Mycelial form of dimorphic fungus Malassezia species dictates the microbial interaction

Abstract

Dandruff is one of the most common clinically manifested and studied scalp disorders. It has been associated with both bacteria and fungi. Bacteria and fungi inhabiting the scalp are known to influence each other and manifestation of dandruff. Fungal and bacterial isolates from scalp epithelial flakes (dandruff) were identified by rDNA sequencing. Local oils were tested for fungal and bacterial inhibition, interaction and biofilm formation, cell–cell interactions were studied by auto aggregation and surface thermodynamics studies. The isolates Bacillus sp.C2b1 (MK036745) and Malassezia sp. C2y1 (MK036746) were inhibited by Mahabhrungraj oil. The fungal morphological switch was evident and dependent on nutrition. Cell aggregation studies suggested the interaction of bacteria with yeast (non-pathogenic) phase of the fungus. Bacterial and yeast cells were found to be compatible for biofilm formation. The fungal mycelial surfaces were found to be conducive for interaction with both bacterial cells and yeast forms. The results here indicate the significance of mycelial phase of scalp-isolated fungus in interaction with the bacterial surfaces and also with self-yeast phase surface. This is the first report of the interaction between scalp-isolated microorganisms with respect to their surface interaction capabilities.

Electronic supplementary material

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Introduction

Human skin is a complex ecosystem that includes bacteria, fungi, archaea and viruses directly influencing the disorders and health of the host. Sebum can act as an importance food source for both bacterial and fungal growth [1]. Flaking of the scalp epithelial cells with itchiness are common symptoms of both dandruff and seborrheic dermatitis, which are common scalp disorders. Dandruff can be considered as a chronic inflammatory disorder related to human skin leading to flaking and pruritus [2].

Bacteria are known to produce free fatty acids to the human skin promoting bacterial adherence and are reported to be of higher severity in relation to fungi in dandruff [1]. Malassezia species are commensal and pathogenic skin microflora on the surfaces of animals including humans. They are (M. furfur, M. globosa and M. restricta) associated with human skin affections includings Pityriasis versicolor [3]. Malassezia furfur is a dimorphic fungus that grows in yeast phase (unaffected skin) and also in mycelial phase (affected skin) [4].

Bacteria and fungi are known to directly or indirectly influence each other’s growth and/or physiology, behavior and survival. This polymicrobial encounter between the two influences their virulence. There are reports of interactions like bacterial cell contact and aggregation on fungal (mycelial and yeast) cells to organize bacterial biofilms on the fungal surfaces. These interactions can be mutually beneficial or antagonistic but have a profound effect on the host health and immune response [5].

In this manuscript, bacterial and fungal isolates from scalp epithelial flakes (dandruff) were isolated and characterized. Interaction studies were done between the dandruff isolates theoretically by using DLVO theory (Derjaguin-Landau-Verwey-Overbeek). This is the first report of use of thermodynamics for the study of interactions between bacterial and fungal isolates from dandruff. Here we also report the possible association of Bacillus cereus in the dandruff microflora.

Materials and Methods

Sample Collection

Scalp-epithelial flakes (Dandruff) was collected from a female scalp (age 22 years, suffering with chronic infection with profuse shedding from crown part of scalp which is the sampling point) using a sterile cotton swab. Sample collection was performed using swab technique.

Isolation

100 mg of sample was inoculated separately into Sabouraud and Nutrient broths for fungal and Nutrient agar (NA) medium for bacterial isolation respectively. Bacterial and Fungal enrichment was carried out with 37 °C for 72 h and 24 h respectively in shaking (180RPM), streaked onto their respective agar plates and further incubated (37 °C). NA plates were incubated for only 24 h while for fungi 120-144 h incubation was checked.

Morphological Characteristics

Isolated fungi (Sabouraud medium at 37 °C for 5–6 days) were stained with Lactophenol cotton blue for characterizing the morphology while bacteria (NA medium at 37 °C for 24 h) were Gram stained (data not shown).

Tests for the Dimorphic Fungus

Fermentation (Carbohydrate) Test

Characterization of fungal isolates was done by using their carbohydrate fermentation profiles. Isolates were inoculated in 10 ml PDA medium and incubate at 37 °C for 2 to 3 days. Carbohydrate fermentation broths were prepared with 10% (w/v) sugars and inverted Durham’s vials and addition 1% inoculum (37 °C for 24–48 h). Potential Fungal-bacterial partners were selected and studied further.

