Antimicrobial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay

International Journal of Pharmaceutics 436 (2012) 851–856

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Antimicrobial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay Susanne Finger a,∗ , Cornelia Wiegand a , Hans-Jürgen Buschmann b , Uta-Christina Hipler a a b

Department of Dermatology, University Medical Center Jena, Germany Deutsches Textilforschungszentrum Nord-West e.V. Krefeld, Germany

a r t i c l e

i n f o

Article history: Received 16 April 2012 Received in revised form 3 July 2012 Accepted 9 July 2012 Available online 31 July 2012 Keywords: Antimycotic Bioluminescence Candida albicans Cyclodextrin-complex Laser nephelometry Malassezia pachydermatis

a b s t r a c t Antimicrobial effects of substances can be determined with different methods that measure distinct parameters. Thus, a comparison of the results obtained can be difficult. In this study, two in vitro methods were employed to determine concentration and time dependent effects of cyclodextrin (CD)-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) on Candida albicans and Malassezia pachydermatis. Using both, microplate laser nephelometry and the ATP bioluminescence assay, it could be shown that CD–antiseptics-complexes tested exhibited significant antifungal effects with the exception of ␥–CD–CHX in the case of C. albicans. Microplate laser nephelometry (MLN) is an optical method and enables a quantitative determination of particle concentrations in solution. By means of this method, microbial growth under influence of potential antimicrobial substances can be monitored over a prolonged time period. In addition, the antimicrobial activity was analyzed by measurement of the microbial adenosine triphosphate (ATP) content with a bioluminescent assay. The luminescent signal is directly proportional to the amount of ATP, and thus, a linear function of the number of living microbial cells present. Both methods were compared according to the half maximal inhibitory concentration (IC50 ) calculated and the statistical evaluation of Pearson’s correlation coefficient (r). In summary, it could be demonstrated that both methods yield similar results although they differ in the parameter. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Microbial growth can be observed over a prolonged time period with the help of photometric instruments. This technique measures the light, which is transmitted through a suspension of particles. Hence, turbidimetry requires relatively high concentrations of particles due to the effects of reflection and absorption. In contrast, laser nephelometry is a direct method for measuring light scattered by particles suspended in solution at right angles up to 80◦ . Already low concentrations of particles in a suspension, as they are found at the beginning of microbial growth, can be measured using this technique. Thus, this method yields a higher sensitivity than regular transmission readers, which detect the reduction of the intensity of light passing through a suspension (Hipler et al., 2003). It is also a highly reliable technique for the application in 96-well plate format (Bevan and Lloyd, 2000; Fouda et al., 2006). The light source used is a red laser diode (633 nm). The intensity of the scattered light is

∗ Corresponding author at: Department of Dermatology, University Medical Center Jena, Erfurter Straße 35, 07743 Jena, Germany. Tel.: +49 3641 937331; fax: +49 3641 37437. E-mail address: [email protected] (S. Finger). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.07.009

directly proportional to the particle concentration (e.g. yeast) in a suspension for up to three orders of magnitude (Bevan and Lloyd, 2000; Hipler et al., 2003). Fouda et al. (2006) and Joubert et al. (2010) described laser nephelometry as an efficient technique for monitoring the growth of microorganisms like fungi and state its applicability for the evaluation of antifungal activity. Another approach for determining the reaction of microorganisms to substances is the quantification by measurement of the cellular adenosine triphosphate (ATP) content. ATP is found in all living and metabolic active cells. Thus, it can be used to determine the amount of viable microbial cells present. ATP can be quantified using a bioluminescence assay comprised of the enzyme luciferase from Photinus pyralis, which has got a high sensitivity to ATP, and d-luciferin, the enzyme’s substrate. Luciferin is converted into oxyluciferin in an ATP-, Mg2+ - and oxygen dependent reaction which generates yellow-green light (Marques and Esteves da Silva, 2009; Nazari and Hosseinkhani, 2011). The amount of emitted light is directly proportional to the ATP content (Han et al., 2011; Marques and Esteves da Silva, 2009), and hence, a linear function of the number of living cells in the suspension. Moreover, like microplate laser nephelometry, the ATP bioluminescence assay is applicable for high-throughput screening.

