Eukaryote culturomics of the gut reveals new species.

The repertoire of microeukaryotes in the human gut has been poorly explored, mainly in individuals living in northern hemisphere countries. We further explored this repertoire using PCR-sequencing and culture in seven individuals living in four tropical countries. A total of 41 microeukaryotes including 38 different fungal species and three protists were detected. Four fungal species, Davidiella tassiana, Davidiella sp., Corticiaceae sp., and Penicillium sp., were uniquely detected by culture; 27 fungal species were uniquely detected using PCR-sequencing and Candida albicans, Candida glabrata, Trichosporon asahii, Clavispora lusitaniae, Debaryomyces hansenii, Malassezia restricta, and Malassezia sp. were detected using both molecular and culture methods. Fourteen microeukaryotes were shared by the seven individuals, whereas 27 species were found in only one individual, including 11 species in Amazonia, nine species in Polynesia, five species in India, and two species in Senegal. These data support a worldwide distribution of Malassezia sp., Trichosporon sp., and Candida sp. in the gut mycobiome. Here, 13 fungal species and two protists, Stentor roeseli and Vorticella campanula, were observed for first time in the human gut. This study revealed a previously unsuspected diversity in the repertoire of human gut microeukaryotes, suggesting spots for further exploring this repertoire.

Introduction

The human gut microbiota is a diverse ecosystem comprising of bacteria, archaea, virus and eukaryotes referred to as the gut microbiota. It has been observed that the composition of gut microbiota depends on environmental factors [1], [2]. Numerous studies focused on gut bacteria, but the repertoire of gut microeukaryotes has been poorly explored [3]–[8]. Previous studies indicated that components of the gut microbiota, including gut microeukaryotes, were interacting one with each other [9], [10]. Recently, high-throughput sequencing and clone library sequencing of gut microeukaryote community indicated that fungi and Blastocystis were the two dominant components of gut microeukaryote community [4]–[6], [8], [11]. Interestingly, fungal abundance was found to be significantly associated with recently consumed foods: in particular Candida spp. abundance significantly correlated with recent consumption of diet rich in high carbohydrates [12]. Likewise, our previous studies on eukaryote community in an obese individual and in an anorexic individual and revealed fungi diversity related to diet [4], [13]. A diversity of eukaryotic fungi was detected in healthy individuals and infants with low weight [5], [6], [8]. Despite evidence for the gut microeukaryote community being influenced by the environment, a few studies have been reported from a limited number of individuals, mainly living in the northern hemisphere countries. Indeed, of twelve studies, three issued from individuals in Europe [4], [7], [8], three from the USA [6], [12], [14], two from China [3], [15], one from India [16], one from Turkey [17], one from Korea [11] and one from Senegal [5]. Therefore, the current body of knowledge may not be representative of the actual diversity of this repertoire, as no data issued from individuals living in southern hemisphere countries such as Polynesia and Amazonia. Here, in an effort to broaden knowledge on gut microeukaryotes, we investigated microeukaryotes in seven individuals living in four tropical countries.

Materials and Methods

Fecal sample collection

The study was approved by the local ethics committee of the Institut Fédératif de Recherche 48 (IFR 48, Marseille, France; agreement number 09–022). After the participants' written consent was obtained, a stool sample was collected from three individuals from Polynesia (Iles Raietea, rural area), two individuals from Amazonia (Manaus, urban area, forest area), one individual from Senegal (N'Diop, rural area) and one individual in India (New Dheli) (Table 1). No specific pathology was reported in any of these individuals. Each stool sample was preserved as 1-g-aliquots in sterile microtubes stored at −80°C until use.

Table 1

Clone library of microeukaryotes in seven stool samples collected in four different countries. The number of positive clones / total number of clones is given into brackets.

