 103–104 ) (0Æ40 mmol l 1). The hierarchy in natural product ICs greater in antifungal activity than the natural products differed in the C. neoformans strain from Candida strains (Table 3). In general, IC50 levels of Group A natural prod- )1 with highest to lowest IC50 levels at 2,3- > 2,5-DBAs ucts for the Candida ranged from 4 lmol l (2,3-DBA, ) > cinnamic > thymol > benzoic > salicylic. Similar to Ca. glabrata)to2Æ5 mmol l 1 (2,5-DBA, Ca. tropicalis)

Candida strains, 2,3-DBA had the highest fungicidal activ- (Table 2). By comparison, the highest IC50 for an antifun- ) ) ity against C. neoformans of all natural products. gal drug was 26 lmol l 1 (8 mg l 1, FLU, Ca. parapsilosis

No claim to US Government works Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology 509 Natural antifungal chemosensitizers N.C.G.Faria et al.

Table 3 Inhibitory concentrations (IC; mg l)1) for antifungal drugs, amphotericin B (AMB), fluconazole (FLU) and itraconazole (ITR) at 25, 50, 75 and 90 levels against reference strains of Candida and Cryptococcus neoformans

Candida Candida Candida Candida Candida Candida albicans parapsilosis glabrata tropicalis krusei lusitaniae C. neoformans

ATCC ATCC PYCC ATCC PYCC PYCC PYCC PYCC PYCC CBS 90028 10231 3436T 22019 2545 2418T 3097T 3341 2705T 132T

AMB

IC25 0Æ65 0Æ33 0Æ15 0Æ13 0Æ14 0Æ16 0Æ19 0Æ61 0Æ32 0Æ06

IC50 1Æ14 0Æ56 0Æ47 0Æ45 0Æ43 0Æ35 0Æ72 1Æ06 0Æ89 0Æ29

IC75 2Æ00 0Æ97 1Æ50 1Æ60 1Æ34 0Æ77 2Æ65 1Æ86 2Æ48 1Æ30

IC90 3Æ30 1Æ58 4Æ27 5Æ02 3Æ71 1Æ57 8Æ59 3Æ09 6Æ17 5Æ11 Ave. 1Æ77 0Æ86 1Æ59 1Æ80 1Æ40 0Æ71 3Æ04 1Æ65 2Æ46 1Æ69 FLU * IC25 0Æ53 3Æ53 0Æ13 2Æ40 0Æ34 n ⁄ c 0Æ12 1Æ71 n ⁄ c0Æ10

IC50 1Æ48 7Æ84 0Æ67 8Æ01 1Æ74 n ⁄ c0Æ99 3Æ17 n ⁄ c0Æ51

IC75 4Æ11 17Æ39 3Æ43 26Æ77 8Æ98 n ⁄ c0Æ82 5Æ89 n ⁄ c2Æ75

IC90 10Æ32 35Æ62 15Æ01 79Æ33 39Æ40 n ⁄ c55Æ82 10Æ27 n ⁄ c12Æ45 Ave. 4Æ11 16Æ09 4Æ81 29Æ13 12Æ61 n ⁄ c14Æ44 5Æ26 n ⁄ c3Æ95 ITR IC25 0Æ02 <0Æ01 nd nd nd 0Æ04 nd <0Æ01 0Æ01 nd

IC50 0Æ06 0Æ01 <0Æ01 nd nd 0Æ25 <0Æ01 0Æ01 0Æ05 <0Æ01

IC75 0Æ20 0Æ05 0Æ02 <0Æ01 <0Æ01 1Æ75 0Æ01 0Æ03 0Æ21 0Æ06

IC90 0Æ58 0Æ26 0Æ13 0Æ01 <0Æ01 10Æ13 0Æ12 0Æ12 0Æ79 0Æ92 Ave. 0Æ22 0Æ08 0Æ04 <0Æ01 <0Æ01 3Æ04 0Æ03 0Æ04 0Æ26 0Æ24

*n ⁄ c, Not calculated, technical problem. nd, Not determined, below lowest concentration tested.

