Desertifilum sp. EAZ03 cell extract as a novel natural source for the biosynthesis of zinc oxide nanoparticles and antibacterial, anticancer and antibiofilm characteristics of synthesized zinc oxide nanoparticles.

AIMS: The use of cyanobacterial cell extracts for the synthesis of zinc oxide nanoparticles (ZnO NPs) seems to be superior to other methods of synthesis because of its a green, environmentally friendly and low-cost approach. In this study, the cell extract of a newly characterized cyanobacterial strain Desertifilum sp. EAZ03 was used for the biosynthesis of ZnO NPs. The antimicrobial, antibiofilm and anticancer activities of the biosynthesized ZnO NPs (hereinafter referred to as CED-ZnO NPs) were examined as well. METHODS AND RESULTS: UV-Vis spectroscopy analysis of CED-ZnO NPs showed an absorbance band at 364 nm, and powder X-ray diffraction analysis confirmed the purity of the synthesized nanoparticles. The analyses of scanning electron microscopy and transmission electron microscopy images revealed that CED-ZnO NPs were rod-shaped with a size of 88 nm. The study of the biological features of CED-ZnO NPs showed a significant antimicrobial potential against the bacterial strains tested. CED-ZnO NPs were able to impede the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa up to 80%, 89% and 85%, respectively. The nanoparticles also showed 69%, 70% and 62% degrading activity against S. aureus, E. coli and P. aeruginosa 1-day-old biofilms, respectively. The antibiofilm activity of the synthesized nanoparticles was investigated by confocal laser scanning microscopy. The MTT assay showed that CED-ZnO NPs, at a concentration of 100 µg/ml, had less cytotoxicity towards normal lung (MRC-5) cells, at the half, compared to cancerous lung alveolar epithelial (A549) cells. The minimum inhibitory concentration and minimum bactericidal concentration values of CED-ZnO NPs against E. coli, P. aeruginosa and S. aureus were 1500, 2000 and 32 µg/ml, and 2500, 3500 and 64 µg/ml, respectively. CONCLUSIONS: The multifunctional CED-ZnO NPs seem to be promising for possible applications in the therapeutic and pharmaceutical industries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes a new approach for the biosynthesis of zinc oxide nanoparticles using a newly characterized cyanobacterial strain Desertifilum sp. EAZ03. The considerable antimicrobial, antibiofilm and anticancer activities of the biosynthesized zinc oxide nanoparticles further emphasize the emerging role of microbial systems in the green synthesis of metal oxide nanoparticles.

Growth Optimization of Lactobacillus acidophilus for Production of Antimicrobial Peptide Acidocin 4356: Scale up from Flask to Lab-Scale Fermenter.

BACKGROUND: Antibiotic-resistant bacteria are a major threat to global health. Older antibiotics have become more or less ineffective as a result of widespread microbial resistance and an urgent need has emerged for the development of new antimicrobial strategies. Acidocin 4356 is a novel antimicrobial bacteriocin peptide produced by Lactobacillus acidophilus ATCC 4356 and capable of confronting the Pseudomonas aeruginosa ATCC 27853 infection challenges. According to our previous studies, the production of Acidocin 4356 is in parallel with cellular biomass production. OBJECTIVES: Given the costly production of Acidocin 4356, the development of a beneficial approach for increasing productivity of the cellular biomass has been targeted in the lab-scale fermenter for scale-up production of this bacteriocin. Therefore, in this study, we developed an inexpensive optimal culture medium based on the whey feedstock, evaluating this medium for scaling-up of the bacteriocin production from flask to fermenter. MATERIAL AND METHODS: In the first step, the optimization of the process parameters and medium components was carried out using the Plackett-Burman (PB) design and Response surface methodology (RSM) in flask culture. After optimization of the medium, bacteriocin production in the optimum culture medium was compared with de Man, Rogosa and Sharpe (MRS) medium by analyzing the intensity of the peptide band. Intensity analysis has been conducted on the PAGE band of the peptide using Image J software. Finally, the scale- up of bacteriocin production in the optimum culture medium was evaluated by batch fermentation in a 3-liter fermenter. RESULTS: In this study, a medium containing whey (40 g.L-1) and sodium acetate (5 g.L-1) was used as basal medium, and the effect of other factors were then evaluated. According to the PB design, three factors of peptone concentration, yeast extract concentrations and cultivation temperature were selected as the most effective factors which improve the growth of L. acidophilus. The condition providing the highest growth capacity for bacteriocin production were predicted based on the results of RSM as following: temperature 40 ° C, yeast (4 g.L-1), and peptone (8 g.L-1). Finally, the dry cell weight was obtained after incubation for 12 h as 2.25 g.L-1. Comparison of cell growth and bacteriocin production between MRS medium and optimized medium confirmed the efficacy of these optimal conditions for the cost-effective production of Acidocin 4356 in the flask. Besides, the scale- up of bacteriocin production has made under optimal condition in the 3-L fermenter. CONCLUSIONS: In this study, for the first time, scale- up production of Acidocin 4356 was presented by using a low-cost method based on whey feedstock to tackle P. aeruginosa infections.

Effects of cefazolin-containing niosome nanoparticles against methicillin-resistant Staphylococcus aureus biofilm formed on chronic wounds.

