Synergistic broad-spectrum antibacterial activity of Hypoxis hemerocallidea-derived silver nanoparticles and streptomycin against respiratory pathobionts

Respiratory tract infections arise due to the introduction of microbes into the airway, disrupting the normal, healthy, complex interdependent microbiome. The selective disruption of this community can be either beneficial or dangerous. Nanoparticles are a potential tool for modifying this population. Coated silver nanoparticles (AgNPs) were synthesized using ethanolic extracts of Hypoxis hemerocallidea (EEHH), a Southern African plant used extensively in traditional medicine and the source of many bioactive secondary metabolites. The room temperature reaction between silver nitrate and EEHH forms largely spherical AgNPs with an average diameter of 6–20 nm. These nanoparticles show similar levels of antibacterial activity as the broad-spectrum antibiotic streptomycin against Bacillus cereus, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeuroginosa, and Moraxella catarrhalis. However, the AgNPs synergistically increase the antibacterial activity of streptomycin when they are applied in combination (30–52%). AgNPs are reiterated to be promising dual-function antibiotics, synergistically enhancing activity while also acting as delivery agents for small molecules.

www.nature.com/scientificreports/ Biological Sciences, North-West University, Mafikeng, South Africa, and the University of Windsor, Windsor, Canada. The corms were then extensively washed under running water, dried at room temperature (24 °C), and pulverized into a powder with a commercial blender. Pulverized corms (500 g) were soaked in absolute ethanol (1.5 L) with mechanical shaking for 72 h. The extract was filtered to remove the powder, then concentrated under reduced pressure using a rotary evaporator at 70.1 °C to obtain a dark brown powder. After drying under high vacuum for an additional 16 h to constant mass, 23.6 g of a dark brown powder was obtained 18 . This fraction, the ethanolic extract of Hypoxis heamerocallidea is referred to as EEHH.

Synthesis of AgNPs-HH.
A stock solution of EEHH was prepared by suspending 2 g of the crude powder in 50 mL of 70% ethanol (v/v). Next, it was sonicated for 10 min to solubilize the solution to a translucent deep yellow solution with no observable turbidity. To a 40 mL aliquot of this solution was then added 400 µL of 0.1 M AgNO 3 in one portion with vigorous stirring. The reaction mixture immediately changed to a dark brown colour, indicative of the formation of the AgNPs. The process was carried out at ambient room temperature open to the atmosphere, and progress was monitored by measuring the UV-Vis spectrum of the reactant mixture at hourly intervals over 4 h according to our previously reported procedure 24 . With no further changes being observed between 3 and 4 h, the particles were purified by dialysis (against water, cut-off of 3.5 kDa), then dried by lyophilization and stored until resuspended when needed.
Phytochemical screening. A qualitative phytochemical analysis of the EEHH (independent of the presence of the silver) was carried out using standard procedures [25][26][27]  Characterization of the nanoparticles. The absorption spectra of the AgNPs were measured between 300 and 700 nm using a PerkinElmer (Germany) 365 UV-Vis spectrometer at 24 °C. The morphology of the AgNPs was examined on a JEOL 3010 high-resolution transmission electron microscope equipped with energydispersive X-ray (EDX) functionality (Bruker, Germany). The FTIR spectra of the crude extract and the AgNPs were obtained on a Bruker Alpha-P FTIR spectrophotometer (Germany) from 500 to 4000 cm −1 . The structural characterization of the AgNPs was carried out using an X-ray diffractometer. XRD analysis was conducted using Bruker equipment with monochromatic Cu kα radiation (λ = 1:5406 Å) at 40 kV. Scanning was conducted in the region of 20-100 2θ angles. Dynamic light scattering (Malvern Zetasizer Nano-ZS) was used to analyze the zeta potential of the synthesized AgNPs. For the DLS measurements, powder AgNPs were resuspended in distilled water and sonicated for 15-20 min to properly disperse the particles in water. Zeta potential, and hydrodynamic diameter values were obtained from the triplicate analysis of the nanoparticles in the aqueous media 24 29 . Using the disc diffusion technique, pure cultures of these microorganisms were sub-cultured on nutrient agar and incubated at 37 °C for 24 h. Fresh overnight cultures were inoculated on Mueller Hinton agar (MHA) plates using sterile swabs and allowed to stand for 20 min. Wells of 6-mm diameter were made on MHA plates with the bacterial lawn. Each well was filled with 50 μL of different concentrations (50, 100, and 150 μg/mL) EEHH in distilled water and HH AgNPs in dimethyl sulfoxide (DMSO) prepared from 10 mg/mL stock. DMSO (5%) was used as the negative control, and streptomycin (10 μg/mL) served as the reference standard. The plates were incubated at 37 °C for 24 h, and the diameters of the inhibition zones around the wells were measured. Experiments were carried out in triplicate to reduce error 30 . The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) of green synthesized AgNPs were determined using the modified method described in the CLSI guideline (2012) 31 . The MIC test was performed in a 96-well round bottom microtiter plate using standard broth microdilution methods while the MBC test was performed on the MHA plates. The bacterial inoculums were adjusted to the concentration of 10 6 CFU/mL. For the MIC test, 100 μL of the synthesized AgNPs stock solution (500 μg/mL) was added and diluted twofold with the bacterial inoculums in 100 μL of MHB started from column 12 to column 3. Column 12 of the microtiter plate contained the highest concentration of AgNPs, while column 3 contained the lowest concentration. Column 1 served as negative control (only medium) and the column 2 served as positive control (medium and bacterial inoculums). Each well of the microtiter plate was added with 30 μL of the resazurin solution and incubated at 37 °C for 24 h. Any colour changes were observed. Blue/purple colour indicated no bacterial growth while pink/colourless indicated bacterial growth. The MIC value was taken at the lowest concentration of antibacterial agents that inhibits the growth of bacteria (colour remained in blue).
Streptomycin (Sigma, St. Louis) treatments were all conducted at 10 μg/mL. This was prepared as needed from a stock solution by diluting tenfold with the assay media. The stock solution was prepared by transferring 10 mg of streptomycin into a 1 mL volumetric flask, and making up the volume with a 1:1 ethanol: water solution, providing a 10 mg/mL solution. This was then diluted 100-fold by transferring 10 μL of this solution to a new 1 mL volumetric flask, and making up the volume with the 50% ethanolic solution. This provided the 100 μg/mL stock.
The MBC was defined as the lowest concentration of the antibacterial agents that completely kill the bacteria. MBC test was performed by plating the suspension from each well of microtiter plates that exhibited no colour change into MHA plate. The plates were incubated at 37 °C for 24 h. The lowest concentration with no visible growths on the MHA plate was taken as MBC value. The log of reduction RF value were enumerated accordingly.

