Analysis Of Volatile Fingerprints: A Rapid Screening Method For Antifungal Agents For Efficacy Against Dermatophytes

Kamran Naraghi, Natasha Sahgal, Beverley Adriaans, Hugh Barr, and Naresh Magan Citation: AIP Conf. Proc. 1137, 191 (2009); doi: 10.1063/1.3156504 View online: http://dx.doi.org/10.1063/1.3156504 View Table of Contents: http://proceedings.aip.org/dbt/dbt.jsp?KEY=APCPCS&Volume=1137&Issue=1 Published by the American Institute of Physics.

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Analysis Of Volatile Fingerprints: A Rapid Screening Method For Antifungal Agents For Efficacy Against Dermatophytes
Kamran Naraghi, Natasha Sahgal, Beverley Adriaans*, Hugh Barr*, Naresh Magan
Cranfield Health Applied Mycology Group, Vincent building, Cranfield University, Beds. MK43 0AL, * Gloucestershire Hospitals NHS Trust, Great Western Road, Gloucester GL1 3NN, U.K.
Abstract. The potential of using an electronic nose (E.nose) for rapid screening dermatophytes to antifungal agents was studied. In vitro, the 50 and 90% effective concentration (EC) values of five antifungal agents for T.rubrum and T.mentagrophytes were obtained by mycelial growth assays. Then, the qualitative volatile production patterns of the growth responses of these fungi to these values were incorporated into solid medium were analysed after 96-120 hrs incubation at 25C using headspace analyses. Overall, results, using PCA and CA demonstrated that it is possible to differentiate between various treatments within 96-120 hrs. This study showed that potential exists for using qualitative volatile patterns as a rapid screening method for antifungal agents for microorganism. This approach could also facilitate the monitoring of antimicrobial drug activities and infection control programmes and perhaps drug resistance build up in microbial species. Keywords: Volatile fingerprints, anti-fungals, dermatophytes, screening tools

INTRODUCTION
Dermatophytoses are common among skin diseases worldwide. Even in Europe, infections such as tinea capitis are an increasing problem (Hackett et al., 2006). Although, resistance to antibiotics among dermatophyte species is very uncommon, determination of the in vitro susceptibility of dermatophytes, particularly for management of treatment failures, may prove helpful [1]. Many techniques, such as agar-based methods (dilution and diffusion) and broth dilution, have been used for antifungal susceptibility testing (AST) but there is no standard method for AST of dermatophytes [2]. However, dermatophytes grow slowly and thus agarbased methods are time-, labour-, and resourceintensive. There is a need for novel and quick alternative laboratory approaches. All micro-organisms produce by-products as a result of their normal metabolism [3]. Some of these metabolic by-products, including alcohols, aliphatic acids, ketones and terpenes are volatile at low temperature and are known as volatile organic compounds (VOCs). Many VOCs have characteristic

odours. Since the production patterns of VOCs are unique to certain micro-organisms (or disorders), they can potentially be used as biomarkers [4]. Quantitative analysis of VOCs has almost exclusively been based on gas chromatography-mass spectrometry which is relatively expensive, and requires skilled operators. However, rapid qualitative analysis of volatile fingerprints using sensor arrays including electronic nose devices for early detection/discrimination of infections has yielded promising results [5]. The aims of this work were to study the potential of using an E-nose as a rapid screening method for antifungal agents for controlling dermatophytes using the drugs and antioxidants by using volatile fingerprints. The objectives were thus to (a) identify the growth responses of two Trichophyton species, T. rubrum and T. mentagrophytes, to 50% and 90% effective concentrations values (EC50 and EC90 values) of five antifungal agents (itraconazole, griseofulvin, butylated hydroxyanisole, octyl gallate and n-propyl-phydroxybenzoate) and (b) use of an E.nose for discrimination between treatments for these fungal species.

CP1137, Olfaction and Electronic Nose: Proceedings of the 13th International Symposium, edited by M. Pardo and G. Sberveglieri C 2009 American Institute of Physics 978-0-7354-0674-2/09/S25.00

