Phylogenetic Identification, Diversity, and Richness of Aspergillus from Homes in Havana, Cuba

Aspergillus is one of the most common fungal genera found indoors; it is important because it can cause a wide range of diseases in humans. Aspergillus species identification is based on a combination of morphological, physiological, and molecular methods. However, molecular methodologies have rarely been used for the identification of environmental isolates of Aspergillus in Cuba. Therefore, the objective of this work was to identify the species of the genus Aspergillus obtained from houses in Havana, Cuba, through the construction of phylogeny from a partial sequence of the benA gene region, and to analyze the diversity and richness of Aspergillus in the studied municipalities. Isolates of Aspergillus spp. included in this study presented the typical macro- and micromorphology described for the genus. According to this polyphasic characterization, A. niger, A. flavus, A. welwitschiae, A. heteromorphus, A. sydowii, A. tamarii, A. fumigatus, A. clavatus, and A. tubingensis were the most abundant species. Most of the identified species constitute new records for outdoor and indoor environments in Cuba and contribute to the knowledge of fungal biodiversity in the country. These results constitute an alert for the health authorities of the country, since prolonged exposure of the inhabitants to Aspergillus spores can cause severe persistent asthma, among other diseases.


Introduction
Indoor environments have unique fungal communities that are adapted to the specific carbon, temperature, and humidity restrictions of these environments [1,2]. The most frequently isolated fungi belong to the Aspergillus, Cladosporium, and Penicillium genera [3,4]. However, Aspergillus has particular importance because it can produce a wide range of diseases in humans [5], as the genus Aspergillus' members are opportunistic pathogens that pose major threats to immunocompromised individuals, who can acquire the spores of Aspergillus spp. present in the environment through inhalation. However, the spores are not effectively eliminated and can remain in the airways, causing a variety of infections, including allergic bronchopulmonary aspergillosis (ABPA), aspergilloma (chronic aspergillosis), and invasive aspergillosis (IA) [6,7].
This genus includes a large number of species that may be responsible for allergic syndromes, intoxications, chronic infections, and acute invasive diseases, particularly in people with compromised immune systems [8][9][10]. The ability to grow and sporulate on various organic substrates allows the widespread development of Aspergillus in many geographical A total of 153 Aspergillus isolates were collected for this biodiversity study. From the indoor environments, 122 isolates were analyzed: 30 from dust samples, 8 from walls, and 84 from the air. For the analysis of the outdoor samples, 31 isolates were collected from homes with a history of moisture in the municipalities of Cerro (23 • 05 17.0 N; 82 • 23 42.0 W) and Arroyo Naranjo (23 •

Sampling Procedure
The air fungal propagules were collected using a SAS Super 100 ™ volumetric biocollector (VWR International Srl, Milano, Italy). The sampler was placed at a height of 1.0 m from the floor, in the center of each sampling area (terrace, patio, or balconies of houses). The medium used was malt extract agar (MEA). Dust was sampled using a sterile brush, whereas the walls were sampled with a swab in places where there was evident fungal growth. Samples were suspended in saline solution and 0.05% Tween 80. An aliquot of each sample's suspension was cultured on MEA.

Monosporic Culture Procedure
From each isolate grown in potato dextrose agar (PDA) (Bioxon, Ciudad de México, Mexico) for 2-4 days at 28 • C, a conidial suspension was prepared with 1 mL phosphate buffer (pH 7.4) and 0.5% Tween 20 (PBST). This suspension was diluted 1:1000 with PBST, and 50 µL was taken and inoculated in Petri dishes with PDA (Bioxon, Ciudad de México, Mexico). The Petri dishes were then incubated at 28 • C. An isolated colony was selected from each plate and grown in a tube with the same medium at 28 • C. Conidia from the monosporic cultures were stored in sterile distilled water at 4 • C. In addition, all the isolates were stored in tubes with agar at 4 • C or in distilled water and glycerol with skim milk at −30 and −70 • C, respectively, in the Microbial Cultures Collection of the Faculty of Biology, University of Havana, and in the Collection of the Molecular Mycology Laboratory, Mycology Unit, Department of Microbiology and Parasitology, Faculty of Medicine, National Autonomous University of Mexico.

