Distribution of Malassezia species on healthy human skin in Bosnia and Herzegovina: correlation with body part, age and gender.

BACKGROUND AND OBJECTIVES: The genus Malasezia currently includes fourteen species that have been isolated from healthy and diseased human and animal skin. However, there were differences with respect to the species most commonly isolated, not only in patients with various skin diseases but also between healthy individuals. The aim of this study was to analyze the prevalence of Malassezia species from clinically normal skin of the scalp and trunk of healthy individuals and to examine if the range of species varies according to body site, gender and age.
MATERIALS AND METHODS: The study was conducted at the Department of Dermatovenerology, University Clinical Center in Sarajevo, Bosnia and Herzegovina from December 2012 to May 2013. One hundred healthy men and women with no skin diseases and aged from <1 to 82 years were studied. The samples were obtained by scraping the skin surface from the upper and middle part of trunk and from scalps of all subjects and then incubated on modified Dixon agar. The yeasts isolated were identified by their morphological and physiological properties according to Guillot et al. method.
RESULTS: M. sympodialis was the predominant species on trunk skin in older subjects, M. restricta on scalp skin in age groups 21-35 years, while M. globosa was identified as common species in adults (36-50 years), both from scalp skin and trunk skin. From the trunk skin M. furfur was the most frequent in children.
CONCLUSION: This study confirmed that cutaneous Malassezia microbiota in healthy subjects varies by body part and age but not by gender.

INTRODUCTION

Malassezia yeasts have been recognized for more than 150 years (1) as members of the normal human cutaneous commensal flora. However, under the influence of predisposing factors these lipolphilic yeasts may become pathogenic and associated with several diseases such as pityriasis versicolor, folliculitis, seborrheic dermatitis, confluent and reticulate papillomatosis, and even systemic infections (2,3).

The taxonomy of Malassezia has been confusing since their early description, because yeasts are dimorphic, existing in both yeast and mycelial phases (4). Today, the genus Malassezia includes 14 lipophilic species which have been identified with a conventional culture method or using molecular methods. Most human isolates belong to the species M. globosa, M. restricta, M. sympodialis, M. furfur, M. slooffiae (5), M. dermatis (6), M. japonica (7) and M. yamotoensis (8).

In humans Malassezia yeasts inhabit sebum-rich areas of the skin, including the trunk and the head region, with population densities peaking between 20-45 years (9). However, there is a great variation regarding presence and density in various skin locations (9-11), in children compared with adults (11-13) and in normal skin compared with diseased skin (2).

Although the role of Malassezia in the pathogenesis of cutaneous diseases is not fully understood, recent studies have shown that decreased density of Malassezia led to improvement of these diseases. There is also some controversy as to whether specific species cause different skin diseases (2,3).

Several studies have been carried out worldwide on the epidemiology of Malassezia species isolated from healthy individuals (11,14-24). The findings vary within the same body sites and same age groups depending on the researcher as different methodologies, isolation media and identification procedures have been employed. The difference in the results may also be attributable to the various internal and external factors such as racial and geographic differences and inevitable errors in each research. Therefore, it is important to recognize the existence of a difference in the distribution of Malassezia species in various ages and different body sites.

The aim of this study was to isolate and identify Malassezia species from the healthy human skin by using morphological, biochemical and physiological criteria and to examine if the range of species varies with different body areas, gender and age groups.

PATIENTS AND METHODS

Patients

The study was conducted from December 2012 till May 2013 at the Department of Dermatovenerology, University Clinical Center, Sarajevo, Bosnia and Herzegovina.

One hundred healthy individuals (50 females and 50 males; age range <1 to 82 years, mean 32) with no skin diseases and without any known underlying disease were included in the study. Subjects were selected among medical students (45), medical staff (20) and their children (17) or relatives (18).

All participants were instructed not to take a shower or use emollients on the day of investigation. Only those subjects who had not used any topical and oral treatment or ultraviolet phototherapy in previous two months were included in the study. All the subjects gave their written informed consent with the requirements of the Institutional Ethics Committee.