ATCC 22019). Compared to those of natural products, IC to FLU than C. neoformans. Similar to the Candida, ITR levels of antifungal drugs varied among Candida strains. had highest potency of the drugs to the C. neoformans. For example, ranking of sensitivities of all Candida strains to natural compounds was generally similar. However, Drug ⁄ natural product interactions Ca. parapsilosis ATCC 22019 was the least susceptible ) strain to FLU (average ICs: 29Æ1mgl 1), but one of the Group A natural product and drug combinations were ) more sensitive strains to ITR (average ICs: <0Æ01 mg l 1). mainly additive or synergistic against the Ca. albicans, Alternatively, Ca. glabrata PYCC 2418T was least suscepti- and neutral, additive or synergistic against C. neoformans ) ble to ITR (average ICs: 3 mg l 1), but most susceptible to (Table 4). With Ca. albicans, benzoic acid, thymol and ) AMB (average ICs: 0Æ7mgl 1). ITR demonstrated the 2,5-DBA were synergistic when combined with one or highest antifungal activity being 10 to >100 times more more of the antifungal drugs, with thymol synergistic potent than AMB and FLU, respectively, depending on the with all three drugs. The most dramatic enhancement of types of strains tested. An exception was with the Ca. glab- antifungal drug activity was thymol and ITR. This combi- rata strain, where AMB was, on average, c. 5· more potent nation inhibited growth of the Ca. albicans by 96% with than ITR. each only at an IC25 level in the matrix assay (data not The range in IC levels of antifungal drugs for the shown). Benzoic acid and 2,5-DBA were synergistic with C. neoformans strain fell within that of many of the Can- AMB and ITR. ITR and AMB were most amenable to the dida reference strains (Table 3). For example, the IC90 enhancement of antifungal activity against the Ca. albi- levels for AMB, FLU and ITR, 5Æ11, 12Æ45 and cans in combination with natural products, showing syn- ) 0Æ92 mg 1 1, respectively, were similar to those of ergism with three of the natural products. Additionally, Ca. parapsilosis ATCC 22019, Ca. albicans ATCC 90028 with the Ca. albicans, FLU was synergistic with one of the and Ca. lusitaniae PYCC 2705T, respectively. However, natural products, while cinnamic and salicylic acids and unlike the Candida strains, C. neoformans was not more 2,3-DBA were either neutral or additive with all drugs. sensitive to the drugs than to the natural products. A few There were some major differences in antifungal drug Candida strains (i.e. Ca. albicans ATCC 10231, two and natural product interactions for the C. neoformans Ca. parapsilosis and Ca. tropicalis) showed higher IC levels strain compared with the Ca. albicans (Table 4). The

No claim to US Government works 510 Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology N.C.G.Faria et al. Natural antifungal chemosensitizers

Table 4 Fractional Inhibitory Concentration Indices (FICI90) and defined interactions of drug ⁄ natural product combinations against Candida albi- cans ATCC 90028 and Cryptococcus neoformans CBS 132T

Antifungal drug

Amphotericin B Itraconazole

Natural prod. Ca. albicans C. neoformans Ca. albicans C. neoformans Ca. albicans C. neoformans

Cinnamic acid 0Æ54 add* 1Æ06 neu 0Æ71 add 2Æ00 neu 0Æ64 add 0Æ32 syn Benzoic acid 0Æ27 syn 0Æ56 add 0Æ83 add 1Æ01 neu 0Æ20 syn 1Æ00 add Salicylic acid 0Æ53 add 1Æ01 neu 1Æ40 neu 1Æ16 neu 0Æ62 add 1Æ00 add Thymol 0Æ50 syn 0Æ40 syn 0Æ37 syn 1Æ01 neu 0Æ21 syn 1Æ00 add 2,5-DBA 0Æ46 syn 0Æ12 syn 0Æ71 add 0Æ35 syn 0Æ47 syn 0Æ35 syn 2,3-DBA 1Æ61 neu 0Æ09 syn 2Æ0 neu 1Æ01 neu 0Æ79 add 0Æ31 syn