852

S. Finger et al. / International Journal of Pharmaceutics 436 (2012) 851–856

Both methods were used to analyze concentration and time dependent effects of Cyclodextrin–antiseptics-complexes on Candida albicans and Malassezia pachydermatis. C. albicans is a prominent facultative human pathogen, which can be found in healthy humans as a commensal of the gastrointestinal tract without harmful effects (Odds, 1987). Systemic fungal infections have emerged in immune-compromised patients (AIDS, cancer chemotherapy, organ or bone marrow transplantation) as important cause of morbidity and mortality. Its pathogenicity is due to the ability to grow as yeast cell or as hyphae (Hashash et al., 2011; Odds, 1994) and can be modulated by the adhesion molecules on the cell surface (McLain et al., 2000; Odds, 1994). M. pachydermatis can be found in patients with granulomatus skin infection (Fan et al., 2006), pityriasis versicolor (Rasi et al., 2010), seborrhoeic dermatitis (Prohic, 2010) and has also been isolated from patients who received parenteral nutrition (Cannizzo et al., 2007). Even isolations of M. pachydermatis from neonates have been reported (Chryssanthou et al., 2001; Guého et al., 1987; Welbel et al., 1994). To battle these fungal infections substances are needed which completely inhibit microbial growth. Antiseptics, that are also used to prevent and cure bacterial infections, may be a promising alternative to widespread use of antimycotica like fluconazole, itraconazole or ketoconazole. For antimicrobial substances such as chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) the packaging into cyclodextrins could achieve a better skin compatibility, higher antimicrobial activity, and increased storage stability (Fouda et al., 2006). Cyclodextrins (CD) are ring-shaped degradation products of starch. The most important CDs are composed of 6, 7, or 8 glucose molecules and are named ␣-CD, ␤-CD, and ␥-CD (Szejtli, 1998). They are able to form inclusion complexes with other molecules such as antiseptics (Stella and He, 2008; Szejtli, 1998). In the present study, the antimicrobial activity of ␣-, ␤-, and ␥CD-complexes with CHX, IOD and PHMB has been studied using both, microplate laser nephelometry and ATP bioluminescence assay. Subsequently, both methods were compared according to the results. 2. Materials and methods 2.1. Materials The following antiseptics have been used in this study: polihexanide (20% polyhexamethylene biguanide; Fagron, Barsbüttel, Germany), chlorhexidine diacetate (Fagron, Barsbüttel, Germany) and iodine (Fagron, Barsbüttel, Germany). The Cyclodextrin–antiseptics-complexes were provided by Deutsches Textilforschungszentrum Nord-West e.V. Krefeld, Germany. Stock solutions were performed in sterile water. All solutions were sterilized by pass through a 0.2 ␮m filter (Sartorius Stedim Biotech, Göttingen, Germany). Candida albicans DSM 1386 and Malassezia pachydermatis DSM 6172 were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). For cultivation of yeast, Sabouraud dextrose medium was obtained from Oxoid (Basingstoke, England). Sabouraud dextrose agar plates were purchased from bioMérieux (Nürtingen, Germany). 2.2. Elementary analysis An elementary analysis of the CD-complexes was carried out by SEWA GmbH (Laborbetriebsgesellschaft; Essen, Germany). The analysis of CD–CHX-complexes occurred in solid by total content of chlorine (in %) according to DIN EN 24260. The content of solid iodine (in %) of CD–IOD-complexes was analyzed according to DIN

