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

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ~700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.     

Is Histoplasma capsulatum a native inhabitant of Japan?

Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat‐inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma‐specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.     

Environmental implications of pollen spectra in bat droppings from southeastern Spain and potential for palaeoenvironmental reconstructions

Pollen was analysed from bat guano from nine caves in southeastern Spain and surface soils in their immediate surroundings. We compare the pollen spectra of 34 modern dung samples from the nine caves with one modern surface pollen sample from each cave. The contents suggest reasonable pollen diversity and richness, including anemophilous and zoophilous pollen types. Since the latter is usually under-represented in atmospheric pollen, the guano spectra therefore appear to reflect the vegetation more effectively than normal surface soil samples. Despite health hazards such as histoplasmosis, the difficulties of obtaining bat guano in deep caves and possible interpretational concerns relating to behaviour and feeding habits of different bat species, this material can be very useful in palaeoecological research provided that the dung was fossilized under favourable environmental conditions that allowed the preservation of pollen.     

Isolation of fungi from bats of the Amazon basin

A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi. From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered. Seven known pathogenic species were present: Candida parapsilosis, C. guilliermondii, C. albicans, C. stellatoidea, C. pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata. Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ. The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats. Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates. Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi.     

Isolation of fungi from bats of the Amazon basin.

A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi. From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered. Seven known pathogenic species were present: Candida parapsilosis, C. guilliermondii, C. albicans, C. stellatoidea, C. pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata. Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ. The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats. Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates. Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi.     

Identification and Molecular Analysis of Pathogenic Yeasts in Droppings of Domestic Pigeons in Beijing, China

Feral pigeons are known as reservoirs of pathogenic yeasts that cause opportunistic infections in human. In the outskirts of Beijing, China, pigeons are more frequently raised at homes than are encountered in public areas. Many studies have focused on the presence of pathogenic yeasts in the excreta (fresh or withered) of a variety kinds of birds, pigeon crop and cloacae. One hundred and forty-three samples of fresh droppings were collected from three suburban pigeon-raising homes in an area of northern Beijing, China. The internal transcribed sequences (ITS) of all strains (except for 8 strains of Rhodotorula sp. ) were sequenced and compared with those of the databases of the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov ) using the Basic Local Alignment Search Tool (BLAST). Yeasts representing 8 genera, Cryptococcus, Filobasidium, Rhodotorula, Candida, Debaryomyces, Saccaromyces, Trichosporon and Sporidiobolus, were identified from 120 isolates. Cryptococcus was the most prolific genera represented by eight species. The populations of yeast species isolated from fresh pigeon droppings were different among homes. Although it is well established that Cryptococcus neoformans exists mainly in old pigeon guano, several C. neoformans strains were still isolated from fresh pigeon excreta, providing a clue that live cryptococcal cells could move through the gastrointestinal tract of the pigeons. Eight genera identified from fresh droppings of domestic pigeons further confirm that pigeons serve as reservoirs, carriers and even spreaders of Cryptococcus species and other medically significant yeasts. The proportion of pathogenic yeasts in all isolates is more than 90 %.     

Guano exposed: Impact of aerobic conditions on bat fecal microbiota

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large‐scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost‐effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12 hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environment—much faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non‐exposed samples could still be recovered after 12 hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.     

Leptogenys khammouanensis sp. nov. (Hymenoptera: Formicidae). A possible troglobitic species of Laos,with a discussion on cave ants

The new species Leptogenys khammouanensis sp. nov. is described from two caves of the Khammouan karst (Laos). It is characterized by a set of striking morphological characters (reduced eyes, light pigmentation, slender body and very elongated legs and antennae), which recall the troglobiomorphic traits of cave arthropods. Relations between caves and ants are discussed at this occasion, in the light of the recent biological explorations of caves in Southeast Asia. The classical view that ants are rare and unimportant in caves is challenged. Ants are actually major and regular components of guano assemblages in many caves of the region, but none of these guano species exhibit cave-related adaptation in its external morphology. Conversely, ants are very rare in low-resources habitats, where only accidental occurrence of outside species are reported in Southeast Asia. Leptogenys khammouanensis has been found only in such an oligotrophic environment, very deep in the cave and far from any guano deposits. Its presence there, together with its troglobiomorphic traits, support the idea that Leptogenys khammouanensis might be the first truly troglobitic ant.     

<Emphasis Type="Italic">Chrysosporium speluncarum</Emphasis>, a new species resembling <Emphasis Type="Italic">Ajellomyces capsulatus,</Emphasis> obtained from bat guano in caves of temperate Europe

A new species, Chrysosporium speluncarum, obtained from bat guano in caves in the Czech Republic, Slovakia, and Romania, is described, based on phenotype and rDNA sequences. It possesses tuberculate conidia similar to the Histoplasma anamorph of Ajellomyces capsulatus, but it is not dimorphic, does not grow at 37°C, lacks microconidia, and is nonpathogenic and nonkeratinolytic. All studied characteristics suggest a close relationship with Renispora flavissima. The morphologically similar C. chiropterorum, another species known from the bat environment, is a relative of C. lobatum and Polytolypa hystricis, but is distinct from A. capsulatus and C. speluncarum. The affinity for bats and the presence of conidia with prominent tuberculations has originated convergently in several groups of onygenalean fungi. In addition, the occurrence of fungi mimicking A. capsulatus in caves of temperate Europe is reviewed.     

