Trichosporon Species Isolated from Guano Samples Obtained from Bat-Inhabited Caves in Japan

Yeasts from caves have rarely been examined. We examined yeastscollected from bat guano samples from 20 bat-inhabited limestoneand volcanic caves located in 11 prefectures in Japan. Of $~$ 700yeast-like colonies, nine Trichosporon species were recoveredfrom 15 caves. Two of these were known species, and the remainingseven are potentially novel species, based on molecular phylogeneticanalyses. In addition to Trichosporon species, identifiablestrains of eight ascomycetous yeasts and one basidiomycetousyeast were recovered at frequencies of 5 to 35%. Our findingssuggest that Trichosporon spp. are the major yeast species inbat guano in Japan and that bat guano is a potentially richsource of previously undescribed yeast species.

INTRODUCTION

Top

Abstract

Introduction

Sample collection.

Isolation of yeasts.

Caves usually are nutrient-limited sites that are sequesteredfrom the outside environment and may contain novel, diversemicrobial populations. Bat biologists, speleologists, and touristssometimes develop clinical symptoms, such as fever or cough,after visiting bat-inhabited caves. A pulmonary disease (histoplasmosis)that is commonly contracted in bat-inhabited caves of Northand Latin America is caused by Histoplasma capsulatum (3, 10,20). Histoplasmosis is rare in Japan, and it is generally assumedthat most of the cases that do arise are from infections contractedin other countries (7, 18). The excreta of feral birds and animals,including bats, contain medically significant fungi, such asCryptococcus neoformans and Cryptococcus laurentii (5, 12, 23),and locations that contain large amounts of such excreta arepotential sites of human infection. In this presentation, weanalyze yeasts from bat guano from bat-inhabited caves in Japan.

Sample collection.

Top

Abstract

Introduction

Sample collection.

Isolation of yeasts.

Sixty-two bat guano samples were collected between December2003 and February 2004 from 20 bat-inhabited caves of speleologicalinterest (1 to 13 samples per cave), located in 11 Japaneseprefectures (Table 1).

View this table:
[in this window]
[in a new window]
TABLE 1. Yeast isolation from bat guano samples in various Japanese caves

Isolation of yeasts.

Top

Abstract

Introduction

Sample collection.

Isolation of yeasts.

Approximately 0.5 to 1.0 g of each sample of bat guano was suspendedin YM broth (3 g yeast extract, 3 g malt extract, 5 g peptone,10 g glucose, 20 g agar; Becton Dickinson, Paramus, NJ) thatcontained 50 µg/ml chloramphenicol (Sankyo, Tokyo, Japan),400 IU/ml penicillin (Meijiseika, Tokyo, Japan), and 400 IU/mlstreptomycin (Meijiseika). Aliquots of 100 µl of the brothsupernatants were then inoculated onto YM agar plates (at least10 plates per sample) that contained the three antibiotics listedabove, and the plates were incubated at 27°C until coloniescould be seen.

Identification of yeast isolates.

Top

Identification of yeast...

Each yeast isolate was identified with rRNA sequence analysis.Genomic DNA was extracted (11), and the D1/D2 26S rRNA and theinternal transcribed spacer (ITS) region, which includes the5.8S rRNA, were sequenced directly from the PCR products withprimer pairs NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') plus NL-4(5'-GGTCCGTGTTTCAAGACGG-3') (9) and pITS-F (5'-GTCGTAACAAGGTTAACCTGCGG-3')plus pITS-R (5'-TCCTCCGCTTATTGATATGC-3') (17), respectively.The PCR products were sequenced with an ABI 310 DNA sequencerand the Big Dye Terminator Cycle Sequencing Ready kit (Perkin-ElmerApplied Biosystems, Foster City, CA), according to the manufacturer'sinstructions. Strains with a $≥$ 99% ITS or D1/D2 26S rRNA sequencesimilarity were defined as conspecific (15, 17). The sequencedata were analyzed with the National Center for BiotechnologyInformation (NCBI; Bethesda, MD) BLAST system (http://www.ncbi.nlm.nih.gov/BLAST/).

Molecular phylogenetic analysis of new species.

Top

Molecular phylogenetic analysis...

DNA sequences were aligned using CLUSTAL W (19). For the neighbor-joininganalysis (16), distances between the sequences were calculatedusing Kimura's two-parameter model (8). A bootstrap analysiswas performed using 1,000 replications (4).