Samples

All samples consisted of scales and scrapings from the upper and middle part of trunk and from scalps in all participants. Collected samples were inoculated into Sabouraud dextrose agar and into modified Dixon agar (mDA) consisting of 3.6% malt extract, 0.6% mycological peptone, 2.0% desiccated ox bile (Sigma Chemical Co. Ltd, Dorset, UK), 1% Tween 40, 0.2% glycerol, 0.2% oleic acid, 0.05% chloramphenicol, 0.05% cycloheximide, and 1.2% agar (pH 6.0). The medium was always used within one week of preparation and the cultures were inoculated at 32°C for seven days.

Identification of Malassezia yeasts

Malassezia species were identified according to their macroscopic and microscopic features and physiological characteristics. The macroscopic features of the predominant colonies included their shape, size, color, consistency, and the characteristics of medium around them. Microscopic features of the yeast cells in culture were described after lactophenol staining and included the predominant morphology, size and budding base of the yeasts.

To assess the physiological properties of the yeasts catalase reaction was determined by using a drop of hydrogen peroxide (30%) onto a culture smear on a glass slide. The production of gas bubbles, indicative of release of oxygen, was considered a positive reaction. Utilization of Tween compounds was done according to the test originally described by Guillot et al. (25) and later modified by Gupta et al. (26). Yeast suspension, obtained by inoculating 5 mL of sterile water with a loopful of actively growing yeasts, was inoculated on Sabouraud glucose agar. The inoculum was then spread evenly. Each plate was divided into four sections and 5 mL of Tween 20, 40, 60 and 80 were added into a hole made in center of each section and incubated for a week at 32°C. Utilization of Tweens was assessed by the degree of growth and/or reaction of the lipophilic yeasts around individual holes.

The ability of the various Malassezia species to growth on mDA at 38°C was studied. A sample of actively growing cultures was transfered to mDA and incubated at 38°C for 7 days after which the ability to grow was investigated.

The β-glucosidase activity (spliting of esculin) of different Malassezia species was assayed using method described by Mayser et al. (27). Briefly, a loop of fresh yeast was inoculated deeply in the esculin agar tube and incubated for 5 days at 32°C. The splitting of esculin is revealed by darkening of the medium.

Statistics

Chi-squared test with Yates’ correction for a small sample size was carried out to determine the statistical significance of differences in proportions. We used a statistical software package Minitab 13.0. Significance level was set at P < 0.05.

RESULTS

Cultures

Malassezia yeasts were found in 65% samples taken from healthy scalp skin. The most frequently isolated species was M. restricta found in 33 patients, followed by M. globosa (16%) M. sympodialis (8%), M. slooffiae (5%), M. furfur (2%) and M. obtusa in one case.

The results of culture obtained from healthy trunk skin were positive for Malassezia yeasts in 69 cases. The predominant species was M. sympodialis found in 30 patients and the prevalence of other species was 20% for M. globosa and 14% for M. furfur. Other species were less frequently isolated: M. restricta 3%, M. slooffiae 2%.

Statistically significant differences were found in the distribution of the species isolated from healthy scalp and trunk skin – M. restricta was more commonly positive in healthy scalp skin cultures (ratio 11.0), while M. furfur and M. sympodialis were more frequent in the healthy trunk skin cultures (ratio 7 and 3.7, respectively) (P < 0.05) (Table 1).