The ability of biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) causes significant mortality and morbidity in wound infections. Nanoparticles because of the drug concentration increment at the point of contact of nanoparticles and bacteria, and slower release of the drug at the desired location are considered as proper tools to overcome the therapeutic problem of antimicrobial-resistant infections. This study was aimed to evaluate the anti-biofilm activity of cefazolin-loaded nanoparticles against MRSA isolates. The 27 clinical isolates of MRSA were collected from patients with pressure sores and diabetic ulcers referred to Loghman Hospital in Tehran-Iran. MRSA isolates were detected by polymerase chain reaction (PCR) and biochemical tests. Cefazolin-loaded niosome was synthesized using the thin-film hydration method and were characterized by zeta potential measurement and transmission electron microscopy (TEM). The round-shaped cefazolin-loaded niosomes had a diameter of 100 nm and a -63 mV zeta potential. The cefazolin-containing niosomes removed 1, 3, and 5 d old biofilms at the concentration of 128 µg ml-1, 128 µg ml-1, and 256 µg ml-1, respectively. Histological results indicated that BALB/c mice receiving cefazolin-loaded niosomes were treated effectively faster than those treated by cefazolin or untreated group. In conclusion, the cefazolin-loaded niosome could be considered as a promising candidate for the treatment of biofilm-mediated infections of MRSA.

A bio-inspired strategy for the synthesis of zinc oxide nanoparticles (ZnO NPs) using the cell extract of cyanobacterium Nostoc sp. EA03: from biological function to toxicity evaluation.

Cyanobacteria, as one of the largest groups of phototrophic bacteria, have a high potential as an excellent source of fine chemicals and bioactive compounds, including lipid-like compounds, amino acid derivatives, proteins, and pigments. This study aimed to synthesize ZnO nanoparticles using the cell extract of the cyanobacterium Nostoc sp. EA03 (CEN-ZnO NPs) through a rapid and eco-friendly approach. The biosynthesized nanoparticles, CEN-ZnO NPs, were characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA), FTIR, SEM, TEM, and EDX spectroscopy. The UV-Vis spectrum showed an absorption peak at 370 nm. The star-shaped CEN-ZnO NPs, as observed in the TEM and SEM images, had an average diameter of 50-80 nm. MIC and MBC values for E. coli, P. aeruginosa and S. aureus, were determined to be, respectively, 2000, 2000, and 64 µg ml-1, and 2500, 2500 and 128 µg ml-1. Further analysis through confocal laser scanning microscopy (CLSM) provided the observable confirmation that the CEN-ZnO NPs stunted the bacterial growth, preventing the formation of exopolysaccharides. The AFM analysis of surface topography of bacterial biofilm samples treated with CEN-ZnO NPs showed a rugged topography in some parts of the biofilm surface, indicating the destruction of biofilms. In contrast, in the untreated control samples, the structured biofilms were flat and prominent. MTT assay indicated that CEN-ZnO NPs had less cytotoxicity on the MRC-5 lung fibroblast cells compared with the cancerous treated A549 cells. As the concentration of the CEN-ZnO NPs increased, the amount of ROS produced in the tested bacterial strains also increased. Analyzing the data obtained from flow cytometry showed that the higher concentrations of CEN-ZnO NPs lead to a reduction in the viability of P. aeruginosa PAO1, E. coli and S. aureus. The biosynthesized ZnO nanoparticles using Nostoc cell extracts exhibited different attributes, inspiring enough to be considered for further investigation.

Limnothrix sp. KO05: A newly characterized cyanobacterial biosorbent for cadmium removal: the enzymatic and non-enzymatic antioxidant reactions to cadmium toxicity.

In this study, we isolated five indigenous cyanobacterial strains from different aqueous environments, with heavy metals contamination, in East Azerbaijan Province (northwest portion of Iran). A strain was identified by morphological and 16S rRNA sequence analysis as Limnothrix sp. KO05 and selected for further studies as having the greatest potential for cadmium uptake. Scanning electron microscopy (SEM) demonstrated cyanobacterium Limnothrix sp. KO05 forms filamentous structures and is straight or curved to some extent. The utmost biosorption capacity was found to be 82.18±1.22mgg-1 at a Cd (II) concentration level of 150mgL-1. Langmuir adsorption isotherm indicated a better fit to the experimental data. Response surface methodology (RSM) on the basis of four independent variables and the predicted maximum biosorption efficiency was 98.7% under the optimum condition. FT-IR spectroscopy profile of the Cd treated sample as demonstrated in confirmation of the benefits of various functional groups of proteins and polysaccharides of cyanobacterial biomass, involved in surface binding of Cd. The determination of catalase (CAT) activity in strain KO05 exposed to Cd (II) concentrations of 2, 5 and 10mgL-1 showed an increase in enzyme activity after 24h exposure compared to unexposed cells. Correspondingly, CAT activity showed a significant decrease after 48h of treatment with Cd (II) concentrations of 5 and 10mgL-1. CAT activity was decreased significantly at all concentrations within 72h after exposure to Cd. On the contrary, while ascorbate peroxidase (APX) gave the expected lower activity compared to the CAT within 24h after Cd treatment, its activity lasted up to 72h. Limnothrix sp. KO05 cells treated with 5 and 10mgL-1 Cd (II) over 72h exposure showed a reduction in chlorophyll a contents compared to the controls. However, following exposure to Cd, chlorophyll a and carotenoid contents is reduced and after overcoming stress and deployment of an adaptation mechanism, the amounts of these pigments is gradually increased in the cells. The reduction was slower for chlorophyll a pigment compared to carotenoids that may be an indication of the physiological importance of chlorophyll pigment for the phtosynthetic cells. Results related to lipid peroxidation in Limnothrix sp. KO05 represent a significant increase of MDA in the first 24h after exposure to the different concentrations of Cd (2, 5 and 10mgL-1). However, the MDA levels were decreased over time and no significant difference attained after 72h exposure to Cd concentrations of 2 and 10mgL-1 compared to control.