The World Health Organization calls to assess possible health risks from emerging fungi originating not only from hospitals but also from the natural environment. Fungal contamination in oligotrophic water systems represents a public health concern due to the potential for the emergence of antifungal-resistant strains. This study focused on the identification of Aspergillus spp. and Candida spp. isolated from different water sources and materials in contact with water. Isolated strains have been tested against nine antifungals to assess the prevalence of resistance in these strains. Only one strain of Aspergillus protuberus was resistant to amphotericin B. On the other hand, all Candida strains were intermediately resistant to anidulafungin and micafungin, 5.8% were borderline resistant to 5-flucytosine and fluconazole, and 3% to voriconazole. Candida parapsilosis sensu stricto isolated from water samples had statistically higher minimal inhibitory concentration (MIC) for anidulafungin than clinical strains and clinical strains had statistically higher MIC for itraconazole. Statistical analysis pointed out habitat to be significant for higher MIC in C. parapsilosis. Our findings show that borderline-resistant strains can be transferred by water; thus, potable water should be considered as a possible source of resistant strains in hospitals and healthcare units.

  • World Health Organization calls to assess health risks from environmental fungi.

  • Aspergillus and Candida were isolated from 42 and 16% of drinking water samples.

  • Candida strains showed intermediate resistance to echinocandines and resistance to azoles.

  • Primary habitat is statistically significant for higher minimal inhibitory concentration in Candida strains.

  • Potable water could be a source of resistant fungal strains.

Water is crucial for life on Earth, yet the sources of fresh water are limited (Musie & Gonfa 2023). Global warming, the growing human population, and the need for bigger food production led to a phenomenon particularly observed in the last decade – the rise of fungi (Neabore 2024). Fungi are ubiquitous, important for the circulation of matter in the environment, and can be isolated from diverse habitats, including habitats with extreme growth conditions (Hyde et al. 2019). As such, they are being regularly reported on also from different water sources worldwide (Novak Babič et al. 2017). Despite the rapid development of water treatment and disinfection methods, certain fungi can survive chlorination and form biofilms on materials inside water networks (Novak Babič et al. 2023). The fungi are protected from environmental stresses in the biofilm, and the interactions between fungal species in the biofilm may contribute to antifungal resistance (Costa et al. 2022). In addition, it has been shown that colonization with certain fungi depends on the type of building material. For instance, dematiaceous moulds and yeast-like fungi more often colonize materials made of metal and rubber, while yeasts are more associated with plastics (Novak Babič et al. 2023; Černoša et al. 2024). Biofilm formation in an otherwise oligotrophic water environment can significantly affect the quality of material used for the transport and storage of water (Erdei-Tombor et al. 2024). Moreover, mature biofilm represents the continuous source of microbes released into the water and transported to drinking water consumers; thus, the ability of fungi to colonize oligotrophic water systems raises concerns about the potential transmission of opportunistic pathogens to humans (Novak Babič & Gunde-Cimerman 2021). Particularly the genera Aspergillus and Candida came under the spotlight in a healthcare environment due to their ubiquity and the antifungal resistance reported for clinical strains (Arendrup 2014). Their resistance in hospitals has been speculated to originate from the environment as the consequence of fungicides used on crops (Hollomon 2017). Although the presence of these genera is well documented in water, there is still only little data on possible resistance to antifungals in waterborne strains (Monapathi et al. 2021; Shittu et al. 2022), particularly in drinking water. The present study thus seeks to characterize the response of Aspergillus spp. and Candida spp. isolated from samples of Slovenian raw and drinking water to nine commercial antifungal agents. The results of this study contribute to a broader understanding of antifungal resistance in oligotrophic water systems, providing valuable insights for public health interventions and guiding future research efforts in the field of environmental mycology.
Figure 1

MICs for echinocandins and 5-flucytosine depending on strains’ primary environment. Statistically significant differences at significance level α = 0.01 between group means were determined for anidulafungin, pointing out the possibility of waterborne strains being more likely to have higher MIC than clinical strains.

Figure 1

MICs for echinocandins and 5-flucytosine depending on strains’ primary environment. Statistically significant differences at significance level α = 0.01 between group means were determined for anidulafungin, pointing out the possibility of waterborne strains being more likely to have higher MIC than clinical strains.

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Figure 2

MICs for azoles depending on strains’ primary environment. Statistically significant differences at significance level α = 0.01 between group means were determined for itraconazole, pointing out the possibility for clinical strains being more likely to have higher MIC than waterborne strains.

Figure 2

MICs for azoles depending on strains’ primary environment. Statistically significant differences at significance level α = 0.01 between group means were determined for itraconazole, pointing out the possibility for clinical strains being more likely to have higher MIC than waterborne strains.

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Figure 3

MICs for fluconazole depending on strains’ primary environment. No statistically significant differences at significance level α = 0.01 between group means were determined. Strains from both habitats have similar resistance levels to fluconazole.