EN 24260. The determination of completely bounded nitrogen (in g mL−1 ) of CD–PHMB-complexes occurred according to DIN 38409 H27. 2.3. Preparation of yeast suspensions C. albicans DSM 1386 and M. pachydermatis DSM 6172 were inoculated on Sabouraud dextrose agar plates. Candida yeasts were grown overnight at 37 ◦ C. M. pachydermatis was incubated at 30 ◦ C for 48 h. An inoculation loop with cultural material of each yeast was suspended in Sabouraud dextrose medium and shaken at 37 ◦ C or 30 ◦ C for 24 h or 48 h. The overnight cultures of C. albicans were then counted using CASY® 1 Cell Counter (Schärfe System, Reutlingen, Germany) and adjusted to a final inoculum size of 5–7 × 103 cells mL−1 . M. pachydermatis suspensions were diluted to comply with McFarland standard 0.1 (bioMérieux, Nürtingen, Germany) (∼5–6 × 104 cells mL−1 ). 2.4. Microplate laser nephelometry (MLN) Yeast growth under influence of CD–antiseptics-complexes was monitored using the NEPHELOstar Galaxy (BMG LABTECH, Offenburg, Germany). Serial dilutions of the test substances were prepared in Sabouraud dextrose medium. 100 ␮L each were put in triplicate into the respective wells of a sterile, clear 96-well microplate (GreinerBioOne, Frickenhausen, Germany). Blanks for each substance concentration tested were run at every assay. 100 ␮L of the corresponding yeast suspension were put in the respective wells of the 96-well microplate containing the prepared antiseptics’ dilutions. Microplates were covered with a clear adhesive film (GreinerBioOne, Frickenhausen, Germany). The adhesive film was punctured with a 25-gauge needle at the right brim of the well to allow gas exchange. Microplates were then placed in the microplate lasernephelometer (NEPHELOstar Galaxy, BMG LABTECH, Offenburg, Germany) and incubated for 24 h and 48 h, respectively, at 37 ◦ C or 30 ◦ C. During incubation, microplates were shaken in the instrument except for the duration of the hourly measurement. Parameter settings for the nephelometer have been described previously (Seyfarth et al., 2008). The half maximal inhibitory concentrations (IC50 ) of the substances under the experimental conditions used were calculated from the growth curves. The ‘area under the curve’ was determined from the results for each antiseptic concentration tested and calculated as percentage of the untreated control. This was used to realize a dose–response curve for each antiseptic tested from which the IC50 was calculated using following logistic fit function (A1: upper limit, A2: lower limit, x0 : IC50 , p: slope of the curve; Origin® 7.5, OriginLab; Northhampton, U.S.). y=

A2 + (A1 − A2) 1 + (x/x0 )

p

2.5. ATP bioluminescence assay The effect of the test substances on microbial viability was determined by measurement of the cellular ATP content using the BacTiter-GloTM Assay (Promega, Mannheim, Germany), which is based on the detection of light generated by the ATP dependent enzymatic conversion of D-luciferin to oxyluciferin by firefly luciferase. After MLN measurements, microplates were equilibrated at room temperature under agitation. 10 ␮L of the contents of each well were transferred into a new white 96-well microplate (NUNC MaxiSorpTM ; Thermo Fisher Scientific, Langenselbold, Germany) and mixed with 90 ␮L Saubouraud dextrose medium. Afterwards, 100 ␮L BacTiter-GloTM Reagent was added to each well.

S. Finger et al. / International Journal of Pharmaceutics 436 (2012) 851–856

The microplates were further incubated under agitation for 7 min (M. pachydermatis) and 15 min (C. albicans), respectively. Thereafter, luminescence was recorded using the LUMIstar Galaxy (BMG LABTECH, Offenburg, Germany). The ATP concentration of viable cells was calculated using an ATP standard curve.

853

C. albicans, where distinctly higher concentrations were needed to inhibit yeast growth (Fig. 2e and f). Additionally, high Pearson’s correlation coefficients (r > 0.650) in case of M. pachydermatis and the CD–PHMB-complexes were calculated, indicating a very good comparability between results from MLN and ATP bioluminescence assay (Table 3).