Results and discussion
Phytochemical screening. The results from the qualitative analysis of the EEHH are provided as Table 1.
The EEHH ethanolic extract was positive for alkaloids, flavonoids, steroids, phenols, terpenes, glycosides, carbohydrates, saponins, and tannins as expected, but the tests were negative for cardiac glycosides 32 .
GC-MS profiling. The volatile phytochemicals present in the ethanolic crude extract were subjected to GC-MS analysis ( Table 2). The extract is dominated with high molecular mass unsaturated fatty acid palmitoleic acid, unusual sugar d-allose, and the unusual chlorinated long-chain hydrocarbon 2-chloroethyl linoleate. Considerable amounts of siloxanes were observed, these are likely environmental contaminants from the growing conditions, but are not naturally produced by the plant.
UV-Vis analysis. The UV-vis absorption spectra of the AgNPs as a function of time during synthesis are provided as Fig. 2A. EEHH-AgNPs produced surface plasmon resonance (SPR) peaks at 430-434 nm, which is FTIR analysis. The FTIR spectrum is consistent with the incorporation of the secondary metabolites and their degradation products into the AgNPs as the same functional groups are present in both the extract and the nanoparticles. As noted by others, many classes of phytochemicals can reduce silver salts to metallic silver, while others can act as capping agents. This will, of course, affect the chemical structure of these organic compounds, and these transitions are noted by the changes in the relative intensity of the vibrational bands. The pronounced peaks of the extract were at 3284, 2920, 1591, 1507, 1246, 1037, and 586 cm −1 , whereas those of the AgNPs were at 3225, 2910, 1620, and 990 cm −1 . The broad vibration at 3284/3225 cm −1 is typical of hydroxyl groups on carbohydrates, flavonoids, and saponins. The peaks at 2920/2910 cm −1 arise from aliphatic  35,36 . These alcohols and phenols are probably involved in the reduction of ionic silver to zero-valent AgNPs and decrease upon reaction (Fig. 2B) 37 . The reduction of the relative intensity of the aliphatic C-H stretching region during AgNPs synthesis suggests conversion of C-H bonds in the phytocompounds to multiple bonds or oxidation 38 . The FTIR analysis is consistent with the formation of AgNPs through reduction of Ag(I) to Ag (0) with concomitant oxidation of the HH phytochemicals.
Phytochemical screening of crude EEHH confirmed the presence of flavonoids and carbohydrates. Flavonoids release free reactive hydrogen during their tautomeric transformations (keto-enol rearrangement), which can assist in the reduction of AgNPs 39 . Furthermore, alcohols facilitate the reduction of ionic silver to zero-valent AgNPs as they oxidize to carbonyls 40 . TEM, SAED, and EDX analysis. TEM analysis supports a largely spherical morphology for the AgNPs (Fig. 3A). Through sizing 30 randomly selected particles observed by TEM we observed the mode was between 12 and 14 nm, with the mean value being 13.3 nm (Fig. 3B). As noted above, these are core-shell structures; this is suggested in the TEM images, but the distinct rings found in the SAED patterns (Fig. 3C) of the AgNPs confirm the polycrystalline property of the as-synthesized AgNPs 41 . Elemental composition analysis by EDX supports the contention that the nanoparticles comprise a metallic core with an organic shell: strong silver (Ag) signals and weaker signals from C and O atoms are consistent with this hypothesis. There is no significant contamination from the nitrogen in the nitrate or from other opportunistic metals. The position of the signal at 13cps/eV suggest that the silver core is crystalline rather than amorphous (Fig. 3D) 41 .