Location	Individual (sex, age)	ITS1F/ITS4R	Euk1A/Euk516r	AmiF/AmiR	MAlF/MalR
Amazonia 1 (Manaus, urban area)	M, 49	Candida albicans (36/48) Clavispora lusitaniae (12/48)		Alternaria alternata (2/48)	Malassezia sp. (40/48) Malassezia restricta (4/48)
Amazonia 2 (Amazonian forest area)	M, 39	Candida albicans (30/48) Trichosporon asahii (12/48)	Vorticella campanula (1/48) Saccharomyces cerevisiae (2/48) Phytophthora pinifolia (1/48) Trichosporon asahii (10/48) Pleosporales sp.(3/48) Puccinia poarum (1/48) Sclerotinia sclerotiorum (2/48) Rhodosporidium babjevae (2/48)	Filobasidium capsuligenum (6/48) Penicillium chrysogenum (2/48) Vorticella campanula (1/48) Exophiala equina (1/48)	Malassezia sp. (22/48) Malassezia restricta (10/48) Malassezia globosa (2/48)
Polynesia1 (Rural area)	M, 48	Trichosporon asahii (12/48) Candida albicans (30/48) Trichosporon faecale1/48	Westerdykella cylindrica (2/48) Rhodotorula mucilaginosa (2/48) Blastocystis sp. (1/48)		Malassezia sp.(22/48) Malassezia restricta (6/48) Malassezia globosa (2/48)
Polynesia 2 (Rural area)	M, 32	Trichosporon asahii (18/48) Candida albicans (20/48) Galactomyces geotrcichum (1/48)	Clitopilus prunulus (1/48)		Malassezia sp.(20/48) Malassezia restricta (12/48)
Polynesia 3 (Iles Raiatea)	M, 49	Saccharomycetales sp. (2/48) Clavispora lusitaniae (20/48) Fomitopsis pinicola (1/48) Fomes fomentarius (1/48)	Candida glabrata (6/48) Bispora christiansenii (1/48) Yarrowia lipolytica (1/48)
India (New Delhi)	M, 28	Candida albicans (12/48) Trichosporon asahii (20/48 Geotrichum candidum (1/48)	Debaryomyces hansenii (5/48) Filobasidium globisporum (1/48) Candida dubliniensis (1/48) Stentor roeseli (1/48) Rhodotorula mucilaginosa (5/48) Westerdykella cylindrica (2/48)		Malassezia pachydermatis (28/48) Malassezia restricta (10/48) alassezia globosa (5/48)
Senegal (N'Diop)	M, 20	Galactomyces geotrichum (7/48)v Dipodascaceae sp. (2/48) alactomyces candidum (8/48)		Saccharomyces cerevisiae(4/48) Aspergillus restrictus (10/48)	Malassezia restricta (10/48) Malassezia sp.(25/48)

DNA-based analyses

Total DNA was extracted using the Qiamp stool mini kit (Qiagen, Courtaboeuf, France) as previously described using mechanic and enzymatic lyses [4]. Potential PCR inhibitors were tested by mixing Acanthamoeba castellanii DNA with DNA extracted from stool specimen prior to PCR, as previously described [4]. A set of 35 eukaryotic PCR primer pairs obtained from the literature were used independently to target the 18S rRNA gene and the internal transcribed spacer (ITS) on all seven specimens as previously described [4]. The PCR reaction (50 µL final volume) contained 5 µL of dNTPs (2 mM of each nucleotide), 5 µL of DNA polymerase buffer (Qiagen), 2 µL of MgCL2 (25 mM), 0.25 µL HotStarTaq DNA polymerase (1.25 U) (Qiagen), 1 µL of each primer (Eurogentec, Seraing, Belgium) and 5 µL of DNA. PCR included a 15-min initial denaturation at 95°C followed by 40 cycles including denaturation at 95°C for 30-sec and extension at 72°C for 1 min. A 5-min final extension was performed at 72°C. All PCRs were performed using the 2720 thermal cycler (Applied Biosystems, Saint Aubin, France). PCR buffer without DNA was used as a negative control for each PCR run. PCR products were visualized by electrophoresis using a 1.5% agarose gel. The PCR products were purified using the Nucleo- Fast 96 PCR Kit (Marcherey-Nagel, Hoerdt, France) according to the manufacturer's instructions. PCR products were cloned separately using the pGEM -T Easy Vector System Kit as described by the manufacturer (Promega, Lyon, France). Forty-eight white colonies were collected from each PCR product and sequenced using the Big Dye Terminator V1.1 Cycle Sequencing Kit (Applied Biosystems) and M13 primers on ABI PRISM 3130 automated sequencer (Applied Biosystems). Chimeras were eliminated by analyzing sequence with ChromasPro Software. Sequences were compared with sequences available in the GenBank database using BLAST. A 97% sequence similarity and 95% coverage with a described species were used for molecular identification [7]. All the sequences obtained in this work have been deposited in GenBank database with accession number KF768259-KF768340.

