Mussin et al. AMB Expr (2019) 9:131 https://doi.org/10.1186/s13568-019-0857-7 ORIGINAL ARTICLE Open Access Antifungal activity of silver nanoparticles in combination with ketoconazole against Malassezia furfur Javier Esteban Mussin1* , María Virginia Roldán2, Florencia Rojas1, María de los Ángeles Sosa1, Nora Pellegri2 and Gustavo Giusiano1 Abstract Malassezia furfur is lipophilic and lipid-dependent yeast, inhabitant of human skin microbiota associated with several dermal disorders. In recent years, along with the advances in nanotechnology and the incentive to fnd new antimi- crobial drugs, there has been a growing interest in the utilization of nanoparticles for the treatment of skin microbial infections. This work aimed to study the in vitro inhibitory activity of silver nanoparticles (AgNP) against 41 M. furfur clinical isolates, visualize the interaction between AgNP-Malassezia, evaluate the synergism with ketoconazole (KTZ) and to produce an antimicrobial gel of AgNP–KTZ. The synthesized AgNP were randomly distributed around the yeast surface and showed a fungicidal action with low minimal inhibitory concentration values. AgNP showed no antago- nistic efect with KTZ. The broad-spectrum antimicrobial property with fungicidal action of AgNP and its accumula- tion in afected areas with a sustained release profle, added to the great antifungal activity of KTZ against Malassezia infections and other superfcial mycoses, allowed us to obtain a gel based on carbopol formulated with AgNP–KTZ with the potential to improve the topical therapy of superfcial malasseziosis, reduce the number of applications and, also, prevent the recurrence. Keywords: Malassezia, Nanoparticles, Antifungal activity, Synergy Introduction papillomatosis, and neonatal pustulosis (Giusiano 2006; Yeasts of Malassezia genus are normal inhabitants of the Boekhout et al. 2010; Saunders et al. 2012; Rojas et al. human skin microbiota and other warm-blooded verte- 2014; Rudramurthy et al. 2014; Prohic et al. 2016). Cuta- brates. Since they are unable to synthesize fatty acids, all neous diseases associated with Malassezia are often Malassezia species are lipophilic and most of them lipid- chronic and recurrent. In these cases, the results of anti- dependent, requiring an external source of lipids. For fungal therapy, both topical and systemic, are not always this reason, they prevail in body areas rich in sebaceous efective due to high relapse rates (Giusiano et al. 2010; glands (Boekhout et al. 2010). Carrillo-Muñoz et al. 2013; Prohic et al. 2016; Rojas et al. Tese yeasts are considered to be the etiological agent 2016). of pityriasis versicolor and Malassezia folliculitis, associ- Topical antifungal medications are the frst-line treat- ated agents in seborrheic dermatitis/dandruf and a con- ment for Malassezia infections, and ketoconazole (KTZ) tributory factor that exacerbate other skin disorders such is one of the most efective antifungal agents. KTZ as atopic dermatitis, psoriasis, confuent and reticulate is a fungistatic imidazole that inhibits the lanosterol 14α-demethylase, an enzyme that regulates the synthe- *Correspondence: [email protected] sis of ergosterol. Te disruption of ergosterol biosynthe- 1 Mycology Department, Instituto de Medicina Regional, Universidad sis alters cell membrane structure, thus compromising Nacional del Nordeste, Consejo Nacional de Investigaciones Científcas membrane integrity and permeability and consequently y Tecnológicas (CONICET), Av. Las Heras 727, 3500 Resistencia, Chaco, Argentina interfering with cellular growth and reproduction. KTZ Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Mussin et al. AMB Expr (2019) 9:131 Page 2 of 9 was the frst broad-spectrum antifungal used in the treat- [N-[3-(trimethoxysilyl) propyl] diethylenetriamine] ment of superfcial mycoses (Gupta and Foley 2015). (ATS, Aldrich) was dissolved in ethanol absolute under Nanotechnology is an important feld of modern N2 atmosphere and magnetic stirring. Ten, both solu- research which deals with synthesis and manipulation tions were mixed and homogenized obtaining a fnal of structures of matter ranging from approximately 1 solution of 12 mM of AgNO3 and 0.197 M of ATS. Tis to 100 nm in size, commonly called nanomaterials (Liz- solution was placed at a temperature bath at 40 °C under Marzán and Kamat 2003; Rao et al. 2004). Te noble N2 atmosphere for 4 h. Te color of the solution changed metal nanoparticles showed unique and considerably from uncolored to bright yellow. diferent physical and chemical properties