Nanomaterials in the Management of Fungal Udder Inflammation in Cattle as an Effective Preventive Strategy Based on In Vitro Studies

Simple Summary

The incidence of fungal udder inflammation is increasing, negatively affecting the welfare of dairy cattle and reducing milk production. This disease is often associated with the intensive use of antibiotics, whereas effective methods of controlling fungal infections remain limited. The aim of this study was to evaluate the antimicrobial activity of nanoparticles against fungi responsible for udder inflammation. The activity of several types of metal nanoparticles and their complexes against isolated fungal strains was evaluated. The results showed that most of the tested nanoparticles effectively inhibited fungal growth even at low concentrations. Importantly, some strains resistant to standard antifungal drugs remained sensitive to nanoparticles. The most effective combinations reduced fungal survival by up to 90%. The results indicate that nanomaterials may be a valuable tool in the future for preventing and supporting the treatment of fungal mastitis, reducing losses in livestock farming and the excessive use of traditional drugs.

Abstract

Fungal mastitis is rare but poses a significant problem for dairy farmers. It is often underestimated and under-researched, with most studies and treatments focusing on bacterial infections. Antibiotics are ineffective against fungi, and they exacerbate fungal mastitis. This study aimed to determine the antifungal properties of silver (Ag), gold (Au), copper (Cu), iron with a hydrophilic carbon coating (FeC), and platinum (Pt) nanoparticles (NPs) at five different concentrations, as well as their complexes, on the survival of fungal strains such as Pichia kudriavzevii, Wickerhamiella pararugosa, Saccharomyces cerevisiae, Cutaneotrichosporon mucoides, Wickerhamomyces anomalus, Coniochaeta hoffmannii, and Kluyveromyces marxianus. The strains’ susceptibility to 8 standard antifungals, along with MIC (minimal inhibitory concentration) and MFC (minimal fungicidal concentration) after NP treatment, was assessed. Clotrimazole and ketoconazole (10 µg) were most effective, while fluconazole (10 µg) and flucytosine (1 µg) showed the weakest activity. The AgCuNP complex demonstrated the strongest biocidal activity against all isolated strains, while FeCNPs and PtNPs showed very weak or no biocidal properties. The study’s results provide a basis for further in vivo research, indicating the great potential of nanoparticles in combating fungal mastitis, providing an innovative solution against infections caused by drug-resistant pathogens.

1. Introduction

Mastitis is mainly caused by bacteria of various origins. It is the main etiological agent with which mastitis is mainly associated. The bacteria’s distribution and frequency of occurrence depend on many factors, including the country’s climate, type of housing system, the feed’s nutritional level, whether milking hygiene is used and properly maintained, and overall animal health [1], age, breed, and milk yield [2]. Therefore, the distribution varies significantly; however, it is possible to introduce a categorization by which pathogens isolated from the diseased udder can be classified. There are two categories, environmental and contagious pathogens, based on bacterial origin. Environmental pathogens are present in the immediate environment in which the animal resides and include Escherichia coli, Streptococcus uberis, Klebsiella spp., Enterobacter spp., Pseudomonas spp. [3], Serratia spp., Streptococcus equinus, Enterococcus faecalis, and Enterococcus faecium [4]. Meanwhile, infectious pathogens, i.e., those that colonize the cow’s udder or teats, include Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, and Corynebacterium bovis [5]. Another division allows mastitis to be classified into three further forms: clinical, subclinical, and chronic. The main determinant that classifies the stage of mastitis is the somatic cell count (SCC) in the milk. It is estimated that an SCC of more than 200,000 in 1 mL is evidence of the subclinical form, while a value exceeding 400,000 indicates the clinical form. The chronic form is indicated when mastitis recurs with high frequency and when SCC levels remain consistently high, but there is no increase in the number of pathogens. A diagnosis of mastitis is also possible by visually assessing the health of the udder and the organoleptic quality. In the case of clinical mastitis, the cow’s udder can be red, there is visible swelling, and there may be changes in the appearance of the milk, such as a change in color or structural changes and the presence of lumps. Subclinical inflammation is more challenging to diagnose visually because it is not characterized by any external symptoms. Therefore, a mastitis diagnosis should be based mainly on an evaluation of SCC levels and an examination of the microbiological quality of the milk. However, it is estimated that subclinical conditions in the herd are far more common than clinical ones, but their detection is decisively lower.

The second etiological factor that leads to mastitis is fungi. Fungal strains are classified as environmental pathogens found in the animal’s direct environment. The literature reports that they are isolated, e.g., from bedding, floors, dirty milking equipment, and from the hands of personnel [6]. The literature reports many different species isolated from diseased udders, including those of the Cryptococcus genus: C. neoformans, C. albidus, C. flavus, C. laurentii, and C. luteolus; of the Rhodotorula genus: R. glutinis, R. minuta, and R. rubra; and of the Trichosporon genus: T. cutaneum [7]; as well as Aspergillus amstelodami, Aspergillus fumigatus, Geotrichum candidum, and Saccharomyces spp. (e.g., S. fragilis) [8]. Various yeast species are commonly isolated, with Candida spp. being the most frequently identified species in milk from cows diagnosed with mastitis [9]. In the literature, as well as in the diagnostic field, the main focus is often on the specific species, which can be limiting in terms of a full, comprehensive diagnosis and understanding of the entire spectrum of pathogens that can contribute to the development of mastitis. This approach is due to the fact that in different parts of the world, these particular species are the most commonly isolated. It is estimated that the most common isolates include strains such as C. kefyr, C. krusei, C. rugosa, C. albicans, and C. parapsilosis [6]. These are the strains on which further testing and analysis are most often carried out to study their mechanisms of activity. Unfortunately, this approach leads to the neglect of other pathogens that, although less common, can also cause infections that are more difficult to treat.

