Biogenic nanosilver bearing antimicrobial and antibiofilm activities and its potential for application in agriculture and industry.

Due to the increasing resistance of bacteria and fungi to antimicrobials, it is necessary to search for effective alternatives to prevent and treat pathogens causing diseases in humans, animals, and plants. In this context, the mycosynthesized silver nanoparticles (AgNPs) are considered as a potential tool to combat such pathogenic microorganisms. AgNPs were synthesized from Fusarium culmorum strain JTW1 and characterized by Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS) and Zeta potential measurement. The minimum inhibitory (MIC) and biocidal concentrations (MBC) were determined against 13 bacterial strains. Moreover, the combined effect of AgNPs with antibiotics (streptomycin, kanamycin, ampicillin, tetracycline) was also studied by determining the Fractional Inhibitory Concentration (FIC) index. The anti-biofilm activity was examined by crystal violet and fluorescein diacetate (FDA) assays. Furthermore, antifungal activity of AgNPs was evaluated against a panel of phytopathogenic fungi viz., Botrytis, Colletotrichum, Fusarium, Phoma, Sclerotinia, and an oomycete pathogen Phytophthora by agar well-diffusion and micro-broth dilution method to evaluate the minimal AgNPs concentrations that inhibit fungal spore germination. Fungi-mediated synthesis resulted in the formation of small (15.56 ± 9.22 nm), spherical and stable (zeta potential of - 38.43 mV) AgNPs with good crystallinity. The results of FTIR spectroscopy indicated the presence of various functional groups, namely hydroxyl, amino, and carboxyl ones, from the biomolecules on the surface of AgNPs. The AgNPs showed antimicrobial and antibiofilm formation activities against Gram-positive and Gram-negative bacteria. The values of MIC and MBC ranged between 16-64 and 32-512 μg mL-1, respectively. The enhanced effect of AgNPs in combination with antibiotics was confirmed against human pathogens. The highest synergistic effect (FIC = 0.0625) was demonstrated by the combination of AgNPs with streptomycin against two strains of Escherichia coli (ATCC 25922 and ATCC 8739), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (FIC = 0.125). Enhanced effects of AgNPs with ampicillin were also shown against Staphylococcus aureus ATCC 25923 (FIC = 0.125) and P. aeruginosa (FIC = 0.25), as well as kanamycin against S. aureus ATCC 6538 (FIC = 0.25). The crystal violet assay revealed that the lowest concentration of AgNPs (0.125 μg mL-1) reduced the development of biofilms of Listeria monocytogenes and Salmonella enterica, while the maximum resistance was shown by Salmonella infantis, its biofilm was reduced after exposure to a concentration of 512 μg mL-1. A high inhibitory effect on the activity of bacterial hydrolases was observed by the FDA assay. AgNPs at a concentration of 0.125 μg mL-1 reduced the hydrolytic activity of all biofilms formed by the tested pathogens, except E. coli ATCC 25922, P. aeruginosa, and Pectobacterium carotovorum (efficient concentration was 2-fold higher, at 0.25 μg mL-1), while the hydrolytic activity of E. coli ATCC 8739, Salmonella infantis and S. aureus ATCC 6538 was suppressed after treatment with AgNPs at concentrations of 0.5, 2 and 8 μg mL-1, respectively. Moreover, AgNPs inhibited fungal growth and spore germination of Botrytis cinerea, Phoma lingam, and Sclerotinia sclerotiorum. MIC and MFC values of AgNPs against spores of these fungal strains were determined at 64, 256, and 32 μg mL-1, and zones of growth inhibition were 4.93, 9.54, and 3.41 mm, respectively. Fusarium culmorum strain JTW1 was found to be an eco-friendly biological system for an easy, efficient and inexpensive synthesis of AgNPs. In our study, the mycosynthesised AgNPs demonstrated remarkable antimicrobial (antibacterial and antifungal) and antibiofilm activities against a wide range of human and plant pathogenic bacteria and fungi singly and in combination with antibiotics. These AgNPs could be applied in medicine, agriculture, and food industry to control such pathogens that cause numerous human diseases and crop losses. However, before using them extensive animal studies are required to evaluate the toxicity, if any.

