Anticandidal Effects of Zinc Oxide Nanoparticles on Fluconazole-Resistant Candida Isolates Causing Diarrhea in Calves, in vitro

1. Introduction

Candidiasis is one of the most common fungal diseases in warm-blooded animals and humans, which has spread significantly in recent years. The causative agent of the disease is the opportunistic fungus from the genus Candida, which is part of the normal microflora in the gastrointestinal tract, mucosa, and skin of animals and humans. When the host resistance is reduced locally or systemically, primary or secondary, due to predisposing factors, this agent can cause diseases in various areas of the body as superficial infections (e.g., oral, vaginal, and cutaneous candidiasis) or as deep-seated infections (e.g., candidemia and pulmonary, gastrointestinal, and urinary tract candidiasis) ( 1 ). Although Candida albicans is the main pathogenic species, non-albicans species such as Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Candida krusei, and Candida famata are also important. Although often isolated from air, soil, water, plants, and animal and human feces, C. famata (also known as Debaryomyceshansenii and Torulopsis candida) is recognized as a rare etiologic agent of diseases in humans and animals ( 2 , 3 ). In animals, C. famata has been isolated from the breast tissue of cows with mastitis, ruminants’ vaginal discharge, the mouth of pigs with vitamin A deficiency, horses with arthritis, and fecal samples of dogs with diarrhea.

Hydrocarbon-rich diets, strong antibiotics, and metabolic diseases predispose factors for cattle candidiasis. Candidiasis in cattle often affects the gastrointestinal tract, causes thrush lesions in the mouth, esophagus, and foregut, and causes enteritis and diarrhea when affecting the intestines. The disease is often seen in calves receiving long-term antibiotic therapy. Clinical signs depend on the location of the lesions, so in the case of pre-gastric and intestinal involvement, watery diarrhea followed by anorexia, dehydration, weakness, and severe lethargy are seen, which may ultimately progress to paralysis and death ( 4 ).

Various antifungal drugs, including azoles, are used to treat these infections. Fluconazole is an oral azole that inhibits ergosterol biosynthesis in fungi. Studies have indicated resistance of Candida species to antifungal drugs ( 5 ). In addition, due to the side effects of drugs and the risk of drug interactions, the use of alternative antimicrobial compounds, including nanoparticles, with less toxicity and side effects, has recently received much attention. Zinc oxide (ZnO) nanoparticles (NPs) are of great importance due to their diverse industrial and medical applications. An essential characteristic of ZnONPsis their antimicrobial properties at normal pH and in the absence of light. This NP has selective toxicity and can be considered a potential alternative to some antibiotics ( 6 ). This study aimed to determine the frequency of enteropathogenic Candida strains isolated from calves with diarrhea and to compare the antifungal effects of fluconazole and ZnONPs on the isolates obtained from livestock farms.

2. Materials and Methods

This study was performed on a herd of calves (n=231) aged less than three months in livestock farms around Gorgan (northern Iran) in 2020. After examining vital signs such as respiratory rate, heart rate, and a number of normal rumen contractions, fecal samples from 94 calves with diarrhea were taken and sent to the microbiology laboratory. After ensuring a lack of viral contamination, 1 g of each fecal sample was cultured in 9 ml of peptone water and incubated for 24 hours at 37 °C. The cultivated colonies were sub-cultured on blood agar, MacConkey agar,eosin-methylene blue agar, and in selenite F broth and examined by microbiological and differential tests in order to determine bacterial genus. To isolate fungal species, the samples were cultured on sabouraud dextrose agar with chloramphenicol (Merck, Germany). After 48 hours of incubation at 35 °C, yeast-like fungi were identified based on conventional biochemical techniques using VITEK 2 system (Biomerieux India Private Limited) with YST-ID ( 7 ).