Fungal mastitis in dairy cows is often underdiagnosed and undertreated because routine diagnostics and research tend to focus primarily on bacterial infections. Infections caused by yeasts can occur in subclinical, chronic, or acute forms, and their under-recognition may result in subtle clinical signs or misinterpretation as bacterial mastitis. Consequently, these infections may persist unnoticed, leading to chronic udder inflammation, reduced milk yield, and challenges in herd management. Highlighting fungal mastitis is therefore essential for comprehensive diagnosis and for developing effective preventive and therapeutic strategies [10].

The next rare etiological agent is an algae of the genus Prototheca. Although cases of infection caused by this pathogen are relatively rare, they should not be underestimated. Protothecal mastitis poses a serious therapeutic challenge, mainly due to the high resistance of this microorganism to available antimicrobials [11].

In the treatment of mastitis, which is most often caused by bacteria, the predominant method of treatment is antibiotic therapy. Unfortunately, the use of antibiotics leads to an imbalance of the bacterial microflora, including the elimination of protective bacteria that shield the organism from infection. As a result, fungi multiply, increasing the problem of mastitis and making it more difficult to treat effectively. It is also worth noting that, in the context of global antibiotic resistance, antibiotics have become less effective in the treatment of mastitis. Their use may not only be ineffective but also contribute to the intensification of the fungal mastitis problem [12].

An increase in the incidence of fungal infections most often occurs in late winter and early spring, when there are high humidity and high temperatures [13]. It is estimated that, of all cases of mastitis, the incidence of fungal mastitis is about 1–12% [14]. However, there are also reported cases where fungal mastitis occurred in 22% of cases [15]. This value varies depending on the country, prevailing weather conditions, maintenance of milking hygiene, and conditions in the barns. For this reason, there are also differences in the composition of fungal strains. In order to reduce rates of mastitis, it is necessary to maintain hygiene levels in the immediate environment in which the animals are located, as well as the hygiene of the teats. This includes litter hygiene and the general cleanliness of the housing, as well as maintaining milking hygiene practices: disinfection of the teats, disinfection and proper use of milking apparatuses, protection of the teats after milking, and general maintenance of the udder’s health [2].

Mastitis is a global problem, and despite constant developments in livestock production, there is still a lack of effective alternatives for the prevention and treatment of this disease, which reduces animal welfare and exposes dairy producers to financial losses [16]. Resistance of pathogens to antimicrobial agents is increasing and is a major problem in the treatment of basic infections in not just animals, but also in humans to whom resistant pathogens spread. It is now a global problem that scientists worldwide are working to solve. With antibiotic-associated problems and the difficulty in treating fungal diseases, metal nanoparticles (NPs) are being discussed in the context of substances that have biocidal potential. They have many properties (antibacterial, antifungal, antiviral) [17]; however, one of the most useful properties, in terms of their biocidal activity, is their high affinity for pathogenic cells. Metal NPs can disrupt microbial cells by inducing reactive oxygen species (ROS) production. Elevated ROS levels damage cellular components, particularly lipids in the cell membrane, leading to loss of membrane integrity, increased permeability, and ultimately cell death. Assessment of membrane integrity is therefore an important measure of the fungicidal effect of NPs, as it reflects both direct physical interactions of NPs with the membrane and oxidative damage caused by ROS [18].

Most research studies have focused on combating bacterial mastitis, while fungal mastitis—particularly cases caused by rarely isolated and poorly characterized fungal strains—remains largely neglected. This represents a significant knowledge gap, as these rare pathogens, like bacterial ones, pose a threat to animal health and may have implications for human health. Moreover, there is limited research on effective antifungal strategies for these uncommon strains. Recently, the biocidal properties of nanoparticles, especially metal nanoparticles, have gained attention, offering a promising avenue to address this unmet need in antifungal therapy. The above-mentioned issues constitute a research problem that necessitated further investigation. Nanoparticles hold considerable promise for further investigation, particularly in the context of in vivo testing. However, in order to confirm their high biocidal potential, it was necessary to conduct tests.

Therefore, the primary objective of this study was to determine the effect of silver (AgNPs), gold (AuNPs), copper (CuNPs), platinum (PtNPs), iron with hydrophilic carbon coating (FeCNPs), and their combinations (AgCuNPs, AgAuNPs, AuCuNPs) on the survival of seven fungal strains isolated from bovine mastitis, including Pichia kudriavzevii, Wickerhamiella pararugosa, Saccharomyces cerevisiae, Cutaneotrichosporon mucoides, Wickerhamomyces anomalus, Coniochaeta hoffmannii, and Kluyveromyces marxianus. These fungal species are rarely isolated in routine diagnostics, partly due to their low prevalence; however, they are clinically relevant, as they have been associated with bovine mastitis. Therefore, further research focusing on these rarely isolated pathogens is necessary to better understand their role in the disease and to improve diagnostic and therapeutic strategies. The concentrations of nanoparticles tested were 1.56, 3.125, 6.25, 12.5, and 25 mg/L, and for PtNPs 0.625, 1.25, 2.5, 5, and 10 mg/L. The concentrations of the nanoparticles were selected based on ranges commonly reported in the literature to allow meaningful comparison with previous studies.

