Green synthesis of zinc oxide nanoparticles (ZnO-NPs) by Streptomyces baarnensis and its active metabolite (Ka): a promising combination against multidrug-resistant ESKAPE pathogens and cytotoxicity.

Various eco-friendly techniques are being researched for synthesizing ZnO-NPs, known for their bioactivity. This study aimed at biosynthesizing ZnO-NPs using Streptomyces baarnensis MH-133, characterizing their physicochemical properties, investigating antibacterial activity, and enhancement of their efficacy by combining them with a water-insoluble active compound (Ka) in a nanoemulsion form. Ka is a pure compound of 9-Ethyl-1,4,6,9,10-pentahydroxy-7,8,9,10-tetrahydrotetracene-5,12-dione obtained previously from our strain of Streptomyces baarnensis MH-133. Biosynthesized ZnO-NPs employing Streptomyces baarnensis MH-133 filtrate and zinc sulfate (ZnSO4.7H2O) as a precursor were purified and characterized by physicochemical investigation. High-resolution-transmission electron microscopy (HR-TEM) verified the effective biosynthesis of ZnO-NPs (size < 12 nm), whereas dynamic light scattering (DLS) analysis showed an average size of 17.5 nm. X-ray diffraction (XRD) exhibited characteristic diffraction patterns that confirmed crystalline structure. ZnO-NPs efficiently inhibited both Gram-positive and Gram-negative bacteria (MICs: 31.25-125 µg/ml). The pure compound (Ka) was combined with ZnO-NPs to improve effectiveness and reduce dose using checkerboard microdilution. Niteen treatments of Ka and ZnO-NPs combinations obtained by checkerboard matrix inhibited Klebsiella pneumonia. Eleven combinations had fractional inhibitory concentration index (FICi) between 1.03 and 2, meaning indifferent, another five combinations resulted from additive FICi (0.625-1) and only one combination with FICi of 0.5, indicating synergy. In the case of methicillin-resistant S. aureus (MRSA), Ka-ZnO-NPs combinations yielded 23 treatments with varying degrees of interaction. The results showed eleven treatments with indifferent interaction, eight additive interactions, and two synergies with FICi of 0.5 and 0.375. The combinations that exhibited synergy action were transformed into a nanoemulsion form to improve their solubility and bioavailability. The HR-TEM analysis of the nanoemulsion revealed spherical oil particles with a granulated core smaller than 200 nm and no signs of aggregation. Effective dispersion was confirmed by DLS analysis which indicated that Ka-ZnO-NPs nanoemulsion droplets have an average size of 53.1 nm and a polydispersity index (PI) of 0.523. The killing kinetic assay assessed the viability of methicillin-resistant Staphylococcus aureus (MRSA) and K. pneumonia post-treatment with Ka-ZnO-NPs combinations either in non-formulated or nanoemulsion form. Results showed Ka-ZnO-NPs combinations show concentration and time-dependent manner, with higher efficacy in nanoemulsion form. The findings indicated that Ka-ZnO-NPs without formulation at MIC values killed K. pneumonia after 24 h but not MRSA. Our nanoemulsion loaded with the previously mentioned combinations at MIC value showed bactericidal effect at MIC concentration of Ka-ZnO-NPs combination after 12 and 18 h of incubation against MRSA and K. pneumonia, respectively, compared to free combinations. At half MIC value, nanoemulsion increased the activity of the combinations to cause a bacteriostatic effect on MRSA and K. pneumonia after 24 h of incubation. The free combination showed a bacteriostatic impact for 6 h before the bacteria regrew to increase log10 colony forming unit (CFU)/ml over the initial level. Similarly, the cytotoxicity study revealed that the combination in nanoemulsion form decreased the cytotoxicity against kidney epithelial cells of the African green monkey (VERO) cell line. The IC50 for Ka-ZnO-NPs non-formulated treatment was 8.17/1.69 (µg/µg)/ml, but in nano-emulsion, it was 22.94 + 4.77 (µg/µg)/mL. In conclusion, efficient Ka-ZnO-NPs nanoemulsion may be a promising solution for the fighting of ESKAPE pathogenic bacteria according to antibacterial activity and low toxicity.

Genetic Stability, Phenolic, Flavonoid, Ferulic Acid Contents, and Antioxidant Activity of Micropropagated Lycium schweinfurthii Plants.

Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L-1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L-1 Indole-3-butyric acid (IBA). The Jaccard's similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL-1 of MiP dried leaves' methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.

Optimization of spore laccase production by Bacillus amyloliquefaciens isolated from wastewater and its potential in green biodecolorization of synthetic textile dyes.

The spore laccase enzyme production by B. amyloliquefaciens was optimized. It was characterized and tested for its textile dye decolorization potential. LB medium was found to be the most promising growth medium with addition of glucose (1-2%), yeast extract (0.1%), FeCl3 (0.01 mM) and MnCl2 (0.001 mM). The optimum spore laccase production was at pH 8, 30 °C, 1:5 medium to air ratio, 2% inoculum size and 7 days incubation. The characterization study of the enzyme showed the maximum activity at 60 °C and pH 6-7.5. It was induced by Ca+2, Mg+2, Fe+3, Zn+2, Cu+2 and Na+ at 1 mM concentration. Also, it was stable in the presence of methanol, ethanol, acetone and chloroform. In addition, it enhanced about 34% by 5 mM H2O2 and it was nearly stable at 10-20 mM H2O2. Furthermore, mediators such as ABTS, syrengaldazine and 2, 6 dimethyl phenol enhanced the spore laccase activity. The spore laccase enzyme efficiently decolorized direct red 81 and acid black 24 after 24 h. Phytotoxicity of the direct red 81 solution after decolorization by tested spore laccase was lower than that of the untreated dye solution. Finally, this study added a promising spore laccase candidate for ecofriendly and cost-effective dye wastewater bio-decolorization.