2.6. Statistics Experiments were performed in triplicate and each sample was measured in six replicates. Data are presented as mean ± standard deviation. For statistical evaluation Pearson’s correlation coefficient (r) was determined with SPSS 18 (IBM, Munich, Germany). Pearson’s correlation coefficients r ≥ 0.5 were chosen for a good correlation between both methods. These values exhibit a distinct linear correlation. Statistically significant results are indicated as follows: [*] p ≤ 0.01 or [**] p ≤ 0.05. 3. Results 3.1. CD-complexes with chlorhexidine (CHX) All CD–CHX-complexes exhibited a significant antimycotic activity against M. pachydermatis in vitro (Figs. 1 and 2b). Fig. 1 shows the effect of ␤–CD–CHX on growth of M. pachydermatis. For both methods, MLN and ATP bioluminescence assay, a decrease of growth was observed at a concentration of 1000 ␮g mL−1 and higher. To analyze the relation of the results from both methods, the Pearson’s correlation coefficient was calculated showing a highly significant correlation (r = 0.939) between the results from both methods. Moreover, results of MLN and ATP bioluminescence assay indicated a distinct antimycotic effect of the tested ␣- and ␤CD–CHX-complexes against C. albicans (Fig. 2a). Merely, ␥-CD–CHX had no effect on C. albicans growth. ␣-CD–CHX showed the strongest activity against both yeast strains compared to the complexes with ␤- or ␥-CD, although, higher concentrations were needed to influence C. albicans in contrast to M. pachydermatis. Thus, the IC50 for M. pachydermatis was found to be app. 200 ␮g mL−1 while C. albicans exhibited an IC50 that was 10 times higher (Table 1). 3.2. CD-complexes with iodine (IOD) CD-complexes with IOD had significant influence on the growth of C. albicans and M. pachydermatis (Fig. 2c and d). Again, the ␣–CD-complex exhibited the highest antimycotic activity (Table 2). However, higher amounts of ␣-CD–IOD were needed to inhibit the growth of M. pachydermatis (MLN: 695 ± 46 ␮g mL−1 ; ATPba: 723 ± 38 ␮g mL−1 ) compared to C. albicans (MLN: 437 ± 29 ␮g mL−1 ; ATP-ba: 498 ± 10 ␮g mL−1 ). Furthermore, it could be shown that ␤- and ␥-CD–IOD-complexes act in higher concentration fungicidal. Thus, the IC50 for M. pachydermatis was twofold (␤CD–IOD) or 10 times (␥-CD–IOD) higher than for ␣-CD–IOD. While, for C. albicans 4 times (␤-CD–IOD) or nearly 30 times (␥-CD–IOD) higher concentrations were needed to inhibit 50% of fungal growth. 3.3. CD-complexes with polihexanide (PHMB) CD–PHMB-complexes showed the highest antimycotic activity of the substances tested (Table 3). Efficacy was found to increase from ␣- to ␥-CD–PHMB-complexes. For example, app. 5 ␮g mL−1 ␣-CD–PHMB and 1 ␮g mL−1 ␥-CD–PHMB inhibited 50% of M. pachydermatis growth. In case of C. albicans the IC50 for ␣CD–PHMB was about 600 ␮g mL−1 and for ␥–CD–PHMB 30 ␮g mL−1 . Furthermore, M. pachydermatis exhibited a significantly higher sensitivity towards the CD–PHMB-complexes compared to