Macro-and Micromorphology
The morphological characterization was performed following the procedures of Klich and Pitt [28] and Samson et al. [19]. The isolates were inoculated in Czapek yeast autolysate (CYA) extract agar and incubated at 5, 25, and 37 • C and in MEA and CYA with 20% sucrose (CYA20S) at 25 • C. The isolates were inoculated at three points on each medium's plates and incubated at 25 and 37 • C in the dark for 7 days. For micromorphological observations, microscopic mounts were constructed in lactic acid from MEA colonies, and a drop of alcohol was added to remove air bubbles and excess conidia [19].

Obtaining DNA
The isolates were grown in PDA (Bioxón, Ciudad de México, Mexico) for 3 days at 28 • C. From these cultures, a conidial suspension was obtained by adding 1 mL PBS buffer with 0.5% Tween ® 20. Subsequently, the 1% yeast extract, 2% peptone, and 2% dextrose (YEPD) was seeded in the liquid medium and incubated at 37 • C with stirring for Microorganisms 2021, 9, 115 4 of 12 2 days or until mycelial growth was observed. The mycelial biomass of each isolate was filtered (with 2 washes performed using sterile milliQ ® water, (Merck Millipore, Darmstadt, Germany) under a vacuum with sterile filter paper in a Büchner funnel until the moisture was removed.
DNA was extracted with a Wizard ® genomic DNA purification kit (Promega, Madison, WI, USA) as follows: 100 mg of dry mycelial mass from each isolate was deposited in 1.5 mL microtubes containing 0.2 g glass beads, 600 µL lysis solution was added, and the tubes were placed in a FastPrep ® 24 device (MP Biomedicals, Santa Ana, CA, USA). Four periods of cell disruption (40 s at 6 m/s) were applied, with 5 min of cooling between each one after breaking apart the mycelium. We followed the kit's manufacturer's instructions. The concentration of the DNA obtained was quantified by spectrophotometry (spectrophotometer DS-11, DeNovix, Wilmington, DE, USA) and gel electrophoresis (1% agarose) and compared against different concentrations (10, 30, and 50 ng/µL) of phage λ (Gibco BRL ® , San Francisco, CA, USA). Each DNA sample was mixed with 2 µL GelRed ™ nucleic acid gel stain 10,000× (Biotium Inc., Fremont, CA, USA) before being loaded into the gel benA PCR.
The following conditions were followed for the PCR: The reaction mixture (25 µL) con-

Phylogenetic Analysis
The amplified fragments obtained from the 153 Aspergillus isolates were sent to be sequenced in both directions (Macrogen, Rockville, MD, USA). The electropherograms of the obtained sequences were edited using the BioEdit program [30]. The sequences (forward and reverse) for each sample were checked, verified, and used to create consensus sequences, which were subsequently deposited in the GenBank database (http://www. ncbi.nlm.nih.gov) (Supplementary Table S1).
Each edited sequence was compared with all the nucleotide sequences belonging to fungi deposited in GenBank (URL4) through the Basic Local Alignment Search Tool (BLAST) program [31] to evaluate the similarity percentages, identities, and expectation values. The reference sequences identified with the highest similarity and identity percentages, as well as the expectation values closest to zero, were chosen to perform the phylogenetic analysis.
The maximum likelihood method was used to identify the Aspergillus species. The support values of the internal branches were assessed using a bootstrap method with 1000 replications (values equal to or higher than 70% were considered significant) and the GTR G+I evolutionary model; the nearest neighbor interchange (NNI) heuristic method was applied. Maximum likelihood (ML) analysis was conducted with MEGA v.10.1.7 software [32].

Richness, Abundance, and Diversity of Species
Species richness (S = the total number of species), abundance (the sum of the number of isolates of each species), and diversity were evaluated through the Shannon diversity index (H' = -Σ pi Ln pi, where pi is the proportion of individuals of species i) and the Simpson diversity index (D = Σ(n/N) 2, where n is the total number of organisms of a particular species, and N is the total number of organisms of all species). The analyses were performed independently for each municipality (Cerro and Arroyo Naranjo) [33,34].