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EXPERIMENTAL AND METHODS

Inocula and growth medium Two Trichophyton species, T. rubrum (No. 115) and T. mentagrophytes (No. 224), were used in this study. These were human isolates and obtained from National Collection of Pathogenic Fungi (NCPF), Bristol, UK. Sabouraud dextrose agar (SDA) was prepared with 10 g l-1 mycological peptone (Oxoid, UK), 40 g l-1 glucose (Acros Chemicals, Belgium), and 15 g l-1 agar (No.3, Oxoid, UK). At the end, 0.05 g l-1 chloramphenicol (Sigma, UK) was added. Antifungal agents Two antifungal drugs, itraconazole (Janssen Pharmaceuticals, Belgium) and griseofulvin (Darou Pakhsh Co., Iran), and three antioxidants including butylated hydroxyanisole (BHA; C11H16O2) (Sigma, US), octyl gallate (O-G; C15H22O5) (Fluka Chemie GmbH, Germany) and n-propyl-p- hydroxybenzoate (P-P; C10H12O3) (Sigma, US) were used in this study. All antioxidants are Generally Recognized As Safe (GRAS) compounds. For each drug, a stock solution with the concentration of 100 g ml-1 was prepared by dissolving 2mg of anti-fungal agent in 20 ml of 99.5% dimethyl sulfoxide (DMSO) (Sigma, US). Stock antioxidant solutions (1 mM) in 20 ml of 99% ethanol was made. These were stored at 4C. E.nose system An AppliedSensor 3320 E.nose (AppliedSensor Group, Sweden) was employed in this study. The core sensor technology is based on a hybrid array of 10 metal-oxide-silicon field-effect-transistor (MOSFET) sensors and 12 metal oxide sensors (MOS), and one humidity sensor. Test procedure Stock solutions were added to molten SDA (itraconazole, griseofulvin; 0.25, 0.50, 1.00 and 2.00 g ml-1). SDA was also amended with two antioxidants combinations (BHA + P-P, O-G + P-P) to obtain 20 and 40 mM. Triplicates of each treatment and each species were inoculated with a 0.5 mm diameter agar plug taken from growing cultures of T.rubrum and T.mentagrophytes on SDA. Plates were incubated at 25C in dark and mycelial extension diameters measured daily for 14 days. However, no mycelial extension was detected on griseofulvin and antioxidants-amended plates after 14 days. Therefore, the test was repeated at 0.025-0.200 g ml-1

griseofulvin. The radial extension rates were plotted against time. The EC50 and EC90 values of all antifungal agents against T.rubrum and T. mentagrophytes were calculated by linear regression of the temporal extension rates and then plotting the relative growth rates (mm day-1) to compare treatments. For each treatment molten SDA media were amended with the calculated EC50 and EC90 values per species and poured in 9 cm plastic Petri plates. At least 30 replicate Petri plates of plain and amended SDA were inoculated with 0.25 ml of a 10 6 spore mL-1 suspension of each species, and 10 replicate blank SDA plates used as negative controls. They were incubated at 25C for 96-120 hours. At each time point, four 2 cm diameter agar plugs from 5 replicate plates of each treatment were destructively sampled using a cork borer and placed in sample vials which were sealed with a septum and lid. After 1 hr equilibration, the headspaces were analysed by the 3320 E-nose. The 96-120 hours period represents the early stages of microscopic and visible growth, when identification is difficult. Data analysis Data were analysed using NSTSenstool (software package in the AppliedSensor 3320) to perform principal components analysis (PCA) on response parameters (mean-centred data) using the maximum peak response for the various sensors. These data were analysed by cluster analysis (CA) using Statistica 8.

RESULTS
Effective concentration values Growth rates relative to the controls (data not shown) were used to determine the EC50 and EC90 values for antifungal agent concentrations. For all treatments the EC90 values were the highest concentrations used. Early differentiation between inoculated antifungal treatments and controls The main purpose of this research work was to rapidly (after 96-120 hours) study the growth responses of T. rubrum and T. mentagrophytes to EC50 and EC90 of the antifungal agents by analysing the volatile fingerprints. There was very good reproducibility of the response of 10 MOSFET sensors to replicates of T. mentagrophytes grown on SDA.

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Figure 1 shows the PCA of the response of the hybrid sensor E-nose to treatments of inoculated amended SDA with EC values of itraconazole and antifungal-free SDA (as positive controls) together with blank SDA (as a negative control) and uninoculated amended SDA at 2.00 g ml-1 of itraconazole after 96 hours. There was clear separation between inoculated and un-inoculated itraconazole treatments, and negative controls implying that the Trichophyton species on amended SDA plates, as on the itraconazole treatments there was no growth after 96 hours. This accounted for 97.6% of the data described by the first two principal components (PC). After 120 hours growth there was also distinct clusters of the two dermatophytes growing on un-amended SDA clearly differentiated from other treatments (Figure 2). Effect of anti-oxidant treatments Analysis of the data related to the response of the E.nose sensors to different BHA + P-P treatments by PCA after 96 hours showed three clusters of T. rubrum and T. mentagrophytes growing on SDA, and T. mentagrophytes growing on amended SDA at the EC50 concentration of the antioxidants combination distinguished from other treatments which grouped together. This accounted for 94.9% of the data described by PCs 1, 2 and 9 (Figure 3). However, positive controls and replicates of T. mentagrophytes growing on SDA at EC50 of BHA + P-P were clearly differentiated from negative controls, and samples of un-inoculated SDA amended with antioxidants and inoculated SDA at EC90 of BHA + P-P which formed a mixed group. After 120 hours, examination of the PCA map showed four groups of dermatopyte species growing on SDA and that amended with the EC50 concentration of BHA+P-P per species, clearly separated from other treatments (Figure 4).