DNA Extraction and Sequencing of the Isolates

DNA was extracted from the selected bacterial and fungal isolate colonies by using QIAamp (Qiagen, Valencia CA, USA) and Macherey–Nagel DNA extraction kit (Macherey–Nagel, Düren, Germany) using the manufacturer instructions. The DNA extracted was amplified for conserved bacterial 16S rDNA using universal primers 27F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492R (5′ TACGGYTACCTTGTTACGACTT 3′); fungal 26S rDNA primers- LR7 (5′ TACTACCACCAAGATCT 3′) and LROR (5′ ACCCGCTGAACTTAAGC 3′). For removal of dNTPs and unutilized primers from the PCR products, Montage PCR Clean up kit (Millipore) was used. Further sequencing was done using PCR primers in ABI PRISM^® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq^® DNA polymerase (FS enzyme) (Applied Biosystems).

Bioinformatics Protocols

The rRNA sequences were checked online using GenBank BLAST tool. MUSCLE 3.7 program was used for multiple sequence alignment and curing was done using Gblocks 0.91b program (eliminating divergent regions and poorly aligned positions). The evolutionary analysis of the sequences was conducted using MEGA6 [6]. Using their closest BLAST matches, the evolutionary history of isolates was got using Neighbor-Joining method. Using bootstrap test (5000 replicates) from the clustered associated taxa are drawn to optimal tree (sum of branch length = 30.28) is shown as percentage replicates. The analysis included 17 nucleotide sequences with elimination of gaps and missing data.

Preparation of Isolated Cultures for Interaction Studies

The cultures (grown in standard nutrient broth (NB) and Potato dextrose broth (PDB) for 24 and 48 h for bacterial and yeast cultures from 1^st passage of isolated cultures respectively) were centrifuged at 5000 rpm for 10 min, washing the pellet with 0.5 ml sterile saline (0.8% NaCl) and resuspended in the same (Till OD 0.4 for both yeast and Bacterium).

Cell Count Method

Total bacterial count was done by spore count method after heating the suspension to 60° for 15 min and staining the suspension with 0.1% malachite green (and 1% saffranine as secondary stain). While the yeast cell count was done by using 1% crystal violet and observing the cells at 40 ×  (bacterial cells being smaller could be differentiated). Both the cell counts were done by using Neubauer chamber.

Measurement of Fungal Growth by Colony Diameter Method

0.5 μL suspension (prepared as given above) put on the center of potato dextrose agar (PDA) plate. After incubating the plates at 37 °C for 1 week, the fungal colony diameter was measured everyday with the help of ruler and the results were recorded.

Effect of Oils on Microbial Growth

The bacterial and yeast isolates were checked for their ability to survive in commonly used hair-oils. The regular hair-oils of south Gujarat region-Olea europaea (Olive oil), Cocos nucifera (Coconut oil), Ricinus communis (Mahabhrungraj oil), Eclipta prostrate (Castor oil), Prunus dulcis (Almond oil) oils were procured fresh from government sanctioned farmers market (Agricultural Produce Market Committee, or APMC), Surat,

Plate assay: The oils were applied at 50 µL/well in agar plate (Mueller–Hinton agar (MHA) and Sabouraud dextrose agar (SDA) for bacteria and yeast respectively) spread with 100 µL bacterial and 50 µL yeast suspension prepared as given before [7].

Broth assay: The bacterial and yeast cultures were inoculated (1%) into Mueller–Hinton broth (MHB) and Sabouraud dextrose broth (SDB) respectively in a 24 well microtitre plate. The effect of oil on the cell count was checked after 24 h incubation at room temperature with gentle shaking (50RPM).

The antibacterial and antifungal activity of the oils was checked by growing 100 µL of 10⁻² cells (diluted in normal saline) from above mentioned cell suspension preparations as lawn culture on NA and PDA plates respectively. Wells were bored into the corresponding agar plates (0.4 mm diameter) and 25 µL of the five oils were introduced in the wells separately. The plates were incubated at 37 °C for 24 h and the zone diameters were measured (n = 3).