Circumdati
Colony on CYA, 25 • C, 7 days: Yellow to ochre colonies, cream, or pale yellow; smooth texture; yellow, orange, or cream reverse. Presence of pink to purple-brown sclerotia in some isolates. Predominantly biseriate, rough stems, smooth or finely rough globose conidia.

Phylogenetic Analysis
The tree obtained through the maximum likelihood method showed a topology of XXVII groups (Supplementary Figure S1 and

Richness, Abundance, and Diversity of Species
The results showed higher species richness in the municipality of Cerro, from which 20 species were identified, whereas in the municipality of Arroyo Naranjo, 18 species were identified. Likewise, the results showed that in the municipality of Cerro, A. niger (14.77%), A. welwitschiae (12.5%), A. flavus (12.5%), A. heteromorphus (11.36%), A. clavatus (7.96%), and A. tamarii (7.96%) were the most abundant species (Figure 1  The diversity, estimated through the Shannon and Simpson indices, was slightly greater in the municipality of Cerro than in Arroyo Naranjo (Table 3). The diversity, estimated through the Shannon and Simpson indices, was slightly greater in the municipality of Cerro than in Arroyo Naranjo (Table 3).

Discussion
A high prevalence of allergic diseases has been observed in Cuba. de la Vega Pazitková et al. [35] showed that 9% of young people under the age of 15 in Havana might suffer from undiagnosed asthma. Venero Fernández et al. [36] estimated that allergic diseases have a prevalence of 32%, although the triggering factors were not well-established in both studies. However, this high prevalence can be explained by the climatic conditions in Havana, as it has an average annual temperature of 25.5 • C, average annual precipitation of 1510 mm, and a relative humidity of 78%. These weather conditions are shared by other tropical countries, which contribute to fungal development, associated with an increased number of spores in the environment [37].
An indoor and outdoor air myco-biological study performed in housing environments located in Havana, Cuba, showed that A. flavus, A. niger, and A. clavatus were the most frequently isolated species [37]. In addition, other studies have focused on fungi identification in indoor or outdoor environments in Havana [38,39]; however, none used genotypic methods for identification at the species level. Therefore, identifying Aspergillus isolates at the species level through molecular methods represents a significant contribution, since these may be associated with allergic conditions.
Here, we identified Aspergillus isolates obtained from indoor and outdoor housing environments in Havana, Cuba, belonging to the following sections: Versicolores, Usti, Flavi, Aspergillus, Cremei, Fumigati, Clavati, Nigri, Candidi, Flavipedes, and Circumdati. Of these, species from the sections Fumigati, Flavi, and Nigri may cause allergic diseases [40]; they were also found to be the most abundant.
The species identified in the indoor housing environments coincided with those detected in the outdoor environments, which is consistent with the results described by Rahmawati et al. [2]. The movement of spores can explain this coincidence from outdoor environments; spores enter houses through open windows and doors, on the clothing of inhabitants and visitors, or through the entry and exit of pets [5]. Initially, in this work, the isolates were identified according to their macro-and microscopic characteristics. However, these methods have limitations due to the morphological similarity and variability shown by these fungi; therefore, it was only possible to identify the Aspergillus isolates at the section level. Thus, genotypic methods were needed to correctly identify the Aspergillus species, and the benA marker was used to construct a phylogenetic analysis through a maximum likelihood method.
According to this polyphasic identification, A. welwitschiae (section Nigri), A. flavus (section Flavi), A. niger (section Nigri), A. heteromorphus (section Nigri), A. sydowii (section Versicolores), A. tamarii (section Flavi), A. fumigatus (section Fumigati), A. clavatus (section Clavati), and A. tubingensis (section Nigri) were the most abundant species. Section Nigri was the most represented among the isolates evaluated. These results are different from those reported by Richardson et al. [41], who most frequently isolated A. fumigatus among the species of this genus. Hashimoto et al. [42], when identifying clinical and environmental isolates of the Nigri section, reported that this set of isolates included three species, A. niger, A. welwitschiae, and A. tubingensis, of which A. welwitschiae had the highest number of isolates identified, which is consistent with our results. This may correspond to the members of these sections being xerotolerant and thermotolerant, so they grow in a wide range of temperatures [43]. In other studies, A. niger was identified and showed a frequency similar to that obtained in this study, but in hospital settings [44]. This supports the importance of the presence of species from the section Nigri in the environment, as that section includes species that cause pulmonary aspergillosis and otomycosis in humans, as well as localized and disseminated diseases in domestic and wild animals [45].
