The occurrence of potentially pathogenic filamentous fungi in recreational surface water as a public health risk

Microfungi occurring in surface water may represent an important health risk. Recreational water reservoirs are a potential reservoir of pathogenic fungi. The aim of the study was to assess the diversity of mycobiota in selected artificial bathing reservoirs with regard to its biosafety for the human population. The studies were conducted during the summer of 2016 in three research seasons (June (I), July and August (II), and September (III)), taking into account the various periods of recreational activities. Filamentous fungi were isolated from water samples collected at five different ponds utilized for recreation. From 162 water samples, 149 fungal taxa of filamentous fungi were identified: 140 were classified to species level and only nine to genus level. Aspergillus fumigatus was the dominant species. The highest species richness (S) was noted in June, with 93 fungal taxa (Menhinick’s index from 2.65 to 4.49). Additionally, in season I, the highest diversity of fungal species was revealed (Simpson’s diversity index from 0.83 to 0.99). The average number of CFU/1 mL sample ranged between 0.4 and 4.6 depending on the time of sampling and ponds. Of all the isolated species, 128 were clinically relevant (11 from RG-2 and 117 from RG-1), highlighting the need to introduce seasonal mycological monitoring of such reservoirs. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2020.096 ://iwaponline.com/jwh/article-pdf/18/2/127/709059/jwh0180127.pdf Katarzyna Góralska (corresponding author) Joanna Błaszkowska Department of Biology and Parasitology Medical University of Lodz, Pomorska 251, 92-213 Lodz, Poland E-mail: katarzyna.goralska@umed.lodz.pl Magdalena Dzikowiec Department of Biomedicine and Genetics Medical University of Lodz, Pomorska 251, 92-213 Lodz, Poland This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.


INTRODUCTION
Water reservoir contamination occurs when substances and chemical compounds, and allochthonous organisms, not present under natural conditions are discovered in reservoirs (Libudzisz et al. ). Microbiological contamination of the aquatic environment can come from natural sourcesmainly soil in the immediate vicinity but also the transmission of microorganisms through air currentsor through anthropogenic sources, including wastewater, surface and ground runoff from industrial and agricultural areas, and landfills (Lucyga et al. ). The presence of potentially pathogenic fungi in water reservoirs, as well as the lack of sanitary and epidemiological supervision over such objects, poses a risk of acquiring waterborne infections.
Throughout the past 30 years in Europe, more than 400 different fungal species have been found in groundwater, surface water, and drinking water; among these species, 46 were classified as Biosafety Level-2 (Novak-Babičet al. ), meaning they cause various diseases, such as allergies and mycoses, in humans and animals.
In natural water environments, autochthonous species are most often represented by microscopic fungi from the following classes: Chytridiomycetes, Oomycetes, Trichomycetes, and Mucoromycetes (which were once named Zygomycetes). Of the millions of estimated fungal species, only 3,000-4,000 are classified as aquatic fungi (Grossart & Rojas-Jimenez ) for which the water environment is a natural place of existence. The so-called water fungi belong to various taxonomic units and occur abundantly in water reservoirs in the form of vegetative mycelium, producing zoospores or other types of spores adapted to spread in water. The optimal temperature for the growth of most temperate aquatic fungi is 25 C, but the fungi can also grow relatively well at temperatures as low as 10 C. Genera such as Alternaria, Aureobasidium, Cladosporium, and Penicillium detected in aquatic environments are classified as secondary freshwater fungi since they originate from terrestrial habitats (Krauss et al. ).
Although it is very difficult to prove the existence of a Rhizopus, and Mucor) in lake water and the incidence of fungal infections in the associated population of Kashmir, India: a higher incidence of fungal infection (9.84%) was found in people using lake water than people using only tap water (4.16%). Serious fungal infections (Aspergillus spp., Scedosporium spp., and Rhizopus spp.) of the lungs and brain resulting from the aspiration of contaminated water have been observed in people who have experienced near-drowning episodes (Leroy et

MATERIALS AND METHODS
The research area included five artificial water reservoirs used as public bathing places, located in recreational areas in Lodz. The city is situated in Central Poland, in a temperate climate zone with the following characteristics: four distinct seasons, a mean annual air temperature of 7.5 C, and a mean annual relative air humidity of 80%. The area of the city is 293.25 km 2 , making it the fourth largest city in Poland; it is inhabited by 690,422 people (2,354 people/ km 2 ). Within the city, there are 19 rivers and streams, partly covered with urban infrastructure.  Research was conducted during the summer of 2016 in three research seasons: I in June (before the start of the bathing season), II in July and August (peak bathing season during the holidays), and III in September (after the bathing season). The time of sampling was selected to align with the greatest use of these water reservoirs by people. Weather conditions in the first and second seasons of the research period were similar. Due to frequent rainfall, water levels were very high. However, before the third season, there was a period of drought, significantly lowering the water level; this was especially noticeable in the SJ reservoir, where the coastline moved by more than 2 m. Samples were always taken at the same time (between 6 and 8 AM) and weather conditions were similar (windless and rainless).
The samples were collected in places most frequently used by people (gang-boards, beaches, designated bathing areas, and harbours).
Water samples were taken in triplicate 1.5-2 m from the shore of the water reservoirs at a layer 15 cm below the surface; the sampling was performed using Whirl-Pak ® bags (Nasco, USA) with a volume of 500 mL. The number of sampling locations was dependent on the size of the water body: three sampling points on SJ, three on M, four on S, three on J, and five on A. In total, 162 samples were collected.
During each collection, the air and water temperatures and water pH were measured (pHep Tester, Pocket pH Tester, HANNA instruments, Romania). The collected samples were delivered to the laboratory within an hour. The samples (500 mL) were concentrated by centrifugation to a volume of 10 mL, and then 1 mL of each sample was seeded on Sabouraud dextrose agar (SDA, Biomerieux, France) with chloramphenicol (repeated three times) and Czapek-Dox medium (Biomerieux, France) (repeated three times).
As the study focused on fungi that could pose a potential threat to human health, Sabouraud's dextrose agar was chosen for the growth of clinically relevant species, whereas Czapek-Dox medium was used to obtain sporulation of moulds (mostly of genera Aspergillus and Penicillium) to facilitate identification. Incubation was carried out for 5-7 days at 24 C. After 7 days of incubation, the number of colonies was counted and expressed as the number of colony-forming units (CFU)/1 mL for water samples. For slow-growing fungi, incubation was prolonged for 14-21 days to achieve sporulation. From the obtained filamentous fungal isolates, microscopic preparations were made according to the Gerlach technique by pressing the adhesive tape to the mycelium and then transferring it to a microscope slide, using lactophenol and aniline blue staining (Gerlach ).
Simpson's diversity index is a measure of diversity which takes into account the number of species present (n), as well as the relative abundance of each species (N): The obtained data were analysed using the χ 2 test and the Spearman rank correlation. Additionally, for comparison of fungal counts obtained from media, the non-parametric Kruskal-Wallis ANOVA was used. All calculations were performed using the STATISTICA 13.2 software. For all test, the significance level was assumed to be α 0.05.