Interaction Studies

Compatibility Assay

The bacteria and yeast were checked for their compatibility by using Cross streak antagonism assay by streaking the freshly grown bacteria and yeast on the nutrient agar (NA) plate at right angles to each other [8], the plates were incubated (24 h at room temperature) and results were noted.

Biofilm Assay

The above mentioned culture suspensions (10 µL) were inoculated into 1/10^th of the diluted NB and SDB medium (100 µL/well) individually and as a mixture (incubation 24 h). The biofilm formation was checked according to Markande and Nerurkar [9]. Planktonic cells were washed and were counted for yeast and bacterial cells as given before. The biofilm formed was washed with NS and stained with 2% crystal violet for 10 min, washed with NS (till colour stops coming into discard), checked for biofilm formation at 595 nm with Methanol.

Aggregation Studies

The aggregation studies were according to Markande and Nerurkar [9]. The suspension of yeast and bacteria prepared as before (OD 0.6) were checked for their cell-aggregation. The isolates individually and as a mixture were checked in a sterile flask where the cell-suspension was allowed to stand undisturbed at room temperature. The upper most suspension was checked for A₆₀₀ every 60 min. The auto aggregation percentage was calculated by using the formula,

$$Autoaggragation=1-\left(\frac{A_1}{A_0}\right)\times 100$$

where A₁ = A₆₀₀ at time t = 1, 2, 3, 4, 5 and 6 h; A₀ is the A₆₀₀ at time t = 0.

Contact Angle Measurements

The bacterial and yeast suspension was prepared as given before. The suspensions were spread on 45 mm bacterial filter (0.45 µm pore size, Pall India Pvt Ltd) in sterile plates prepared in NA and PDA for bacteria and yeast respectively. The system was incubated for 6 h at room temperature (for actively growing cultures on lawn). The microbial lawn thus achieved was segregated into two groups of bacterial and yeast layers (n = 4 for two groups of bacteria and yeast) and one set of membranes were dried in empty sterile petriplates (at 50 °C for 2 h). Contact angles of the specific liquids (2 μL of Water, formamide and hexane) were checked on both the group of microbial lawns and photographs were taken. The contact angle were calculated using Image J software and results were tabulated. θ thus achieved was used in the Young’s equation and Lifshitz–Van der Waals equation.

$$\begin{array}{cc}\hfill \mathrm{\gamma }\text{s}& =\mathrm{\gamma }\text{lcos}\mathrm{\theta }+\mathbf{\gamma }\text{sl}\hfill \\ \hfill \mathrm{\Delta }\text{Gsl}& =-2\sqrt{{\mathrm{\gamma }}_s^{\mathit{lw}}{\mathrm{\gamma }}_l^{\mathit{lw}}}-2\sqrt{{\mathrm{\gamma }}_s{\mathrm{\gamma }}_l^{-}}-2\sqrt{{\mathrm{\gamma }}_s^{-}{\mathrm{\gamma }}_l^{+}}\hfill \end{array}$$

Contact angle was used to check the hydrophobicity of solid surface and further calculate the interaction of the two surfaces under study. On basis of data graph were plotted [9].

Results and Discussion

Human skin is a complex ecosystem, thus studying the microbial interaction in this niche is difficult [10]. Severity of dandruff has been reported to be associated with bacterial partner than fungi while host-microbe interaction is a major factor. The disequilibrium between bacterial–fungal partners of dandruff has also been reported with respect to the severity of Dandruff [1, 2]. Fungal dimorphism is a unique quality that few fungi possess. Hibbet et al. [11] reported exhaustive study on the fungal classification with respect to dimorphism.

Isolation and Identification of Bacteria and Fungi from Dandruff

The bacterial and fungal isolates were maintained in their respective media. The isolates were confirmed for their identity with fungal and bacterial staining (data not shown). Isolates were characterized according to their fermentation profiles against different carbohydrate substrates. Glucose, Sucrose, Lactose was positive for assimilation and negative for gas formation, and Xylose was negative for both assimilation as well as gas formation. The isolates were also checked for their lipase activity.

