A total of 84 fungal isolates was examined from the coarse outer hair of nine living individuals of the three-toed sloth (Bradypus variegatus) encountered along Pipeline Road in Soberanía National Park, Republic of Panama. Hair samples were transported to the lab in sterile Falcon tubes which had been half filled with silica gel, a desiccant that is effective for storing fungal tissue [31],[32]. Every piece of sloth hair placed on agar yielded multiple fungal isolates representing a variety of morphotypes. Phylogenetic analyses of axenic strains revealed a diverse group of fungi, some of which appear to be novel relative to previously observed or sequenced taxa (Table 1; Figure 1). Many of these isolates display bioactivity in vitro against parasites that cause malaria and Chagas disease, breast cancer cells, and both Gram-positive and Gram-negative human pathogenic bacteria (Table 2; Table 3).

Topology of each tree reflects ML analysis, and values above branches indicate ML bootstrap values and Bayesian posterior probabilities (>0.50 and >0.75, respectively). Outgroups and taxon sampling for each tree were validated by literature surveys (see methods). Taxonomic conclusions are presented in Table 1 . Figure 1(A) : Placement of F5073 in group 17; (B) F4847 in group 18; (C) F4831a in group 19; (D) F4819 in group 20; (E) F4801 in group 5; (F) F4806 in group 7; (G) F4886 in group 8; (H) F5071 in group 15; (I) F4812–F4816, F4830, F4831, F4845, F4852–F4856, F4860, F4873, F4882, F4883 and F4900–F4902 in group 1; (J) F4803, F4817, F4820, F4823, F4824, F4826, F4827, F4829, F4837, F4841, F4842, F4846, F4848, F4857, F4858, F4861, F4862, F4870, F4872, F4875, F4878, F4879, F4894–F4896, F4908, F4909, F5069 and F5074 in group 2; (K) F4818 and F4839 in group 10; (L) F4877, F4890 and F4897 in group 11; (M) F4828 and F4898 in group 12; (N) F4876 and F4881 in group 13; (O) F4863 and F4884 in group 6; (P) F4821, F4874, F4889 and F4913 in group 3; (Q) F4802, F4807, F4825, F4844, F4906 and F5068 in group 4; (R) F4850 and F4891 in group 9; (S) F4904 and F4905 in group 14; and (T) F5070 and F5072 in group 16.

Eighty-four fungal isolates from the coarse outer hair of nine individuals sloths (B. varieagatus), their top BLAST matches, and maximum identity value from BLAST searches; group ID and tree ( Fig. 1 ) revealing phylogenetic placement; and taxonomic placement based on phylogenetic analyses at the family (order) and genus levels ( Fig. 1 ).

Isolate F4891 ( Figure 1(R) ) did not appear to be closely related to any named sequences in GenBank and could only be identified as possibly a member of the order Pleosporales (Dothideomycetes). Isolate F4850 ( Figure 1(R) ) had 96% sequence similarity to its closest match in GenBank, an uncultured soil fungus clone ( Table 1 ; [35] ). Further analyses are warranted in order to confirm whether isolates F4891 and F4850 are novel fungal species. In general, identifications assigned by phylogenetic analyses closely matched those of the top BLAST hits for each isolate ( Table 1 ).

Eighty isolates could confidently be assigned to 15 genera ( Table 1 ; Figure 1 ). Two isolates were given more tentative phylogenetic placements. F4886 ( Figure 1(G) ) was identified as Robillarda sp. or as a member of a closely related genus within the Amphisphaeriaceae. Isolate F4831a ( Figure 1(C) ) was identified as a member of Paraconiothyrium sp. or a closely related genus within the Montagnulaceae. The two most common genera, Pestalotiopsis sp. and Hypocrea sp., were isolated from 7 of 9 and 5 of 9 sloth hair samples, respectively.

Following phylogenetic analyses, high level identities (class, order, family) could be confidently assigned to 82 fungi representing 15 families and 10 orders ( Table 1 ; Figure 1 ). The majority (81.7%) of these were Sordariomycetes. Fungi from this class are well documented sources of bioactive metabolites (e.g., [33] , [34] ). The remaining isolates were Dothideomycetes (15.9%) and Eurotiomycetes (2.4%).