The secondary objectives were to determine MIC (minimal inhibitory concentration) and MFC (minimal fungicidal concentration) after application of NPs. The study also tested the effect of common standard mycotic agents—flucytosine (1 g/L), econazole, ketoconazole, clotrimazole, fluconazole (10 g/L), amphotericin B (20 g/L), fluconazole (25 g/L), and nystatin (100 g/L) on the survival of isolated strains—in order to assess the biocidal potential of NPs. These findings provide a strong foundation for future in vivo studies to evaluate the clinical applicability of these nanoparticles in treating bovine mycotic mastitis.

2. Materials and Methods

2.1. Biological Material

For the study, quarter milk was collected from Polish Holstein–Friesian cows with diagnosed subclinical mastitis (above 200,000/mL somatic cell count in a particular teat). Cows were kept at the Wilanów-Obory Agricultural Experimental Station in Poland (Mazowieckie county), which is an integral part of the Warsaw University of Life Sciences. The animals included in the study come from a dairy farm where routine breeding practices are carried out as an inherent part of the production cycle. To maintain lactation, reproduction is conducted, and calves are a natural part of this process. Some of the female calves remain on the farm as breeding stock used to replenish the herd. Therefore, the animals used in the study are an integral part of the farm’s resident population and were not sourced externally specifically for the purposes of the experiment. Samples were taken in the spring. The milk was collected during evening milking when the milking apparatus detached from the cow’s teats. Then, before the milk sample was taken, the teat was disinfected with 70% ethanol and wiped dry. Milk was collected into sterile tubes, which were then placed into a portable refrigerator. The samples were transported within 30 min at 5 °C to the Warsaw University of Life Sciences microbiology laboratory.

2.2. Isolation of Fungi Strains and Identification

Immediately after transport to the laboratory, microbiological inoculations were performed. Previously prepared microbiological media dedicated to fungal culture, such as Czapek Dox Modified Agar, Oxytetracycline Yeast Extract Agar Base (Pol-Aura, Warsaw, Poland), Sabouraud Chloramphenicol Agar (Bio-Rad, Hercules, CA, USA), DG-18 Agar (Graso-biotech, Owidz, Poland), and DRBC Lab-Agar (Biomaxima, Lublin, Poland), were used. The cultures were then incubated at 37 °C for 24–48 h. Fungal colonies that differed morphologically were selected from the performed cultures and then inoculated onto new media. The cultures were then sent to an external laboratory for identification using a MALDI-TOF MS apparatus (Bruker, Poznan, Poland).

2.3. Determination of the Nanoparticles’ Morphology

The nanoparticles used in the experiment were purchased from Nano-tech (Warsaw, Poland), except for the iron nanoparticles, which were purchased from 3D-nano (Krakow, Poland). The manufacturer of the nanoparticles indicates that they were synthesized using physical methods. In the experiment, nanoparticles from different production batches were used: AuNPs (batch no. 40Z/03/2023), CuNPs (batch no. 49M/02/2023), AgNPs (batch no. 160E/10/2025), PtNPs (batch no. 22P/06/2021), and FeCNPs (batch no. 551312). To determine the morphology of the nanoparticles, a mixture of NPs was prepared with distilled water, which was then sonified. After that, this mixture was applied to TEM Formvar on 3 mm 200 mesh Cu grids (Agar Scientific, Stansted, UK). The formvar grids were then allowed to dry, and images were taken with a JEM-2000EX transmission electron microscope (JEOL, Akishima, Tokyo, Japan) with a voltage of 80 keV.

2.4. Determination of the Physicochemical Properties of the Nanoparticles

The experiment, the zeta potential and hydrodynamic size of the nanoparticles were determined using a Zetasizer Nano ZS apparatus (ZEN3500, Malvern Instruments, Malvern, UK). A 10 mg/L mixture of nanoparticles diluted with distilled water was prepared. The zeta potential was measured in four independent measurements (n = 4), while the hydrodynamic size distribution was determined by dynamic light scattering (DLS) based on three independent measurements (n = 3).

2.5. Determination of Fungal Viability After Treatment with Nanoparticles and Their Complexes

Fungal suspensions were prepared with OD = 1 McF from the isolated strains using NaCl. The prepared suspension was pipetted into 96-well plates in a volume of 50 µL per well. 50 µL of sterile water were added to the wells that constituted the control groups (100% survival rate). To the remaining wells, 50 µL of nanoparticles were added so that the concentrations in the wells were 1.56, 3.125, 6.25, 12.5, and 25 mg/L for Au, Ag, Cu, and FeC NPs, and 0.625, 1.25, 2.5, 5, 10 mg/L for Pt NPs. The range of nanoparticle concentrations was selected to cover a broad spectrum of potential biological effects and to allow the assessment of dose–response relationships. Each concentration was pipetted in six replicates. The prepared plates were placed at 37 °C for 24 h, and then 20 µL of XTT Cell Proliferation Assay Kit reagent (Merck, Darmstadt, Germany) was added to each well of the control and experimental groups. The plates were incubated again at 37 °C for 24 h. After this time, the survival results were read using spectrophotometric absorbance at a measurement wavelength of 450 nm and a reference wavelength of 690 nm (ELISA Infinite M200 reader, Tecan, Durham, NC, USA).