Figure 3

MICs for fluconazole depending on strains’ primary environment. No statistically significant differences at significance level α = 0.01 between group means were determined. Strains from both habitats have similar resistance levels to fluconazole.

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Selection and identification of test strains

The study analysed environmental strains of the Candida parapsilosis species complex and Aspergillus spp. that were isolated during the previous studies of Slovenian waters. These included 24 groundwater samples and 135 drinking water samples, and sampled material in contact with water (Novak Babič et al. 2016, 2023; Novak Babič & Gunde-Cimerman 2021). All water samples were taken according to SIST ISO 5667-5:2007, using sterile containers. Biofilms from material in contact with water were sampled from 1 cm2 surfaces with sterile cotton swabs (Golias, Ljubljana, Slovenia).

In addition, the Medical Faculty (University of Ljubljana, Slovenia) provided seven strains of C. parapsilosis species complex from clinical material.

Isolated strains were taxonomically identified according to the sequences of the complete internal transcribed spacer region (ITS = ITS1, 5.8S ribosomal DNA (rDNA), ITS2) using primers ITS5 and ITS4 (White et al. 1990). Aspergillus species were determined by sequencing the partial beta-tubulin gene exons and introns (benA; primers Ben2F and Bt2b (Glass & Donaldson 1995)). Species within the C. parapsilosis species complex were additionally distinguished by restriction fragment length polymorphism (RFLP) analysis of the secondary alcohol dehydrogenase (SADH) gene fragment. The SADH amplicons were obtained using primer set S1F and S1R (Tavanti et al. 2005) and restricted with BanI (ThermoFisher Scientific, Waltham, Massachusetts, USA).

All identified strains were stored in a genetically stable form at the Ex-Culture Collection (Infrastructural Centre Mycosmo, MRIC UL, Slovenia (http://www.ex-genebank.com/, accessed on 20 July 2024)), at the Department of Biology, Biotechnical Faculty, University of Ljubljana.

Determination of C. parapsilosis sensu stricto phenotypes

Following molecular identification and determination of species within the C. parapsilosis complex, we additionally determined phenotypes of C. parapsilosis sensu stricto strains with the method described in Zupančič et al. (2019). Strains were inoculated on Malt Extract Agar, and incubated at 4 °C for 4 weeks. To obtain consistent observation phenotypes of the colonies were checked each week.

Antifungal testing and MIC determination

To perform antifungal susceptibility testing, we used the YeastOne YO10 Kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). Each plate contains nine antifungal agents in dried form. The dilution ranges of antifungals were as follows: amphotericin B (AB, 0.12–8 μg/mL), caspofungin (CAS, 0.008–8 μg/mL), fluconazole (FZ, 0.12–256 μg/mL), itraconazole (IZ, 0.015–16 μg/mL), 5-flucytosine (FC, 0.06–64 μg/mL), voriconazole (VOR, 0.008–8 μg/mL), anidulafungin (AND, 0.015–8 μg/mL), micafungin (MF, 0.008–8 μg/mL), and posaconazole (PZ, 0.008–8 μg/mL).

Aspergillus spp. strains were prepared by dilution of conidia in YeastOne broth according to the manufacturer's instructions to reach the final inoculum of 0.5–5 × 104 CFU/mL. Each well was rehydrated with 100 μL of single-strain suspension. The plates inoculated with strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus westerdijkiae, and Aspergillus udagawae were incubated at 35 °C, and read after 24 and 48 h. Strains of Aspergillus protuberus and Aspergillus creber were incubated at 25 °C and read after 24, 48, and 72 h.

Candida spp. strains were prepared by dilution of 24 h old yeast cultures in broth according to the manufacturer's instructions to reach the final inoculum of 1.5 − 8 × 103 CFU/mL. Each well was rehydrated with 100 μL of single-strain suspension. The plates inoculated with Candida spp. strains were incubated at 35 °C, and read after 24 h.

Growth in each well was determined due to colour change from blue (negative) to pink (positive). Minimal inhibitory concentration (MIC) was determined as the first concentration at which the growth indicator in the well remained negative.

Statistical analysis

Statistical evaluation of the results for C. parapsilosis sensu stricto strains was performed with XLSTAT (Data Analysis and Statistical Solution for Microsoft Excel, Addinsoft, Paris, France, 2017) and in GraphPad Prism, version 9.3.1 for Windows, GraphPad Software, Boston, Massachusetts, USA (www.graphpad.com). First, the Kolmogorov–Smirnov test was used to determine the distribution of samples. The equality of variances was then tested with an F-test for comparison of clinical strains vs. waterborne strains, and Levene's test was performed for evaluation of variances between four C. parapsilosis phenotypes.

Determination of the significance of the results for the populations of clinical and environmental strains with normal distribution and equal variances was done with Student's t-test while populations following normal distribution with unequal variances were subjected to Welch's t-test.

Data on phenotypes and antifungals were all normally distributed; thus, one-way ANOVA was used to determine the significance of the obtained results for data with equal variances, while Welch's ANOVA was used for data with unequal variances.

