Investigation of Antimicrobial and Synergistic effects of doped Zinc Oxide Nanoparticles against Bacillus cereus

Metal oxide nanoparticles have unique physical and chemical properties. These components have shown antimicrobial effects against a wide range of microorganisms. In order to improve the physical properties of metal oxide nanoparticles, doping other elements with metal oxide nanoparticles is an effective way. Bacillus cereus is a gram-positive bacteria causing food-borne diseases. In this study, the antimicrobial effects of doped zinc oxide nanoparticles with manganese or iron on Bacillus cereus have been studied. To investigate the synergistic effects of the combined nanoparticles with two common biocides, including hydrogen peroxide and sodium hypochlorite, have been used. Co- precipitation method was used to prepare nanoparticles of manganese-zinc oxide and iron-zinc oxide. In this method, zinc sulfate and manganese sulfate were used to prepare manganese-zinc oxide  and iron sulfate and zinc sulfate are used for Zn- Fe doped nanoparticles. After preparing the sulfate solutions, the sulfate solutions were mixed and placed in an ultrasonic apparatus at a frequency of 57 kHz for 2 hours at 50ºC. Then, it was stirred at 80°C. A solution of NaOH was added until the pH of the solution reached 12. In these conditions, the mixing was done for 30 minutes. The solution was placed at ambient temperature for 18 hours. Then the centrifuge was performed to separate the sediment. Purification was done through washing with distilled water and ethanol. The precipitates were dried in the vacuum oven. In this way, the doped nanoparticles of manganese-zinc oxide and iron-zinc oxide were obtained. The Fourier transform infrared spectrum (FTIR) was carried out by the Perkin-Elmer apparatus of the Spectroma2 model, using a dry potassium bromide tablet at a frequency range of 4500-4000 cm-1. The X-ray diffraction was tested using the Phillips PW1820 from 2º to 80º. Structure of produced nanoparticles was assessed by the HITACHI electron microscope, the H-7500 model, by placing a drop of nanoparticles dissolved in methanol on a special lining with carbon coating and air drying, and performing microscopic images using an electron microscope in 100kv. The bacteria used in this study included Bacillus cereus (PTCC 1665) was purchased from the Iranian Scientific and Industrial Research Center and was transferred to the BHI medium in sterile condition and incubated for 32 hours at a temperature of 32°C. Microbial cells were isolated by centrifugation at 4000 rpm. McFarland's method was used for determining the bacterial population. Dilution was carried out to reach a population of about 106 CFU/ml. Agar disc diffusion method was used for assessing the antimicrobial effect of the doped nanoparticles alone or in combination with tested biocides (hydrogen peroxide, sodium hypochlorite). At first, 106 CFU/ml of Bacillus cereus were inoculated on the surface of Blood Agar. Then, 5, 10, 20, 30, 50, 100 and 200 mg/L of each of the nanoparticles were placed on the surface of the culture medium and then the plates was incubated at 37°C for 24 hours. Inhibition zone was considered as antibacterial activity. In order to investigate synergistic effects, inhibitory fraction index test was calculated. All experiments were performed in three replications. Statistical analyzes were performed using STATISTICA software. Results obtained from X-ray and FTIR analysis of doped nanoparticles confirmed that co- precipitation is a suitable method for producing doped nanoparticles of zinc oxide. TEM analysis of produced nanoparticles also affirm formation of doped nanoparticles of zinc oxide with manganese and iron. The results of antimicrobial tests showed that Mn-Zn oxide nanoparticles have more antimicrobial effects on Bacillus cereus than zinc oxide (32mm inhibition zone) whereas Fe- Zn oxide nanoparticles cause inhibition zone about 12 mm. In addition, both doped nanoparticles have more antimicrobial effects than zinc oxide nanoparticles alone, resulted in doping process improves antimicrobial properties of zinc oxide. The synergistic effects of synthetic nanoparticles in the combination of two common antimicrobial agents, including hydrogen peroxide and sodium hypochlorite, have been identified. Both nanoparticles show synergistic effects in combination with two tested biocides (especially in high concentrations). A mixture of two biocides with nanoparticles increases their antimicrobial properties. Manganese-zinc oxide nanoparticles with hydrogen peroxide and sodium hypochlorite showed a partial synergistic effect at low concentrations (5 + 20) and a complete synergistic effect at higher concentrations. In the case of iron-zinc oxide, combination of this nanoparticle with hydrogen peroxide and sodium hypochlorite, has complete synergistic effects at high concentration (100 + 200) and at other conditions, shows partial synergistic effects.