2.6. Determination of the Minimum Inhibitory Concentrations (MIC) and Minimum Fungicidal Concentrations (MFC)

For MIC determination, sterile 24-well plates were used, into which Glucose Chloramphenicol Broth (GCB) liquid medium (Pol-Aura, Warsaw, Poland) was pipetted at 1 mL per well. The nanoparticles were then pipetted using serial dilution. In this way, the final concentrations of nanoparticles in each individual well were the same as for the 96-well plate experiment, i.e., for AgNPs, AuNPs, CuNPs, PtNPs, FeCNPs, AgCuNPs, AgAuNPs, and AuCuNPs: 1.56, 3.125, 6.25, 12.5, 25 mg/L, while for PtNPs, concentrations of 0.625, 1.25, 2.5, 5, 10 mg/L were used. Then, 10 µL of fungal suspension (OD = 0.5 McF) was added to each well. The positive control was a well containing 1 mL of liquid medium along with 10 µL of the fungal suspension to verify that the medium was sufficient for pathogen growth. On the other hand, the negative control contained only 1 mL of liquid medium to determine the working environment’s sterility and assess the risk of contamination of the plate. The plate was then incubated for 24 h at 37 °C. Subsequently, the occurrence of MIC was assessed as the concentration at which the well was free of turbidity at the bottom.

On the day the MIC results were read, an experiment was conducted to determine the MFC by performing inoculations from suspensions contained in the wells of a 24-well plate. The cultures were performed on solid microbiological media. Such cultures were then incubated for 24 h at 37 °C. MFC was the concentration for which no growth occurred on the media.

2.7. Evaluation of the Susceptibility to Antifungal Agents Using the Disk Diffusion Method

On the dedicated fungal culture media mentioned earlier in the methodology, carpet inoculations were performed using 100 µL of fungal suspension OD = 0.5 McF. Disks containing the following antifungal substances were then placed on the Petri dishes: flucytosine (1 g/L), econazole, ketoconazole, clotrimazole, fluconazole (10 g/L), amphotericin B (20 g/L), fluconazole (25 g/L) and nystatin (100 g/L) (MASTDISCAST^®, MAST GROUP, Liverpool, UK). The prepared Petri dishes were incubated for 24 h at 37 °C with the medium lying down. The size of the zone of growth inhibition was then determined.

2.8. Statystical Analysis

The statistical analysis specifically concerned the survival tests of microorganisms after treatment with nanoparticles and their complexes (determination of fungal viability). Data were analyzed using GraphPad Prism 9 software (version 9.2.0, San Diego, CA, USA). Differences between groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for group comparisons. Graphs and visualizations were generated in GraphPad Prism 9. Mean standard deviation is indicated in the figures. The differences at p ≤ 0.05 were considered statistically significant and are marked with an asterisk on the figures.

3. Results

3.1. Identification of the Isolated Fungi

The results of pathogen identification indicated that the isolated strains were P. kudriavzevii, W. pararugosa, S. cerevisiae, C. mucoides, W. anomalus, C. hoffmannii, K. marxianus. Fungal identification results are shown in Table S1.

3.2. The Nanoparticles’ Morphology, Physicochemical Properties Analysis of the Nanoparticles: Hydrodynamic Diameter (nm) and Zeta Potential (mV)

The basic physicochemical properties of the tested nanoparticles are presented in Figure 1, Figure 2 and Figure 3. Figure 1 illustrates the morphology of the NPs, Figure 2 presents their size distribution, and Figure 3 shows the zeta potential.

The morphology (Figure 1) of the tested nanoparticles showed the proper shape, which is in accordance with the standard normal characteristics of morphological features. Only in the case of CuNPs (Figure 1C) was a presence of impurities observed, probably related to the nanoparticle production process.

The zeta potential (Figure 2) of the tested NPs differed from each other. The most stable was FeCNPs (Figure 2D), while slightly larger measurement fluctuations were shown by PtNPs (Figure 2E). In contrast, Ag, Au, and CuNPs (Figure 2A–C) showed the largest measurement fluctuations.

As for the size distribution of NPs (Figure 3), differences in the amounts of size fractions were observed. Two size fractions were evident for Ag, FeC, and Pt NPs (Figure 3A,D,E). A single fraction was observed for AuNPs (Figure 3B), while as many as three different size fractions occurred for CuNPs (Figure 3C).

3.3. Fungicidal Properties of the Tested Nanoparticles

All tested nanoparticles differed in terms of showing biocidal properties and were dependent on their type and concentrations. The survival rates observed in the experimental groups (i.e., after nanoparticle treatment) were directly compared with the control group, which represented 100% pathogen survival. A synergistic effect of NPs was observed due to which the most fungicidal complex was AgCu NPs, followed by slightly less biocidal properties exhibited by AgAu and CuAu NPs, followed by significantly less biocidal properties exhibited successively (with a decreasing trend of biocidal properties) by NPs of Ag, Cu, Au, FeC, Pt NPs.

In some cases, the survival rates exceed 100%, which may indicate growth-promoting effects at low nanoparticle concentrations. This phenomenon is called hormesis, where a substance exhibits biocidal properties at high concentrations but stimulates microbial defense mechanisms at low concentrations.

Since the nanoparticles were commercially obtained and production methods were unspecified, differences in surface chemistry, coatings, or residual stabilizers may affect the observed antifungal activity.