FIGURE 1. PCA score plot differentiating Trichophyton species grown on plain SDA and itraconazole treatments after 96 hours.(Key: B,blank SDA; ITZ, SDA amended at 2 g ml-1 itraconazole; M, T. mentagrophytes; M50, T.mentagrophytes and EC50 itraconazole; M90, T.mentagrophytes and EC90 itraconazole; R, T.rubrum; R50, T.rubrum and EC50 itraconazole; R90, T. rubrum and EC90 itraconazole).

DISCUSSION
This is the first study to examine the potential of using an E.nose for qualitative screening of the responses of dermatophytes to antifungal agents. It was possible to discriminate between T.rubrum and T. mentagrophytes growing on un-amended solid media (SDA) from those inoculated on antifungal-modified media as early as 96 hrs after incubation by analysing their volatile production patterns. In the presence of itraconazole, the species grown on control media and those amended with the EC50/EC90 values were successfully differentiated after 96-120 hrs. This supports previous studies with four Trichophyton species which showed differentiation Figure 2. PCA score plot (120 hrs) separating positive controls and from itraconazole and blank SDA treatments. (Key: B, blank SDA; ITZ, SDA amended at 2 g ml-1 itraconazole; M, T.mentagrophytes; M50, T.mentagrophytes/EC50/itraconazole;M90,T.mentagroy tes/EC90/itraconazole;R,T.rubrum;R50,T.rubrum/EC50/ itraconazole;R90,T.rubrum/EC90/itraconazole). within 96 hours using qualitative volatile fingerprints [5]. The results also revealed a similarity in the volatile production patterns of controls and fungi inoculated on antifungal-modified media where growth was inhibited

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growing on antifungal-free media and those modified with the EC50 values. The clear separation of negative controls and inoculated treatments at EC90 values from others within 96-120 hours indicated inhibition of growth similar to existing drugs (itraconazole, griseofulvin). The E.nose was able to differentiate between closely related treatments, notably those growing on controls and amended media at the EC50 value of the combination per species. Previous studies showed that the nature of the culture media influences the production of volatiles by micro-organisms [4]. In contrast, Sahgal et al. [5] found that good discrimination by analysing the volatile fingerprints of four Trichophyton species growing on various solid media, regardless of the medium used, required at least 96 hours. Figure 3. Non-linear PCA score plot (96 hrs) showing three clusters of Trichophyton species on amended SDA + antioxidant combination of BHA+P-P. (Key: B, blank SDA; BP, un-inoculated SDA at 0.250 mM BHA+PP;M,T.mentagrophytes;M50,T.mentagrophytes /EC50 BHA+P-P; M90, T.mentagrophytes/EC90 of BHA+P-P;R,T.rubrum;R50,T.rubrum/EC50of BHA+PP; R90, T.rubrum/EC90 of BHA+P-P).

CONCLUSIONS
Most susceptibility testing of dermatophytes use two protocols (1, 2) which are time-consuming. In contrast, detection of VOCs by sensor arrays has many advantages. It is non-invasive, sensitive, and relatively inexpensive. This study showed the potential for rapid screening of novel antifungal compounds using volatile fingerprints and monitoring the build up of resistance to anti-microbial drugs. By examining the PCA maps it would be possible to identify when poor control is being achieved by having both positive and negative controls on a regular basis for comparison.

ACKNOWLEDGMENTS
We are grateful to Gloucestershire NHS Foundation Trust for financial support to Dr. Naraghi.

Figure 4. PCA score plot (120 hrs). Clusters of T. rubrum and T. mentagrophytes on various treatments. (Key: B, blank SDA; BP, un-inoculated SDA at 0.250 mM/BHA+PP;M,T.mentagrophytes;M50,T.mentagrop hytes/EC50 BHA+P-P; M90, T.mentagrophytes/EC90 BHA+P-P;R, T.rubrum; R50, T.rubrum/EC50 BHA+PP;R90,T.rubrum/EC90/BHA+P-P). by the drugs within the same time. The results of the griseofulvin test suggest that culture age may affect the discrimination achieved. Differences were not clear after 96 hrs, but 120 hrs gave differentiation between EC90 treatments of griseofulvin and negative controls. . Alternative antioxidants for dermatophytes control have not been studied. Working with a combination of BHA+P-P, the E.nose could successfully discriminate between 3-4 treatments of Trichophyton species

REFERENCES
1. Z.Cetinkaya, N.Kiraz, S.Karaca et al., European Journal of Dermatology 15, 258-261 (2005). 2. W.G.Merz, and R.J. Hay. Medical Mycology. In: Topley & Wiilsons Microbiology and microbial infections, 10 th ed. London: Hodder Arnold Ltd (2005). 3. J.M.Scotter, V.S.Langford, P.F.Wilson et al.,Journal of Microbiological Methods 63, 127-134 (2005). 4. A.P.F.Turner and N. Magan, Nature Reviews. Microbiology 2, 161-166 (2004). 5. N.Sahgal, B.Monk, M.Wasil et al., British Journal of Dermatology 155, 1209-1216 (2006).

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