RESULTS
In total, 149 different taxa of filamentous fungi were identified: 140 classified to the species and only nine to genus level (Supplementary Table S1). In water reservoirs (SJ, M, and A), fungi belonging to Oomycota, Zygomycota, Ascomycota, and Basidiomycota were identified. No representatives of the Zygomycota were found in S, while no fungi from the Basidiomycota were found in J (Table 1 and Figure 2). The greatest species richness (S) was found in SJ (79 species), while the smallest number of species was found in M (47) and A (42) (Figure 3). Significant differences in species richness (S) were observed between research seasons and examined ponds (χ 2 ¼ 31.20; df ¼ 8; p ¼ 0.0001). Statistically significant differences between species richness and reservoirs were noted for season I (χ 2 ¼ 14.61; df ¼ 4; p ¼ 0.0056) and for season III (χ 2 ¼ 22.5; df ¼ 4; p ¼ 0.0002); no significant difference was observed for season II (χ 2 ¼ 9.06; df ¼ 4; p ¼ 0.0595).
The highest number of species (93)  number of CFU/1 mL ranged between 0.4 and 4.6. For pond A, the highest differences between CFU/1 mL were noted in relation to seasons; in season III, the value of CFU (2.9/1 mL) was over 3.5 and 7 times higher than in season I and II, respectively. More detailed data regarding CFU/1 mL are given in Table 2.

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Chrysosporium X/1 X/1 X/1 X/1 X/2 X/1 X/1 2 2 36 Trichophyton X/1 X/1 X/1 X/1 X/1 X/1 X/ In the examined ponds, depending on the sampling date, differences for water pH and water and air temperature were noted. The physicochemical parameters of the analysed tanks are presented in Table 3.
A statistically significant negative correlation (Spearman rank correlation) was found between the number of species from the SJ reservoir and the pH of its water (r ¼ À0.737) (Figures 4 and 5). There was no correlation between the number of species in the M, J, and A reservoirs and pH and water temperature, while positive correlations were found between the number of species in the S pond and water pH (r ¼ 0.59) and water temperature (r ¼ 0.75) ( Figure 4). No significant correlation was observed between the number of colonies and air temperature in any pond ( Figure 5).
In the study, dominated A. fumigatus was detected in all reservoirs. Alternaria alternata, Chrysosporium inops, and Penicillium aurantiogriseum were also frequently isolated (Supplementary Table S1). In research season I, A. fumigatus and P. chrysogenum predominated, but Bjerkandera adusta was also frequent. In research season II, A. fumigatus, followed by A. alternata and C. inops, were most commonly isolated. In the third season, Aspergillus fisheri and A. fumigatus were most often identified. In the SJ pond, the most frequently recorded species was A. fumigatus, but P. waksmanii, P. citrinum, and P. aurantiogriseum were also very often found. In the M, dominated A. fumigatus, however, Trichoderma harzianum was also frequently identified. In the S reservoir, A. fumigatus and A. alternata were most frequently recorded. In pond J, the most frequently identified species was A. niger, but A. fumigatus, P. waksmanii, and C. inops were often noted as well.
The most common species in reservoir A was A. fumigatus, but A. niger and P. chrysogenum were also very common (Supplementary Table S1).
Almost 86% of all identified fungal species are known to be associated with human infections. Table 2   Species classified as BSL-1 and BSL-2 were most common in SJ (65 and 16, respectively), but similar frequencies were observed in other ponds; however, statistically significant differences were found between water reservoirs with regard to the number of species classified as BSL-1 (χ 2 ¼ 11.91, df ¼ 4, p ¼ 0.0180). A significantly higher total much fewer studies concern fungi that secondarily colonize     10-24 C, pH; 6.5-8.5), it seems that these fluctuations did not appear to have a significant impact on the abundance of the fungal population. It should be emphasized that high air temperatures associated with strong solar radiation were recorded (mean 18 C; maximum 33 C) during the sampling period, both in June and July, that could modulate the abundance fungal communities. The abundance of fungi was found to be low in the samples, varying from 0.4 to 4.6 CFU/1 mL according to the time of sampling. Comparable CFU values were obtained for three lakes used for recreational purposes located in Olsztyn, northern Poland

SUPPLEMENTARY MATERIAL
The Supplementary Material for this paper is available online at https://dx.doi.org/10.2166/wh.2020.096.