Aspergillus spp. from Slovenian water are susceptible to antifungals

Fungi from the genus Aspergillus were isolated from 19 groundwater samples (79.2%) and 57 drinking water samples (42.2%). The most common species in groundwater and on material in contact with groundwater were A. fumigatus, A. flavus, and A. creber. The majority of A. creber, and species A. protuberus and A. westerdijkiae were isolated from already chlorinated drinking water samples.

Representative strains were selected depending on the location of the main water source and tested against nine common antifungals (Table 1). Among all tested strains, only one strain of A. protuberus (EXF-16259) showed possible resistance to amphotericin B (AB, 4 μg/mL). All other strains were susceptible to tested antifungals when compared with the available literature.

Table 1

MIC for Aspergillus spp. strains after 48 h of incubation

SpeciesEXF No.HabitatMIC (μg/mL)
ANDABMFCASFCPZVORIZFZ
A. creber 16946 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.06 256 
16773 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
16938 Tap water 0.015 0.008 0.008 64 0.06 0.06 0.06 128 
16759 Tap water 0.015 0.008 0.015 64 0.06 0.12 0.06 256 
16276 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
16251 Material in contact with groundwater 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.03 0.03 0.03 >16 0.5 0.5 No data 
A. flavus 17188 Groundwater 64 0.03 0.12 0.06 128 
ECOFF data S ≤ IE IE IE No data 0.25 No breakpoint 
R > IE IE IE No data 0.5 No breakpoint 
A. fumigatus 17187 Groundwater 0.5 64 0.015 0.12 0.06 256 
17049 Groundwater 0.25 64 0.03 0.12 0.12 256 
ECOFF data S ≤ IE IE IE No data 0.125 No breakpoint 
R > IE IE IE No data 0.25 No breakpoint 
A. protuberus 16259 Material in contact with tap water 0.015 4a 0.008 0.015 32 0.015 0.06 0.03 32 
14928 Tap water 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
14929 Tap water 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.03 0.03 0.03 >16 0.5 No data 
A. udagawae 17061 Groundwater 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Alastruey-Izquierdo et al. (2014)  MIC90 0.05 0.12 No data 0.25 No data 
A. westerdijkiae 16761 Tap water 0.015 0.008 0.03 64 0.06 0.06 0.06 256 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.5 >16 0.06 0.06 No data 0.5 0.5 No data 
SpeciesEXF No.HabitatMIC (μg/mL)
ANDABMFCASFCPZVORIZFZ
A. creber 16946 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.06 256 
16773 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
16938 Tap water 0.015 0.008 0.008 64 0.06 0.06 0.06 128 
16759 Tap water 0.015 0.008 0.015 64 0.06 0.12 0.06 256 
16276 Tap water 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
16251 Material in contact with groundwater 0.015 0.008 0.008 64 0.06 0.12 0.12 128 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.03 0.03 0.03 >16 0.5 0.5 No data 
A. flavus 17188 Groundwater 64 0.03 0.12 0.06 128 
ECOFF data S ≤ IE IE IE No data 0.25 No breakpoint 
R > IE IE IE No data 0.5 No breakpoint 
A. fumigatus 17187 Groundwater 0.5 64 0.015 0.12 0.06 256 
17049 Groundwater 0.25 64 0.03 0.12 0.12 256 
ECOFF data S ≤ IE IE IE No data 0.125 No breakpoint 
R > IE IE IE No data 0.25 No breakpoint 
A. protuberus 16259 Material in contact with tap water 0.015 4a 0.008 0.015 32 0.015 0.06 0.03 32 
14928 Tap water 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
14929 Tap water 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.03 0.03 0.03 >16 0.5 No data 
A. udagawae 17061 Groundwater 0.015 0.12 0.008 0.008 0.06 0.008 0.008 0.015 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Alastruey-Izquierdo et al. (2014)  MIC90 0.05 0.12 No data 0.25 No data 
A. westerdijkiae 16761 Tap water 0.015 0.008 0.03 64 0.06 0.06 0.06 256 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Siqueira et al. (2016)  MIC90 0.5 >16 0.06 0.06 No data 0.5 0.5 No data 

EXF No., strain accession number in Ex Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana (Infrastructural Centre Mycosmo, MRIC UL, Slovenia); ECOFF, epidemiologic cut-off values for wild-type strains, as abbreviated by EUCAST (2023); IE, insufficient evidence that the organism or group is a good target for therapy with the agent (EUCAST 2023); S, susceptible; R, Resistant.

aBorderline or truly resistant strain in comparison to ECOFF and other published data (in bold).

Natural and drinking water as a potential source of C. parapsilosis

Altogether, yeasts identified as genus Candida were isolated from two groundwater samples (8.3%) and 22 drinking water samples (16.3%). The most common species was C. parapsilosis sensu stricto, while only three tap water samples harboured Candida orthopsilosis and Candida pseudointermedia.

