Incidence of Fungal Aerosols from Selected Crowded Places in Port Harcourt, Nigeria

The role of aerosols in the spread of infectious diseases cannot be overemphasized in the face of increased environmental pollution from different sources. This study aimed at determining the distribution of fungi occurring in the air around human dwellings. The fungal aerosols were randomly collected from five crowded environments (market, church, school, motor park and crowded home) in Port Harcourt, Rivers State, Nigeria and examined. The sedimentation method was used for the microbiological sampling of air and fungi were identified based on macroscopic and microscopic method. The mean fungal load of the different crowded environments were as follows; market (3.19±0.43 log10 SFU cm−2 min−1), church (2.96±0.33 log10 SFU cm−2 min−1), school (3.22±0.29 log10 SFU cm−2 min−1), motor park (3.25±0.35 log10 SFU cm−2 min−1) and home (3.04±0.46 log10 SFU cm−2 min−1), with motor park having the highest and church having the lowest. A total of 16 fungal isolates belonging to twelve (12) genera were identified. They included Aspergillus spp. (31.25%) and Paecilomyces sp., Mucor sp., Fusarium sp., Aspergillus sp., Rhizopus sp., Colletotrichum sp., Cryptococcus sp., Alternaria sp., Cladosporium sp., Chrysosporium sp. and Lophophyton sp. each having a percentage occurrence of 6.25%. Although there was no significant difference (p≥0.05) in the fungal population in the different sampled locations, Motor Park was observed to have the highest percentage occurrence (34.50%) and church the least (9.35%). The percentage occurrence from all sampled sites was in the order motor park>school>market>home>church. The presence of some potential pathogenic fungi such as Aspergillus flavus pose serious public health risks.


INTRODUCTION
A suspension of tiny solids or liquid particles in air is known as an aerosol (Heo et al., 2014).With sizes ranging from 0.001 to 100 μm, they are easily transferred from an area to another in air ( Jaiyu et al., 2019;Kim et al., 2018).Quite ubiquitous, they are bioaerosols when they have a biological origin and have been classified based on the constituting organism (dead or living) to include viruses, bacteria, fungi and other products from organisms such as spores, toxins, metabolites and pollen ( Jaiyu et al., 2019;Kim et al., 2018;Heo et al., 2014).The route of introduction of bioaero-sols into the air is from plants, soil and most importantly animals including humans (Lee, 2011).Humans present a very important introduction route of bioaerosols due to some reflex actions like coughing and sneezing that are popular among humans (Wang and Du, 2020).These reflexes result from hypersensitivity reactions due to exposure to allergens usually present in their environment introduced by organisms or their products.Further, anthropogenic activities such as composting and other agricultural processes as well as industrial activities have also been highlighted for the introduction of bioaerosols (Kim et al., 2018).This explains the presence of bioaerosols both in outdoor and indoor environments that have human populations (Mahdwal et al., 2020;Jaiyu et al., 2019;Kim et al., 2018;Heo et al., 2017Heo et al., , 2014)).Bioaerosols have severe effects in various ways on human and environmental health including visibility (Heo et al., 2014).
The health impact of bioaerosols or droplets is enormous as they have been implicated for numerous diseases (Tay et al., 2020).These diseases result from aetiologic agents borne in aerosols (Wang and Du, 2020;Afanou et al., 2019;Mbareche et al., 2019;Jaiyu et al., 2019;Kim et al., 2018;Saha et al., 2017;Heo et al., 2017Heo et al., , 2014)).Mostly, these diseases have been linked to environmental exposures to bioaerosols in different places important to these individuals, like occupational and residential environments (Kim et al., 2018).Most remarkable diseases linked to bioaerosols are respiratory diseases including rhinitis and chronic obstructive pulmonary diseases (COPD) (Kim et al., 2018;Heo et al., 2014).Bioaerosols have also been implicated for more severe illnesses such as Severe Acute Respiratory Syndrome (SARS) (Tay et al., 2020;Heo et al., 2014), a group of diseases caused by deadly viruses including the recent 2019 Corona Virus Disease (Covid-19) (Wang and Du, 2020;Heo et al., 2014).
Whole fungal cells as well as fragments and fungal products including spores, toxins and metabolites have been found in bioaerosols from both indoor and outdoor samples (Afanou et al., 2019;Jaiyu et al., 2019;Mbareche et al., 2019;Kim et al., 2018;Saha et al., 2017;Heo et al., 2017Heo et al., , 2014)).These organisms have been found among breathable aerosols (1 to 10 μm) (Heo et al., 2017) as well as those that have been found to be of dermatological health importance; settling on the skin (Kim et al., 2018).Bioaerosols containing fungal constituents have been implicated in patients with allergies as well as irritations and opportunistic infections (Mbareche et al., 2019).Specifically, fungal aerosols cause health effects ranging from irritations to cough and sore throat on short term exposure as well as severe lung diseases due to long term exposure.Severe lung infections in immunocompromised persons have been reported following the inhalation of airborne fungi such as Aspergillus fumigatus (Afanou, 2015).
The issues relating to gap in knowledge of bioaerosols is owing to the minimal study of air flora compared to other ecosystems.Fungal aerosols have been understudied as fungi have generally not been studied in all natural environments; air, water and soil.About 1.5 million fungal species are estimated to be in existence, with only about 120,000 species described (Afanou, 2015).However, fungi colonization of the air environment should be anticipated as most fungi are aerobic ( Jia et al., 2016).The production of spores as adaptive reproductive parts (Jia et al., 2016;Afanou, 2015) have made the introduction and proliferation of fungi in air most likely.
Recently, bioaerosols have received a lot of emphasis due to consequences reputed to them (Kim et al., 2018).This has led to investigations of crowded places in both indoor and outdoor settings recording anthropogenic activities (Afanou et al., 2019;Jaiyu et al., 2019;Mbareche et al., 2019;Kim et al., 2018;Saha et al., 2017;Heo et al., 2017Heo et al., , 2014)).The need to study and monitor the microbial load and quality of air has been highlighted (Jaiyu et al., 2019;Mbareche et al., 2019).This is important for occupational, public health risks assessments and will present a mirror of the prevailing hygiene conditions (Heo et al., 2017).This research sought to study the fungal aerosols in crowded places in Port Harcourt, Rivers State.Specifically, sites comprising indoor (crowded home, school and church) as well as outdoor (market and motor park) environments were sampled.