Yeasts in bat guano.

Top

Yeasts in bat guano.

We identified approximately 700 yeast-like colonies from theYM agar plates. Trichosporon spp. were found in samples from15 of the 20 caves.

Trichosporon laibachii and Trichosporon porosum were isolatedfrom 7 and 5 of the 20 caves, respectively. Based on the molecularphylogenetic analysis, seven new Trichosporon spp. (designatedspecies 1 to 7) were recovered from 10 of the caves. In additionto the Trichosporon spp., eight ascomycetous yeasts (Candidapalmioleophila, Candida lusitaniae, Debaryomyces hansenii, Hanseniasporaspp., Saccharomyces cerevisiae, Saccharomyces kluyveri, Williopsiscalifornica, and Zygosaccharomyces florentinus) and one basidiomycetousyeast (Cryptococcus podzolicus) were isolated from the guanosamples at frequencies that ranged from 5 to 35% for the 20caves (Table 1).

Phylogenetic analysis of the new Trichosporon species.

Top

Phylogenetic analysis of the...

As the level of dissimilarity of the D1/D2 26S rRNA or ITS sequencesbetween Trichosporon species 1 to 7 and the phylogeneticallyclosest species was >1%, we concluded that Trichosporon species1 to 7 were new species. The new species were not located withina single clade on the tree (Fig. 1) but were distributed acrossseveral clades (Gracile, Porosum, Brassicae, and Cutaneum).The Ovoides clade was the only clade for which we did not identifya new species.

View larger version (58K):
[in this window]
[in a new window]
FIG. 1. Phylogenetic trees constructed using the D1/D2 26S rRNA sequences of Trichosporon spp. that were isolated from bat guano samples and from known species of Trichosporon. DDBJ/GenBank accession numbers are indicated in parentheses. The numerals near branch points are confidence limits for 1,000 bootstrap replicates (limits of <50% are not indicated). Knuc, Kimura's parameter (8).

In recent years, research on bio-speleology and microbial diversityhas increased. Pathogenic fungi are potential sources of infectionand allergy for speleologists. A similar investigation in Italy(13) found the pathogenic yeasts Cryptococcus neoformans andCryptococcus laurentii in 3 of 25 cavernicolus fungus-inhabitedcaves. Although C. neoformans colonization of pigeon droppingsis well known (23), there are few reports of this microorganismfrom caves.

We detected nine Trichosporon species, including seven novelspecies, in bat guano samples from 75% of the caves sampled.It is not known whether the Trichosporon spp. are part of thecutaneous or intestinal microbiota of the bats or if they areintroduced into bat guano from other organisms, the soil, orother excreta in the cave. Repeated inhalation of Trichosporoncells may cause summer-type hypersensitivity pneumonitis, withconsequent development of cough, dyspnea, and remittent fever(1). These symptoms are similar to those that may develop inspeleologists after they visit caves.

Trichosporon spp. have four different serotypes (types I, II,III, and I-III) (6, 14). Each serotype is associated with theoccurrence of summer-type hypersensitivity pneumonitis, andthere is a correlation between the serotype of inhalation challenge-positiveantigens with those of Trichosporon spp. from patients' homes(2). These serotypes also correspond to the phylogenetic clades.Trichosporon serotypes I, II, III, and I-III are associatedwith species in the Cutaneum, Ovoides, Brassicae, and Gracile-Porosumclades, respectively (Fig. 1).

In addition to the Trichosporon species, we isolated Candidalusitaniae and Debaryomyces hansenii from the collected batguano. These species have been implicated in opportunistic infectionsof immunocompromised patients (21, 22). Because bats are widelydistributed in the environment, bat guano may mediate the exchangeof these pathogens, just as pigeon excreta mediate the exchangeof Cryptococcus neoformans, the causal agent of cryptococcosis(23).

In conclusion, our analysis provides new, basic informationon potential sources of infection and allergy for speleologists.Japanese bat guano contains several novel yeast species andmay be of significant mycological interest.

ACKNOWLEDGMENTS

We thank the members of the Speleological Society of Japan andthe local and university caving clubs that cooperated in collectingbat guano samples.

This study was supported in part by a Health Science ResearchGrant for "Research on Emerging and Re-emerging Infectious Diseases"from the Ministry of Health, Labor, and Welfare of Japan.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. Phone/Fax: 81-424-95-8762. E-mail: sugita{at}my-pharm.ac.jp.

REFERENCES

Top