Geographical variation in echolocation call and body size of the Okinawan least horseshoe bat, Rhinolophus pumilus (Mammalia: Rhinolophidae), on Okinawa-jima Island, Ryukyu Archipelago, Japan

The Okinawan least horseshoe bat, Rhinolophus pumilus, is a cave-dwelling species endemic to the central and southern Ryukyus, Japan. We analyzed variation in the constant frequency (CF) of the echolocation call and in forearm length (FAL) of this species on Okinawa-jima Island on the basis of data for 479 individuals from 11 caves scattered over the island. CF values in samples from six caves, all located in the southwestern half of Okinawa-jima, were significantly higher than those in samples from five caves in the northeastern half of the island. Also, FAL was significantly greater in the latter group than in the former group, although the ranges of variation in this character substantially overlapped between the two groups. These results suggest substantial differentiation between R. pumilus populations on Okinawa-jima. The implications of our findings for the conservation of this endangered bat species are briefly discussed.     

Two Trichosporon species isolated from Central-European mygalomorph spiders (Araneae: Mygalomorphae)

Trichosporon (Dikarya: Basidiomycota) is a genus of anamorphic yeasts typically associated with soil and water, although many species are causative agents of diseases in animals and man. Here we provide the first compelling evidence that spiders can be occasionally colonized by at least two Trichosporon species. Trichosporon dulcitum (Berkhout) Weijman 1979 was isolated from the exoskeleton of purse-web spider Atypus piceus, while Trichosporon porosum (Stautz) Middelhoven, Scorzetti & Fell 2001 was isolated from the exoskeleton of purse-web spider Atypus affinis. Both of the species were identified based on DNA sequence analysis of the host specimens displaying macroscopic signs of the superficial white mycosis on their exoskeleton. Only two specimens with macroscopic signs of superficial yeast growth were identified among the 125 individuals of A. affinis, A. piceus and Atypus muralis examined that were collected at various sites throughout the Czech Republic. The consistent burrow microclimate, uninterrupted occupancy of the single burrow for several subsequent years, and presence of prey remnants in the burrow below the purse-web may play a role in the course of infection of the mygalomorphs examined. The phylogenetic relationships of Trichosporon species are analyzed, concluding that association with invertebrates clusters predominantly among four groups of closely related species in independent Trichosporon clades.     

Isolation of yeasts from bovine milk in Belgium

A total of 436 milk samples from non-infected and 80 from infected quarters were investigated: 24.5% of the samples collected from non-infected and 55% of those collected from infected quarters were positive. Normal milk yielded not less than 16 different species and among them many potentially pathogenic yeast species such as C. parapsilosis, C. guilliermondii, C. tropicalis, C. glabrata and T. asahii, all five able to grow at 40 ° C. In contrast, the yeasts isolated from infected quarters were from 3 species: C. kefyr, C. catenulata and C. lambica, which were also among the yeasts species recovered from normal milk. Among the three species, only one i.e. Candida kefyr is able to grow above 40 ° C and from there can be considered as potentially pathogenic, even if bacterial association is necessary to cause mastitis.     

Biodiversity of killer activity in yeasts isolated from the Brazilian rain forest

The occurrence of killer activity against a panel composed of 22 industrially and (or) medically important yeasts was investigated in 438 yeast and yeast-like cultures belonging to 96 species, isolated from different environments of the Brazilian rain forest. Altogether, 26% of ascomycetes, 56% of basidiomycetes, and 42% of yeast-like cultures exhibited killer activity against at least one of the panel yeasts. More than 15 species never reported before as toxin producers were found, with Pseudozyma antarctica, Trichosporon asteroides, and Geotrichum klebahnii, showing the broader activity spectra. Plasmid curing did not cure the killer phenotypes of Candida maltosa, Debaryomyces hansenii, G. klebahnii, Tr. asteroides, Cryptococcus laurentii, and Ps. antarctica.     

Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods

A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sterigmatomyces and Trichosporon , and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic activities of 50 psychrotrophic strains were studied by use of skim milk within the temperature range of 0–42°C. All the strains showed various degrees of proteolysis. In particular, Candida lipolytica. Trichosporon pullulans and Candida scottii were active species at low temperatures. Sensory spoilage due to the proteolytic yeasts were observed in mackerel homogenates stored at 10°C. C. lipolytica -inoculated homogenates caused spoilage with ammoniacal odours after 1 week of storage. Values of total volatile basic nitrogen at 10°C were highest with C. lipolytica among 35 strains tested, followed by Tr. pullans. Trichosporon cutaneum, C. scottii, Rhodotorula glutinis and Cryptococcus luteolus. Proteolytic psychrotrophic yeasts were widely distributed in raw seafoods.     