4. Discussion Complexes of ␣-, ␤- and ␥-CD with antiseptics such as CHX, IOD and PHMB were analyzed in respect to antifungal effects on C. albicans and M. pachydermatis employing two different in vitro methods, microplate laser nephelometry (MLN) and luminometric measurement of the microbial ATP content (ATP bioluminescence assay). Although both methods measure different parameters, it could be shown that they yield highly comparable results (Pearson’s correlation coefficient r ≥ 0.5). MLN continuously measures the growth of microorganisms in solution by recording the solution’s turbidity, whereas, the ATP bioluminescence assay is an end-point-measurement based on metabolic processes in viable cells. However, the ability of yeasts like C. albicans to grow in the form of hyphae may present a problem in the MLN. Mostly, lower IC50 concentrations were calculated from the MLN measurements compared to the ATP bioluminescence assay. Only CD–PHMBcomplexes yielded higher IC50 values in the MLN. PHMB and CHX are both biguanides that interact with negatively charged acidic phospholipids in microbial membranes resulting in higher permeability and disruption of these (Ikeda et al., 1984). Koburger et al. (2007) described that PHMB is more effective than CHX due to the structural differences between PHMB and CHX. It can be presumed that this fact would also apply to the CD-complexes with PHMB and CHX. In accordance, a higher antifungal activity of CD–PHMBcomplexes was observed in contrast to CD–CHX-complexes. In addition, lower amounts of CD–PHMB-complexes were able to inhibit yeast growth compared to CD-complexes with IOD. Iodine has a high antimicrobial potential. This halogen is able to denaturise proteins in different ways, e.g. it interacts with S H bonds and N H groups in amino acids leading to dysfunction of enzymes and structural proteins. This may cause changes in the cell wall, the membrane, and the cytoplasm determining the death of the cell (Cooper, 2007). The yeast cell wall is the first barrier and contact point for external factors. One of its main functions is to protect against physical stress or chemical substances. Electron microscope images have shown that it is build of at least two or more layers (Northcote and Horne, 1952) containing polysaccharides, proteins, lipids, and other substances (Gander, 1974). Inner layers are enriched for chitin and polysaccharide matrices while outer layers consist mainly of mannoproteins. Antimycotic active substances, such as CHX, PHMB or IOD, can lead to changes in the cell wall and lead to damage of the barrier function. A higher tolerance of C. albicans in comparison to M. pachydermatis against the CD–antiseptics–complexes was observed for all CD–CHX- and CD–PHMB-complexes and also for ␤and ␥-CD–IOD. Only for ␣-CD–IOD lower inhibitory concentrations for C. albicans (IC50 C. albicans: 400–500 ␮g mL−1 ; IC50 M. pachydermatis: 700 ␮g mL−1 ) were determined. It was shown that C. albicans and M. pachydermatis differ in the number of layers of the cell wall. The cell wall of C. albicans is composed of 4–8 layers (Poulain et al., 1978; Ruiz-Herrera et al., 2006), while for M. pachydermatis only 2–3 layers have been described (Abou-Gabal et al., 1979; Guillot and Bond, 1999). Moreover, C. albicans and M. pachydermatis differ in the cell size. Thus, C. albicans is with 3–8 ␮m × 2–7 ␮m (De Hoog, 2000) bigger than M. pachydermatis with 2–3 ␮m × 4–5 ␮m (Guillot and Bond, 1999). Cell size and number of cell wall layers

854

S. Finger et al. / International Journal of Pharmaceutics 436 (2012) 851–856

a

8000

0 µg/mL 200 µg/mL

turbidity in RNU

7000 6000

300 µg/mL 400 µg/mL

5000

500 µg/mL 1000 µg/mL 2000 µg/mL

4000 3000 2000 1000 0 4

0

8

12

16

20

24

28

32

36

40

44

48

time in h

b

c

800

12000

turbidity in RNU

ATP in nM

10000

600

400

8000 6000 4000

200

2000 0 0

200

300

400

500

µg mL

1000

0

2000

-1

0

100000

200000

300000

400000

500000

600000

luminescence in RLU Fig. 1. Effect of ␤-CD-CHX on the growth of M. pachydermatis. Growth curves determined by microplate laser nephelometry (MLN) over 48 h (a), end-point-measurement of the amount of viable yeast cells after 48 h by ATP bioluminescence assay (ATP-ba; b), and correlation between the results obtained for MLN and ATP-ba (c).