X-ray diffraction. The XRD pattern of the AgNPs shows sharp diffraction peaks corresponding to the (111), (200), (220), and (311) crystal planes (Fig. 4), which are associated with the face-centered cubic lattice of silver. The XRD profile indicates that our materials crystallize in a monoclinic phase, and this spectrum is in line with Dynamic light scattering and Zeta potential analysis of the nanoparticles. The zeta potential value of HH mediated AgNPs in aqueous suspension was established as − 29.2 mV (Fig. 5). This suggests that the surface of the nanoparticles is negatively charged and that the particles are uniformly dispersed in the aqueous medium. The high negative value is evident of the extreme stability of the nanoparticles because of electrostatic repulsive forces between the particles. Zeta potential value of about − 29 mV ensures a high energy barrier for the stabilization of the nanosuspension. DLS suggests a hydrodynamic diameter of 119 nm with a polydispersity index of 0.188. This is an order of magnitude larger than that observed by TEM. Although there are many small particles generated, the TEM images suggest the presence of larger structures. These might move as an assembly in solution together to provide this larger observed size in solution.
Antibacterial susceptibility. The antibacterial activity of the crude extracts, AgNPs, and the AgNPs coadministered with streptomycin, were investigated against both Gram-positive and Gram-negative bacteria. This was quantified using a standard Kirby-Bauer disc diffusion assay with DMSO as the negative control and pure streptomycin as the positive control (Fig. 6).
The extract alone only shows mild activity. The components of EEHH have been a traditional medicine, and like most secondary plant metabolites likely have some role in plant defense against infection. However, the AgNPs show good activity against all bacteria regardless of Gram-status and are similar in potency to streptomycin. However, when streptomycin and the AgNPs are used together, the effect is synergistic: the area of disinfection is considerably greater than a simple addition of their individual activities would indicate. DMSO showed no activity with no area of inhibition (data not shown) confirming the viability of the tested bacteria strains. Silver salts have long been known to be potent antimicrobials, and the development of silver nanoparticles, with their far higher surface area of activated silver metal, has greatly accelerated their investigation 45 . However, the overuse of silver can decrease its efficacy against microorganisms as they develop resistance 46 .
The MIC result (Table 3) shows the bacteriostatic effect of the AgNP at 0.156 µg/mL for Streptococcus pneumonia, Moraxella catarrhalis and Pseudomonas aeruginosa and 0.312 µg/mL upon exposure to Escherichia coli and Bacillus cereus. The bactericidal effect of the AgNP ranges from 0.312 to 5 µg/mL for Streptococcus pneumonia, Moraxella catarrhalis and Pseudomonas aeruginosa and 0.625-5 µg/mL with Escherichia coli and Bacillus cereus. The log of reduction RF value > 4 in all the bacteria challenge irrespective of their Gram status. The nanoparticle thus possessed high efficacy with a percentage greater than 90%.   www.nature.com/scientificreports/ This synergistic effect is expected and has been seen with other silver nanoparticles 47,48 . The mechanism of action of the AgNPs is through adsorption to the bacterial cell membranes, followed by passive penetration into the bacteria. The surface then becomes transiently exposed, and they can cause damage by interacting strongly with essential phosphorous and sulfur-containing compounds such as DNA and proteins, resulting in bacterial cell death 49 . They could also assist in preventing the bacteria from initiating gene expression changes due to the presence of the streptomycin, or streptomycin might better enter the cell by adsorbing to the surface of the nanoparticle and being carried into the cell itself along with the toxic silver particle 50 .

Conclusions
HH corm extract is readily obtained in large amounts from even small amounts of plant material (5% mass recovery from crude material) and can be used to initiate the solution-phase synthesis of low dispersity AgNPs under ambient conditions.
These biosynthesized AgNPs showed no agglomeration and had sizes typically ranging from 6 to 20 nm with a roughly spherical or ovoid shape. These HH-AgNPs show broad spectrum antibacterial activity against common respiratory pathobionts and synergistically enhance the antibacterial activity of streptomycin. We propose that these could be useful agents for transporting largely insoluble antibiotics in the body as potential biologicallyactive drug delivery vehicles, but much more analysis needs to be conducted.