Culture and identification of fungi

Stool samples were diluted in sterile phosphate-buffered saline (PBS) and cultured on potato dextrose agar (PDA) (Sigma-Aldrich, Saint-Quentin Fallavier, France) from potato infusion and dextrose, Czapeck dox agar (Sigma-Aldrich), semi-synthetic solid medium containing sucrose as C-source and sodium nitrate as the sole source of nitrogen supplemented with 0.05 g/L chloramphenicol and 0.1 g/L gentamycin, Sabouraud dextrose agar (BD diagnostic system) and Dixon agar [18] supplemented with 0.05 mg/mL chloramphenicol and 0.2 mg/mL cycloheximide. Dixon agar medium was prepared as previously described [4]. Following previously published protocols [3], [19], [20] the agar plates were kept in plastic bags with humid gas to prevent desiccation and incubated aerobically at room temperature (∼25°C) in the dark [21]. Dixon agar medium plates were incubated aerobically at 30°C. Growth was observed for two weeks. The dilution solution of the sample was spread on the same media and incubated in the same conditions as a negative control. Fungi were identified with ITS 1F / ITS 4R and MalF/Mal R. Purified PCR were sequenced using the ITS1R/ ITS4 and MalF/Mal R primers with the Big Dye Terminator V1,1 Cycle Sequencing Kit (Applied Biosystems). When the peaks of the sequence overlapped, the amplification products were cloned.

Results

Culture-independent methods

In all PCR-based experimental the negative controls remained negative. Among the primers tested, four sets of primers yielded positive amplifications ( ). A. castellanii DNA mixed with stool sample yielded positive amplification. A total of 1,056 clones were sequenced and 528 sequences identified 37 microeukaryotes including 34 fungal species and three protists Stentor roeseli, Vorticella campanula and Blastocystis sp. (). Plant and human DNA sequences were excluded for analysis. Species distribution in stools from geographical locations is shown in . Malassezia spp. and Candida spp. were detected in all stools from different locations except in Polynesia 3 and Senegal, respectively.

Figure 1

Microeukaryotes species distributions in seven stool samples from different geographical locations detected by PCR-based methods.

Culture dependant-methods

While the negative control plates remained sterile, stool specimens grew Candida albicans (4 positive), Trichosporon asahii (4 positive), Malassezia restricta (2 positive), and Candida glabrata, Clavispora lusitaniae, Debaryomyces hansenii, Malassezia sp., Corticiaceae sp., Davidiella tassiana, Davidiella sp., and Penicillium sp. each in one specimen. The four later fungal species were detected uniquely by culture-dependant methods ( ).

Table 2

Fungal species isolated using four culture media in stool samples collected in four different countries.

Location			Medium
	PDA	SDA	CZPEK	DA
Amazonia1	Candida glabrata Candida albicans	Candida glabrata Candida albicans	Candida glabrata Candida albicans	Candida albicans Candida glabrata Davidiella tassiana
Polynesya1		Trichosporon asahii Penicillium sp.
Polynesya2		Candida albicans		Candia albicans Malassezia restricta Trichosporon asahii Davidiella sp.
Polynesya3	Trichosporon asahii Candida albicans	Trichosporon asahii Candida albicans		Debaryomyces hansenii Trichosporon asahii
India	Candida albicans Trichosporon asahii Clavispora lusitaniae	Trichosporon asahii Clavispora lusitaniae Corticiaceae sp.	Penicillium sp.	Malassezia restricta Candida albicans Malassezia sp.

Overall results

Combining of the two approaches yielded a total of 38 different fungal species and three protists including S. roeseli, V. campanula and Blastocystis sp. Thirteen fungal species and two protists were observed for the first time in the human gut ( ). Fourteen fungal species including C. albicans, C. glabrata, C. lusitaniae, D. hansenii, Galactomyces candidum, Galactomyces geotrcichum, Malassezia globosa, M. restricta Malassezia sp. Penicillium sp., Rhodotorula mucilaginosa, Saccharomyces cerevisiae, T. asahii and Westerdykella cylindrica were shared by the seven individuals whereas 27 microeukaryotes were found in only one individula including 11 species in Amazonia, nine species in Polynesia, five species in India and two species in Senegal.