The isolates sequenced were found to be belonging to Bacillus species and Malassezia species respectively. Their sequence similarity seen during NCBI BLAST using GenBank database showed bacterial isolate matching 100% with Bacillus cereus DFT-5 (KY750689.1) and yeast isolate matching 94% with Malassezia furfur AFTOL-ID 855 (AY745725.1).The sequences have been submitted to NCBI GenBank (MK036745 for bacterium Bacillus sp. strain C2b1 and MK036746 for yeast Malassezia sp. strain C2y1 respectively). Phylogenetic tree constructed shows the top NCBI BLAST matches with isolates (Supplementary figure S1). The top matches for bacteria were all associated with aquatic B. cereus strains, while all the strains matched with yeast showed Malassezia species associated with skin diseases.

Measurement of Fungal Growth by Colony Diameter Method

The fungal growth was studied with respect to the colony diameter method. The fungus being dimorphic, showed a specific two step growth. The first yeast growth was halted at 5th day where mycelia were observed for the first time. 7th day onwards, the yeast phase was completely converted to mycelial phase (Fig. 1). Frischmann et al. [12] reported the unique phenomenon of hydrophobins increasing the polarity of surfaces and solutions. Hence this property of fungus has been reported to be influencing the fungal spread on a surface.

Fig. 1.

Fungal Growth study by colony diameter method

There have been reports of pathogens like, reported the change in dimorphism in Ustilago maydis depending on the medium pH changes due to its own growth. They suggested that the growth continuation in change in pH overcome the control processes governing the heterologous expression of dimorphism.

Effect of Oils on Microbial Growth

Plant/herbal oils have been extensively reported to be antimicrobial, but their use and activity assays differ and a standard assay is a requirement. There have been reports of antibacterial activities (minimum inhibitory activity) and antifungal activities (minimum fungicidal concentration) from oils namely-olive oil [7], coconut oil [13], mahabhrungraj oil [14], castor oil [15] and almond oil [16]. Uma et al. [17] reviewed the activities of different plant extracts and essential oils and reported the unclear mode of studies followed till now on their effects with respect to pathogens under study. Castor and olive oil have been reported extensively for Malasezzia cultivation with species specific growth.

The use of oils for controlling was checked (Fig. 2). The control organisms taken were E. coli, an enteric microorganism (associated with enteric microflora and also enteric discomforts) and S. aureus, a normal skin flora (associated with skin infections). All the oils showed some amount of microbial inhibition at 24 h. The effects of Mahabhrungraj Oil was significant (p < 0.05) on all the microorgasnisms tested while coconut oil exhibited least activity. The effect of oils on dandruff associated microorganisms (Bacillus sp. C2b1 and Malassezia sp. C2y1) in a mixture was more than the effect on organisms individually. Bacillus sp. C2b1 exhibited lowest sensitivity to the Olive oil, Coconut oil and Mahabhrungraj oil with respect to Malassezia sp. C2y1 tested in this study. While it showed higher sensitivity to Castor oil and Almond oil with respect to Malassezia sp. C2y1. Many reports have reported higher anti-microbial (MIC and MFC) activities of essential oils than organic extracts from the herbal plants [18].

Fig. 2.

Inhibition studies of oils against microorganisms

Interaction Studies

As stated by Soares et al. [2], there is need for further analysis of fungal interactions with respect to the diversity of scalp microflora providing new perspectives on the dandruff pathogenesis further enhancing our knowledge of healthy skin microflora and their interaction for sustained human health.

When checked in biofilm formation (Fig. 3, Bacillus sp.C2b1 showed higher biofilm formation in Olive oil, Coconut oil, Mahabhrungraj oil and in Castor oil with respect to Malassezia sp. C2y1 while its ability was found to be reduced in Almond oil. Bacillus sp.C2b1 also showed higher planktonic cells with respect to all the oils tested. The mixed culture (C2y1 and C2b1) showed relatively lower biofilm formation than compared to Bacillus sp.C2b1 but high with respect to Malassezia sp. C2y1 and lower planktonic cell presence in all the oils tested. The planktonic cell presence in yeast and bacterial population of the mixed biofilm showed relatively lower numbers than individually tested.

Fig. 3.