2.1. Determination of Drug Susceptibility

After preparing suspensions from 48-hour cultures of Candidaisolatesand confirmation of 530 nm wavelength and 77-75% light transmission in a spectrophotometer, a primary suspension containing 10⁶cfu/ml yeast cells was diluted with 1640 RPMI medium (Sigma, USA) (1:100) to obtain the final concentration of 10³cfu/ml. Drug susceptibility testing was performed using the broth microdilution method according to the Clinical & Laboratory Standards Institute(CLSI-M27) guideline ( 8 ). After preparing the fluconazole solution (Gibco, Germany) in water, dilutions (0.06-512 µg/ml) were made and poured into wells of a 96-well plate containing RPMI 1640 medium (without bicarbonate and with pH indicator and glutamines) and MOPS buffer (Sigma, USA). After inoculating yeast suspensions (1×10³cfu/ml) into the wells, the microplate was incubated at 35°C for 48 hours. To determine the minimum fungicidal concentration (MFC), 100 µl from the content of each well with a concentration higher than the minimum inhibitory concentration (MIC), together with 100 µl of the positive control (the wells with yeast suspension and RPMI), were separately inoculated on sabouraud dextrose agar and were incubated for 48 hours at 35°C.

2.2. Determination of Antimicrobial Properties of ZnONPs

To prepare the NPs, 5 g of zinc acetate powder (Merck, Germany) was mixed with 50 ml of deionized distilled water at 80 °C. The mixture was then placed at 100±5 °C for 12 consecutive hours and finally at 300±20 °C for 24 hours to form NP crystals. Morphology and size of the formed ZnONPs were examined by transmission electron microscopy using the Leo 906 microscope (KV model, Zeiss, Germany) with an acceleration voltage of 120 kV (Figure 1). In order to determine the crystallographic structure of ZnONPs, X-ray crystallography was performed using an XRD device with CuKa radiation (Olympus, Japan) in the range of 0 to 110 degrees.

Figure 1. Transmission Electron Microscopy

To determine the antimicrobial properties, serial dilutions (1.85-238 μg/ml) were prepared by mixing ZnO powder with distilled water. After centrifugation at 3800 rpm, the precipitate was dissolved in distilled water. Absorption spectra in the 700-600 nm range were detected using spectroscopy to ensure binding. Next, 10 μl of the colloidal ZnONPs (diameter: 20 nm) solution and 200 μl of sabouraud dextrose broth were separately added to wells of a 96-well plate. After inoculating the yeast suspension (10³cfu/ml) and 48 hours of incubation, 10 μl were taken from each well and inoculated onto sabouraud dextrose agar with chloramphenicol. Finally, the number of colonies (cfu) was determined.

To determine the minimum fungicidal concentration (MFC), 10 μl were collected from wells where no fungal growth was observed and poured onto sabouraud dextrose agar. The lowest concentration at which no growth was observed was determined as the MFC. To confirm the antifungal effects of ZnONPs, the isolates were treated with sub-inhibitory concentrations of the NPs.

2.3. Statistical Analysis

Data were analyzed with the GraphPad Prism statistical software using Chi-Squared (χ²) test. A p-value of less than 0.05 was considered statistically significant.

3. Results and Discussion

In the clinical picture of the studied calves, involvement of oral and gastrointestinal mucosa in the form of enteritis and diarrhea was observed so that among 231 calves with the same pattern of vital signs and diet, 94 (40.7%) calves had watery diarrhea. Examination of the fecal samples of infected animals did not reveal any viral contamination. Examination of the grown yeasts indicated that diarrhea was predominantly caused by C. albicansin in 41 cases (43.61%) and C. famata in 8 cases (51.8%). The remaining positive cultures contained bacterial contamination, so 14 samples (14.9%) were infected with Salmonella, and 31 samples (32.98%) were infected with Escherichia coli.

