Background: Although Malassezia genusare part of the skin normal flora, under certain conditions, they become pathogenic. Catheter-related fungemia, caused by Malassezia, which is associated with biofilm formation, is considered a nosocomial infection. Objectives: The aim of this study was to evaluate the ability of Malassezia globosa and Malassezia restricta in biofilm formation. Methods: Biofilm formation was carried out using catheter segments in 12-well plates. Results were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay in 96-well plates. The data was analyzed using univariate Analysis of Variance (ANOVA) or Repeated Measures ANOVA. P values of â�¤ 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics version 22.0 software. Results: Both M. globosa and M. restricta species were able to form biofilms in vitro. Malassezia restricta was more capable than M. globosa to form biofilms, yet, significant differences were not observed (P = 0.192). Conclusions: Over time, Malassezia biofilms matured. Due to the above species ability in forming biofilm, they could play an important role in fungemia that should be considered in therapeutic procedures. Â© 2017, Archives of Clinical Infectious Diseases

Borujeni, Z.B.

Mohammadi, S.R.

Roudbary, M.

Shahbazi, S.

Zareei, M.

eprints Iran University of Medical Sciences

Arch Clin Infect Dis. 2018 August; 13(4):e62223.Published online 2017 July 31.doi: 10.5812/archcid.62223.Research ArticleEvaluation of the Ability of Malassezia Species in Biofilm FormationMahdi Zareei 1, Shahla Roudbar Mohammadi 1, *, Shirin Shahbazi 2, Maryam Roudbary 3 and ZeinabBorjian Borujeni 41Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran2Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran3Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran4Department of Medical Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran*Corresponding author: Shahla Roudbar Mohammadi, Faculty of Medical Sciences, Department of Medical Mycology, Tarbiat Modares University, Jalal Al Ahmad St, Tehran,Iran. Tel: +98-2182884540, E-mail: sh.mohammadi@modares.ac.irReceived 2016 July 31; Revised 2017 March 11; Accepted 2017 March 14.AbstractBackground: Although Malassezia genusare part of the skin normal flora, under certain conditions, they become pathogenic.Catheter-related fungemia, caused by Malassezia, which is associated with biofilm formation, is considered a nosocomial infection.Objectives: The aim of this study was to evaluate the ability of Malassezia globosa and Malassezia restricta in biofilm formation.Methods: Biofilm formation was carried out using catheter segments in 12-well plates. Results were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay in 96-well plates. The data was analyzed usingunivariate Analysis of Variance (ANOVA) or Repeated Measures ANOVA. P values of ≤ 0.05 were considered statistically significant.Statistical analyses were performed using IBM SPSS Statistics version 22.0 software.Results: Both M. globosa and M. restricta species were able to form biofilms in vitro. Malassezia restricta was more capable than M.globosa to form biofilms, yet, significant differences were not observed (P = 0.192).Conclusions: Over time, Malassezia biofilms matured. Due to the above species ability in forming biofilm, they could play an im-portant role in fungemia that should be considered in therapeutic procedures.Keywords: Biofilm, Fungemia, Catheter, Malassezia1. BackgroundMalassezia biofilm is produced by a community ofthese bacteria, alone or mixed with other microorganisms,which are attached to the living and non-living surfacesand are difficult to separate from surfaces. This structurehas physical and chemical resistance, and resistance to an-timicrobial agents is almost 2000 times higher comparedwith planktonic cells. Biofilm of Malassezia is a structurethat is organized as single-layer or multi-layer of blasto-conidia. Biofilms, with the ability of producing an extracel-lular matrix, acts as a barrier against the influence of drugs(1-5). Although Malassezia genus is part of the skin nor-mal flora, under certain conditions, becomes pathogenicand in the recent years has been reported as an emergingpathogen (1, 6-9). The ability of these fungi in coloniza-tion on the catheter is associated with biofilm formation.Catheters are frequently used in new medical devices, es-pecially at the Intensive Care Unit (ICU) and their use maylead to local or systemic infection (1, 5, 10-12). Since noso-comial infections effect approximately 10% to 30% of pa-tients at the ICU and are associated with significant mortal-ity, it should be noted that a significant proportion of theseinfections are due to biofilm formation (5, 10-12); how-ever, fungal biofilms have been studied less than bacterialbiofilms. Most studies are about Candida (13-16), however,there have been only a few studies on Malassezia biofilms(1, 4, 5, 13, 17).2. ObjectivesThe aim of this study was to evaluate the abilityof Malassezia globosa and Malassezia restricta species inbiofilm formation.3. MethodsAfter preparing the standard species of M. restricta (CBS7877) and M. globosa (CBS 7966) from the Institute (Cen-traalbureau voor Schimmelcultures, Utrecht,The Nether-lands), the study was conducted at the Medical mycologylaboratory, faculty of Medical Sciences, Tarbiat ModaresUniversity.Copyright © 2017, Archives of Clinical Infectious Diseases. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided theoriginal work is properly cited.Zareei M et al.3.1. Evaluation of Biofilm FormationCatheter segments of Certofix® Mono V 420 central ve-nous catheter (B. Braun, Milano, Italy) with 1 × 1 cm di-mensions were prepared in 12-well plates (Nalge Nunc In-ternational, NY, USA). After suspension of the Malasseziayeast in a concentration of 106 CFU/mL Phosphate BufferedSaline (PBS, PH = 7.2 - 7.4) biofilm was formed using themedium modified Dixon broth (made of 3.6 g malt extract(Merck, Germany), 0.6 g mycological peptone, 2 g driedbeef bile (Sigma, UK), 1 mL Tween 40 (Merck, Germany), 0.2mL glycerol (Merck, Germany), 0.2 mL oleic acid (Merck,Germany), 5 mg chloramphenicol, 50 mg cycloheximideand 100 mL distilled water) (13). The test wells were selectedand catheter segments were divided in doubled form andincubated in fetal bovine serum (FBS) for 24 hours (h) at37°C on a rocker. Next, catheter segments were placed innew 12-well plates and 4 mL of culture medium mDixonbroth containing Malassezia yeast at a concentration of 106CFU/mL, was poured in each well to submerge catheter seg-ments and then they were incubated in a 75 rpm (round perminute) shaking incubator for 24 hours at 32°C for cell at-tachment to the catheter surface. After a period of time,to separate the loose and non-binding cells (planktoniccells), catheter segments were washed with PBS and 4 mLof culture medium mDixon broth containing 100 mM ofglucose was added to the wells (4, 18). After incubation at32°C for 48, 96, and 144 hours in a 75 rpm shaking incu-bator (5), catheter segments were removed from wells andformed biofilms on the surfaces were scraped by a plasticspatula (cell scraping) and were poured in 96-well micro-plates (Nalge Nunc International, NY, USA). After additionof 50 µL of distilled water and 10 µL MTT (Sigma Chemi-cal Co., St Louis, MO, USA) at a concentration of 5 mg/mL,they were incubated for 4 hours at 32°C. Finally, by adding50 µL of Dimethyl Sulfoxide (DMSO) solution to all wellsand incubation for 10 minutes at 32°C, the percentage ofattached cells on the surface of the catheter segments wasmeasured by using the enzyme linked immunosorbent as-say (ELISA) absorbance reader (Stat Fax 2000, USA) at 540nm. In this method, the well containing catheter segmentswith mDixon broth medium and without yeast cell suspen-sion was used as a negative control.3.2. Evaluation of Biofilm Formation on the Catheter SegmentsThrough Scanning Electron Microscopy (SEM)Two catheter segments with biofilms formed on themafter 96 hours were studied with a scanning electron mi-croscope according to a previously described method (4)(Leo, Cambridge, UK).3.3. Statistical AnalysisNormality of data was assessed using the One-SampleKolmogorov-Smirnov Test (with Lilliefors SignificanceCorrection). After collecting and classifying the data,they were analyzed using univariate analysis of variance(ANOVA) or repeated measures ANOVA. A value of P ≤0.05 was considered statistically significant. Statisticalanalyses were performed using IBM SPSS Statistics version22 software (19).4. ResultsBoth M. globosa and M. restricta species were able toform in vitro biofilms. At three time points, Malasseziabiofilm matured and had a linier increase. Optical den-sity (OD) results of biofilm formation after periods of 48,96 and 144 hours are shown in Figure 1. Malassezia restrictawas more capable than M. globosa to form a biofilm, yet, be-tween them, a significant difference was not observed (P =0.192). Figure 2 shows the SEM results of two M. globosa andM. restricta species.Figure 1. Comparison of the Ability to Form Biofilms by Malassezia restricta andMalassezia globosa at Three Time PointsM. restrictaM. rlobosaTime Period (Hours)48 hrs              96 hrs            144 hrs1.41.210.80.60.40.20Absorption (Optical Density = OD)Significant differences were not observed between the two species during the abovetime points (P = 0.192).5. DiscussionCatheter-related fungemia caused by Malassezia is re-ported in premature infants, immune compromised chil-dren, adults with parenteral nutrition, and individualswith immune deficiency (13). Reports show the role ofMalassezia in catheter-related biofilm formation and itspathogenicity in etiology of infections, such as fungemia(5, 13, 20-22). In this study, M. globosa