1 Study Area
This study was carried out in Port Harcourt, the capital of Rivers State, Nigeria.Precisely, we studied chosen locations ( Samples were collected from selected locations, consisting of market, church, school, crowded home and Motor Park in Diobu and Nkpor village, Rumuolumeni, all in PHALGA.This local government area is the city centre in Port Harcourt and centre of most activities in the state.This location was chosen based on the dense population and level of economic activities recorded. All sample locations were within Port Harcourt metropolis and consistent with already reported meteorological conditions.Sampling was done between May and July with wind speed between 2 and 3.18 m s -1 (Edokpa and Nwagbara, 2017;Umoh et al., 2014).

2 Media Preparation
The solid medium used for the fungal analysis of the aerosol was the Sabouraud Dextrose Agar (SDA).Sabouraud Dextrose Agar is a selective medium used for the identification, isolation and characterization of fungi and yeasts.The acidic pH 5.6 of this medium inhibits many species of bacteria.Selectivity of the medium was enhanced by the addition of chloramphenicol, a broad spectrum antibiotic, effective against both Gram negative and Gram positive bacteria (Harrigan and McCance, 1990).

3 Sample Collection
The settle plate method with passive monitoring was used as previously described (Saha et al., 2017).Briefly, sterile Petri dishes of 9 cm diameter, containing culture medium were placed at strategic points at the sampling locations.Each plate was placed on a stool of approximately 1 m above the ground level and exposed for a period of thirty (30) minutes and then covered.This was done to allow fungi to impinge on the agar surfaces.Sampling was done at 11:00 am from all locations, once a week for 10 weeks consecutively, from May-July, 2018 at all five (5) sampling sites.Samples were transported to the Microbiology Laboratory, Rivers State University, for analysis.

4 Enumeration and Isolation of Pure Fungi
Colonies After incubation at 30°C for 3 to 5 days, distinct spore forming units were counted and expressed as spore forming unit per cubic centimetre per minute (SFU cm -2 min -1 ) taking into account the following equation described by Omenliansky (Hayleeyesus and Manaye, 2014) with minimal adjustments; Where N is the fungal SFU cm -2 min -1 of air; a = the number of colonies per Petri dish; b = the dish surface area (cm 2 ); t = the exposure time (min) To obtain pure fungi colonies, discrete spores were subcultured onto freshly prepared Sabouraud dextrose agar plates and incubated at 30°C for 3 to 5 days.

