**2.1 Colonization by the fungus** *Malassezia* **in patients with atopic dermatitis**

The lipophilic yeast *Malassezia* is the predominant fungus on human skin. Morphologically, these microorganisms are ovoid, elongate, and cylindrical (Fig. 1). Their genome is smaller than that of other fungi (Xu *et al*. 2007). As *Malassezia* species require lipids for growth, they preferentially colonize sebum-rich areas such as the head, face, or neck, rather than the limbs or trunk. Specific IgE antibodies against *Malassezia* are present in the serum of patients with AD, and antifungal therapy can improve the symptoms of AD by decreasing the degree of colonization by *Malassezia*; thus, this fungus is believed to be an exacerbating factor in AD (more details are provided in a later chapter). In contrast to *S. aureus*, *Malassezia*  species colonize both AD patients and healthy individuals. In addition to AD, *Malassezia* species are responsible for seborrheic dermatitis, folliculitis, and ptiryasis versicolor (Gupta *et al*. 2004; Ashbee 2007). Currently, 14 species are recognized within the genus *Malassezia* (Table 1), and five of these (*M. caprae*, *M. cuniculi*, *M. equina*, *M. nana*, and *M. pachydermatis*) show affinity for nonhuman animals.


AD, atopic dermatitis; SD, seborrheic dermatitis; SI, systemic infection; PV, pityriasis versicolor

Table 1. Currently accepted *Malassezia* species

A number of epidemiological studies have been conducted during the past decade to elucidate the role of *Malassezia* as an exacerbating factor in AD. The first was carried out by Nakabayashi *et al*. (2000) in Japan and detected *M. furfur*, *M. globosa*, *M. sympodialis*, and *M.* 

The lipophilic yeast *Malassezia* is the predominant fungus on human skin. Morphologically, these microorganisms are ovoid, elongate, and cylindrical (Fig. 1). Their genome is smaller than that of other fungi (Xu *et al*. 2007). As *Malassezia* species require lipids for growth, they preferentially colonize sebum-rich areas such as the head, face, or neck, rather than the limbs or trunk. Specific IgE antibodies against *Malassezia* are present in the serum of patients with AD, and antifungal therapy can improve the symptoms of AD by decreasing the degree of colonization by *Malassezia*; thus, this fungus is believed to be an exacerbating factor in AD (more details are provided in a later chapter). In contrast to *S. aureus*, *Malassezia*  species colonize both AD patients and healthy individuals. In addition to AD, *Malassezia* species are responsible for seborrheic dermatitis, folliculitis, and ptiryasis versicolor (Gupta *et al*. 2004; Ashbee 2007). Currently, 14 species are recognized within the genus *Malassezia* (Table 1), and five of these (*M. caprae*, *M. cuniculi*, *M. equina*, *M. nana*, and *M. pachydermatis*)

**Host Species Species implicated in skin** 

*Malassezia furfur* SI

*Malassezia globosa* AD, SD, PV

*Malassezia sympodialis* AD, SD *Malassezia restricta* AD, SD, PV

**disease in human** 

**2. The fungal microbiome in patients with atopic dermatitis** 

show affinity for nonhuman animals.

Nonhuman animal associated

species *Malassezia caprae* 

Table 1. Currently accepted *Malassezia* species

**2.1 Colonization by the fungus** *Malassezia* **in patients with atopic dermatitis** 

Human associated species *Malassezia dermatis* AD

*Malassezia japonica Malassezia obtusa Malassezia slooffiae* 

*Malassezia yamatoensis*

*Malassezia cuniculi Malassezia equina Malassezia nana Malassezia pachydermatis*  AD, atopic dermatitis; SD, seborrheic dermatitis; SI, systemic infection; PV, pityriasis versicolor