3.4. Survival Rate of Fungal Strains After Application of AgNPs

As shown in Figure 4, the highest resistance to the applied AgNPs was observed in the case of S. cerevisiae, whose survival rate at 25 mg/L reached 119% compared to the control group, and this increase was statistically significant (p ≤ 0.05). The survival rate at 3.125 mg/L also showed a statistically significant difference compared to the control (p ≤ 0.05), while for the other concentrations, the survival rate settled around 90–96% with no statistically significant differences (p > 0.05). These nanoparticles also showed a weak biocidal effect against C. hoffmannii, where, at the highest concentration, the survival rate was 50%, and 35% compared to the control group at the lowest concentration; however, these differences were not statistically significant (p > 0.05). In the case of W. pararugosa, the survival rate for the applied concentrations of 25 and 1.5 mg/L ranged from 20% to 26%, and these reductions were statistically significant compared to the control group (p ≤ 0.05). C. mucoides and W. anomalus were most susceptible to the biocidal effects of these NPs, showing statistically significant reductions in survival rate at all tested concentrations compared to the control group (p ≤ 0.05).

3.5. Survival Rate of Fungal Strains After Application of AuNPs

Figure 5 illustrates that the applied nanoparticles showed the weakest antifungal properties against S. cerevisiae, with a statistically significant decrease in survival compared to the control group only at 6.25 mg/L (p ≤ 0.05). The survival rate of K. marxianus for concentrations of 6.25–25 mg/L decreased to 16–18%, and these reductions were statistically significant for all tested concentrations, while for 3.125–1.56 mg/L, there was a rapid increase in survival rate compared to the control group. A similar trend was observed for C. hoffmannii, with an increase from 36% (6.25 mg/L) to 148.8% (3.125 mg/L); all concentrations except 3.125 mg/L were statistically significant (p ≤ 0.05). The effect of AuNPs on the survival of C. mucoides was effective for concentrations of 12.5–25 mg/L (7–9% survival, p ≤ 0.05), while for a concentration of 6.25 mg/L, there was an increase to 94%, which was not statistically significant (p > 0.05) compared to the control group, and further increases at lower concentrations were statistically significant (p ≤ 0.05). The same was observed for W. anomalus, which showed statistically significant reductions at all tested concentrations (p ≤ 0.05); however, the increase for a concentration of 3.125 mg/L was not as pronounced as with the previously mentioned strains. At concentrations of 6.25–25 mg/L, the survival rate of W. anomalus was 10%, while a concentration of 3.125 mg/L showed a lower biocidal effect, resulting in 54% survival, and the lowest concentration of 1.56 mg/L resulted in an increase to 145%, both statistically significant (p ≤ 0.05) compared to the control group. In the case of W. pararugosa, survival was not statistically significantly different from the control at any concentration (p > 0.05). P. kudriavzevii survival was 7% across all concentrations; statistically significant reductions were observed at all concentrations except 1.56 mg/L, which was not significant (p > 0.05).

3.6. Survival Rate of Fungal Strains After Application of CuNPs

As presented in Figure 6, W. pararugosa was the most susceptible pathogen to CuNPs, with statistically significant reductions in survival at all tested concentrations (p ≤ 0.05) compared to the control group, where for the highest concentration the survival rate was 5% and 19.5% for the lowest concentration. In the case of C. mucoides and P. kudriavzevii, high concentrations (12.5 and 25 mg/L) reduced the survival rate to 1–7% compared to the control group, and these reductions were statistically significant (p ≤ 0.05), while for low concentrations, survival increased; for C. mucoides at 3.125 mg/L and for P. kudriavzevii at 3.125 mg/L, the differences were not statistically significant (p > 0.05). In contrast, the survival rate of K. marxianus and W. anomalus ranged from 3% to 8% for concentrations of 6.25–25 mg/L, with statistically significant reductions at all tested concentrations (p ≤ 0.05) compared to the control group. A strain that did not show such low survival rates for high concentrations was S. cerevisiae, which showed statistically significant reductions at all tested concentrations (p ≤ 0.05); although high concentrations (25–12.5 mg/L) of NPs reduced the survival rate of P. kudriavzevii to 2%, the survival rate for low concentrations was 100%.

3.7. Survival Rate of Fungal Strains After Application of FeCNPs

The results shown in Figure 7 confirm that only in the case of P. kudriavzevii did FeC NPs show notable biocidal activity, with statistically significant reductions in survival at all tested concentrations (p ≤ 0.05) compared to the control group. P. kudriavzevii was susceptible to the highest concentration, with a survival rate of 16%, which then increased with decreasing concentrations, reaching 41% at the lowest concentration. A slightly lower biocidal effect was observed for W. pararugosa, with statistically significant reductions at all concentrations (p ≤ 0.05) compared to the control group; survival at 25 mg/L reached 28%, while at the lowest concentration, it was 40%. The most resistant pathogens to FeC NPs were C. hoffmannii, C. cerevisiae, and C. mucoides.

3.8. Survival Rate of Fungal Strains After Application of PtNPs

As demonstrated in Figure 8, the worst results of all the tested nanoparticles regarding fungicidal activity were observed for PtNPs. Only in the case of W. pararugosa was a decrease in survival rate observed, with statistically significant reductions at all tested concentrations (p ≤ 0.05), up to 49–68% compared to the control group, depending on the concentration of nanoparticles used.

3.9. Survival Rate of Fungal Strains After Application of AgAuNPs

As depicted in Figure 9, P. kudriavzevii was the most sensitive to this complex, with statistically significant reductions in survival at all tested concentrations (p ≤ 0.05), ranging from 5 to 8% compared to the control group. The survival rate of C. hoffmannii was 25–35%, depending on the concentration, with statistically significant reductions at all concentrations (p ≤ 0.05). Slightly lower biocidal activity, but still comparable to P. kudriavzevii, was observed for C. mucoides and W. anomalus, with both showing statistically significant reductions at all concentrations (p ≤ 0.05) compared to the control group. The most resistant strain to this complex was S. cerevisiae, which showed statistically significant reductions at all concentrations except 1.56 mg/L, where the decrease in survival was not statistically significant (p > 0.05).