5 Identification of Fungi
All isolates were identified based on macroscopic and microscopic characteristics using standard methods.Macroscopic identification used morphological features including shape, size and colour.Following macroscopic identification, the isolates were subjected to microscopic identification by staining all the isolates with Lactophenol cotton blue.Stained samples were viewed for spores arrangement and hyphae structure using × 40 objective lens (Dugan, 2006).To ensure clear results were obtained, the popular cellotape flag method as previously described was used (Harris, 2000).Briefly, a small wooden applicator stick and clear cellotape of 2 cm width were used to make a 2 × 2 cm cellotape flag.Sterile technique was used by gently pressing the sticky side of the flag onto  the surface of the culture.Then the tape was removed and a 95% alcohol drop applied to the flag.This alcohol drop had the dual functions of a wetting agent and to dissolve the glue and release the flag from the applicator stick.A drop of lactophenol cotton blue was then placed in the flag on a grease free slide.After discarding the applicator stick, another drop of stain was added, covered with a coverslip, gently pressed and excess stain moped up.The slide was placed on the microscope and viewed using × 40 objective lens.

6 Statistical Analysis
The software package SPSS version 17.0 was used to analyze fungi properties.Means were calculated and compared.The one-way analysis of variance (ANOVA) was used to find the levels of significance (p≤0.05) for the considered parameters.