According to the CLSI guidelines ( 8 ), 21(51.2%) C. albicans isolates and 3 (37.5%) C. famata isolates had MIC values of ≥64 µg/ml and were therefore considered resistant to fluconazole. The results indicated that ZnONPs had inhibitory effects on fluconazole-resistant Candida isolates in a dose-dependent manner. At a 119 μg/ml concentration, the ZnONPs inhibited the growth of 97% of fluconazole-resistant C. albicans isolates. There was a significant difference between the MIC of fluconazole, which inhibited the growth of 50% of C. albicans isolates (MIC50=7.43µg/ml), and the MIC of ZnONPs, which inhibited the growth of 90% of C. albicans isolates (MIC90=29.75µg/ml) (P=0.01). The results showed that all fluconazole-resistant isolates were eliminated in the presence of 119 μg/ml of ZnONPs (Table 1). As shown in table 2 and figure 2, after treatment of Candida isolates with ZnONPs, the absorbance at 570 nm reduced significantly (P=0.02) compared to the control group (untreated isolates).

Figure 2. Mean absorption of Candida strains before and after treatment with ZnO nanoparticles

Diarrhea is one of the most important causes of death in livestock. In addition to bacteria, viruses, and protozoa, fungi can cause diarrhea in these animals ( 3 ). In this study, the prevalence of diarrhea in calves was 40.7%, of which 52.1% was confirmed as candidal diarrhea. Previous studies in Brazil ( 9 ), Hungary ( 10 ), and the Netherlands ( 11 ) reported the prevalence of diarrhea at 40%, 4%, and 2.6%, respectively. The difference in the prevalence rates might be due to the diversity of the geographical area under study and the microbial agents involved, including viruses, bacteria, and fungi.

In the present study, C. albicans was calves' predominant causative agent of gastrointestinal diarrhea. As part of the gastrointestinal flora of humans and animals, C. albicans is often considered the main causative agent of gastrointestinal infections; however, numerous studies have reported non-albicans species such as C. glabrata, C. guilliermondii, C. krusei, and C. tropicalis as the causative agents for diarrhea and severe enteritis ( 12 , 13 ).

In 2015, C. famata was reported for the first time as the predominant cause of gastrointestinal infection in a dairy herd in Iran ( 14 ). Epidemiological studies have shown that severe fungal infections are generally caused by drug-resistant fungal species, particularly fluconazole-resistant ( 15 ). In the present study, 24 C. albicans isolates (49%) were resistant to fluconazole.

The role of ZnONPs in inhibiting drug-resistant microorganisms has long been proven ( 16 ). However, the antifungal effects of these NPs have been less exploited. In a study on solar photocatalytic disinfection of a group of microbial aqueous suspensions, ZnO could effectively inhibit the growth of fungi ( 6 ).

In the present study, increasing the concentration of ZnONPs to 119 μg/ml significantly increased the antifungal effects. Similarly, previous studies demonstrated that ZnONPs exerts antifungal effects in a dose-dependent manner ( 17 , 18 ). Researchers reported that ZnO NPs significantly inhibit biofilm formation in C. tropicalis at very low concentrations ( 19 ). In line with our findings, a study reported that the antibacterial effect of ZnONPs depends on their size and shape, so a smaller-sized NP exerts greater antifungal activity ( 20 ). In conclusion, the prevalence of yeast diarrhea in calves is relatively high. Given their favorable antifungal properties, ZnONPs can be used as a suitable option for the treatment of gastrointestinal infections in calves caused by drug-resistant Candida strains.

Authors' Contribution

Study concept and design: L. F.

Acquisition of data: M. T.

Analysis and interpretation of data: L. F.

Drafting of the manuscript: L. F.

Critical revision of the manuscript for important intellectual content: L. F.

Statistical analysis: L. F.

Administrative, technical, and material support: M. T.