Aspergillus niger, A. welwitschiae, and A. tubingensis are distributed worldwide, whereas the rest of the species identified in this work have only been reported in contaminated food, in the soil, and in the air in some countries [22,46]. A. heteromorphus, A. neoniger, and A. aculeatinus have been described as new global records in the air of indoor and outdoor environments, and A. heteromorphus and A. violaceofuscus in dust [1]. A. aculeatus and A. violaceofuscus have only been previously detected in the indoor air of libraries in Brazil [22].
The detection of the species in the section Nigri is important since, as several authors have stated, A. welwitschiae and A. niger have the ability to produce ochratoxin A (OTA), a potent nephrotoxin that causes carcinogenic effects and fumonisin [47]. Although few of the species in the section Nigri are involved in mycoses associated with humans or animals, there are reports of IA caused by A. niger [46], onychomycosis caused by A. welwitschiae, A. tubingensis, and A. niger [48] and external otitis caused by A. niger and A. tubingensis [8].
The species A. flavus, A. tamarii, A. oryzae, and A. nomius identified in this study belong to the section Flavi; they are considered cosmopolitan, although A. nomius has only been previously recorded in indoor environments in Brazil [1,22]. A. nomius has been reported to produce several aflatoxins and have a carcinogenic effect [49]. A. flavus produces aflatoxins B1 and G, aflatrem, cyclopiazonic acid, and 3-nitropropionic acid; A. tamarii produces tenuazonic acid, cyclopiazonic acid, and 3-nitropropionic acid; whereas A. nomius produces tenuazonic acid, and aflatoxins B1 and G [50]. However, reports of aspergillosis due to A. tamarii and A. nomius are scarce, since their conidia tend to be deposited in the sinuses and in the upper respiratory areas [51]. Aspergillus oryzae is the domesticated form of the aflatoxigenic species A. flavus [50]. A. flavus is considered the etiologic agent responsible for 10% of bronchopulmonary aspergillosis worldwide and the second most important agent of IA. The high concentrations of its conidia in the outdoor and indoor environments of houses and hospitals have been correlated with the diseases it causes in tropical countries [15].
Aspergillus ochraceus, A. westerdijkiae, and A. subramanianii, belonging to the section Circumdati, represent the first records of these species in environmental studies in the Caribbean. A. melleus was detected by Rojas et al. [21] in the library of the Faculty of Biology in Havana. A. ochraceus and A. westerdijkiae are of clinical importance, since they have been reported as causing chronic granulomatous diseases and non-dermatophyte onychomycosis, respectively [52,53]. Aspergillus subramanianii has been reported as a causal agent of IA, exclusively for patients with inborn defects in the host antifungal defense pathways [53].
The species A. sydowii and A. versicolor (section Versicolores) are commonly isolated from indoor environments, and A. versicolor has been associated with sick building syndrome [1]. These species have also been recognized as emerging pathogens in onychomycosis [54,55]. In addition, A. versicolor can cause invasive pulmonary aspergillosis and produce OTA [15].
The presence of the species A. clavatus and A. giganteus (section Clavati) is common in tropical, subtropical, and Mediterranean regions, whereas A. giganteus has not been reported in tropical or subtropical regions. Several studies have reported that A. clavatus is the cause of endocarditis, extrinsic allergic alveolitis, and toxic syndromes with neurological disorders [56].
Notably, records in the Caribbean of the species A. micronesiensis and A. templicola are limited. These species included in the Flavipedes section were first described by Visagie et al. [1] in the dust of homes in Mexico and Thailand. Likewise, Visagie et al. [1] reported A. micronesiensis in the dust of North American homes. However, in our study, except for the CCMFBH-917 isolate from an indoor environment, the majority of the isolates were obtained from dust.
The species A. calidoustus (section Usti) constitutes a new record for Latin America, since it has only been reported from indoor environments in the United States, West Asia, and Europe, and in the dust of North American homes [1,57]. A. calidoustus, usually confused with A. ustus, is an emerging pathogen that causes IA [8,58].
