Evaluation of the effect of zinc oxide nanoparticles prepared by green synthesis on Pseudomonas aeruginosa biofilm

BACKGROUND AND OBJECTIVESThe ability of bacteria to build cellular assemblies on biotic and abiotic surfaces, immersed in a matrix of extracellular polymeric materials as a biofilm, has been demonstrated in recent years. This biofilm helps microbial populations in better nutrient uptake, protection from harmful circumstances, predation, desiccation, and exposure to antibacterial agents. It also plays a key role in the progression of bacterial pathogen disease. According to reports, bacteria that reside in biofilms are responsible for around ۸۰% of bacterial infections. Pseudomonas aeruginosa is a pathogenic rod-shaped, Gram-negative bacterium that is widely distributed in water, plants, soil, and animals. It rarely causes infections in healthy people, but it is far more likely to do so in immune-compromised people. Previous investigation has indicated that nanotechnology is a viable strategy for treating infections caused by biofilms. There are many benefits to using nanoparticles (NPs) as drug delivery systems because they ensure continuous medication release and reduce unwanted side effects. A lot of research is also being done on the possible antibacterial properties of certain NPs. For instance, metallic oxide nanoparticles with minimal cytotoxicity and high drug delivery potential include zinc oxide (ZnO NPs) and copper oxide (CuO- NPs). It has been demonstrated that a variety of elements, including shape and surface charge, can significantly influence a nanomaterial's antibacterial and antibiofilm characteristics. In this study, we intend to reduce the biofilm formed by Pseudomonas aeruginosa by using a green synthesized nanoparticle.MATERIALS AND METHODSTo investigate the inhibitory effect of green synthesized nanoparticles on biofilm formation, we first prepared an overnight culture of the desired bacterium Pseudomonas aeruginosa. Then we prepared the ۴۸-hour biofilm in ۹۶-well plates. We fill the wells with a volume of ۲۰۰μl of Mueller-Hinton broth (MHB). Then we prepare a bacterial suspension with a dilution of ۱۰۸ CFU/mL and add ۲۰μl to the wells containing the culture medium and incubate for ۴۸ hours at ۳۷°C. After this period, the wells were washed ۳ times with phosphate buffer and then the same culture medium containing nanoparticles in the same initial volume was added to the wells. Then, it was re-incubated with the nanoparticle used in this research, which was prepared from orange peel by the researchers of Tabriz University Research Center, and to check the antimicrobial effect of nanoparticles, optical density at ۶۰۰nm was measured by spectrophotometer. In this research, the MIC of nanoparticles was also prepared by the broth۱۷۵microdilution method. This was repeated in a ۹۶-well plate, where ۱۸۰μl of Mueller-Hinton broth containing bacteria at a concentration of ۲×۱۰۵ CFU/mL was added to the first well, and ۱۰۰μl of culture medium and bacteria were added to the other ۵ wells. Then it was prepared from the initial concentration of zinc oxide nanoparticles (۱۰۰۰μg/ml) and serial dilution (۵۰۰, ۲۵۰, ۱۲۵, ۶۲.۵, ۳۱.۲۵μg/ml). twenty μl of nanoparticles with a concentration of ۱μg/ml were poured into the first well, then ۱۰۰μl from the first well was pipetted into the second well, and this work was repeated until the fifth well, then incubated the plate at ۳۷°C for ۲۴ hours to check the minimum inhibitory concentration.RESULTS AND DISCUSSIONThe morphological study of the green synthesized zinc oxide nanoparticles with orang was performed by SEM. The particles show a semispherical shape, and these particles are in a highly agglomerated form. The particle size ranged from ۳۵.۳ to ۸۳.۱ nm, respectively. The MIC results showed a weak antibacterial effect and growth inhibition occurred at a concentration of ۵۰۰μg/ml. The results obtained from the spectrophotometer showed that the wells treated with nanoparticles compared to the control wells that only contained bacteria and were filled with empty medium after ۴۸ hours, had a lower absorption number, and in the replicates, there was a ۴۶% reduction in biofilm formed by Pseudomonas after ۴۸ hours, so the nanoparticle prepared as a green synthesis was able to reduce the biofilm formed by Pseudomonas aeruginosa. In line with our study in ۲۰۱۴, Vincent et al. reported good anti-biofilm activity for zinc oxide nanoparticles in inhibiting the biofilm formation of strong biofilm-forming Pseudomonas aeruginosa bacteria at concentrations of ۱۰۰, ۲۰۰, and ۵۰۰μg/ml, and the highest inhibition at the concentration They announced ۵۰۰μg/ml. The results of the study by García-Lara and colleagues in ۲۰۱۵ showed that zinc oxide nanoparticles at a concentration of ۱ millimolar have a strong inhibitory effect on biofilm formation by ۲۶ to ۱۰۰% in clinical and environmental strains of Pseudomonas aeruginosa and in the standard strain PAO۱, at the rate of ۹۴%.CONCLUSIONThe results of the study showed that the biosynthesis of zinc oxide nanoparticles using orange peel without the need to spend energy and expensive raw materials is economically more economical than chemical and physical methods and is compatible with the environment and has many advantages. Such as the simplicity of the manufacturing method, good stability, less time consumption, non-toxic wastes, and large-scale synthesis capability.

کلیدواژه ها:

Pseudomonas aeruginosa ، Biofilm ، Nanoparticles ، Green synthesis