Table 1 Summary of the IC50 values (in ␮g mL−1 ) and the Pearson’s correlation coefficients (r) for CD–CHX-complexes. C. albicans

MLN ATP-ba r

M. pachydermatis

␣–CD–CHX

␤–CD–CHX

␥–CD–CHX

␣–CD–CHX

␤–CD–CHX

␥–CD–CHX

2121 ± 177 2351 ± 141 0.762*

5653 ± 285 6865 ± 518 0.664*

/ / /

205 ± 10 211 ± 14 0.893*

817 ± 112 727 ± 185 0.939*

1035 ± 12 1094 ± 43 0.826*

Table 2 Summary of the IC50 values (in ␮g mL−1 ) and the Pearson’s correlation coefficients (r) for CD–IOD-complexes. C. albicans

MLN ATP-ba r

M. pachydermatis

␣-CD–IOD

␤-CD–IOD

␥-CD–IOD

␣-CD–IOD

␤-CD–IOD

␥-CD–IOD

437 ± 29 498 ± 10 0.603*

1739 ± 167 2735 ± 400 0.246*

11,860 ± 1108 21,085 ± 1504 0.545*

695 ± 46 723 ± 38 723 ± 38

1565 ± 30 1822 ± 101 0.572*

6670 ± 783 6959 ± 15 0.579*

Table 3 Summary of the IC50 values (in ␮g mL−1 ) and the Pearson’s correlation coefficients (r) for CD–PHMB-complexes. C. albicans

MLN ATP-ba r

M. pachydermatis

␣-CD–PHMB

␤-CD–PHMB

␥-CD–PHMB

␣-CD–PHMB

␤-CD–PHMB

␥-CD–PHMB

619 ± 123 198 ± 41 0.562*

244 ± 65 132 ± 15 0.747*

27.4 ± 3.7 21.4 ± 0.9 0.453**

4.99 ± 0.21 3.42 ± 0.38 0.741*

4.22 ± 2.76 4.94 ± 1.73 0.652*

0.95 ± 0.10 1.45 ± 0.01 0.773*

S. Finger et al. / International Journal of Pharmaceutics 436 (2012) 851–856

855

Fig. 2. Comparison of the IC50 values for CD-complexes with chlorhexidine (a and b), iodine (c and d), and polihexanide (e and f) against C. albicans (on the left side, graphs a, c, and e) and M. pachydermatis (on the right side, graphs b, d, and f) by means of nephelometric (MLN) and luminometric assay (ATP-ba).

might play a role in resisting antimycotic substances. Therefore, C. albicans may be able to sustain higher concentrations of the tested CD–antiseptic-complexes than M. pachydermatis. Moreover, the antifungal activity of CD–PHMB-complexes was found to increase from ␣- to ␥-CD. In contrast, CD-complexes with CHX and IOD showed a decrease in the antifungal efficacy in this range. CDs are able to form inclusion complexes with guest molecules. This depends on the relative size of the CD to the size of the guest molecule. Hence, ␣-CD can complex low molecular weight molecules while ␥-CD can accommodate larger molecules (Hipler et al., 2007; Szejtli, 1982). According to this, it was assumed that small molecules like CHX and IOD form a better complex

with ␣-CD, whereas, ␥-CD shows the highest binding capacity for large molecules like PHMB. This reactivity was observed for all CDcomplexes tested. Thus, for ␣–CD–CHX and ␣-CD–IOD the lowest IC50 values in comparison to the ␤- and ␥-CD-complexes were determined. In contrast, ␥-CD–PHMB inhibited yeast growth at lower concentrations compared to the other CD–PHMB-complexes. The results of the elementary analysis confirm these findings. The analysis showed, e.g. for ␣-CD–IOD, 0.2 mol IOD per 1 mol ␣-CD, while ␥-CD–IOD showed 0.006 mol IOD per 1 mol ␥-CD. In case of CD-complexes with PHMB a ratio of 0.04 mol PHMB per 1 mol ␣-CD and 2.21 mol PHMB per 1 mol ␥-CD were determined.