Two isolates identified as A. chevalieri (section Aspergillus) were obtained from indoor environments. Júnior et al. [22] showed that their presence is common in these environments. Recently, Siqueira et al. [59] reported this species as a causal agent of fatal cutaneous and cerebral aspergillosis following traumatic inoculation.
The species A. fumigatus (section Fumigati) was the only species identified in the Fumigati section. This fungus produces large numbers of conidia (asexual spores), which easily become airborne and are efficiently dispersed through the air due to their small size (2-3 µm diameter) and inherent hydrophobicity. Additionally, these conidia persist in the air for long periods due to the complex nature of the cell wall, which protects them from several physical and chemical stressors [60]. This species, due to its multiple pathogenic attributes, can cause IA, aspergilloma, and ABPA in immunocompromised individuals [61]. In addition, prolonged exposure to A. fumigatus conidia in indoor housing environments can cause occupational asthma [10].
The sections Cremei and Candidi were each represented by a single species: A. wentii and A. tritici, respectively. A. wentii was previously reported in the indoor environment of the Central Library of Havana University [21]. A. tritici was not found on the walls of houses in any geographical region. There are few reports of A. tritici associated with medical pathologies, possibly due to its morphological similarity to A. candidus. Hubka et al. [62] reported the first case of A. tritici associated with onychomycosis. They suggested that it could have been the same agent previously associated with onychomycosis, and that it was possibly misidentified as A. candidus in other reports due to a lack of sequencing analysis.
The benA marker has been previously used to study the phylogeny within the Aspergillus genus and other related species [16] because it is a conserved, slow-evolving gene with a high degree of interspecific variability in the intronic regions. In the present study, the benA marker was used to classify the environmental isolates of Aspergillus spp., corroborating its value as a phylogenetic marker for species identification. In another study, Duarte-Escalante et al. [63] carried out the phenotypic identification of isolates of A. fumigatus from Mexico, Argentina, France, and Peru, assigning all isolates to the Fumigati section, based on their macro-and micromorphology. The phenotypic classification was confirmed by the phylogenetic analysis with sequences of the benA gene region, which showed that all isolates belonged to the species A. fumigatus. Likewise, when Montenegro et al. [64] characterized the phenotyping and genotyping of isolates of the Fumigati section, they used several conserved genes, including the gene that encodes for the b-tubulinencoding gene (benA), the mitochondrial cytochrome b-encoding gene (mtcytb), the gene that encodes for rodlet (rodA), the salt-responsive gene, the internal transcribed spacer (ITS) 1-5.8S-ITS2 region, the genes encoding calmodulin (CaM), and actin. They used partial sequences of these genes to assess the phylogenetic relationships among the Aspergillus species; they found that the most relevant genes for differentiating the Aspergillus species are benA and rodA.
Finally, the municipality of Cerro is slightly richer than the municipality of Arroyo Naranjo in the number of Aspergillus species and diversity; this small difference can be explained by the two regions having different microenvironmental conditions. In addition, the Cerro municipality is characterized as one of the most industrialized and with the highest vehicular traffic in the province of Havana and is therefore one of the most complex from the environmental point of view.

Conclusions
Fifteen species identified in this study constitute new records for outdoor and indoor environments in Cuba (A. calidoustus, A. heteromorphus, A. nomius, A. giganteus, A. violaceofuscus, A. aculeatus, A. neoniger, A. welwitschiae, A. tritici, A. aculeatinus, A. micronesiensis, A. templicola, A. subramanianii, A. melleus, and A. ochraceus). This is relevant, since our findings contribute to the knowledge of fungal biodiversity and ecology. In addition, it constitutes an alert for the sanitary authorities of Cuba, since prolonged exposure of the inhabitants to the spores and humidity in houses can aggravate symptoms of asthma and allergic rhinitis in susceptible individuals.
Supplementary Materials: The following are available online at https://www.mdpi.com/2076-260 7/9/1/115/s1, Figure S1: Molecular phylogenetic analysis by the maximum likelihood method. The evolutionary history was inferred using the maximum likelihood method based on the general timereversible model. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed; Table S1: Groups identified in the phylogenetic tree obtained by maximum likelihood from benA gene sequences.

Conflicts of Interest:
The authors declare no conflict of interest.