A number of epidemiological studies have been conducted during the past decade to elucidate the role of *Malassezia* as an exacerbating factor in AD. The first was carried out by Nakabayashi *et al*. (2000) in Japan and detected *M. furfur*, *M. globosa*, *M. sympodialis*, and *M.*  *slooffiae* in 21.4, 14.3, 7.1, and 3.6% of samples from Japanese AD patients, respectively. A study conducted in Sweden in 2005 produced similar results (Sandström *et al*. 2005). However, a Canadian study by Gupta *et al*. (2001) reported the predominant species to be *M. sympodialis*, which was detected in 51.3% of the samples from AD patients. All of these studies were performed using culture-dependent methods. In all cases, scale samples were collected by an appropriate method, e.g., swabbing, scratching, or stripping, and were incubated in medium containing several types of fatty acids. The recovered microorganisms were identified based on biochemical or physiological characteristics, including assimilation of Tween compounds and esculin, catalase reaction, and maximum growth temperature (Guého-Kellermann 2010; Kaneko *et al*. 2007). However, culture-dependent methods may not provide accurate and reliable results for *Malassezia*. The efficiency of culturing *Malassezia* strains depends on the isolation medium used, and the growth of some species, such *M. obtusa* and *M. restricta*, is slower than that of others.

Magnification is x5,000.

Fig. 1. Morphology of *Malassezia restricta* by scanning electron microscope

To overcome the difficulties of culture-dependent methods, including scale sampling methods, culturing conditions, and isolation techniques, Sugita *et al*. (2001) developed the first molecular analytical method for *Malassezia*. For this method, scale samples are collected by stripping with medical transparent dressing, and skin *Malassezia* DNA is directly extracted from the dressing. The *Malassezia* microbiota is then analyzed by realtime PCR, specific detection by PCR with a species-specific primer, or an rRNA clone method (Sugita *et al*. 2011). Although more expensive than culture-dependent methods, a

Atopic Dermatitis and Skin Fungal Microorganisms 127

ranged from 4 to 11 for (CT1)n, 3 to 10 for (CT2)n, and 3 to 11 for (CT3)n, with 4 (CT1)n repeats in 50% of the samples, 8 (CT2)n in 60%, and 9–11 (CT3)n in 80%. For (GT)n, the respective numbers of repeats in 70–80% of the SSRs in the IGS 1 region were 9–11 in samples from AD patients and 15–19 in samples from healthy individuals. A phylogenetic tree constructed from 52 IGS 1 sequences is shown in Fig. 4. The tree consists of four major groups, which correspond to the sources of the samples (AD patients or healthy individuals). Two groups are from AD patients, and one is from healthy individuals. The remaining group included samples from both AD patients and healthy individuals. The IGS 1 sequences were more diverse in the samples from healthy individuals compared with AD patients. The IGS 1 sequence similarity was 94.5 ± 3.5% among the AD patient samples and 89.9 ± 3.5% among the samples from healthy individuals. The IGS 1 sequences of *M. restricta* are divided into two

major groups, corresponding to AD patients and healthy individuals.

Fig. 3. DNA sequences of the IGS 1 region of *M. globosa.* 

*restricta* were almost identical (*p* > 0.05) in the severe patients (Fig. 5B).

The *Malassezia* microbiota of the skin is also associated with the severity of AD. Fifty-six adult neck and head AD patients (21 mild, 18 moderate, and 17 severe cases) and 32 healthy individuals were examined for skin *Malassezia* microbiota, using a real-time PCR assay (Kaga *et al*. 2011). The level of colonization by *Malassezia* was almost identical among the mild and moderate AD patients and the healthy individuals, while *Malassezia* colonization in the severe AD cases was approximately 2- to 5-fold that in the mild and moderate AD patients and healthy individuals (Fig. 5A). Two major species, *M*. *globosa* and *M*. *restricta*, accounted for more than 80% of all *Malassezia* colonization in AD patients of all severities, but their proportions differed with severity. In the mild and moderate cases, *M. restricta* predominated over *M. globosa* (*p* < 0.05), whereas the proportions of *M. globosa* and *M.* 

**2.2** *Malassezia* **colonization and severity of AD** 

molecular-based, non-culture approach appears to be the most reliable and appropriate for analysis of the skin *Malassezia* microbiota (Sugita *et al*. 2001; Morishita *et al*. 2006; Takahata *et al*. 2007a, 2007b; Tajima *et al*. 2008; Amaya *et al*. 2007). In all scale samples from AD patients, both *M. globosa* and *M. restricta* were detected by the molecular-based method, with the level of colonization by *M. restricta* being approximately 1.6 times that of *M. globosa* (Sugita *et al*. 2006a). *Malassezia sympodialis* was the second most predominant species (detected in 58% of the cases), and *M. dermatitis*, *M. furfur*, *M. obtusa*, and *M. slooffiae* were detected in less than 30% of the cases (Fig. 2). These results suggest that both *M. globosa* and *M. restricta* may significantly exacerbate AD.