*Interactions: syn = synergistic; add = additive; neu = neutral DBA, dihydroxybenzaldehyde. Bold font indicates synergistic (syn) interactions between compounds. starkest was 2,3-DBA, being synergistic with AMB and chemosensitizing agent (Niimi et al. 2004). However, its ITR against the C. neoformans. Alternatively, cinnamic fate and toxicity in an in vivo system have not since been acid, which was additive with all three drugs against the reported. Ca. albicans, was neutral with FLU and synergistic with 2,5-DBA was previously found to synergize a commer- ITR against the C. neoformans. cial agricultural fungicide (Kim et al. 2008b) against In summary, 2,5-DBA had the broadest synergism with Aspergillus fumigatus, an agent of invasive aspergillosis all drugs against both strains, being synergistic with all (Walsh et al. 2008). However, two other benzoic ana- three drugs against C. neoformans, and AMB and ITR logues (cinnamic acid, 2,3-DBA) that enhanced agricul- against Ca. albicans. Thymol also had a synergistic inter- tural fungicides by targeting the response action with one or more of the drugs with both strains. system in that study had only a neutral interaction with Except with salicylic acid, ITR was synergistic with all an antifungal drug in this study. natural compounds with one or the other strain. AMB Thymol, a natural product of the common spice, also showed predominantly favourable interactions, syner- thyme, was used as a traditional, natural antifungal agent gistic with four natural products. FLU, contrastingly, was in Chinese medicine, but has far lower antifungal activity synergistic with only two compounds. than commercially available (Nong et al. 1999). However, our endeavour is not to use natural products directly as antifungal agents, but use them to enhance Discussion activity of available antifungal agents. Synergism by thy- The increase in incidence of candidiases and cryptococcosis mol may result from disruption of the cell wall ⁄ mem- combined with resistance and stagnation in development brane integrity mitogen-activated protein kinase (MAPK) of antifungal agents is a major medical issue. Combination system (Kim et al. 2008a) or by creating lesions in the therapy with available drugs has only achieved moderate plasma membrane in combination with disruption of recognition in view of potential deleterious side effects ergosterol biosynthesis by FLU (Guo et al. 2009). (Mougdal et al. 2005; Clemons et al. 2006). Variability in drug ⁄ natural product interaction can be The initial discovery of natural products as chemosen- affected by type of yeast strain. In our matrix assays, we sitizing agents involved commercial agricultural fungicides used strains that had not been exposed to the drugs, and (Kim et al. 2006, 2007, 2008b). The mode of action of ICs of these strains to the drugs were similarly suscepti- the fungicides was disruption of redox homeostasis. ble. Alternatively, these strains responded to natural prod- Accordingly, the most effective chemosensitizing agents ucts differently. The Ca. albicans strain originated from were found to target the oxidative stress response system. human blood, whereas the C. neoformans was collected Certain phenylpropanoids have been reported to have from fruit juice. Fruit juices contain a number of differ- antifungal activity through induction of oxidative stress ent phenolic compounds (Hernandez et al. 1997), and (Ahmad et al. 2010; Khan et al. 2010). A synthetic pep- MAPK stress response pathways among different strains tide, 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted of yeast can respond quite differently to treatment by d-octapeptide, KN20, that disrupted an ABC-transporter phenolics (Kim et al. 2008a). This may be reflected by the associated with FLU resistance, was reported as a C. neoformans strain having seven neutral phenolic ⁄ drug