Smooth phenotype of colonies prevails in drinking water samples

All strains identified as C. parapsilosis sensu stricto were tested for phenotypic growth of colonies. Both strains isolated from groundwater (100%) and four strains from drinking water (18.2%) had a crepe phenotype of colonies, seven strains from drinking water (31.8%) had a crater phenotype, three had a concentric phenotype (13.6%), and eight were characterized with smooth colonies (36.4%).

C. parapsilosis sensu stricto from water samples are intermediate resistant to anidulafungin and micafungin

Representative strains were tested against nine common antifungals and compared with seven clinical strains (Table 2). All tested clinical and waterborne strains were intermediate resistant to anidulafungin and micafungin. Strains EXF-10240 and EXF-9623 from drinking water with the crater phenotype were borderline resistant against 5-flucytosine (MIC = 4 μg/mL), and clinical strain EXF-10193 with the crepe phenotype was borderline resistant against voriconazole (MIC = 0.25 μg/mL). Two strains, one from clinical material with a concentric phenotype (EXF-10099) and another from drinking water with a smooth phenotype, were borderline resistant against fluconazole (MIC = 4 μg/mL). The rest were susceptible to these antifungals. Additionally, all tested strains were susceptible against amphotericin B (MIC ≤ 1 μg/mL), posaconazole (MIC ≤ 0.064 μg/mL), and itraconazole (MIC ≤ 0.125 μg/mL) (Table 2).