RESULTS AND DISCUSSION
Fungi in bioaerosols were investigated using standard microbiological techniques.The result of fungal population as shown in Fig. 1 showed no difference (p> 0.05) across the locations.However, the motor park (outdoor site) recorded the highest count of 3.25± 0.35 log 10 SFU cm -2 min -1 while the least was the church (indoor) 2.96±0.33log 10 SFU cm -2 min -1 (Fig. 1).
We report a marked fungal contamination of the bioaerosol in all the studied sites.The spores present varied according to locations probably in response to variation in hosts, environmental conditions as well as substrates available in such sites (Table 4).This is in consonance with the study of Durugbo et al. (2013) in Ogun State, Nigeria.The present study was conducted between May and July when the humidity is expected to be high.Port Harcourt is mostly humid in the wet season when the study was conducted as shown in Table 2 (Umoh et al., 2014).High relative humidity and temperatures have been reported to improve fungal growth (Manna and Kim, 2018).The factors have also been shown to affect the sedimentation method used for sample collection in  the present study based on difference in the travel speed of suspended particles (Pasquarella et al., 2000).The Port Harcourt environment presents high relative humidity values (Table 2).Nastasi et al. (2020) established a significant relationship between relative humidity and fungal growth; high RD values improved fungal growth.
The current study found an abundance of spore-forming Fungi in some sites.Air is a difficult environment for microbial colonization, thus organisms inhabiting air are those with adaptive features (Jerdan et al., 2019).Fungal spores are adaptive features for surviving harsh environments.This may explain the high counts of spore-forming fungal species in the present study.Sporulating fungi have also been reported in previous studies (Rawdan and Abdel-Aziz, 2019;Patel et al., 2018;Durugbo et al., 2013).Aspergillus spp. with a percentage occurrence of 31.25% (Table 4) were the most dominant species reported in this study which is in agreement with another study (Durugbo et al., 2013;Al-doory, 1980).
While A. niger was present at all the sites, A. flavus was present in 4 out of the 5 sites.The least occurring fungal genera were species of Aspergillus tereus, and Mucor spp.This may be due to the prevailing conditions at the time of this study as A. tereus (Pang et al., 2020) and Mucor spp.(Morin-Sardin et al., 2016) have shown to grow under severe conditions.
In terms of percentage occurrence, Aspergillus spp.were most prevalent at 31.25% while all other species recorded accounted for 6.25% of all reported fungi (Table 4).Al-doory (1980) also reported similarly in an earlier investigation in Philadelphia, USA.The similarity in the results of both studies could be tied to similar weather conditions especially RD (Umoh et al., 2014;Hemler et al., 1997).Motor parks recorded the highest percentage occurrence (34.50%) while it was least in Church.Douglas and Lumati (2018) also reported a high incidence of fungal species in motor parks.This high incidence was explained by the high density of people in motor parks and the level of activity compared to calm church environment.
The fungal species isolated from bioaerosols in this study are similar to those reported from previous studies.Njokuocha and Osayi (2005) as well as Agwu et al. (2004) reported similar results in Nsukka, Nigeria, Adekunle (2001) also reported from Lagos.Similarly, Ogunlana (1975) did their study in Ibadan, Nigeria and recorded similar results.Also, Rao et al. (2009) in Kajachi, Pakistain, Saha et al. (2019) in India, Madsen et al. (2016) in Denmark and Mbareche et al. (2019) in Canada all found similar fungal species in bioaerosols.
Fungal spores have been associated with air pollution and can cause adverse effects on biota including humans (Mbareche et al., 2019;Shelton et al., 2002).The fungi reported in the present study present severe public health risks.This risk is linked to reported allergen and opportunistic pathogens (Afanou et al., 2019;Mbareche et al., 2019;Madsen et al., 2016;Durugbo et al., 2013).Remark able diseases attributed to these reported fungi include lung diseases allergic rhinitis, asthma and other diseases of the respiratory tracts (Armstrong-James et al., 2017;Durugbo et al., 2013).Globally, fungal infections  et al., 2017).Further, fungal materials such as spores and other particles may cause allergies and respiratory illnesses especially in hypersensitive and immunosuppressed individuals (Heo et al., 2017;Durugbo et al., 2013;Singh et al., 1994;Kurup and Kumar, 1991).This may explain the severe consequences of inhaling air contaminated with fungal spores of Mucor, Aspergillus, Rhizopus, Penicillium (Afanou et al., 2019;Armstrong-James et al., 2017;Schmiedel and Zimmerli, 2016).Pathogenic fungi such as A. flavus reported in this study, are implicated for several diseases of animals and plants (Mbareche et al., 2019;Rudramurthy et al., 2019).For example, A. flavus is the second leading cause of aspergillosis in humans, especially in immunocompromised individuals such as neonates (Rudramurthy et al., 2019;Robbins et al., 1995).Others such as Mucor and Rhizopus are allergens and opportunistic pathogens (Taj-Aldeen et al., 2017;Ekindayo, 1986), posing appreciable health risks.Mucor causes sinusitis as well as infections of the GIT and lungs (Taj-Aldeen et al., 2017).Rhizopus spp.cause opportunistic infections in neutropenic persons and ketoacidic diabetics (Durugbo et al., 2013;Adekunle, 2001).Further, the Penicillium spp.detected are known to synthesize mycotoxins.These mycotoxins are a leading cause of lung infections (Martin, 2017).Kakefried (2006) also reported that spores from Penicillium spp.are responsible for skin irritations leading to itching in individuals.
The indoor assemblage was dominated successively by Rhizopus arrhizus, Mucor spp., Aspergillus spp.while the outdoor assemblage reported Mucor spp., Aspergillus spp.Our results are similar to the reports of Durugbo et al. (2013) and Nayar et al. (2007), with appreciable difference in the indoor and outdoor environments in Redemption City, Ogun State, Nigeria and Kerala, India respectively.This difference may be explained by the meteorological parameters and hygiene conditions in Port Harcourt as compared with these other cities.The overall climatic conditions of a place as represented by the meteorological parameters, determine the vegetation of a place.Zeng et al. (2020) reported similar fungal colonization in forest grassland regions of China.
The total fungi isolated from the five sampled sites revealed an interesting trend.A total of 12 fungal species were isolated from the 5 sampling sites (Table 4).The species were Mucor spp., Rhizopus spp., Aspergillus spp., Penicillium spp., Alternaria spp., Chrysosporium spp., Cladosporium spp., Colletotrichum spp., Cryptococcus spp., Fusarium spp., Lophophyton spp., Paecilomyces spp.Researchers indicated a variation in the concentration of fungal spores in air with respect to times of the day or seasons (Heo et al., 2014;Huang et al., 2002;Singh et al., 1994;Ogunlana, 1975).Accordingly, Ogunlana (1975) working on the fungal spores in air in Ibadan, Nigeria, observed a variation due to seasons (wet and dry seasons), Huang et al. (2002) reported higher fungal aerosols in winter while studying in southern Taiwan.Similarly, Singh et al. (1994) recorded the highest fungal spores between June and September Delhi, India while Heo et al. (2014) reported a significant increase in aerosols in monsoon areas during the rainy season.The present study further confirms that fungal proliferation may be higher in the wet season with high relative humidity values (Table 2).

CONCLUSION
This study isolated a total of 12 fungal species from aerosols collected from both indoor and outdoor environments in Port Harcourt, Rivers State, Nigeria.Many of the fungi species identified have been linked to health care concerns both on short and long term exposure, especially in immunocompromised individuals.The presence of these fungal species is an indication of the high level of pollution in the environments studied and poses serious public health risks.Further studies involving individuals in these environments will reveal the extent of infection by these fungi species.
Key: RH = Relative Humidity, RF = Rainfall, T = Difference between maximum and minimum Temperature and W = Wind speed.

Fig. 1 .
Fig. 1.Variation of total heterotrophic fungi population in the different study locations.

Table 1 .
Global positioning system (GPS) for the different sampling sites.

Table 3 .
Characteristics of fungal isolates from market, church, school, Motor Park and crowded home.

Table 4 .
Percentage occurrence of fungal species at the various sites.