Table 1

Malassezia species isolated from scalp and trunk of healthy subjects

No	Age (years)	Gender	Isolated species
			Scalp	Trunk
1.	0 (10 months)	F	Negative	M. furfur
2.	2	F	M. restricta	Negative
3.	3	F	M. restricta	M. globosa
4.	5	M	M. restricta	M. sympodialis
5.	6	M	M. restricta	M. slooffiae
6.	6	F	M. restricta	M. sympodialis
7.	7	M	Negative	M. sympodialis
8.	9	F	Negative	M. furfur
9.	9	F	Negative	M. sympodialis
10.	9	F	Negative	M. furfur
11.	10	F	M. globosa	M. sympodialis
12.	10	F	M. globosa	M. furfur
13.	10	F	M. sympodialis	M. furfur
14.	10	F	M. sympodialis	M. furfur
15.	10	F	M. furfur	M. furfur
16.	11	M	M. restricta	M. sympodialis
17.	13	F	Negative	Negative
18.	15	F	M. restricta	Negative
19.	16	F	M. restricta	Negative
20.	16	F	M. globosa	Negative
21.	17	M	M. restricta	M. restricta
22.	18	F	M. restricta	M. resticta
23.	18	M	M. sympodialis	M. furfur
24.	19	M	M. restricta	Negative
25.	19	M	M. sympodialis	Negative
26.	19	M	M. restricta	M. sympodialis
27.	19	F	M. globosa	M. globosa
28.	19	F	M. restricta	Negative
29.	20	F	M. slooffiae	Negative
30.	20	M	M. restricta	Negative
31.	20	M	M. furfur	M. furfur
32.	20	M	M. restricta	M. furfur
33.	20	F	M. obtusa	Negative
34.	20	M	M. restricta	M. sympodialis
35.	21	F	M. restricta	Negative
36.	21	F	M. restricta	Negative
37.	21	M	M. globosa	Negative
38.	23	M	M. restricta	Negative
39.	23	F	Negative	M. sympodialis
40.	23	M	M. restricta	Negative
41.	24	M	Negative	M. sympodialis
42.	24	F	M. restricta	Negative
43.	24	F	Negative	M. slooffiae
44.	24	M	Negative	Negative
45.	24	F	M. restricta	Negative
46.	25	M	Negative	M. furfur
47.	25	F	M. restricta	Negative
48.	25	M	M. restricta	M. sympodialis
49.	25	M	M. restricta	M. furfur
50.	26	F	M. globosa	M. globosa
51.	26	M	M. restricta	M. restricta
52.	26	M	M. slooffiae	Negative
53.	27	M	Negative	Negative
54.	28	M	M. restricta	M. globosa
55.	28	M	Negative	Negative
56.	28	M	M. slooffiae	Negative
57.	29	M	Negative	M. globosa
58.	29	M	M. restricta	Negative
59.	30	F	Negative	M. restricta
60.	31	M	M. sympodialis	Negative
61.	32	M	Negative	M. furfur
62.	33	M	M. sympodialis	Negative
63.	35	F	M. restricta	M. sympodialis
64.	35	M	Negative	Negative
65.	36	M	M. restricta	M. globosa
66.	37	M	M. globosa	M. globosa
67.	37	F	M. globosa	M. sympodialis
68.	38	F	M. slooffiae	M. sympodialis
69.	39	M	M. globosa	M. globosa
70.	39	F	Negative	M. sympodialis
71.	40	M	M. globosa	M. globosa
72.	40	F	M. sympodialis	M. globosa
73.	41	F	Negative	M. sympodialis
74.	42	F	Negative	M. globosa
75.	43	F	M. sympodialis	M. sympodialis
76.	44	F	M. globosa	M. sympodialis
77.	45	M	M. globosa	M. globosa
78.	47	F	Negative	M. globosa
79.	48	M	M. globosa	M. globosa
80.	49	F	Negative	Negative
81.	49	M	Negative	M. sympodialis
82.	50	F	M. globosa	M. sympodialis
83.	50	F	M. globosa	M. sympodialis
84.	50	M	M. restricta	M. globosa
85.	51	M	M. restricta	M. sympodialis
86.	54	F	Negative	M. globosa
87.	56	M	Negative	M. globosa
88.	59	F	Negative	M. sympodialis
89.	59	F	Negative	Negative
90.	61	M	Negative	M. sympodialis
91.	66	M	Negative	M. furfur
92.	66	M	M. restricta	M. sympodialis
93.	67	M	Negative	M. globosa
94.	67	M	Negative	M. sympodialis
95.	68	M	Negative	M. sympodialis
96.	70	F	M. slooffiae	M. sympodialis
97.	74	F	Negative	M. globosa
98.	78	F	Negative	M. globosa
99.	80	M	M. globosa	M. sympodialis
100.	82	F	Negative	M. sympodialis

Distribution of Malassezia species isolated according to demographic parameters

Gender

The same numbers were woman and man (50%). No statistically significant differences were found between the genders in the species isolated from scalp and trunk skin (P > 0.05) (Fig. 1).

Fig. 1

Malassezia species distribution from scalp and trunk skin of healthy subjects according to gender

Age

Subjects were grouped according to age as follows: 0-10 (15%), 11-20 (19%), 21-35 (30%), 36-50 (20%), and ≥51 years of age (16%).