Table 3

List of microeukaryotes detected by culture and PCR-cloning-sequencing in the gut of seven individuals.

Individual	Species
	Culture	PCR-based
Amazonia 1	C. albicans C. glabrata D. tassiana	C. albicans Clavispora lusitaniae Alternaria alternata Malassezia sp. Malassezia restricta
Amazonia 2		C. albicans T. asahii V. campanula S. cerevisiae P. pinifolia Pleosporales sp. P. poarum S. sclerotiorum R. babjevae F. capsuligenum P. chrysogenum V. campanula E. equine Malassezia sp. M. restricta M. globosa
Polynesia 1	T. asahii Penicillium sp.	T. asahii C. albicans T. faecale1 Malassezia sp. M. restricta M. globosa
Polynesia 2	C. albicans M. restricta T. asahii Davidiella sp.	T. asahii C. albicans G. geotrichum C. prunulus Malassezia sp. M. restricta
Polynesia 3	D. hansenii C. albicans T. asahii	Saccharomycetales sp. C. lusitaniae F. pinicola F. fomentarius C. glabrata B. christiansenii Y. lipolytica
India	T. asahii C. lusitaniae Corticiaceae sp. Penicillium sp M. restricta C. albicans Malassezia sp.	C. albicans T. asahii G. candidum D. hansenii F. globisporum C. dubliniensis S. roeseli R.mucilaginosa W. cylindrica M. pachydermatis M. restricta M. globosa
Senegal		G. geotrichum Dipodascaceae sp. G. candidum S. cerevisiae A. restrictus M. restricta Malassezia sp.

Discussion

Here, further exploration of gut microeukaryotes in stool specimens collected in seven individuals living in four tropical countries yielded new data about this poorly explored component of the gut microbiota. Fifteen species were observed for the first time in the human gut. S. roeseli detected in stool from India and V. campanula in stool from Amazonia, are two ciliates previously described from environment in particular in freshwater [22], [23]. Moreover, environmental fungi Puccinia poarum, Rhosporidium babjevae, Phytophthora pinifolia, Alternaria alternata, Aspergillius restrictus, Bispora christiansenii, D. tassiana, Davidiella sp. and W. cylindrical were previously described as plant pathogen or from fresh water [24]–[30]. F. capsuligenum was previously found in fruit, brewery and in soil [31]. These fungal species have not been previously reported in the human gut.

Some opportunist pathogenic fungi including C. albicans, C. glabrata, Filobasidium globisporum, T. asahii, C. lusitaniae, Rhodotorula mucilaginosa, M. restricta, M. globosa and Yarrowia lipolytica were previously described in the human gut [3], [4], [7], [15]. Geotrichum candidum and Saccharomyces cerevisiae were encountered in the human gut and associated with the consumption of cheese and brewery [16], [32], [33]. Similar study found a correlation between diet and fungi detected in gut [12]. Exophiala equina is an environmental fungi previously reported in dialysis water and subscutaneous abcesses [34], [35].

Previous studies on eukaryotes diversity in people from Korea, the United Kingdom and Senegal detected some fungal species different from our study and this could be related of individuals location or diet. Here, the high diversity of microeukaryotes observed in Amazonia, India and Africa could be related to individual environment. Similar study on gut bacteria microbiota found the bacteria to be related to host environment and diet [1], [2], [36]. Our findings are in the same line with previous observation that found that fungal species and protists are dominant components of gut microeukaryotes [8].

Conclusions

A diversity of 41 microeukayotes species including 38 fungal species and three protist was detected in stool samples collected from four different tropical locations. A total of 13 fungal species and two protists Stentor roeseli and Vorticella campanula were observed in the human gut for the first time. Indeed, these microeucaryotes have not been detected among the 249 fungi and the 36 protists cultured and detected from the human gut so far. These data plea for more extensive studies being performed in specimens collected from various geographical regions to further establish the human gut microeukaryote repertoire.