Sequences that aligned well to one another were partitioned into clusters to create 20 groups of apparently similar species ( Table 1 ; Figure 1 ). One group contained 29 sequences from sloth-hair fungi, one group contained 20 sequences, and the remaining 18 groups contained between 1 and 6 sequences each. Between 34 and 165 sequences from closely related taxa were compiled for each group to make non-redundant datasets to which one or two appropriate outgroups were added based on literature review.

BLAST comparisons with GenBank provided preliminary estimations of taxonomic placement and similarity to previously sequenced fungi ( Table 1 ). All isolates were Ascomycota. Fourteen isolates had a top match to uncultured fugal clones and 23 had a top match to cultured but unidentified fungi. The remaining 47 isolates had matches to named strains that tentatively suggested placement in the Sordariomycetes (Xylariales, Glomerellales, Hypocreales), Dothideomycetes (Botryosphaeriales and Pleosporales), and Eurotiomycetes (Eurotiales). To more confidently determine taxonomic placement, sequences were analyzed using maximum likelihood and Bayesian methods.

Bioactivity of sloth hair isolates

Sloth hair fungi that were cultivated in liquid medium and extracted with ethyl acetate were tested for bioactivity. We considered extracts to be highly bioactive if they caused at least 50% inhibition of the growth (i.e., ≥50% IG) of parasites or cancer cells in vitro.

Overall, two of 70 (2.5%) extracts tested were highly active against P. falciparum, eight of 62 (12.9%) extracts tested were highly active against T. cruzi, and 15 of 73 (20.6%) extracts tested were highly active against the MCF-7 breast cancer cell line (Table 2). Most extracts with bioactivity were active in only one assay (Table 2). Bioactive strains were isolated with equal frequency on MEA and PDA (Table 2). Notably, closely related isolates often differed in bioactivity: for example, three of 20 isolates identified as Hypocrea sp. (Table 1) had bioactivity only against MCF-7 breast cancer cells; three were active against MCF-7 breast cancer cells and T. cruzi (Table 2); and one was active only against methicillin-resistant S. aureus (Table 3).

Of the 16 fungi that had high % IG in at least one in vitro assay, 13 belonged to the Hypocreales (Hypocreaceae, Bionectriaceae and Nectriaceae). The remaining three fungi belonged to the Valsaceae (Diaporthales), Amphisphaeriaceae (Xylariales) and Trichocomaceae (Eurotiales). Anti-malarial and anti-cancer activities have been previously reported in the literature from these relatively well-studied fungal lineages (e.g., [36],[37],[38],[39],[40],[41],[42],[43]). However our results represent, to the best of our knowledge, the first reports of anti-trypanosomal activity in Cytospora (Valsaceae) and Bionectria (Bionectriaceae).

The number of isolates highly bioactive against T. cruzi (Table 2) is of particular interest as we so rarely encounter microbes with bioactivity in this assay: only 104 of 2698 fungal endophytes (3.9%) from our overall culture collection are highly bioactive against that parasite [29]. Currently the only treatments available for this illness are nitrofurane and benznidazole, both of which are associated with such toxic side effects that treatment is often abandoned [44].

Of 50 fungal extracts screened by BioMAP, 20 were bioactive against at least one test organism (Table 3). The majority of isolates (16 of 20) active against bacteria in the BioMAP assay belonged to the families Lasiosphaeriaceae (Sordariales), Glomerellaceae (Glomerellales), Valsaceae (Diaporthales), Botryosphaeriaceae (Botryosphaeriales), and Amphisphaeriaceae (Xylariales). The remaining four fungi belonged to the Nectriaceae, Bionectriaceae and Hypocreaceae (Hypocreales). Activity against Gram-positive bacteria was detected far more frequently than against Gram-negative bacteria (Table 3).

Five isolates (Colletotrichum sp. F4806 (Figure 1(F)); Cytospora sp. 1, strain F4818 (Figure 1(K)); Fusarium sp. 1, strain F4898 (Figure 1(M)); Lasiodiplodia sp. 1, strain F4807 (Figure 1(Q)); and Lasiodiplodia sp. 1, strain F4844 (Figure 1(Q)) had particularly potent activity in the BioMAP assay and were selected for further testing. Serially diluted extracts were re-tested for activity against the panel of 15 bacteria and bioactivity profiles were constructed using normalized MIC values (Table 4).