Table 2

MIC for Candida spp. after 24 h of incubation

SpeciesEXF No.HabitatPhenotypeMIC (μg/mL)
ANDABMFCASFCPZVORIZFZ
C. parapsilosis sensu stricto 10095 Clinical material Crepe 0.5 0.5 0.5 0.25 0.25 0.015 0.03 0.06 
10098 Clinical material Crepe 0.5 0.5 0.5 0.12 0.03 0.06 0.06 
10192 Clinical material Crepe 0.25 0.5 0.5 0.06 0.12 0.06 
10193 Clinical material Crepe 0.12 0.5 0.06 0.25a 0.06 
10096 Clinical material Crater 0.5 0.12 0.5 0.5 0.06 0.12 0.06 
10099 Clinical material Concentric 0.5 0.03 0.12 0.06 4a 
10097 Clinical material Smooth 0.5 0.5 0.5 0.008 0.015 0.03 
8460 Groundwater Crepe 0.5 0.12 0.5 0.25 0.015 0.015 0.015 0.5 
8247 Groundwater Crepe 0.12 0.5 0.5 0.03 0.03 0.03 
8404 Tap water Crepe 0.5 0.5 0.25 0.5 0.015 0.06 0.015 
8405 Tap water Crepe 0.5 0.5 0.25 0.5 0.015 0.06 0.03 
8452 Tap water Crepe 0.5 0.25 0.5 0.03 0.06 0.03 
10048 Tap water Crepe 0.5 0.25 0.25 0.5 0.015 0.015 0.03 0.5 
9872 Tap water Crater 0.5 0.25 0.12 0.008 0.015 0.015 0.5 
10133 Tap water Crater 0.5 0.5 0.5 0.25 0.06 0.015 0.03 0.06 
10144 Tap water Crater 0.5 0.5 0.25 0.015 0.03 0.015 
10179 Tap water Crater 0.25 2a 0.25 0.5 0.015 0.03 0.015 
10240 Tap water Crater 0.5 0.25 4a 0.015 0.03 0.03 
9623 Tap water Crater 0.5 0.5 0.5 0.5 4a 0.008 0.015 0.015 
9693 Tap water Crater 0.5 0.5 0.12 0.06 0.008 0.008 0.015 0.12 
5670 Tap water Concentric 0.25 0.5 0.25 0.25 0.015 0.03 0.03 
8411 Tap water Concentric 0.5 0.5 0.25 0.5 0.03 0.06 0.03 
8248 Tap water Concentric 0.25 0.25 0.5 0.25 0.12 0.008 0.008 0.015 0.25 
8406 Tap water Smooth 0.12 0.5 0.12 0.015 0.03 0.03 
9899 Tap water Smooth 0.25 0.5 0.25 0.008 0.008 0.015 0.5 
10058 Tap water Smooth 0.12 0.5 0.12 0.015 0.015 0.03 
10067 Tap water Smooth 0.12 0.25 0.25 0.015 0.015 0.015 0.5 
10174 Tap water Smooth 0.12 0.5 0.25 0.12 0.008 0.015 0.015 
9691 Tap water Smooth 0.5 0.5 0.25 0.12 0.008 0.008 0.015 0.5 
9694 Tap water Smooth 0.25 0.5 0.25 0.25 0.015 0.015 0.03 
9697 Tap water Smooth 0.5 0.015 0.06 0.06 4a 
ECOFF data S ≤ 0.002 0.002 No data 0.064 0.125 0.125 
R > No data 0.064 0.25 0.125 
C. orthopsilosis 8409 Tap water 0.12 0.25 0.25 0.03 0.06 0.015 0.03 0.015 2a 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Borman et al. (2020)  MIC90 0.5 0.25 No data No data ≤0.125 No data ≤0.03 0.06 0.5 
C. pseudointermedia 9894 Tap water 0.015 0.5 0.03 0.03 0.12 0.015 0.015 0.12 
8410 Tap water 0.06 0.5 0.06 0.12 0.03 0.06 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
SpeciesEXF No.HabitatPhenotypeMIC (μg/mL)
ANDABMFCASFCPZVORIZFZ
C. parapsilosis sensu stricto 10095 Clinical material Crepe 0.5 0.5 0.5 0.25 0.25 0.015 0.03 0.06 
10098 Clinical material Crepe 0.5 0.5 0.5 0.12 0.03 0.06 0.06 
10192 Clinical material Crepe 0.25 0.5 0.5 0.06 0.12 0.06 
10193 Clinical material Crepe 0.12 0.5 0.06 0.25a 0.06 
10096 Clinical material Crater 0.5 0.12 0.5 0.5 0.06 0.12 0.06 
10099 Clinical material Concentric 0.5 0.03 0.12 0.06 4a 
10097 Clinical material Smooth 0.5 0.5 0.5 0.008 0.015 0.03 
8460 Groundwater Crepe 0.5 0.12 0.5 0.25 0.015 0.015 0.015 0.5 
8247 Groundwater Crepe 0.12 0.5 0.5 0.03 0.03 0.03 
8404 Tap water Crepe 0.5 0.5 0.25 0.5 0.015 0.06 0.015 
8405 Tap water Crepe 0.5 0.5 0.25 0.5 0.015 0.06 0.03 
8452 Tap water Crepe 0.5 0.25 0.5 0.03 0.06 0.03 
10048 Tap water Crepe 0.5 0.25 0.25 0.5 0.015 0.015 0.03 0.5 
9872 Tap water Crater 0.5 0.25 0.12 0.008 0.015 0.015 0.5 
10133 Tap water Crater 0.5 0.5 0.5 0.25 0.06 0.015 0.03 0.06 
10144 Tap water Crater 0.5 0.5 0.25 0.015 0.03 0.015 
10179 Tap water Crater 0.25 2a 0.25 0.5 0.015 0.03 0.015 
10240 Tap water Crater 0.5 0.25 4a 0.015 0.03 0.03 
9623 Tap water Crater 0.5 0.5 0.5 0.5 4a 0.008 0.015 0.015 
9693 Tap water Crater 0.5 0.5 0.12 0.06 0.008 0.008 0.015 0.12 
5670 Tap water Concentric 0.25 0.5 0.25 0.25 0.015 0.03 0.03 
8411 Tap water Concentric 0.5 0.5 0.25 0.5 0.03 0.06 0.03 
8248 Tap water Concentric 0.25 0.25 0.5 0.25 0.12 0.008 0.008 0.015 0.25 
8406 Tap water Smooth 0.12 0.5 0.12 0.015 0.03 0.03 
9899 Tap water Smooth 0.25 0.5 0.25 0.008 0.008 0.015 0.5 
10058 Tap water Smooth 0.12 0.5 0.12 0.015 0.015 0.03 
10067 Tap water Smooth 0.12 0.25 0.25 0.015 0.015 0.015 0.5 
10174 Tap water Smooth 0.12 0.5 0.25 0.12 0.008 0.015 0.015 
9691 Tap water Smooth 0.5 0.5 0.25 0.12 0.008 0.008 0.015 0.5 
9694 Tap water Smooth 0.25 0.5 0.25 0.25 0.015 0.015 0.03 
9697 Tap water Smooth 0.5 0.015 0.06 0.06 4a 
ECOFF data S ≤ 0.002 0.002 No data 0.064 0.125 0.125 
R > No data 0.064 0.25 0.125 
C. orthopsilosis 8409 Tap water 0.12 0.25 0.25 0.03 0.06 0.015 0.03 0.015 2a 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 
Borman et al. (2020)  MIC90 0.5 0.25 No data No data ≤0.125 No data ≤0.03 0.06 0.5 
C. pseudointermedia 9894 Tap water 0.015 0.5 0.03 0.03 0.12 0.015 0.015 0.12 
8410 Tap water 0.06 0.5 0.06 0.12 0.03 0.06 0.12 
ECOFF data S ≤ IE IE IE IE IE IE IE IE IE 
R > IE IE IE IE IE IE IE IE IE 

EXF No., strain accession number in Ex Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana (Infrastructural Centre Mycosmo, MRIC UL, Slovenia); ECOFF, epidemiologic cut-off values for wild-type strains, as abbreviated by EUCAST (2023); IE, insufficient evidence that the organism or group is a good target for therapy with the agent (EUCAST 2023); S, susceptible; R, Resistant.

*Borderline or truly resistant strain in comparison to ECOFF and other published data (in bold).

Effect of environment on resistance to antifungals

Populations of clinical and environmental strains tested against anidulafungin, amphotericin B, micafungin, caspofungin, 5-flucytosine, itraconazole, and fluconazole followed a normal distribution and had equal variances. Thus, the Student's t-test was used to determine the significance of the obtained results. Populations tested against posaconazole and voriconazole were normally distributed with unequal variances, thus Welch's t-test was used to determine the significance of the obtained results (Table 3 and Figures 13).