*Malassezia* DNA was detected by nested PCR assay with species-specific primers

Fig. 2. Colonization frequency of *Malassezia* in the scale of patient with atopic dermatitis

Given that *M. globosa* and *M. restricta* commonly colonize both AD patients and healthy individuals, specific genotypes of these microorganisms may play a role in AD (Sugita *et al*. 2003, 2004, 2010). The fungal rRNA gene consists of four subunits: 5S, 5.8S, 18S (small), and 26S (large). Located between the subunits are an internal transcribed spacer (ITS) and an intergenic spacer (IGS). In *M. globosa*, the IGS is 444 to 454 bp long and has four short sequence repeats (SSRs), (CT1)n, (CT2)n, (CT3)n, and (GT)n, which occur at positions 29–49, 278–291, 380–485, and 242–267, respectively, in the IGS sequence of *M. globosa* strain CBS 7996. Alignments of IGS 1 sequences of two *M. globosa* strains are shown in Fig. 3. The number of (CT)n SSRs in the IGS 1 region is more variable in samples from healthy individuals than in those from AD patients. In samples from AD patients, the number of sequence repeats in the IGS 1 region

molecular-based, non-culture approach appears to be the most reliable and appropriate for analysis of the skin *Malassezia* microbiota (Sugita *et al*. 2001; Morishita *et al*. 2006; Takahata *et al*. 2007a, 2007b; Tajima *et al*. 2008; Amaya *et al*. 2007). In all scale samples from AD patients, both *M. globosa* and *M. restricta* were detected by the molecular-based method, with the level of colonization by *M. restricta* being approximately 1.6 times that of *M. globosa* (Sugita *et al*. 2006a). *Malassezia sympodialis* was the second most predominant species (detected in 58% of the cases), and *M. dermatitis*, *M. furfur*, *M. obtusa*, and *M. slooffiae* were detected in less than 30% of the cases (Fig. 2). These results suggest that both

*M. globosa* and *M. restricta* may significantly exacerbate AD.

*Malassezia* DNA was detected by nested PCR assay with species-specific primers

Fig. 2. Colonization frequency of *Malassezia* in the scale of patient with atopic dermatitis

Given that *M. globosa* and *M. restricta* commonly colonize both AD patients and healthy individuals, specific genotypes of these microorganisms may play a role in AD (Sugita *et al*. 2003, 2004, 2010). The fungal rRNA gene consists of four subunits: 5S, 5.8S, 18S (small), and 26S (large). Located between the subunits are an internal transcribed spacer (ITS) and an intergenic spacer (IGS). In *M. globosa*, the IGS is 444 to 454 bp long and has four short sequence repeats (SSRs), (CT1)n, (CT2)n, (CT3)n, and (GT)n, which occur at positions 29–49, 278–291, 380–485, and 242–267, respectively, in the IGS sequence of *M. globosa* strain CBS 7996. Alignments of IGS 1 sequences of two *M. globosa* strains are shown in Fig. 3. The number of (CT)n SSRs in the IGS 1 region is more variable in samples from healthy individuals than in those from AD patients. In samples from AD patients, the number of sequence repeats in the IGS 1 region ranged from 4 to 11 for (CT1)n, 3 to 10 for (CT2)n, and 3 to 11 for (CT3)n, with 4 (CT1)n repeats in 50% of the samples, 8 (CT2)n in 60%, and 9–11 (CT3)n in 80%. For (GT)n, the respective numbers of repeats in 70–80% of the SSRs in the IGS 1 region were 9–11 in samples from AD patients and 15–19 in samples from healthy individuals. A phylogenetic tree constructed from 52 IGS 1 sequences is shown in Fig. 4. The tree consists of four major groups, which correspond to the sources of the samples (AD patients or healthy individuals). Two groups are from AD patients, and one is from healthy individuals. The remaining group included samples from both AD patients and healthy individuals. The IGS 1 sequences were more diverse in the samples from healthy individuals compared with AD patients. The IGS 1 sequence similarity was 94.5 ± 3.5% among the AD patient samples and 89.9 ± 3.5% among the samples from healthy individuals. The IGS 1 sequences of *M. restricta* are divided into two major groups, corresponding to AD patients and healthy individuals.

Fig. 3. DNA sequences of the IGS 1 region of *M. globosa.* 