856

S. Finger et al. / International Journal of Pharmaceutics 436 (2012) 851–856

Furthermore, the CDs alone showed no antifungal effects against C. albicans and M. pachydermatis (data not shown). Thus, the inhibitory effects of the complexes can fully be attributed to the respective antiseptic substance. CDs can be seen as a carrier for the antiseptics. The antiseptics are not bound covalently or very tightly to the CDs. Release curves showed that the CDs released the antiseptics completely into liquid (data not shown). Therefore, the interaction between CD and antiseptic is a guest–host-relationship. 5. Conclusion The aim of this study was to compare the results of two different in vitro methods – microplate laser nephelometry (MLN) and ATP bioluminescence assay. The methods were employed to determine concentration and time dependent effects of CD-complexes with the antiseptics CHX, IOD and PHMB on C. albicans and M. pachydermatis. After all, using MLN and ATP bioluminescence assay it could be demonstrated that ␣–CD–CHX- and –IOD-complexes as well as ␥-CD–PHMB-complexes exhibit a significant antifungal effect against C. albicans and M. pachydermatis. Moreover, the study presented showed that MLN and luminometric measurement of the microbial ATP content (ATP bioluminescence assay) yield highly comparable results although both methods measure different parameters. Acknowledgments The research project (IGF-Nr. 15997BG/3) of the Forschungskuratorium Textil e.V. was funded by the Arbeitsgemeinschaft industrieller Forschungsvereinigungen e.V. (AiF) within the program Industrielle Gemeinschaftsforschung und -entwicklung (IGF) by the Bundesministerium für Wirtschaft und Technologie based on a decision of the Deutsche Bundestag. References Abou-Gabal, M., Chastain, C.B., Hogle, R.M., 1979. Pityrosporum pachydermatis “canis” as a major cause of otitis externa in dogs. Mykosen 22, 192–199. Bevan, C.D., Lloyd, R.S., 2000. A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates. Anal. Chem. 72, 1781–1787. ˜ Cannizzo, F.T., Eraso, E., Ezkurra, P.A., Villar-Vidal, M., Bollo, E., Castellá, G., Cabanes, F.J., Vidotto, V., Quindós, G., 2007. Biofilm development by clinical isolates of Malassezia pachydermatis. Med. Mycol. 45, 357–361. Chryssanthou, E., Broberger, U., Petrini, B., 2001. Malassezia pachydermatis fungaemia in a neonatal intensive care unit. Acta Paediatr. 90, 323–327. Cooper, R.A., 2007. Iodine revisited. Int. Wound J. 4, 124–137. De Hoog, G.S., 2000. Atlas of Clinical Fungi. Centraalbureau voor Schimmelcultures. Fan, Y.M., Huang, W.M., Li, S.F., Wu, G.F., Lai, K., Chen, R.Y., 2006. Granulomatous skin infection caused by Malassezia pachydermatis in a dog owner. Arch. Dermatol. 142, 1181–1184.