3.10. Survival Rate of Fungal Strains After Application of AgCuNPs

As shown in Figure 10, the survival rate of P. kudriavzevii at the highest concentration was about 2% compared to the control group, with statistically significant reductions at all tested concentrations (p ≤ 0.05). As the concentration decreased, the survival rate of the pathogens increased, with statistically significant differences observed at each concentration (p ≤ 0.05). For the previously mentioned pathogens at the lowest tested concentration, the survival rate was 4–7% (p ≤ 0.05) compared to the control group. C. hoffmannii, W. pararugosa, and K. marxianus showed survival rates oscillating between 5% and 27%, depending on the concentration, all statistically significant (p ≤ 0.05) compared to the control group, while the most resistant strain was S. cerevisiae, for which the lowest concentration of 1.56 mg/L showed a statistically significant reduction (p ≤ 0.05), and the highest concentration reduced its survival rate to 44% (p ≤ 0.05).

3.11. Survival Rate of Fungal Strains After Application of AuCuNPs

Figure 11 illustrates that the most sensitive pathogen to this complex was W. pararugosa, with statistically significant reductions in survival at all tested concentrations (p ≤ 0.05), where the highest concentration reduced the survival rate to 1–6%; however, an increase in survival rate at the lowest concentration was also observed (20–41%, p ≤ 0.05) compared to the control group. Strong biocidal properties were observed for K. marxianus at concentrations of 25–6.25 mg/L, with survival rates of 4–10%, all statistically significant (p ≤ 0.05) compared to the control group. P. kudriavzevii showed strong susceptibility to this complex at all concentrations, with statistically significant reductions (p ≤ 0.05), except for the lowest concentration, 1.56 mg/L, for which the survival rate was 100% (p < 0.05). As in the previous nanocomplex tests, S. cerevisiae was the most resistant pathogen to AuCuNPs, with statistically significant reductions at all concentrations (p ≤ 0.05) compared to the control group.

3.12. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of selected NPs tested on the isolated strains.

Pathogen Species	Type of NP	MIC (mg/L)	MFC
Pichia kudriavzevii	Ag	6.25	6.25
	AgAu	12.5	12.5
	AgCu	12.5	12.5
Wickerhamiella pararugosa	Ag	3.125	12.5
	Au	25	-
	AgAu	3.125	12.5
	AgCu	-	25
Saccharomyces cerevisiae	Ag	6.25	12.5
	AgAu	12.5	25
	AgCu	12.5	12.5
Cutaneotrichosporon mucoides	Ag	3.125	25
	AgAu	6.25	25
	AgCu	25	-
Wickerhamomyces anomalus	Ag	25	-
	AgAu	6.25	12.5
	AgCu	12.5	25
Coniochaeta hoffmannii	Ag	1.56	6.25
	Au	25	-
	AgAu	6.25	12.5
Kluyveromyces marxianus	Ag	6.25	12.5
	AgAu	12.5	12.5
	AgCu	12.5	12.5

shows the results indicating the concentrations of individual nanoparticles and their complexes, for which MIC and MFC values were estimated for each isolated strain. If the MIC or MFC for a specific nanoparticle could not be determined, the data for that nanoparticle were excluded. In cases where one of the measurements was not available for a strain, a “-” is indicated in the table.

3.13. Antifungal Disk Results

Table S2 shows the numerical values indicating the diameter of the zone of growth inhibition of isolated fungal strains after the application of standard antifungal agents.

The most effective antimicrobials were clotrimazole 10 µg (CTM10) and ketoconazole 10 µg (KCA10), which showed growth-inhibition zone values of 52 mm and 40 mm against W. pararugosa, respectively. The K. marxianus strain also obtained a zone of 32 mm using CTM10. Similarly, a high inhibition zone value of 30 mm was achieved for the W. pararugosa strain when fluconazole 25 µg (FCN25) was applied. The KCA10 formulation also showed high efficacy against the C. mucoides strain, reaching a zone of 30 mm. High values were also observed for W. anomalus after the application of KCA10, obtaining a zone of 38 mm. These results suggest that the effectiveness of individual antimicrobial preparations varies depending on the fungal strain. The worst-performing agent was fluconazole 10 µg (FCN10) for the strains P. kudriavzevii and W. anomalus, with values of 0 mm. The smallest growth-inhibition zone, 3 mm, was obtained for P. kudriavzevii using amphotericin B 20 µg (AMB20). After AMB20 treatment, K. marxianus had a growth-inhibition zone of 4 mm, while W. pararugosa, using FCN10, also had a zone of 4 mm. In the case of P. kudriavzevii, the application of nystatin 100 µg (NY100) yielded a growth-inhibition zone of 7 mm, while W. anomalus achieved a zone of 6 mm with FCN10. Econazole (ECN10) was slightly effective against P. kudriavzevii, reaching an inhibition zone size of 4 mm. The application of this antifungal against the remaining strains resulted in inhibition zones ranging from 14 mm to 22 mm, reaching the highest value (22 mm) for C. mucoides and W. pararugosa. AMB20 was least effective against the P. kudriavzevii strain, achieving a growth-inhibition zone of only 3 mm. A growth-inhibition zone with a radius of 30 mm was obtained for W. pararugosa using FCN25, which was one of the largest. The most resistant strain to the antifungal substances was P. kudriavzevii, which had the lowest values for its growth-inhibition zones. On the other hand, the least resistant pathogen was W. pararugosa, which had the highest number of large inhibition zones, including a value of 40 mm for KCA10. The highest average for the zones of inhibition was observed for KCA10 against various strains, with average values ranging from 14 mm to 40 mm. The worst average for the inhibition zones was observed for FY1, which repeatedly had a value of 0 mm, meaning no growth inhibition. Based on the above results, it can be suggested that some agents, such as KCA10 and CTM10, were particularly effective against many of the strains, while others, such as FY1 and FCN10, showed less potency. The P. kudriavzevii strain proved to be one of the most challenging pathogens for antifungals, as evidenced by its multiple low inhibition zones.