No claim to US Government works Letters in Applied Microbiology 52, 506–513 ª 2011 The Society for Applied Microbiology 511 Natural antifungal chemosensitizers N.C.G.Faria et al. interactions, whereas the Ca. albicans had only three, the Clemons, K.V., Espiritu, M., Parmar, R. and Stevens, D.A. remaining being additive or synergistic. Some clinical (2006) Assessment of the paradoxical effect of strains of C. neoformans, from blood or other human tis- in therapy of candidiasis. Antimicrob Agents Chemother 50, sues, respond to chemosensitization by these same pheno- 1293–1297. lics (unpublished results). Clinical and Laboratory Standards Institute (2007) Reference Chemosensitization may also greatly depend upon drug Method for Broth Dilution Antifungal Susceptibility Testing employed. Different activities between phenolic drug of Yeasts; Approved Standard M27-A3. Wayne, PA: CLSI. combinations may depend upon the positions simulta- Cuenca-Estrella, M., Lee-Yang, W., Ciblak, M.A., Arthington- neously occupied by hydroxyl groups on the aromatic Skaggs, B.A., Mellado, E., Warnock, D.W. and Rodriguez- Tudela, J.L. (2002) Comparative evaluation of NCCLS rings of the phenolics. This would affect dihydroqui- M27-A and EUCAST broth microdilution procedures for none ⁄ semiquinone ⁄ quinone redox cycling, resulting in antifungal susceptibility testing of Candida species. Anti- different redox activities (Brunmark and Cadenas 1989). microb Agents Chemother 46, 3644–3647. This redox activity could affect response of the yeast to Denning, D.W., Baily, G.G. and Hood, S.V. (1997) Azole the drug or result in a direct chemical interaction resistance in Candida. Eur J Clin Microbiol Infect Dis 16, between natural compound and drug. 261–280. In conclusion, our biologically based study is a proof- Finney, D.J. (1971) Probit Analysis. Cambridge, UK: Cam- of-concept that natural products can enhance antifungal bridge University Press. drugs. We have screened the clinical collection of Candida Galagiani, J.N. and Stevens, D.A. (1978) Turbidimetric studies and Cryptococcus at the Instituto de Higiene e Medicina of growth inhibition of yeasts with three drugs: inquiry Tropical and have identified a number of drug-resistant into inoculum-dependent susceptibility testing, time of strains. Chemosensitization studies of these strains are onset of drug effect, and implications for current and underway, using clinical laboratory protocols. We have newer methods. Antimicrob Agents Chemother 13, 249–254. identified combinations of natural product and drug that Gue´ho, E., Improvisi, L., Christen, R. and de Hoog, G.S. result in loss of drug resistance in these strains (unpub- (1993) Phylogenetic relationships of Cryptococcus neofor- lished results). Indeed, thymol has been shown to be an mans and some related basidiomycetous yeasts determined effective chemosensitizing agent against FLU-resistant from partial large subunit rRNA sequences. Antonie Van clinical strains of Ca. albicans (Guo et al. 2009). An addi- Leeuwenhoek 63, 175–189. tional effort has been initiated using animal models. Guo, N., Liu, J., Wu, X., Bi, X., Meng, R., Wang, X., Xiang, However, our current in vitro results point to a promising H., Deng, X. et al. (2009) Antifungal activity of thymol new approach of improving effectiveness of antifungal against clinical isolates of fluconazole-sensitive and -resis- drug chemotherapy while lowering dosages, costs and tant Candida albicans. J Med Microbiol 58, 1074–1079. unwanted side effects. Heimark, L., Shipkova, P., Greene, J., Munayyer, H., Yarosh- Tomaine, T., DiDomenico, B., Hare, R. and Pramanik, B.N. (2002) Mechanism of azole antifungal activity as Acknowledgements determined by liquid chromatographic ⁄ mass spectrometric monitoring of ergosterol biosynthesis. J Mass Spectrom 37, This research was conducted under USDA-ARS CRIS 265–269. Project 5325-42000-035-00D with partial support from Hernandez, T., Ausn, N., Bartolome´, B., Bengoechea, L., Estrel- the Thomas J. Walsh Award in Clinical Mycology. la, I. and Go´mez-Cordove´s, C. (1997) Variations in the phenolic composition of fruit juices with different treat- References ments. Z Lebensm-Unters -Forsch, A Food Res Technol 204, 151–155. Ahmad, A., Khan, A., Yousuf, S., Khan, L.A. and Manzoor, N. Holmes, A.R., Lin, Y.H., Niimi, K., Lamping, E., Keniya, M., (2010) Proton translocating ATPase mediated fungicidal Niimi, M., Tanabe, K., Monk, B.C. et al. (2008) ABC activity of and thymol. Fitoterapia 81, 1157–1162. transporter Cdr1p contributes more than Cdr2p does to Barrett, D. (2002) From natural products to clinically useful fluconazole efflux in fluconazole-resistant Candida albicans . Biochim Biophys Acta 1587, 224–233. clinical isolates. Antimicrob Agents Chemother 52, 3851– Brunmark, A. and Cadenas, E. (1989) Redox and addition 3862. chemistry of quinoid compounds and its biological impli- Isenberg, H.D. (1992) Clinical Microbiology Procedures Hand- cations. Free Radic Biol Med 7,7. book. Washington, DC: American Society for Microbiol- Charlier, C., Hart, E., Lefort, A., Ribaud, P., Dromer, F., Den- ogy. ning, D.W. and Lortholary, O. (2006) Fluconazole for the Jacob, M.R. and Walker, L.A. (2005) Natural products and management of : where do we stand antifungal drug discovery. Methods Mol Med 118, 83–109. after 15 years? J Antimicrob Chemother 57, 384–410.

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