Table 3

Comparison of means between clinical and environmental strains of C. parapsilosis at significance level α = 0.01

Antifungal agentVariancesStatistical testP(T < =t) two-tailSignificance
Anidulafungin Equal Student's t-test 0.006 Yes 
Amphotericin B Equal Student's t-test 0.89 No 
Micafungin Equal Student's t-test 0.99 No 
Caspofungin Equal Student's t-test 0.87 No 
5-flucytosine Equal Student's t-test 0.95 No 
Itraconazole Equal Student's t-test 4.37 × 10−6 Yes 
Fluconazole Equal Student's t-test 0.98 No 
Posaconazole Unequal Welch's t-test 0.76 No 
Voriconazole Unequal Welch's t-test 0.05 No 
Antifungal agentVariancesStatistical testP(T < =t) two-tailSignificance
Anidulafungin Equal Student's t-test 0.006 Yes 
Amphotericin B Equal Student's t-test 0.89 No 
Micafungin Equal Student's t-test 0.99 No 
Caspofungin Equal Student's t-test 0.87 No 
5-flucytosine Equal Student's t-test 0.95 No 
Itraconazole Equal Student's t-test 4.37 × 10−6 Yes 
Fluconazole Equal Student's t-test 0.98 No 
Posaconazole Unequal Welch's t-test 0.76 No 
Voriconazole Unequal Welch's t-test 0.05 No 

Under testing conditions, statistically significant differences between group means were determined for anidulafungin and itraconazole, pointing out that strains from water are more likely resistant to anidulafungin than clinical strains. On the contrary, strains from clinical specimens are more likely resistant to itraconazole compared to waterborne strains. No statistically significant differences between group means were observed for all other antifungals, meaning that strains isolated from water have similar resistance levels as clinical strains for these antifungals (Table 3) and Figures 13).

C. parapsilosis sensu stricto phenotypes have no significant effect on resistance to antifungals

Populations of different phenotypes tested against all antifungals were normally distributed, and all except 5-flucytosine had equal variances; thus, one-way ANOVA was used to determine the significance of the obtained results. Welch's ANOVA was used for data on phenotypes tested against 5-flucytosine because the determined variances were unequal. At the level of significance α = 0.01, we tested whether the mean MIC for four phenotypes is equal (H0). F-values between all four phenotypes were lower than the corresponding F-critical value (F < F-crit.) for all tested antifungals. There were no statistically significant differences between group means of all four phenotypes (Table 4). The results reported no significant effect of phenotypes on resistance against antifungals in C. parapsilosis sensu stricto strains (Supplementary Figures S1–S3).

Table 4

Comparison of means between different phenotypes of C. parapsilosis colonies at significance level α = 0.01

Antifungal agentVariancesStatistical testF-valueF-critical valueSignificance
Anidulafungin Equal One-way ANOVA 1.49 4.60 No 
Amphotericin B Equal One-way ANOVA 0.43 4.60 No 
Micafungin Equal One-way ANOVA 1.35 4.60 No 
Caspofungin Equal One-way ANOVA 0.67 4.60 No 
Itraconazole Equal One-way ANOVA 0.89 4.60 No 
Fluconazole Equal One-way ANOVA 0.80 4.60 No 
Posaconazole Equal One-way ANOVA 2.22 4.60 No 
Voriconazole Equal One-way ANOVA 1.87 4.60 No 
5-Flucytosine Unequal Welch's ANOVA 0.93 Pr = 0.46* No 
Antifungal agentVariancesStatistical testF-valueF-critical valueSignificance
Anidulafungin Equal One-way ANOVA 1.49 4.60 No 
Amphotericin B Equal One-way ANOVA 0.43 4.60 No 
Micafungin Equal One-way ANOVA 1.35 4.60 No 
Caspofungin Equal One-way ANOVA 0.67 4.60 No 
Itraconazole Equal One-way ANOVA 0.89 4.60 No 
Fluconazole Equal One-way ANOVA 0.80 4.60 No 
Posaconazole Equal One-way ANOVA 2.22 4.60 No 
Voriconazole Equal One-way ANOVA 1.87 4.60 No 
5-Flucytosine Unequal Welch's ANOVA 0.93 Pr = 0.46* No 

*Welch's ANOVA gives Pr value instead of F-critical value. Pr > F means significant result.