4. Discussion

This study is focused on strains that are rarely isolated and, consequently, little known in the scientific literature. The lack of available data on these strains and the small number of analyses that have been carried out indicate a clear gap in existing research, demonstrating the neglect of this area in microbiological research. Although there are many papers in the literature examining similar issues, most of them focus on bacteria [19,20], highlighting how limited the focus has been on less common strains, including those analyzed in this study. This kind of research is a valuable contribution, but it still mostly involves widespread bacteria, leaving a gap in knowledge about rare, infrequently isolated microorganisms. In their paper, Stavrou et al. [21] describe that AMB is an excellent antifungal therapy against most known strains. However, this is not fully consistent with our study results, where no strains were sensitive to this antifungal. In our study, AMB20 resulted in the smallest diameters of growth-inhibition zones for the fungi used in the study, reaching the highest value of 16 mm for W. pararugosa. The effectiveness of this antifungal, however, varied depending on the strain. Stavrou et al. proved in their study (as in our study) that K. marxianus and W. anomalus were relatively resistant to AMB20. In our study, K. marxianus was the most resistant pathogen to AMB20, as shown by the 2 mm zone of inhibition, whereas in the study by Stavrou et al., this strain showed low MIC values for AMB20. In the same study, FY showed low MIC values for K. marxianus strains, averaging 0.752 mg/L, whereas in our study, the zone of inhibition classified the strain as resistant. Literature data report that P. kudriavzevii is resistant to FCN [22], which was also confirmed by our study. Ming et al. [23] conducted a study on the susceptibility of D. rugosa to AMB, FY, and FCN. They proved that D. rugosa showed resistance to AMB, FY, and FCN. In comparison to our study, the sensitivity of D. rugosa was confirmed for FCN25. Kudrinskiy et al. [24] conducted a study on the use of AgNPs and silver ions (Ag+) against S. cerevisiae. AgNPs effectively reduced the survival rate of S. cerevisiae cells; however, this was not consistent with the results obtained in our experiment, where the lowest survival rate obtained for S. cerevisiae was as high as 90.77%. Neculai-Valeanu et al. [25], when testing AgNPs and AuNPs, demonstrated that they did have biocidal activity against mastitic pathogens; however, as mentioned above, in our work AgNPs were not effective against S. cerevisiae and C. hoffmannii. Another study conducted by Motrenko et al. [26] showed that AgNPs obtained by green synthesis showed biocidal properties against P. kudriavzevii and W. anomalus for concentrations of 20 and 10 mg/L, consecutively reducing their survival rate to 0%. Lower concentrations also showed biocidal properties but did not fully destroy the cells of these strains. Comparing these data with the obtained results, the potency of AgNPs differed from the cited article. In the case of P. kudriavzevii, none of the tested concentrations showed full biocidality, which would reduce the survival rate of this strain by 100%. It was, however, a susceptible strain to AgNPs, where the lowest and highest tested concentrations resulted in a similar biocidal effect. In the case of Motrenko’s study, W. anomalus showed high resistance, while in our study, its survival rate was low, comparable to P. kudriavzevii. On the other hand, in the cited article, a concentration of 10 mg/L of W. pararugosa resulted in the complete destruction of 100% of the cells of this strain, while in our study, even a concentration of 25 mg/L did not show such strong properties. These differences may be due to the way NPs are synthesized—those used in our study were synthesized by the manufacturer using physical methods. The observed differences in biocidal activity among the nanoparticles may be attributed to the synthesis methods. Factors such as ion release, nanoparticle aggregation, and interactions with microbial cell walls can all influence efficacy. For example, nanoparticles synthesized by physical methods may differ in size, surface charge, and dispersion stability, which can affect the rate of ion release and the extent of interaction with microbial cells. These physicochemical properties likely contribute to the variations in antifungal activity observed in our study [27]. NPs are strongly influenced by their physicochemical properties, especially their surface modification and aggregation tendency. In studies comparing stabilized and unstabilized AgNPs, antifungal efficacy was associated with increased aggregate stability obtained through stabilization with polymers and surfactants [28]. Among the stabilizing agents tested, surfactants—especially sodium dodecyl sulfate (SDS)—showed the most pronounced enhancement of fungistatic and fungicidal activity, which was attributed to the synergistic effect of NPs stabilization and the intrinsic antifungal activity of the surfactant itself [29]. NPs have been identified as key factors influencing interactions with microbial cells. Smaller NPs and specific particle shapes have been found to exhibit increased antibacterial activity, reflecting more effective contact with and disruption of microbial cell envelopes [28]. The aggregation of AgNPs on the cell surface also contributes to cell membrane damage, further confirming the role of the physicochemical properties of nanoparticles in determining antifungal efficacy [30].