Ethics

The study protocol were approved by the ethics committee of the Islamic Azad University, Gorgan, Iran.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Arendrup MC. Candida and candidaemia. Susceptibility and epidemiology. Dan Med J. 2013; 60(11):4698.
2. Desnos-Ollivier M, Ragon M, Robert V, Raoux D, Gantier JC, Dromer F. Debaryomyces hansenii (Candida famata), a rare human fungal pathogen often misidentified as Pichia guilliermondii (Candida guilliermondii). J Clin Microbiol. 2008; 46(10):3237-42.
3. Krcmery V, Kunova A. Candida famata fungemia in a cancer patient: case report. J Chemother. 2000; 12(2):189-90.
4. Nishikawa A, Tomomatsu H, Sugita T, Ikeda R, Shinoda T. Taxonomic position of clinical isolates of Candida famata. J Med Vet Mycol. 1996; 34(6):411-9.
5. Costa Silva RA, da Silva CR, de Andrade Neto JB, da Silva AR, Campos RS, Sampaio LS, et al. In vitro anti-Candida activity of selective serotonin reuptake inhibitors against fluconazole-resistant strains and their activity against biofilm-forming isolates. Microb Pathog. 2017; 107:341-8.
6. Seven O, Dindar B, Aydemir S, Metin D, Ozinel MA, Icli S. Solar photocatalytic disinfection of a group of bacteria and fungi aqueous suspensions with TiO2, ZnO and Sahara desert dust. Journal of Photochemistry and Photobiology A: Chemistry. 2004; 165(1):103-7.
7. Valenza G, Strasen J, Schafer F, Frosch M, Kurzai O, Abele-Horn M. Evaluation of new colorimetric vitek 2 yeast identification card by use of different source media. J Clin Microbiol. 2008; 46(11):3784-7.
8. Wayne P. Clinical and laboratory standards institute: Reference method for broth dilution antifungal susceptibility testing of yeasts. Wayne, PA, USA: In Approved Standard; Clinical and Laboratory Standards Institute; 2017.
9. Salvadori MR, Valadares GF, Leite DdS, Blanco J, Yano TJBJoM. Virulence factors of Escherichia coli isolated from calves with diarrhea in Brazil. 2003; 34:230-5.
10. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli in veterinary medicine. Int J Med Microbiol. 2005; 295(6-7):443-54.
11. Bartels CJ, Holzhauer M, Jorritsma R, Swart WA, Lam TJ. Prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young Dutch dairy calves. Prev Vet Med. 2010; 93(2-3):162-9.
12. Araujo D, Henriques M, Silva S. Portrait of Candida Species Biofilm Regulatory Network Genes. Trends Microbiol. 2017; 25(1):62-75.
13. Azimpour S, Pourtaghi H. A Case Report of Fungal Diarrhea in a Preweaned Calf in Iran. 2016; 4(2):11-34199.
14. Taghipour Bazargani T, Khosravi A, Sharifzadeh A, Balal A. Diarrhea due to Candida famata in a dairy cattle herd located in the Varamin city. Journal of Veterinary Research. 2017; 72(4):507-10.
15. Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, et al. In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance. J Clin Microbiol. 2008; 46(1):150-6.
16. Fozouni L, Javidmehr S, Barghamadi H, Mazandarani A, Rouhafza S. Antibacterial Effect of Zinc Oxide Nanoparticles on Group A β-Hemolytic Streptococci with Macrolide Resistance Isolated from University Student Carriers in North of Iran. 2019; 6(1):e85732.
17. Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol. 2003; 11(1):30-6.
18. Yamamoto O, Iida Y. Antifungal Characteristics of Spherical Carbon Materials with Zinc Oxide. J Ceram Soc Jpn. 2003; 111(1296):614-6.
19. Jothiprakasam V, Sambantham M, Chinnathambi S, Vijayaboopathi S. Candida tropicalis biofilm inhibition by ZnO nanoparticles and EDTA. Arch Oral Biol. 2017; 73:21-4.
20. Zare M, Namratha K, Byrappa K, Surendra DM, Yallappa S, Hungund B. Surfactant assisted solvothermal synthesis of ZnO nanoparticles and study of their antimicrobial and antioxidant properties. J Mater Sci Technol. 2018; 34(6):1035-43.