. 32 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 32-36 ReseaRch aRticle Investigating the Inhibitory Effect of Silver Nanoparticles against Some Species of Candida and Pathogenic Bacteria Alaa M. Hasan, Sura M. Abdul Majeed, Rusol M. Al-Bahrani Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq ABSTRACT Silver nanoparticles synthesized from aqueous extract of mushroom Pleurotus ostreatus exhibited inhibitory effect at the concentration of 12.5, 25, 50, and 100 mg/ml against some pathogenic bacteria and fungi such as Candida albicans, Candida guilliermondii, Candida krusei, Candida zeylanoides, Geotrichum klebahnii, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The maximum inhibition zone was observed against C. zeylanoides at the concentration of 100 mg/ml was 24.5 mm, while the minimum inhibition zone was observed against Geotrichum at the concentration of 25  mg/ml was 8 mm and the concentration of 12.5  mg/ml was not effective against some species. Keywords: Pathogenic Candida, Pleurotus ostreatus, silver nanoparticles INTRODUCTION A new dimension of the metal microbial interaction has been explored for the synthesis of metal nanoparticles such as gold, silver, cadmium, zirconia, and silica titanium.[1] Silver compounds have been used to treat burns, wounds, and infections as well as in preventing bacterial colonization of prostheses and catheters.[2] Various salts of silver and their derivatives are used as antimicrobial agents. Nanosized silver particles exhibit antimicrobial properties. Nanoparticles of silver have been studied as a medium for antibiotic delivery, and to synthesize composites for use as disinfecting filters and coating materials.[3] The nanoparticles were important due to their emission properties. These nanoparticles are used for wide range of application.[4,5] Antibacterial activity is related to compounds that locally kill bacteria or slow down their growth, without being in general toxic to surrounding tissue.[6] The high bactericidal activity is certainly due to the silver cations released from Ag nanoparticles (AgNPs) that act as reservoirs for the Ag+ bactericidal agent.[7] Severe fungal infections have significantly contributed to the increasing morbidity and mortality,[8] immunocompromised patients who need intensive treatment including broad-spectrum antibiotic therapy,[9,10] and Candida spp. represent one of the most common pathogens which are responsible for fungal infections often causing hospital-acquired sepsis with an associated mortality rate of up to 40%.[11] Currently, the majority of yeast species are resistant the available antifungal therapy[12,13] such as on polyenes (amphotericin B), triazoles (fluconazole, itraconazole, voriconazole, and posaconazole) or echinocandins (caspofungin, micafungin, and anidulafungin) which exhibit their toxicity, adverse effects, and drug interaction.[14,15] The objective of this work is to evaluate the effect of a biosynthesized AgNPs product against some Candida spp. and bacteria species to determine how AgNPs interact with the growth of microbial cells. MATERIALS AND METHODS The mushroom Pleurotus ostreatus was obtained from fruit body grown on peach tree in Salah-Alddin Province. Preparation of Hot Aqueous Extracts of Mushroom The oven dried mushroom was blended; the obtained powder was soaked in distilled water at a ratio of 1:10 (w/v) and boiled with agitation at 60 ± 2°C for 30 min. The boiled mushroom powder was then left covered for 30 min. Residues were then removed by filtration through gauze and further centrifugation (10,000 rpm, 30 min, and Corresponding Author: Sura M. Abdul Majeed, Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. E-mail: Suraalsadik80@gmail.com Received: Apr 04, 2019 Accepted: Apr 24, 2019 Published: Jan 20, 2020 DOI: 10.24086/cuesj.v4n1y2020.pp32-36 Copyright © 2020 Alaa M. Hasan, Sura M. Abdul Majeed, Rusol M. Al-Bahrani. This is an open-access article distributed under the Creative Commons Attribution License. Cihan University-Erbil Scientific Journal (CUESJ) https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/ Hasan, et al.: Inhibitory effect of AgNPs against Candida spp. and bacteria 33 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 32-36 4°C). Supernatants were then collected and filtered through Whatman No. 1 filter paper. After that, freeze dried extract powders were obtained using freeze dryer and stored at 4 ± 2°C.[16] Biosynthesized AgNPs from P. ostreatus Silver nitrate (1 × 10−3) AgNO3 stock solution was prepared in sterile deionised triple - distilled water and the subsequent dilutions were made from this stock solution. The bulk amount of (10) mg/ml of aqueous extract solution of is prepared with sterile distilled water and filtered through syringe filter (0.2) µm. Based on the result of a preliminary trial, 2–7 ml of 10 mg/ml of an aqueous extract of P. ostreatus (P2) were filled with sterile distilled water to a total of 10 ml. After that, the solution is added to 5 ml of (1 × 10−3) M aqueous AgNO3 solution and kept at room temperature and exposed under ultraviolet (UV) (365) nm (long UV). After 24 h incubation, the light yellow color of the mixture solution turned to dark yellow indicating the formation of AgNPs.[17] Candida Isolates All Candida isolates Candida albicans, Candida guilliermondii, Candida krusei, Candida zeylanoides, and Geotrichum klebahnii were procured from AL-Yarmouk Teaching Hospital. Mycological identification was carried out for all samples by commercial carbohydrate assimilation systems such as the API 20 C test kit.