Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil

Abstract Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.


Introduction
Urbanization transforms habitats and landscapes where wild animals live (Stone et al., 2009;Perugini et al., 2011;Francis and Barber, 2013). This process is characterized by rapid intensification of agriculture, socioeconomic changes, and ecological fragmentation and may dramatically impact the epidemiology of infectious diseases (Hassell et al., 2017). Among the infectious disease vectors associated with urban environments, bats are one of the most important.
In Brazil, bats of the suborder Microchiroptera inhabit all the national territory, from the Amazon to the pampas of Rio Grande do Sul, including urban areas. This suborder comprises 174 registered species, 65 genera, and 9 families (Paglia et al., 2012).

Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil
Ministry of the Environment -MMA, Chico Mendes Institute for Biodiversity Conservation -ICMBio (Process No. 53484-1/2016). The bats were captured with 36-mm mesh mist nets measuring 9 × 2.5 m. The nets were opened at ground level along forest edges, on established trails, over water bodies, and close to trees (Esbérard, 2003). The bats were captured throughout the night and in all moon phases. The bat species were characterized according to the identification keys of Reis et al. (2013). After being captured, the bats were anesthetized and killed according to Normative Resolution 13/2013 of the National Council for Control of Animal Experimentation (CONCEA) (Brasil, 2013). The bats and blood samples were identified and transported to the laboratory for processing.

Biological sample processing and identification of genera of filamentous and yeast fungi
Samples of bat intestine, lungs, spleen, liver, and blood were obtained as described by Tencate et al. (2012) with some modifications. The samples were individually macerated in sterile 0.85% sodium chloride solution and vigorously homogenized for 5 min. After 30 min, 100 μL of each supernatant was spread over the surface of modified Sabouraud Dextrose agar plus chloramphenicol (40 mg/L) (SBR) (Sigma-Aldrich, St. Louis-MO, USA) and incubated in B.O.D. chamber (Solab, SL200/300) at 25 °C.
The supernatants (100 μL) were also spread over the surface of Brain Heart Infusion (BHI) agar (Kasvi, Curitiba-PR, Brazil) plus 5% defibrinated sheep blood (Newprov, Pinhais-PR, Brazil) and incubated in bacteriological chamber at 37 °C. Before the cultures were considered negative for the presence of fungi, they were observed daily for 30 days to verify fungal growth. The blood smear was stained in panopticus and observed with a 100X immersion objective (Opton, TNB-01B-INF LED).
The fungal genera were identified by macro-and microscopic observations, namely morphology of hyphae, conidia, or spores and colonies that grew on the culture media. The filamentous fungi were identified by using diagnostic keys and descriptions reported by Pitt and Hocking (2009) and Watanabe (2010). The yeast-like fungi were identified by employing the diagnostic key and descriptions published by Kurtzman and Fell (1998).
Among the 48 bats evaluated via microbiological culture of lungs, spleen, liver, blood, and intestine, 70.83% (34 bats, 18 females and 16 males) were positive for at least one through bat feces or decomposing carcasses (Voyron et al., 2011). Therefore, bats have become an important source of environmental contamination because they can travel long distances (Brilhante et al., 2016).
Sinop, a city in the state of Mato Grosso, Brazil, is located in the region known as the Arch of Deforestation. This region contains a few environmental reserves as well as large deforestation areas due to urbanization and agriculture. The population of this county and region lives in the rural area, close to forest areas. People living in urban areas with forest remnants and forest reserves, or even in fully urbanized areas can be a target for different types of fungal infections as a result of rapid expansion of peripheries, deforestation, extractivism, agriculture, livestock, and ecotourism (Brum et al., 2011).
This study aimed to isolate and to identify fungal genera from biological samples obtained from bats inhabiting urban forest fragments in Sinop in order to understand their role in the epidemiological axis of fungal spread. This understanding is relevant to predict the transmission of infectious diseases in cities located in areas of strong agricultural expansion.