Trzcińska-Wencel J ，Wypij M ，Rai M ，Golińska P ... -  《Frontiers in Microbiology》

Biofabrication of novel silver and zinc oxide nanoparticles from Fusarium solani IOR 825 and their potential application in agriculture as biocontrol agents of phytopathogens, and seed germination and seedling growth promoters.

Introduction: Plant pathogenic microorganisms adversely affect the growth and yield of crops, which consequently leads to losses in food production. Metal-based nanoparticles (MNPs) can be a remedy to solve this problem. Methods: Novel silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) were biosynthesized from Fusarium solani IOR 825 and characterized using Dynamic Light Scattering (DLS), Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-ray diffraction (XRD) and measurement of Zeta potential. Antibacterial activity of NPs was evaluated against four plant pathogenic strains by determination of the minimum inhibitory (MIC) and biocidal concentrations (MBC). Micro-broth dilution method and poisoned food technique were used to assess antifungal activity of NPs against a set of plant pathogens. Effect of nanopriming with both types of MNPs on maize seed germination and seedlings growth was evaluated at a concentration range of 1-256 μg mL-1. Results: Mycosynthesis of MNPs provided small (8.27 nm), spherical and stable (zeta potential of -17.08 mV) AgNPs with good crystallinity. Similarly, ZnONPs synthesized by using two different methods (ZnONPs(1) and ZnONPs(2)) were larger in size (117.79 and 175.12 nm, respectively) with Zeta potential at -9.39 and -21.81 mV, respectively. The FTIR spectra showed the functional groups (hydroxyl, amino, and carboxyl) of the capping molecules on the surface of MNPs. The values of MIC and MBC of AgNPs against bacteria ranged from 8 to 256 μg mL-1 and from 512 to 1024 μg mL-1, respectively. Both types of ZnONPs displayed antibacterial activity at 256-1024 μg mL-1 (MIC) and 512-2048 μg mL-1 (MBC), but in the concentration range tested, they revealed no activity against Pectobacterium carotovorum. Moreover, AgNPs and ZnONPs inhibited the mycelial growth of Alternaria alternata, Fusarium culmorum, Fusarium oxysporum, Phoma lingam, and Sclerotinia sclerotiorum. MIC and MFC values of AgNPs ranged from 16-128 and 16-2048 μg mL -1, respectively. ZnONPs showed antifungal activity with MIC and MFC values of 128-2048 μg mL-1 and 256-2048 μg mL-1, respectively. The AgNPs at a concentration of ≥32 μg mL-1 revealed sterilization effect on maize seeds while ZnONPs demonstrated stimulatory effect on seedlings growth at concentrations of ≥16 μg mL-1 by improving the fresh and dry biomass production by 24% and 18%-19%, respectively. Discussion: AgNPs and ZnONPs mycosynthesized from F. solani IOR 825 could be applied in agriculture to prevent the spread of pathogens. However, further toxicity assays should be performed before field evaluation. In view of the potential of ZnONPs to stimulate plant growth, they could be crucial in increasing crop production from the perspective of current food assurance problems.

Trzcińska-Wencel J ，Wypij M ，Terzyk AP ，Rai M ，Golińska P ... -  《-》

Synthesis, characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from Streptomyces xinghaiensis OF1 strain.

Wypij M ，Czarnecka J ，Świecimska M ，Dahm H ，Rai M ，Golinska P ... -  《-》

Antimicrobial and cytotoxic activity of silver nanoparticles synthesized from two haloalkaliphilic actinobacterial strains alone and in combination with antibiotics.