Oligotrophic water systems, characterized by low nutrient levels, are habitats for diverse microbial communities, including fungi. Over 400 different species have been documented in European waters so far, including species with opportunistic pathogenic potential (Novak Babič et al. 2017). Fungal presence in water is closely associated with the location of the main water source, as well as cleaning procedures and disinfection treatment (Ren et al. 2023). While natural water carries diverse fungi associated with rock-weathering processes, plant material, and anthropogenic waste, chlorinated water, on the other hand, has the lowest fungal count (Cho et al. 2022; Novak Babič et al. 2023; Vaksmaa et al. 2023). However, even after disinfection, emerging pathogens might be present in water networks, as was often documented in hospital environments (Caggiano et al. 2020). Particularly nosocomial infections are one of the main concerns in healthcare settings (Khan et al. 2017). Opportunistic pathogenic fungi, such as Aspergillus and Candida, can colonize hospital water systems and persist in biofilms, creating reservoirs for opportunistic infections (Richardson & Rautemaa-Richardson 2019; Arroyo et al. 2020). The World Health Organization (WHO) has highlighted the growing threat of these genera due to their emerging resistance to antifungal agents and their biofilm-forming capabilities (Cavalheiro & Teixeira 2018; WHO 2022).

Aspergillus species are ubiquitous and were isolated from various natural freshwater and marine sources. The most commonly reported are Aspergillus fumigatus, A. niger, A. flavus, A. terreus, A. versicolor (now Emericella versicolor), A. ustus (now Emericella usta), A. candidus, A. sydowii (now Emericella sydowii), A. clavatus (now Neosartorya clavata), and A. ochraceus (Abdel-Azeem et al. 2019). Slovenian samplings of different water sources and materials in contact with water also yielded A. fumigatus, A. versicolor, and A. flavus, with the additional, less common species A. fischeri, A. creber, A. protuberus, A. udagawae, and A. westerdijkiae being also present (Novak Babič et al. 2023). Their presence was more common in groundwater sources than in chlorinated tap water, which indicates the effectiveness of water disinfection against these species (Novak Babič et al. 2023). Aspergillus species are well known for their role in respiratory and invasive fungal infections, particularly in patients with weakened immune systems (Richardson & Rautemaa-Richardson 2019). The presence of these species in drinking water highlights potential pathways for exposure in hospital settings, including humidifiers, shower aerosols, and contaminated medical devices (Arroyo et al. 2020). Rapidly evolving resistance to antifungal agents makes Aspergillus a growing concern, particularly in light of the increasing use of antifungal treatments in both clinical and agricultural settings. Global surveillance studies have reported varying levels of antifungal resistance in Aspergillus isolates from different regions, reflecting local usage patterns of antifungal agents (Fisher et al. 2022). Resistant strains were already identified in rivers, lakes, and drinking water (Richardson & Rautemaa-Richardson 2019). Resistance mechanisms include mutations in the CYP51A gene, which encodes the enzyme 14-α-demethylase targeted by azoles (Rogers et al. 2022). Also, mutations in the FKS genes, encoding the enzyme 1,3-β-D-glucan synthase (targeted by echinocandins) (Perlin 2015), changes in the ergosterol content of the fungal cell membrane, which is the target of polyenes (Lee et al. 2023), and mutations in genes involved in pyrimidine metabolism (affected by flucytosine) (Delma et al. 2021) can be associated with resistance. Studies most often report resistance to azoles, while resistance to echinocandins is less common, and those against polyene macrolides are relatively rare (Wiederhold 2017). The possible existence of resistances in strains obtained from different water sources in Slovenia was studied using antimycotics from the above-mentioned groups: azoles (e.g., itraconazole, voriconazole, and posaconazole), echinocandins (e.g., anidulafungin, caspofungin, and micafungin), polyene macrolides (e.g., amphotericin B), and 5-flucytosine. Results were compared with either epidemiologic cut-off values for wild-type strains (ECOFF) (EUCAST 2023) or available scientific references testing the same Aspergillus species (Table 1). Only one strain of A. protuberus isolated from a metallic water reservoir in contact with chlorinated water showed possible resistance to amphotericin B. These findings suggest that Slovenian water so far represents little risk for the transfer of resistant Aspergillus strains from the natural environment indoors.

Like Aspergillus, Candida species are also ubiquitous and are isolated from various natural water sources (Monapathi et al. 2020). The most common species reported from water are Candida albicans, C. glaebosa, C. intermedia, C. parapsilosis species complex, C. pseudointermedia, C. saitoana, C. tropicalis, C. glabrata, C. dubliniensis, C. sake, and C. zeylanoides (Novak Babič et al. 2017). These species are often primary colonizers in biofilms and are capable of persisting on medical equipment and surfaces, facilitating patient colonization (Malinovská et al. 2023). Hospital-acquired Candida infections are associated with high morbidity and mortality (Salmanton-García et al. 2024), and the intermediate resistance observed in waterborne strains suggests a potential environmental source for such infections. C. parapsilosis species complex and C. pseudointermedia were the most common in Slovenian water, with the majority of isolates originating from tap water. This could suggest their resilience to chlorination, likely due to their biofilm-forming ability (Erdei-Tombor et al. 2024). The resilience to chlorination and their phenotypic diversity further complicate their management in clinical settings. Key aspects of antimycotic resistance in Candida species include the same mechanisms being targeted by echinocandins, polyenes, and flucytosine as in Aspergillus, while azole resistance can occur due to the overexpression of efflux pumps (e.g., CDR and MDR genes), mutations in the ERG11 gene encoding 14-α-demethylase, and alterations in membrane sterols (Nishimoto et al. 2020). The biggest concern currently represents multiresistant C. auris, a species primarily found in hospitals but also in wastewater (Barber et al. 2023). Besides this, C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis often express resistance against different azoles, and strains of C. glabrata and C. parapsilosis against echinocandins (Pristov & Ghannoum 2019). All strains from our study were intermediately resistant to anidulafungin and micafungin, in accordance with previous studies reporting elevated resistance of this species against echinocandins (Zhai et al. 2022). Additional statistical tests showed significant differences between means of clinical and waterborne strains for anidulafungin, suggesting a higher probability for anidulafungin resistance in waterborne strains. Anidulafungin resistance could develop due to different factors; thus, future research should focus on exploring FKS1 and FKS2 gene mutations, increased chitin content in cell walls, overexpression of efflux pumps, or biofilm formation ability (Perlin 2015).