The Identification of Wild Yeast Colonies on Lysine Agar

The most common wild yeasts infecting pressed baker's yeast in Great Britain are Candida tropicalis, C. krusei, C. mycoderma, Trichosporon cutaneum, Torulopsis candida and Rhodotorula mucilaginosa. Wild yeasts are readily detected and quantitatively estimated by plating infected baker's yeast on lysine agar, which permits of only limited growth of baker's yeast.
Morphology of wild yeast colonies on lysine agar is affected by duration of incubation, location in the agar plate, and sometimes by temperature of incubation, density of infection and numbers of baker's yeast cells present. It is therefore possible to identify each species by at least one characteristic type of colony produced under specified conditions. Ability to grow at 30° and 37° serves to distinguish further between certain species.     

Three new species of Antricola (Acari: Argasidae) from Brazil, with a key to the known species in the genus

Three new species of Antricola (Acari: Argasidae) are described from adult specimens collected on bat guano in different caves in Brazil. The female of Antricola guglielmonei n. sp. is easily determined by the presence of 2 smooth, depressed areas lacking setae in the posterolateral portions of the dorsum of the idiosoma, together with the partial fusion of the tubercles in the anteromedian portion of the idiosoma. The male of this species has a small spiracular plate surrounded by a pattern of tubercles disposed concentrically in its dorsal portion. Both sexes have cervical grooves very slightly marked. Antricola delacruzi n. sp. represents the only Antricola species with the dorsum of the idiosoma devoid of tubercles in both sexes and with scarce and minute setae placed over the smooth cuticle. The female of A. inexpectata n. sp. is known only from a few specimens. In this species, lines of smooth cuticle lacking setae separate the tubercles of the dorsum. In addition, there are 3 clumps of plumose setae close to the spiracular plate, in the ventrolateral portions of the idiosoma, in 3 well-delimited regions over cuticular thickenings. These 3 species share the peculiarity of a Haller's organ with the anterior pit bearing only 7 + 2 setae. The collection of these new species in Brazilian caves greatly expands the known range of the genus. A key to the adults of all known species of the genus is provided.     

Occurrence and dominance of yeast species in naturally fermented milk from the Tibetan Plateau of China

To determine which yeasts are present in the naturally fermented milks of China, 69 samples made by the nomads of Tibet were collected from the Tibetan Plateau in China. From these samples, 225 strains of yeast were isolated and identified using conventional microbiological analysis and gene sequencing analysis of the D1/D2 domain of the large subunit (26S) ribosomal DNA. The results showed that the total concentration of yeasts in these samples ranged from 5.01 to 8.97 log10 colony-forming units (CFU)/mL (6.91?± 1.02 log10 CFU/mL; mean?± SD). The number of cultivable yeasts was higher in the samples from Qinghai (7.55?± 0.75 log10 CFU/mL) than those from Tibet (6.21?± 0.79 log10 CFU/mL, P?< 0.05). Moreover, there were 15 phylotypes in these 69 samples. Among these phylotypes, Kluyveromyces marxianus (49.3%, frequency percentage), Saccharomyces cerevisiae (62.3%), and Pichia fermentans (46.4%) appeared frequently and can be considered the most common culturable species in naturally fermented milk products. Traditional fermented Mongolian cow milk featured a wide diversity of yeast species, including Issatchenkia orientalis, Kazachstania unisporus, Rhodotorula mucilaginosa, Candida pararugosa, Torulaspora delbrueckii, Geotrichum sp., Kazachstania unisporus, Geotrichum fragrans, Debaryomyces hansenii, Yarrowia lipolytica, Trichosporon gracile, and Pichia membranifaciens. This study provides new data on yeast composition in naturally fermented milk and shows the yeast biodiversity of fermented milk products from the Tibetan Plateau of China.     

Occurrence of Dermatophytes in Fresh Bat Guano

Evidence is presented in support of the hypothesis that fresh bat guano serves as a means of pathogenic fungi dissemination in caves. A total of 371 guano samples were collected from caves in southeastern New Mexico. Each sample was agitated in sterile saline and sand. The supernatant fluid was treated with an antibiotic and streaked on differential media. Cultures were incubated at 25 and 37 C and examined at intervals over a 4-week period. For animal inoculation, highly concentrated inoculum was injected intraperitoneally into white Swiss mice. Animals were sacrificed 4 weeks later, and portions of their lung, liver, and spleen were cultured on selective media, incubated at 25 C, and examined at intervals over a 4-week period. Microsporum gypseum was isolated at all 10 collecting stations with an incidence of 22.4%, Trichophyton mentagrophytes at 7 stations with an incidence of 5%, T. rubrum at 3 stations with an incidence of 3%, and T. terrestre at 1 station with an incidence of 0.5%. From a total of 60 pools of liver-spleen-lung suspensions, 6 pools yielded positive cultures of Histoplasma capsulatum and 1 pool yielded T. mentagrophytes. No significant difference was found among the different selective media with respect to recovery of dermatophytes. Among the human pathogenic fungi isolated were Candida sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans, H. capsulatum, M. gypseum, T. mentagrophytes, T. rubrum, T. terrestre, and Sporotrichum sp.     

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.