compared to their macro scaled counterparts (Feldheim and Foss Characterization of silver nanoparticles 2002). As the size of the nanoparticles decreased, their It was carried out by UV–Vis absorption spectroscopy surface-volume ratio and antimicrobial activity increased and transmission electron microscopy (TEM). (Rai et al. 2009; Sharma et al. 2009; Song and Kim 2009; Optical characterization of colloidal suspensions was Bera et al. 2014; Ahmed et al. 2016). Silver (Ag) is one of performed by UV–Vis absorption spectroscopy, using the noble metals with higher antimicrobial activity and UV/Vis spectrophotometer (Jasco V-530). UV–Vis spec- lower toxicity for animal cells (ATSDR 1990; Lansdown tra were acquired by employing ethanol as reference. Te 2010). colloidal stability of AgNP over time was evaluated by Tere has been a growing interest in silver nanoparti- visual observation and UV–Vis spectroscopy. cles (AgNP) over the years due to their potential appli- TEM images were acquired with a Phillips 100 keV. cation in human and animal medicine for treating skin Samples were prepared by dropping the nanoparticles infections including dermatomycosis (Rai et al. 2009; Ge suspension over a carbon coated TEM grid. Several TEM et al. 2014; Aljufali et al. 2015). Likewise, these particles images were processed with ImageJ free software to esti- either alone or in combination with other drugs, would mate mean size and standard deviation. represent a therapeutic alternative against resistant microorganisms, as well as in complications associated Microorganisms with the use of antifungals (Rai et al. 2009, 2012; Bera A total of 40 M. furfur isolates were studied. Isolates were et al. 2014). obtained from human clinical samples with diagnosis of Clinical and Laboratory Standards Institute (CLSI) pityriasis versicolor, seborrhoeic dermatitis/dandruf and document M27-A3, describes a broth microdilution atopic dermatitis. All Malassezia yeasts were deposited method for testing the in vitro antifungal susceptibil- in the culture collection of Mycology Department, Insti- ity for Candida species and Cryptococcus neoformans tuto de Medicina Regional (IMR), Universidad Nacional for the determination of minimal inhibitory concentra- del Nordeste (UNNE), Argentina. Identifcation was per- tions (MIC) (Clinical and Laboratory Standards Institute formed by polymerase chain reaction-restriction frag- 2008). Due to the nutritional requirements of Malasse- ment length polymorphism (PCR–RFLP) (Mirhendi et al. zia yeasts, this method is not applicable for this genus. 2005; Sosa et al. 2013). In addition, the reference strain Rojas et al. (2014) proposed a nutritionally supplemented M. furfur CBS 7019 was included. medium to evaluate the in vitro activity of antifungals Isolates were sub-cultured for 72 h onto modifed against some Malassezia species. Dixon Agar at 32 °C before antifungal susceptibility Te aims of this study were to: (a) study the in vitro testing. inhibitory activity of AgNP synthesized against Malasse- zia furfur clinical isolates, (b) evaluate this activity in Minimum inhibitory concentration (MIC) combination with KTZ, (c) visualize the interaction In order to evaluate the inhibitory activity of synthe- between AgNP-Malassezia and (d) produce and evaluate sized AgNP and KTZ (Sigma-Aldrich, Buenos Aires, the activity of an antimicrobial gel of AgNP–KTZ. Argentina), MIC were determined by broth microdilu- tion method in accordance with CLSI M27-A3 document Materials and methods (Clinical and Laboratory Standards Institute 2008) with Synthesis of silver nanoparticles modifcations proposed by Rojas et al. (2014). AgNP were synthesized by chemical reduction of AgNO 3 All inoculum suspensions were prepared in ster- in ethanol, according to Roldán et al. (2008), with some ile saline solution and turbidity was adjusted to a 1 modifcations as described as follow. AgNO3 (Merck) was McFarland scale by densitometer (DEN-1 densitometer, dissolved in ethanol absolute (Ciccarelli) under ultra- Biosan). Tis inoculum was diluted 1:100 in supple- sound stirring. Ethanol was used as solvent and also as a mented RPMI medium to achieve a fnal concentration of 5 mild reducing agent. On the other hand, the aminosilane 0.5–2.5 × 10 CFU/mL. Mussin et al. AMB Expr (2019) 9:131 Page 3 of 9 AgNP and KTZ solutions were prepared using dime- a fungi is inhibited but not killed by an antifungal agent thyl sulfoxide (DMSO) as solvent (fnal concentration that normally is considered fungicidal, defned by a MFC/ ≤ 1%) and RPMI medium as