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 (ZnSO4.7H(2)O) 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 <mu>g/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 log(10) 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 (mu g/mu g)/ml, but in nano-emulsion, it was 22.94 + 4.77 (mu g/mu g)/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.