Site of infection

Among Malassezia species cultured from scalp skin, M. restricta predominated in age groups 21-35 (39%) and 11-20 (33%). M. globosa was identified as common species in adults (36-50 years), both from scalp skin and trunk skin, in a frequency of 56% and 50%, respectively. From the trunk skin, M. sympodialis was most frequently found in older subjects (>51 years) – 30%, while M. furfur was the most predominant in the group of less than 10 years – 50 (Fig. 2).

Fig. 2

Malassezia species distribution from scalp and trunk skin of healthy subjects according to age

Statistically significant differences were observed in the distribution of the species isolated from scalp and trunk skin according to the age groups (P < 0.05).

DISCUSSION

The Malassezia genus contains a group of lipophilic yeasts that form part of the normal human skin. The colony-formation begins immediately after the birth and increases significantly with age of the neonate. It has been reported that skin colonization by Malassezia yeasts was 5% at the first week and 30% at 2-4 weeks of life (28). Using a molecular analysis, Japanese investigators were able to detect Malassezia from 89% and 100% of neonate samples on days 0 and 1 after birth, respectively. Subsequently, the level of Malassezia colonization of the neonates increased with time, whereas that of the mothers did not change (29). Generally, the carriage of Malassezia is the highest during puberty, which is related to the increase in sebaceous gland activity seen at this time.

In one study from Venezuela, 65% cultures yielded positive specimens for Malassezia (30), while earlier studies have identified even greater carriage rates of Malassezia of healthy pubertal children: 74% on the scalp (31), 93% on the back (32), and 87% on the forehead (33).

In adults, the frequency and density of colonization is related to the age and to the activity of the sebaceous glands in the area studied. The highest population densities were noted from chest, upper back and forehead and men yielded more yeasts in lower back and thigh than women (9). Bergbrant and Faergemann found that the density of Malassezia species on the skin decreased with increasing age, which was probably due to a reduction in the level of lipid on the skin (34).

Several studies have been carried out world-wide on the distribution of the newly defined species of Malassezia on healthy adult human skin in which variable results have been reported from different geographical regions (11,14-24) (Table 2).

Table 2

Results from epidemiological studies of Malassezia species isolated from scalp and trunk skin of healthy normal subjects

Country	Sampling method	Culture medium/ Identification method	Site sampled	Number of samples/ pos cultures	Species recovered (% of samples)	Ref.
Spain	Scraping	mDixons/ By Guillot et al. (1996)	Trunk (shoulders)	75/52	M. sympodialis (96) M. slooffiae (4)	14
Japan	Swabing	mDixons/ By Guillot et al. (1996)	Scalp	35/5	M. globosa (6) M. sympodialis, M. furfur, M. slooffiae (3 each)	15
			Trunk	35/24	M. globosa (51) M. sympodialis (26)
Japan	Tape strip	None (direct DNA extraction)/Molecular-Nested PCR	Scalp	18/14	M. restricta (61) M. sympodialis (50) M. globosa (44)	16
Iran	Tape strip	mDixons/ By Guillot et al. (1996)	Not stated	100/60	M. globosa (42) M. sympodialis (25) M. furfur (23)	17
Canada	Contact plates	LNA/ By Guillot et al. (1996)	MS	245/172	M. sympodialis (57) M. globosa (32)	18
Tunis	Swabing	mDixons/ By Guillot et al. (1996)	Trunk (chest)	30/9	M. globosa + M. sympodialis (10) M. furfur, M. sympodialis (7 each)	19
Sweden	Contact plates	LNA/ By Guillot et al. (1996)	Trunk (upper back)	31/26	M. sympodialis (69) M. obtusa (15) M. globosa (12)	20
Korea	Scrub-wash	LNA/ By Guillot et al. (1996)	Scalp	120/56	M. restricta (39) M. globosa (22.5) M. slooffiae (1.5)	11
			Chest	120/101	M. globosa (49) M. restricta (21) M. sympodialis (10)
Iran	Scraping	LNA/ Molecular – PCR-FLPM	Trunk	95/94	M. globosa (70) M. furfur (15) M. sympodialis (6)	21
Japan	Tape strip	None (direct DNA extraction)/Molecular-Nested PCR	Face	30/30	M. globosa (87) M. restricta (83) M. sympodialis (37)	22
Korea	Scrub-wash	LNA/ Molecular-Nested PCR	MS	110/70	M. globosa (32) M. restricta (30) M. sympodialis (15)	23
India	Scraping	mDixons/ By Guillot et al. (1996)	Trunk (upper back)	45/21	M. sympodialis (48) M. obtusa (19) M. globosa (14)	24

mDixons, modified Dixon agar; LNA, Leeming and Notman agar; FLPA, fragment length polymorphism analysis; PCR, polymerase chain reaction; MS, multiple sites