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larger image TIFF original image Download: Table 4. Minimum inhibitory concentrations (MIC) of 5 sloth hair associated fungal extracts in the BioMAP antibiotic profile screen Minimum inhibitory concentrations (MIC) of 5 sloth hair associated fungal extracts in the BioMAP antibiotic profile screen [30] https://doi.org/10.1371/journal.pone.0084549.t004

The extract from Lasiodiplodia sp. 1 (strain F4807) was of particular interest as it had potent and specific activity against Gram-negative bacteria (Table 4). The bioactivity profile of the isolate did not match that of any of the known antibiotic classes previously tested in the BioMAP assay, suggesting a potentially novel mode of action (see [30]). Infections caused by emerging multidrug-resistant (MDR) Gram-negative bacteria are becoming a major clinical concern worldwide [45]. These infections most commonly include MDR Pseudomonas aeruginosa, extended-spectrum β-lactamase producing Enterobacteriaceae, and MDR Acinetobacter baumannii [46]. Recent data suggest that infections traced to Staphylococcus aureus bloodstream isolates are in decline, but that infections by Escherichia coli, the most common Gram-negative species responsible for infections in human blood isolates, are increasing in frequency [47]. That report [47] also highlights the paucity of antibacterial agents under development for Gram-negative bacteria, with most effort being focused on Gram-positive MRSA. The increasing prevalence of MDR organisms is mainly due to overuse of broad-spectrum antibiotics and poor antibiotic stewardship [48]. Hence the specificity of the extract from strain F4807 for Gram-negative strains is highly valuable and worth pursuing for further analysis.

Fungi isolated in this study were taxonomically consistent with groups of fungi known to occur in soil and in plants as pathogens, saprotrophs, or endophytes [11],[49]. Sloths may encounter such fungi incidentally from air spora, or via direct contact when they descend from trees in order to defecate and urinate, at which time they dig a hole in the soil that they subsequently cover with leaf litter [50]. Strikingly, comparison with a large collection of endophytes from terrestrial plants in Panama ([29]; 1269 strains) revealed that sequences from 29 isolates from sloth hair were identical to strains obtained from plants (100% similarity over the full sequence length). Thus this study extends the known host range and ecological mode of putative endophytes. Some of these fungi may affiliate directly with the green alga that inhabits coarse hair of sloths, much like endolichenic fungi associate with green algae in lichen thalli and are often taxonomically similar to endophytes in the same environments [51],[52].

Potential roles of these fungi in the health of sloths have not been investigated. Araújo Xavier et al. [50] reported fungal infections in B. variegatus caused by the fungal pathogen Microsporum (which also causes dermatitis in humans; [53]), but this genus was not recovered in the present study or in previous work by Suutari et al. [16]. In contrast to the human microbiome, where fungi comprise <0.01% of microbial communites on external surfaces such as skin [54], Suutari et al. [16] reported that fungi represented 8% of the flora in surveys of sloth-hair microbes. The high abundance and diversity of fungi associated with sloth hair, coupled with their bioactivity, may speak to a biological importance to sloths that is yet unexplored.

The pressing need for new medications continues to represent one of humanity's greatest challenges. It is commonly agreed that the vast majority of bioactive microbes remain to be discovered [9] and newly discovered microbial taxa hold an important promise of novel chemistry. Abundant evidence points to novel environments as promising sources of as yet undescribed microorganisms [55]. Here we have demonstrated that hair of the three-toed sloth (Bradypus variegatus) in Panama is a rich source of diverse bioactive fungi. Strains isolated here included members of some well-studied lineages (e.g., Hypocreales; Pleosporales), but also members of understudied or potentially novel groups (e.g., Endomelanconiopsis sp. F4801; Robillarda sp. F4886; Ascomycota sp. F4891; Ascomycota sp. F4850). We anticipate that additional novel taxa could be found on sloth hair by expanding survey methods to diverse types of media and using high-throughput methods to assess community composition. We also anticipate that different taxa may be isolated from sloths in other regions, much as communities of endophytes in tropical plants differ markedly at a regional scale [56],[57]. Thus our work suggests that fruitful exploration of the sloth microbiota is warranted for potential applications in drug discovery.