Study area
The city of Sinop, state of Mato Grosso, Brazil, was founded in 1974. It has approximately 113,099 inhabitants in an area of 3,942,229 km 2 . The climate is warm, and the average annual temperature is 28 °C. The rain regime is equatorial, which is characterized by a dry period in the winter and a rainy period in the summer, mainly between January and March (IBGE, 2010). The bats were captured from April to October 2017 in four urban fragments of the city. More specifically, the bats were captured in roof spaces (under a roof tile or lead flashing, between gaps in mortar, or behind fascias and soffits) and seven forest fragments in Sinop.

Discussion
We isolated genera of opportunistic pathogenic fungi from biological samples obtained from bats captured in the urban forest fragments in Sinop, state of Mato Grosso, Brazil. This region has undergone accelerated urban growth due to agricultural expansion. Urbanization, which led to the emergence of buildings and other human constructions such as bridges and tunnels, must have modified the ecology of bats drastically, as evidenced by the number of species that are currently dominant or exclusive in these structures. Human construction may simulate the structural and functional properties found in caves or trees, so bats may have learned to explore the new artificial roosting environment (Russo and Ancillotto, 2015).
In our study, the most frequent bat species was Molossus molossus, which corresponded to 31 (64.6%) of the captured specimens and are the most prevalent in urban areas (Canteros et al., 2005;Dias et al., 2011). The presence of bats in domiciliary areas of the city of Sinop indicates interspecies contact with man and other animals, which may cause public health problems. Given that the city's illumination attracts a large number of insects, the predominance of this insectivorous species in an urban area can be explained by the shelter and food supply (Pacheco et al., 2010).
Being the most prevalent, the species Molossus molossus presented the greatest diversity of fungi: seven fungal genera were isolated from these bats. In 2017, Paz reported similar findings-this author verified that Molossus molossus was the species with the highest positivity for Histoplasma capsulatum, Paracoccidioides brasiliensis, and Cryptococcus spp. (Paz, 2017). In 2013, Corrêa et al. (2013) reported that Molossus molossus and Artibeus lituratus were the species infected with the highest number of pathogens. Molossids have been described as the most frequent bats in buildings (Pacheco et al., 2010), especially under roofs, in various structures, and in tree cavities (Reis et al., 2007).
Several emerging diseases are zoonoses, and bats play an important role as a source of infection and transmission of infectious agents due to their social, biological, and immunological characteristics (Hance et al., 2006;Han et al., 2015;Zanella, 2016).
Regarding the biological samples obtained from bats captured in Sinop, the most isolated genera of filamentous fungi were Penicillium sp. (26, or 54.2%) and Aspergillus sp. (13, or 27.1%). Similar findings were reported in 2003 by Rezende et al. (2003) in Bonito, state of Mato Grosso do Sul; in 2002 by Tencate et al. (2012) in the Northwestern region of the state of São Paulo; and in 2016 by Misra and Elangovan (2016) at several historical monuments in India.
Here, the most isolated opportunistic yeasts were Candida albicans (4.16%), Candida parapsilosis (8.33%), and Candida glabrata (4.16%), followed by Cryptococcus sp. (8.33%) and Trichosporon sp. (2.08%). In 2011, Botelho et al. (2012) obtained similar results when they isolated fecal samples obtained from urban bats inhabiting the city of Londrina, state of Paraná, to detect five species of the genus Candida: Candida guilliermondii, Candida krusei, Candida  Sugita et al. (2005) evaluated fecal samples obtained from volcanic cave bats captured in 11 cities in Japan and isolated nine species-two known as Trichosporon sp., and seven potentially new species as revealed by molecular phylogenetic analyses. Yeasts of the genera Cryptococcus sp., Candida sp., and Trichosporon sp. can be found in the gastrointestinal microbiota and viscera of chiroptera; these species can infect humans through inhalation of fungal propagules present in the environment (Baltazar and Ribeiro, 2008). This contact causes opportunistic infections in immunocompromised patients exposed to a wide range of risk factors (Peixoto et al., 2014;Gibson and Johnston, 2015;Mattede et al., 2015).
Our results demonstrate that bats captured in urban forest fragments in Sinop can act as hosts of fungi with pathogenic potential. This increases the risk of human and domestic animal exposure and acquisition of opportunistic fungal infections because the bats remain in shelters close to the population. PITT, J.I. and HOCKING, A.D., 2009. Fungi and food spoilage. 3rd ed.