Presently, the effective antimicrobial agents have been limited by the emergence of microbial strains with multidrug resistance and biofilm formation potential. In the present study, we report remarkable antimicrobial activity of silver nanoparticles (AgNPs) synthesized from Streptomyces calidiresistens IF11 and IF17 strains, including inhibition of biofilm formation and synergistic effect of AgNPs and antibiotics against selected bacteria and yeasts. Cytotoxic effect of AgNPs on mammalian cell lines was also evaluated. Analysis of biosynthesized AgNPs by Fourier Transform Infrared Spectroscopy and transmission electron microscopy revealed their spherical shape, small size in the range of 5-50 and 5-20 nm, respectively, as well as the presence of capping agents. Study of antimicrobial activity of AgNPs against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Malassezia furfur evaluated by minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) assays revealed that MICs of AgNPs from IF11 and IF17 strains against bacteria and yeasts were found to be in the range of 16-128 and 8-256 μg ml-1 , while MBCs were in the range of 48-192 and 32-256 μg ml-1 respectively. AgNPs inhibited biofilm formation of microbial strains, which was tested by using crystal violet stain. The highest synergistic effect determined by fractional inhibitory index of AgNPs with antibiotic (kanamycin or tetracycline) was found against Staph. aureus; while in case of yeasts, M. furfur showed highest sensitivity to AgNPs-ketoconazole combination (FIC = 0·12). The cytotoxic activity of AgNPs towards HeLa and 3T3 cell lines was studied by MTT assay. The IC50 of AgNPs estimated against mouse fibroblasts was found to be 8·3 and 28·3 μg ml-1 and, against HeLa cell line, 28·5 and 53·8 μg ml-1 respectively. It can be concluded that AgNPs synthesized from S. calidiresistens IF11 and IF17 strains have potential as an effective antimicrobial and cytotoxic agent, especially when used in combination with antibiotics/antifungal agents. This study indicates potential application of biogenic silver nanoparticles as an antimicrobial agent in nanomedicine.

Wypij M ，Świecimska M ，Czarnecka J ，Dahm H ，Rai M ，Golinska P ... -  《-》

Pullulan-based films impregnated with silver nanoparticles from the Fusarium culmorum strain JTW1 for potential applications in the food industry and medicine.

Introduction: Biopolymers, such as pullulan, a natural exopolysaccharide from Aureobasidium pullulans, and their nanocomposites are commonly used in the food, pharmaceutical, and medical industries due to their unique physical and chemical properties. Methods: Pullulan was synthesized by the A. pullulans ATCC 201253 strain. Nanocomposite films based on biosynthesized pullulan were prepared and loaded with different concentrations of silver nanoparticles (AgNPs) synthesized by the Fusarium culmorum strain JTW1. AgNPs were characterized by transmission electron microscopy, Zeta potential measurements, and Fourier-transform infrared spectroscopy. In turn, the produced films were subjected to physico-chemical analyses such as goniometry, UV shielding capacity, attenuated total reflection-Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy, and their mechanical and degradation properties were assessed. The antibacterial assays of the nanoparticles and the nanocomposite films against both food-borne and reference pathogens, including Listeria monocytogenes, Salmonella infantis, Salmonella enterica, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, were performed using standard methods. Results: AgNPs were small (mean 15.1 nm), spherical, and displayed good stability, being coated with protein biomolecules. When used in higher concentrations as an additive to pullulan films, they resulted in reduced hydrophilicity and light transmission for both UV-B and UV-A lights. Moreover, the produced films exhibited a smooth surface. Therefore, it can be concluded that the addition of biogenic AgNPs did not change the morphology and texture of the films compared to the control film. The nanoparticles and nanocomposite films demonstrated remarkable antibacterial activity against both food-borne and reference bacteria. The highest activity of the prepared films was observed against L. monocytogenes. Discussion: The obtained results suggest that the novel nanocomposite films prepared from biosynthesized pullulan and AgNPs can be considered for use in the development of medical products and food packaging. Moreover, this is the first report on pullulan-based nanocomposites with mycogenic AgNPs for such applications.

Wypij M ，Rai M ，Zemljič LF ，Bračič M ，Hribernik S ，Golińska P ... -  《-》