[18] All yeast isolates were inoculated in a primary isolation medium such as Sabouraud Dextrose Agar (SDA) medium for 2–3 days at 37°C. Antimicrobial Activity of AgNPs (Well Diffusion Method) The synthesized AgNP was tested for antimicrobial activity by agar well diffusion method against pathogenic microbes for all the previously mentioned bacteria and yeast species. The pure cultures of bacteria and yeast were subcultured on nutrient and SDA subsequently. Each strain was swapped homogeneously onto the individual plates using sterile cotton swabs. Wells of 10 mm diameter were done. The concentration of AgNPs was poured on each well. After 24 h of incubation the diameter of inhibition zone was measured. Three replicates of experiments were carried out.[19] RESULTS The formation of AgNPs was confirmed by the UV-visible spectrophotometry, which showed a strong peak within the range of 200–500 nm, “Figure 1.” Intensity of Particle Size Distribution Analysis To know the size of synthesized AgNPs, size distribution analysis was performed using light scattering in aqueous solution. The results showed that the size of the particles ranged from 16 to 104 nm. The average size of AgNPs was determined to be 49 ± 16 nm as in “Figures 2 and 3.” DISCUSSION Antimicrobial mechanisms of nanomaterials are not fully understood, but it is proposed that when they come into contact with cells, they provoke the production of reactive oxygen specie, cell membrane disruption, mitochondrial damage, and DNA damage.[20] Antifungal activity by AgNPs has been proved against different Candida species, including C. albicans, since microbial cells were inhibited using AgNPs.[21] Furthermore, it was reported that AgNPs damage the structure of the cell membrane in C. albicans producing “holes” on the surface of the cells and thus inhibiting the budding process.[22] Figure 1: Ultraviolet-visible absorption spectra of silver nanoparticles after bioreduction by Pleurotus ostreatus mushroom aqueous extract Figure 2: Size of silver nanoparticles Figure 3: Intensity of particle size distribution of Pleurotus ostreatus dried mushroom aqueous extract Hasan, et al.: Inhibitory effect of AgNPs against Candida spp. and bacteria 34 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 32-36 The antifungal properties of AgNPs against C. albicans have been demonstrated in some other studies, although reported minimum inhibitory concentration values are different from the ones we found in this work.[21-24] Such differences could be due to the nature of the particles used, the difference in size being particularly important. It is known that size and shape of metallic nanoparticles influence their chemical, optical, and thermal properties.[25] The effectiveness of AgNPs against bacteria is clearly demonstrated, and in fact, AgNPs were shown to be effective against E. coli, with cells showing formation of “pits” in the cell wall. The AgNPs were found to accumulate in the bacterial membrane and some of them were reported to successfully penetrate into the cells.[26] Similar results were found in E. coli and Vibrio cholera; it was established that AgNPs provoked changes mainly in the cell membrane morphology, producing a significant increase in their permeability, thus affecting the proper transport through the plasma membrane, and resulting eventually in cell death. They also reported that silver NPs with small diameters penetrated into the cells.[27] In our study, Ag NPs were found surrounding C. albicans cells, similar to the results found in bacteria.[27-29] In this study, the aqueous extracts of mushroom (P. ostreatus) gave showed (ve−) results against all microbial species when used as a control [Figures 4 and 5], while the biosynthesized AgNPs gave showed (ve+) result as an inhibition zones for different microbial species [Figures 4 and 5, Tables 1,2]. Table 1: The inhibitory effect of AgNPs (mm) against Candida and bacterial species Candida spp. Inhibition zone (mm) 12.5 mg/ml 25 mg/ml 50 mg/ml 100 mg/ml Candida albicans --- 10 12 16 Candida guillermondii --- 10 15.5 20 Candida krusie --- 14.5 15.5 19 Geotrichum ktebahnii --- 8 14 20 Candida zeylanoides 15 18.5 21 24.5 Staphylococcus aureus 10 11 12 16 Pseudomonas aeroginosa --- 12 13 14 Escherichia coli 10 12 13 15 AgNPs: Silver nanoparticles Table 2: Statistical analysis of the inhibitory effect of AgNPs (mm) on Candida and bacterial isolates Microbial species Control AgNPs (mg/ml) s 12.5 25 50 100 Candida albicans <0.001d 0±0.0d 10±0.57c 12±0.58b 16±0.58a Candida guillermondii <0.001d 0±0.0d 15±0.58c 15.6±0.33b 20±0.57a Candida krusie <0.001d 0±0.0d 14.5±0.28c 15.6±0.16b 19±0.57a Geotrichumktebahnii <0.001d 0±0.0d 8±0.57c 14±0.58b 20±0.58a Candida zeylanoides <0.001e 15±0.57d 18.5±0.28c 21±0.57b 24.5±0.28a Staphylococcus aureus <0.001d 10±0.58c 11±0.57bc 12±0.57b 16±0.58a Pseudomonas aeroginosa <0.001c 0±0.0c 12±0.58b 13±0.58ba 14±0.57a Escherichia coli <0.001d 10±0.57c 12±0.58b 13±0.57b 15±0.58a The findings were described in the table represent the average of three replicates±standard error. Small letters (a, b, c, d, e, ba, and ac) indicate to comparison between means in column, similar letters are non-significantly differences between means at (P≤0.05), using (LSD test). AgNPs: Silver nanoparticles Figure 4: Inhibition zone of silver nanoparticles against several isolates of bacteria; (a) Staphylococcus aureus, (b) Pseudomonas aeroginosa (c), Escherichia coli cba Hasan, et al.: Inhibitory effect of AgNPs against Candida spp. and bacteria 35 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 32-36 However, despite the clear antimicrobial properties of AgNPs, their potential use in the clinic should be carefully evaluated since there is a lack of basic knowledge on the potentially different antimicrobial properties of AgNPs which may vary depending on many factors, including the method of synthesis, size, shape, functionalizing agent, and application method and also their interaction in more complex systems such as plants, animals, and humans. 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