Effect of oils on the biofilm formation and planktonic cell presence of dandruff isolates. Dandruff isolates Bacillus sp.C2b1 and Malassezia sp. C2y1 were checked for a biofilm formation and b planktonic cell presence individually and as a mixed population

Compatibility Assays

Aggregation Studies

As seen in Fig. 4 the cells of Bacillus sp. C2b1 and Malassezia sp. C2y1 showed higher autoaggragation percentage than with respect to the mixed cellular suspension. The yeast + bacterial suspension showed lower compatibility for cellular aggregation.

Fig. 4.

Cell aggregation assay

Contact Angle Measurements

Fungal surface characteristics are known to be modulated by interactive fungi depending on necessity. Secretion of small proteins into the air/water interphases to enhance the surface interactions (by inverting the polarity of surfaces) have been reported previously [12]. Frischmann et al. [12] reported the use of contact angle measurements for studying the fungal hydrophobins and their ability to enhance the fungal surface interactions and also there have been previous reports of using contact angle measurements for studies on interaction between bacterial and fungi [19].

The contact angle measurements showed higher θ values for bacteria (125.4 ± 1.25) than fungi (137.2 ± 21.3) in dry state of the surface. While the yeast phase exhibited lower (41.1 ± 0.3) hydrophobicity than mycelial or bacterial phases. In wet state, fungal mycelia showed higher θ values (145.8 ± 0.5) than bacteria (133.5 ± 13.4) and yeast phase showed lower (24.4 ± 0.7) hydrophobicity.

DLVO method uses the relative impact of capillary, charged, hydrophobic and hydrodynamic forces (at solid-air–water interphases) to study the surfaces (bacterial or fungal) and their unsaturated-porous media deposition mechanism. Using the energy of interaction as a sum of electrostatic and van der Waals forces (DLVO forces), classical DLVO theory describes the colloidal interactions and their physico-chemical attachment onto solid-water interfaces [12, 20].

The results in contact angle measurements show that the bacterial–bacterial and yeast–yeast interactions are not governed by the surface hydrophobicity based attractions and hence show lower ΔG_tot or total free energy of interaction (Fig. 5). This result correlates with the aggregation assay where the interaction between bacterial and yeast cells was also found to be lowest. The interaction of yeast and bacterial cultures with mycelium phase of the fungus was found to be significant as this phase is recognized as the pathogenic morphological phase [4]. The interactions of fungal surfaces with bacterial surface seem to be different in two morphologies of the dimorphic fungus.

Fig. 5.

Free energy of interaction studies between bacterial and fungal (yeast and mycelial) phase in dry and wet surface conditions. a Bacterial interactions with yeast and mycelial/fungal phase while b self-interaction among the fungal phases is shown

Xu et al. [1] studied the intra and inter associations among the dandruff microflora with respect to host and host exudates (sebum). They found that depending on the host age and physiological conditions, the interaction pattern of dandruff microflora changed. Due to the diversity of scalp microbiota and the degree of fungal interaction capabilities with respect to its dimorphic nature increases the chances bacterial interaction and thus shifting the microbial population. Thus dandruff can be an asymmetric process which is not restricted to the lesion site with clinical symptoms [2]. Xu et al. [1], studying the bacterial-fungal interactions in scalp reported that the dandruff bacteria and fungi did not show close association but their intra members were tightly linked. But the bacterial-fungal balance in the scalp-microflora dictated the dandruff severity. Yeast—hyphal transition in dandruff causing fungus M. furfur have been known and the hyphae were more virulent with the abiliuty to invade squamous epithelial sites have been well tested.

In conclusion, the pathogenic trait of dandruff causing fungal mycelium (Malasezzia sp. C2y1) was found to be its interaction capabilities. Malassezia furfur has already been reported to be having a mycelial infecting stage [4]. This property aids the fungal pathogen in establishing and manipulating the niche of scalp and also to co-ordinate the fungal-yeast and fungal-bacterial mixed pathogenesis. This is the first report of thermodynamic studies of a dimorphic fungus. Here we also establish the possibility of inhibition of pathogenic fungal-yeaat and fungal-bacterial co-infection. Here we also have studied the effect of herbal oils on controlling the dandruff associated bacterial and fungal microflora.

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Conflict of interest

The authors declare no conflict of interest in publishing this manuscript.