and M. restricta species2 Arch Clin Infect Dis. 2018; 13(4):e62223.Zareei M et al.Figure 2. Scanning Electron Microscopy Showing Biofilm Structures of A, M. globosa and B, M. restrictawere used to evaluate the in vitro ability of biofilm forma-tion on catheter surfaces.One of the common and standard methods to evaluatebiofilm formation is the assessment of cells viability aftertreatment with antifungal drugs. A vital MTT dye could beused for this purpose. The MTT test is a semi-quantitativecolorimetric method to measure cell viability (23, 24). Thismethod was used in the current study.In this study, both M. globosa and M. restricta specieswere able to form in vitro biofilms. Malassezia restricta wasmore capable than M. globosa to form a biofilm and afterperiods of 48, 96 and 144 hours, the OD results of M. restrictawells were more than M. globosa wells. However, betweenthem, significant differences were not observed (P = 0.192,Figure 1). This study shows that the two M. globosa and M.restricta species could form in vitro biofilm, similar to M.pachydermatis species (4, 5). According to this study andother laboratory and clinical studies (4, 5, 13), it has beenconcluded that Malassezia species are able to form biofilmsin two in vivo and in vitro models and give rise to nosoco-mial infections in children and adults.Biofilm formation by microorganisms, includingfungi varies with time (5, 10, 18, 25). In this study, biofilmmass (OD value) had a linear increase as time increased,which is in accordance with the study of Cannizzo (5).In comparison with mature biofilms of Candida that areformed within 48 hours, the Malassezia biofilm requires 4days to mature and the structure of Malassezia biofilm issimpler than Candida. Attachment and nutritional prop-erties of Malassezia may be the cause of this difference(5).There are a few studies about biofilm formation ofMalassezia species and reports are often about M. pachyder-matis. However, there is not enough information availableregarding other species. In this study, to evaluate biofilmformation, two M. globosa and M. restricta species were usedfor the pilot study. These species were standard strains andin terms of the ability to form biofilms (OD value) couldbe different from results of clinical isolates. According toprevious studies, most of the isolates (95%), regardless ofthe area of isolation being a skin lesion or normal skin,were capable to form biofilms, yet their quantity was vari-able among different strains (4, 5). Also, genotypic stud-ies have shown that the ability to form biofilms and theirquantity could be variable among different genotypes (4).Strain dependence of biofilm formation that has been pre-viously suggested for Candida, could be due to physiologi-cal and pathological characteristics of yeast and in the caseof Malassezia could be dependent on phospholipase activ-ity (4). On the other hand, clinically, Malassezia coloniza-tion on the catheter could vary in different wards of thehospital (13). Therefore, due to differences in the ability andquantity of biofilm formation among Malassezia strainsand genotypes, although, the in vitro model conditions ofbiofilm formation are not very different from in vivo sit-uation (26), future comprehensive studies on clinical iso-lates of Malassezia with more samples are suggested to de-termine whether colonization and biofilm formation is re-lated to clinical findings of patients.5.1. ConclusionBoth M. globosa and M. restricta species were able toform in vitro biofilms. Over time, Malassezia biofilms hadArch Clin Infect Dis. 2018; 13(4):e62223. 3Zareei M et al.matured. Due to the two above-mentioned species abil-ity in forming biofilms, they could play an important rolein fungemia and this should be considered in therapeuticprocedures.AcknowledgmentsThe researchers thank all the members of the depart-ment of Medical Mycology, faculty of Medical Sciences, Tar-biat Modares University, Tehran, Iran.FootnotesAuthors’ Contribution: All of the authors cooperated inthis study.Funding/Support: This study was supported in part by thedepartment of Medical Mycology, Faculty of Medical Sci-ences, Tarbiat Modares University, Tehran, Iran.References1. Figueredo LA, Cafarchia C, Otranto D. Antifungal susceptibility ofMalassezia pachydermatis biofilm. Med Mycol. 2013;51(8):863–7. doi:10.3109/13693786.2013.805440. [PubMed: 23834283].2. Blankenship JR, Mitchell AP. How to build a biofilm: a fun-gal perspective. Curr Opin Microbiol. 2006;9(6):588–94. doi:10.1016/j.mib.2006.10.003. [PubMed: 17055772].3. Chandra J, Zhou G, Ghannoum MA. 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Evaluation of the ability of malassezia species in biofilm formation

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Evaluation of the ability of malassezia species in biofilm formation

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