Fouda, M.M., Knittel, D., Hipler, U.C., Elsner, P., Schollmeyer, E., 2006. Antimycotic influence of beta-cyclodextrin complexes—in vitro measurements using laser nephelometry in microtiter plates. Int. J. Pharm. 311, 113–121. Gander, J.E., 1974. Fungal cell wall glycoproteins and peptido-polysaccharides. Annu. Rev. Microbiol. 28, 103–119. Guého, E., Simmons, R.B., Pruitt, W.R., Meyer, S.A., Ahearn, D.G., 1987. Association of Malassezia pachydermatis with systemic infections of humans. J. Clin. Microbiol. 25, 1789–1790. Guillot, J., Bond, R., 1999. Malassezia pachydermatis: a review. Med. Mycol. 37, 295–306. Han, T., Nazarenko, Y., Lioy, P.J., Mainelis, G., 2011. Collection efficiencies of an electrostatic sampler with superhydrophobic surface for fungal bioaerosols. Indoor Air 21, 110–120. Hashash, R., Younes, S., Bahnan, W., El Koussa, J., Maalouf, K., Dimassi, H.I., Khalaf, R.A., 2011. Characterisation of Pga1, a putative Candida albicans cell wall protein necessary for proper adhesion and biofilm formation. Mycoses 54, 491–500. Hipler, B., Brand, S., Angersbach, S., Rückert, C., 2003. Monitoring des Wachstums von Mikroorganismen mit Hilfe der Nephelometrie. BIOspektrum 9, 648–649. Hipler, U.C., Schönfelder, U., Hipler, C., Elsner, P., 2007. Influence of cyclodextrins on the proliferation of HaCaT keratinocytes in vitro. J. Biomed. Mater. Res. Part A 83A, 70–79. Ikeda, T., Ledwith, A., Bamford, C.H., Hann, R.A., 1984. Interaction of a polymeric biguanide biocide with phospholipid membranes. Biochim. Biophys. Acta 769, 57–66. Joubert, A., Calmes, B., Berruyer, R., Pihet, M., Bouchara, J.P., Simoneau, P., Guillemette, T., 2010. Laser nephelometry applied in an automated microplate system to study filamentous fungus growth. Biotechniques 48, 399–404. Koburger, T., Müller, G., Eisenbeiß, W., Assadian, O., Kramer, A., 2007. Mikrobiozide Wirksamkeit von Polihexanid, vol. 2. GMS Krankenhaushyg Interdiszip, Doc44. Marques, S.M., Esteves da Silva, J.C., 2009. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions. IUBMB Life 61, 6–17. McLain, N., Ascanio, R., Baker, C., Strohaver, R.A., Dolan, J.W., 2000. Undecylenic acid inhibits morphogenesis of Candida albicans. Antimicrob. Agents Chemother. 44, 2873–2875. Nazari, M., Hosseinkhani, S., 2011. Design of disulfide bridge as an alternative mechanism for color shift in firefly luciferase and development of secreted luciferase. Photochem Photobiol. Sci. 10, 1203–1215. Northcote, D.H., Horne, R.W., 1952. The chemical composition and structure of the yeast cell wall. Biochem. J. 51, 232–236. Odds, F.C., 1987. Candida infections: an overview. Crit. Rev. Microbiol. 15, 1–5. Odds, F.C., 1994. Pathogenesis of Candida infections. J. Am. Acad. Dermatol. 31, S2–S5. Poulain, D., Tronchin, G., Dubremetz, J.F., Biguet, J., 1978. Ultrastructure of the cell wall of Candida albicans blastospores: study of its constitutive layers by the use of a cytochemical technique revealing polysaccharides. Ann. Microbiol. 129, 141–153. Prohic, A., 2010. Distribution of Malassezia species in seborrhoeic dermatitis: correlation with patients’ cellular immune status. Mycoses 53, 344–349. Rasi, A., Naderi, R., Behzadi, A.H., Falahati, M., Farehyar, S., Honarbakhsh, Y., Akasheh, A.P., 2010. Malassezia yeast species isolated from Iranian patients with pityriasis versicolor in a prospective study. Mycoses 53, 350–355. Ruiz-Herrera, J., Victoria Elorza, M., Valentín, E., Sentandreu, R., 2006. Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity. FEMS Yeast Res. 6, 14–29. Seyfarth, F., Schliemann, S., Elsner, P., Hipler, U.C., 2008. Antifungal effect of highand low-molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N-acetyl-d-glucosamine against Candida albicans, Candida krusei and Candida glabrata. Int. J. Pharm. 353, 139–148. Stella, V.J., He, Q., 2008. Cyclodextrins. Toxicol. Pathol. 36, 30–42. Szejtli, J., 1982. Cyclodextrins and Their Inclusion Complexes. Akadémiai Kiadó. Szejtli, J., 1998. Introduction and general overview of cyclodextrin chemistry. Chem. Rev. 98, 1743–1754. Welbel, S.F., McNeil, M.M., Pramanik, A., Silberman, R., Oberle, A.D., Midgley, G., Crow, S., Jarvis, W.R., 1994. Nosocomial Malassezia pachydermatis bloodstream infections in a neonatal intensive care unit. Pediatr. Infect. Dis. J. 13, 104–108.