When tested against azoles, only one of the waterborne strains and one from clinical material were borderline resistant to fluconazole, and one clinical strain expressed borderline resistance to voriconazole. However, no statistically significant differences were observed for these antimycotics after comparing waterborne and clinical strains. On the other hand, the statistical analysis pointed out that clinical strains likely have higher MICs for itraconazole than waterborne strains. Generally, the low number of azole-resistant waterborne strains suggests their removal during water preparation, but also healthy primary water sources (e.g., groundwater) (Monapathi et al. 2021). The occurring differences between clinical and waterborne strains could be the consequence of different ERG11 gene mutations, overexpression of efflux pumps (e.g., CDR1, CDR2, and MDR1), or overexpression of ERG3 affecting the sterol biosynthesis pathway (Czajka et al. 2023).

Although rarely recorded in the literature, borderline resistance against 5-flucytosine was noted for two waterborne strains, yet no statistically significant differences were observed between the waterborne group and clinical strains. One of the possible mechanisms leading to increased resistance to 5-flucytosine includes loss-of-function mutations in FCY2, FCY1, and FUR1, encoding a cytosine permease responsible for the uptake of the agent into fungal cells. 5-Flucytosine should thus never be used as monotherapy but is usually combined with amphotericin B (Padda & Parmar 2024).

Besides focusing on primary habitat, our study also tried to link possible resistance in C. parapsilosis with the colony phenotypes. Previous studies suggested higher resistance to antifungals in non-smooth phenotypes, which are usually more common in clinical material than smooth ones (Moreno-Martínez et al. 2021). This is in accordance with our study where the prevalence of smooth phenotype in water contrasted with the predominance of crepe phenotype originating from clinical material. However, statistical analysis did not yield significant differences between phenotypes and their resistance against any of the tested antifungals. The reason might be a small number of tested strains; thus, despite the obtained results, future studies should not dismiss the importance of colony phenotypes when assessing resistances in C. parapsilosis.

Our study aimed to investigate possible resistance against nine antifungals in strains of Aspergillus and Candida, previously isolated from different water sources. Its contribution is to understand the environmental reservoirs of antifungal-resistant strains and their possible implications for nosocomial infections. As these genera were identified from 42 and 16% of drinking water samples, they may pose a health risk, particularly in hospitals, where patients with immune deficiency reside for a longer time. However, only one Aspergillus strain showed resistance to amphotericin B. On the other hand, all Candida strains were intermediately resistant to echinocandines, 16% of strains were borderline resistant to azoles, and 8% to 5-flucytosine. Habitat proved to be statistically important for the development of resistance. Understanding the dynamics of antifungal resistance in waterborne fungi is crucial for assessing the risks associated with environmental exposure and for implementing effective strategies to manage and mitigate these risks. Private homes, water catchments, and natural reservoirs in regions with intensive agriculture should be taken into the future investigation due to the heightened possibility for antifungal resistance. Future research should also expand water monitoring and further research resistance mechanisms in water-transmitted fungi. Additionally, a larger study encompassing different phenotypes from diverse environments could provide valuable insights into the complex interplay between Candida origin, phenotype, and antifungal susceptibility.

The authors thank Prof. Dr Tadeja Matos from the Medical Faculty (University of Ljubljana, Slovenia) for the provision of clinical strains.

M.N.B. conceptualized, visualized, and investigated the project, developed the methodology, rendered support in data curation, and contributed to project administration, wrote the original draft preparation,; M.N.B., N.G.-C. rendered support, M.N.B., N.G.-C. validated the project, contributed in resources, wrote and reviewed and edited the article, rendered support in funding acquisition. All authors have read and agreed to the published version of the manuscript.

This study was supported by funding from the Slovenian Research and Innovation Agency (ARIS) to Infrastructural Centre Mycosmo (MRIC UL, I0-0022). ARIS financially supported the work of Monika Novak Babič through the postdoctoral research project (grant number Z7-2668) and research program, grant number P1-0198. ARIS also found research of Nina Gunde-Cimerman through a research program grant number P4-0432.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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