In general, it seems that the most common Malassezia species cultured from healthy scalp skin is M. restricta (11,16), while M. globosa and M. sympodilais are two most frequently isolated species from the trunk (11,14,15,17-24). Other species are less common, but not completely absent.

Similar to the majority of other investigations, we found M. sympodialis as the most frequent species on normal trunk skin isolated in 30% of cases. This species emerges as the predominant species on healthy skin, especially on the trunk, where it can be recovered in great numbers in more than 50% of individuals (18,20).

By contrast, M. globosa has been reported as the most frequently isolated species in some other investigations (15,17,19,21-23). In our study, M. globosa is less common species found in 20% of healthy individuals which goes well with the studies of Gupta et al. (18) and Kaur et al. (24). This species is reported to be a causative agent of pityriasis versicolor, found in filamentous form in the scales from this skin disorder (14,15,17,19,21,35).

M. furfur was isolated in 14% and this species is found to be less common inhabitant of healthy skin (11,17,19,21). However, M. furfur is confirmed to also be responsible for pityriasis versicolor, particularly under tropical climate (36).

In contrast to healthy trunk skin, M. sympodialis was recovered less frequently from the scalp skin of same subjects (8%), whereas M. restricta was the commonest species (33%). This species is isolated regularly from the scalp and face of patients with seborrheic dermatitis (5,14,22). Oh et al. in Korea also found M. restricta to be particularly associated with scalp and M. globosa and M. sympodialis with the chest, whereas other species (M. furfur, M. obtusa and M. slooffiae) were less frequently recovered (23).

In our study, M. slooffiae and M. obtusa were isolated only from a few samples taken from our subjects. These species are considered to be very rare on healthy skin (20,24) and only infrequently isolated from the cases of pityriasis versicolor (17,35), atopic dermatitis (15,20,26) and seborrheic dermatitis (14,20).

M. pachydermatis was not recovered from any of our samples either from trunk or from healthy scalp skin. This species is confirmed to be clearly adapted to animals, although it has been involved in some systemic human infection. The presence of this species on human skin is rare and transient, occurring possibly by transmission from animal pets and environmental sources (37).

Several studies demonstrated that the distribution of Malassezia species on head and trunk region is parallel with the density and activity of pilosebaceous glands in these areas. The difference in species according to different body areas is presumably to be attributed to lipid content and different lipid components in each body area (11).

The higher detection rate of Malassezia species was observed using molecular determination method than by conventional culture methods. These variations may be attributed to the different sampling technique and inadequate determination of the relative proportion of species on the skin, or the consent ability of the fungus to grow in each specified medium that have impact on the range of species recovered (20).

A correlation between the prevalence of Malassezia species and gender of the subjects has been observed without differences among the woman and man. However, statistical difference was noted among age groups and isolated species. M. globosa was the predominant species in adult subjects regardless of body part, M. restricta was recovered more frequently from the scalp skin in the teens and young adults, while on the trunk sin M. sympodialis was most frequently found in older subjects. Our results are concordant with the majority of studies worldwide which clarified that the cutaneous Malassezia microbiota of healthy subjects differed by age (11,13,38). This was contrary to observation of Gupta et al. who cultured more frequently M. globosa on younger subjects and M. sympodialis on the skin of adolescents and adults (18).

In conclusion, researches recovered not only different species distributed on the same anatomic area, but also the same species in even the same frequency from different body areas. These facts suggest that besides different sampling methods and culture media used, there are probably some internal and external factors as well as geographical and ethnic variations reflected in the predominance of different species on the skin of individuals in different parts of the world.

New molecular approaches to the identification of different species will certainly answer many outstanding questions associated with Malassezia species and their role in human pathophysiology.