The superior antifungal activity of the AgCuNP complex observed in this study may be explained by several potential mechanisms. Ag and Cu NPs are known to exert biocidal effects through ion release, generation of ROS, and disruption of microbial membranes. The combination of Ag and Cu could enhance these effects through synergistic interactions. Although the precise mechanism for the enhanced activity of AgCuNP remains to be understand, previous studies support the idea that dual-metal nanoparticles can display higher antimicrobial efficacy compared to single-metal formulations. A recent synergistic mechanistic study [31] on NPs demonstrated that the presence of Cu in the nanoparticle formulation increases the bioavailability of Ag⁺ ions by both accelerating Ag⁺ release and reducing its sequestration by proteins in the medium. Importantly, the synergistic effect was observed only when nanoparticles were present, indicating that direct interaction between the nanoparticle surfaces and microbial cells contributes to the enhanced activity. These combined effects—ion release, reduced Ag⁺ binding, and nanoparticle-mediated surface interactions—provide a plausible explanation for the superior biocidal efficacy of the AgCuNP formulation.

In the viability tests conducted, it was occasionally observed that the survival rates of microorganisms after exposure to low concentrations of nanoparticles exceeded 100%. This indicates that, in these cases, low nanoparticle concentrations promoted the growth of fungal strains. Such phenomena are not uncommon in tests involving potential biocidal agents and are referred to as hormesis. Hormesis occurs when a microorganism’s growth is stimulated by low (subinhibitory) concentrations of a biocidal substance, while higher concentrations inhibit growth [32]. Exposure to subinhibitory concentrations can lead to evolutionary adaptations that enhance microbial survival and contribute to the development of resistance under adverse environmental conditions [33]. This effect is particularly important to consider in the context of applying nanoparticles in the field. On one hand, low concentrations may be safe for animal cells but could promote the development of microbial resistance. On the other hand, high concentrations effectively prevent resistance by destroying microbial cells; however, it may pose risks to the user.

The aim of the study was to evaluate the potential biocidal properties of selected nanoparticles against specific fungal strains. However, in order to reliably assess the potential for practical application of NPs, it is necessary to also consider aspects related to their safety. A key element in this regard is the analysis of cytotoxicity against animal cells and antifungal properties against pathogen cells. Such studies, using nanoparticles from the same source as those used in the present experiment, have previously been conducted by Wierzbicki et al. [19]. Low concentrations of NPs showed no cytotoxic activity against BME-UV1—human mammary epithelial cells and HMEC—human microvascular endothelial cells. Only CuNPs, along with complexes containing these NPs, showed moderate toxicity against BME-UV1 cells. High concentrations of NPs were correlated not only with biocidal potency against pathogens, but also with cytotoxicity against HMEC cells. Nevertheless, for the higher tested concentrations, cytotoxicity against these cells was observed only at the level of 10–15%. It is worth noting that the cited results on the effects of NPs on cells came from the same source and were synthesized in the same way—using physical methods. Therefore, in order to reliably assess their biocidal potential and safety for cells, it is necessary to take into account possible differences in the effects of nanoparticles obtained and produced by other methods, as well as removing analytical limitations such as the lack of cytotoxicity studies on human and animal cells.

5. Conclusions

The results of this study provide preliminary, in vitro evidence that nanoparticles may exhibit antifungal activity relevant to fungal mastitis. These findings are exploratory and do not allow conclusions regarding their in vivo efficacy or clinical application. Nevertheless, they support the need for further research, including in vivo studies, to better assess the potential of nanoparticle-based formulations as antifungal agents. Continued investigation into the effectiveness of various antifungal compounds, particularly against fungi that are infrequently isolated from mastitis cases, remains essential for the development of future therapeutic and management strategies. It is crucial to notice that further in vivo studies are required to evaluate dosage, delivery route, tissue persistence, and host safety before any therapeutic application can be proposed.

Understanding differences in efficacy between therapies, including traditional and modern antifungal agents, is essential for improving the treatment of fungal infections. A review of the literature confirms that some strains show resistance to conventional treatments while highlighting the potential of nanoparticles as alternative antifungal agents. The study revealed significant variation in nanoparticle effects and demonstrated their synergistic antifungal activity. Their efficacy depended on both the type of nanoparticles used and their concentration. Additionally, differences in the sensitivity of individual strains to these nanoparticles were observed. Among the isolated strains, nanoparticle activity followed the order (from most to least effective): AgCu, AgAu, CuAu, Ag, Cu, Au, Fe, and Pt. The strain S. cerevisiae showed the highest resistance to the nanoparticles, whereas W. pararugosa was the most sensitive.

According to the existing literature, several studies have reported that the application of nanoparticles at low concentrations appears to be well tolerated in various human and animal cell lines. However, these observations are based on previously published data and cannot be directly inferred from the results of the present study. While nanoparticles therefore represent a promising subject for further investigation, particularly in the context of in vivo testing, their safety profile requires careful evaluation in appropriately designed studies. Under laboratory conditions, they exhibit potent biocidal properties, indicating their potential as an alternative to traditional antibiotics and antifungals. Research findings in the literature also highlight their efficacy against both bacteria and fungi. However, as nanoparticles are a relatively new field of study, their use should be approached with caution and under strict supervision. The potential long-term effects of their use remain unclear and must be carefully considered.