Silver nanoparticles were gifted by the Global Infection Unit, National Research Center for Protozoan Diseases, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Japan. Kynurenine standards, DMSO, Ehrlich reagent were products of Sigma Chemicals Co. (St Louis, Missouri, USA). Agar and broth were products of HiMedia (Mumbai, India). All other reagents were of analytical grade and used as supplied.

A total of five bacterial species (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumoniae) were used for the study. The isolates were obtained from the Department of Microbiology, Landmark University, Omu-Aran, Kwara State, Nigeria. Prior to use, the isolates first streaked on nutrient agar plates, to ascertain their purity. The pure isolates were then cultured in sterile nutrient broth and incubated at 35 °C for 24 h. All pure isolates were stored in a refrigerator at 4 °C until when needed. They were purified then streaked on nutrient agar plates and incubated for 24 h for growth at 37 °C, after which the purity of the isolates was determined.

To evaluate the antibacterial potential of the AgNPs, 30 mL of sterile nutrient agar was left to cool to 45 °C, after which 1 mL of overnight culture of a respective nutrient broth cultured bacterium was added and swirled before dispensing in 20 mL quantity in a petri dish and allowed to solidify. The stock concentration (2000 µg/mL) of the AgNPs was prepared in universal bottles. Using a cork borer, three wells were bored in each plate before adding 0.2 mL of the respective AgNPs (1000 µg/mL) in a well and allowed to diffuse. Following diffusion of the AgNPs, the plates were incubated at 37 °C for 24 h. The zones of inhibition were observed and measured using a ruler and then recorded in millimeters appropriately. In each case, a control well that contained only the diluent used for preparation of the respective AgNPs was added.

To determine the minimum inhibitory concentration (MIC) of the AgNPs, different concentrations of the AgNPs (0-1000 µg/mL) were prepared. Using the agar well diffusion method and following incubation, the lowest concentration of the AgNPs solution at which the growth of the respective isolates is inhibited (identified by zone of inhibition) was recorded as the MIC.

To accomplish the biochemical experiment, S. aureus and E. coli were selected as representative.

AgNPs only:

The S. aureus was treated with AgNPs at concentrations of 700 µg/mL (MIC), 1400 µg/mL (2x MIC), 2100 µg/mL (3x MIC).

AgNPs and co-treatment with ascorbic acid:

The treatments were as described above except for the simultaneous treatment with ascorbic acid (AA) at 1000 µg/mL. From our preliminary studies, ascorbic acid at 1000 µg/mL did not limit the growth of the bacteria isolates.

A growth rate curve was prepared for both E. coli and S. aureus cells, representing Gram-negative and Gram-positive bacteria. Briefly, flasks containing 150 mL of nutrient broth were prepared according to the manufacturer's instruction and labeled according to the treatments before inoculating with 500 µL of the respective bacteria culture. Following inoculation, 10 mL of the test compounds (quercetin only and/or with 1000 µg/mL of ascorbic acid) were added to the labeled flasks before incubating in a rotary incubator at 37 °C at 120 rpm. Immediately after inoculation, and every one hour interval for the first 8 h and after 24 h of incubation, 5 mL of broth was withdrawn from each flask for the measurement of optical density at a wavelength of 750 nm, using a spectrophotometer (Jenway, Staffordshire, United Kingdom). Growth rate was calculated using the formula;

where C0 and C1 represent initial and final absorbance, respectively

t0 and t1 represent initial and final time, respectively.

Harvesting of cells for biochemical assays was as reported previously (Dong and Cmarik, 2002[11]). Briefly, the bacteria isolate namely E. coli and S. aureus were treated in nutrient broth with quercetin only at concentrations; 0x MIC (control), MIC, 2x MIC and 3x MIC values, or in combination with ascorbic acid (1000 µg/mL). After 24-h incubation at 37 °C, the cells were harvested by centrifugation at 5,000 g for 10 min (model C5, LW scientific, USA). Aliquot of the supernatant was taken for kynurenine assay. The pelleted cells were washed with normal saline three times, re-suspended and thereafter homogenized and stored frozen until required for analysis.

Biochemical determinations in cell lysates were carried out on a UV/Vis spectrophotometer (Jenway, Staffordshire, United Kingdom) where applicable. Determination of kynurenine in bacteria culture suspension was performed according to established protocol (Adeyemi et al., 2017[5]). Nitric oxide concentration was measured as the nitrite level according to the method described elsewhere (Adeyemi and Sulaiman, 2014[8]). Total antioxidant capacity (TAC) of cell lysates was determined as described previously (Adeyemi et al., 2018[3]). Total protein concentration was estimated according to a method described previously by Gornall et al. (1949[13]) with slight modification. Potassium iodide was added to the biuret reagent to prevent precipitation of Cu²⁺ ions. The total thiol level was determined by the method described elsewhere (Adeyemi and Orekoya, 2014[7]). Lipid peroxidation was estimated as malondialdehyde (MDA) using the method described previously (Adeyemi et al., 2017[4]). DNA fragmentation was determined using the diphenylamine (DPA) assay as described elsewhere (Adeyemi et al., 2017[5]). The superoxide dismutase (SOD) activity was also determined using previously reported protocol (Misra and Fridovich, 1978[16]).

Results were analyzed by using one-way analysis of variance (ANOVA) on a GraphPad Prism 6 (GraphPad Software Inc., San Diego, California, USA). Data are expressed as mean of three replicates ± standard error of mean (SEM). Comparisons among group mean values were performed by Tukey's post-hoc test and p< 0.05 was considered to indicate a significant difference.

AgNPs showed antibacterial activity against all the strains (Table 1(Tab. 1)). MIC analysis also showed considerable antibacterial effect of AgNPs (Table 2(Tab. 2)). The growth rate curve of E. coli and S. aureus showed evidence of bactericidal effect of AgNPs (Figures 1(Fig. 1) and 2(Fig. 2)). Treatment with DMSO and ascorbic acid showed no antimicrobial effect. The ascorbic acid was included to determine whether oxidative stress by AgNPs was culpable in the antibacterial action.

To determine if oxidative stress contributes to the antibacterial action of AgNPs, we included an antioxidant (ascorbic acid - AA) in the assay medium. Results showed that AgNPs and its co-exposure with AA caused an increase in the protein content of the microorganisms after 24-hour incubation, p<0.05 (Figure 2A and B(Fig. 2)). In order to evaluate whether, the activation of kynurenine might be involved in the action mechanism of AgNPs, we determined the level of kynurenine in culture supernatant. Results showed inconsistent effect of AgNPs on the kynurenine pathway in both microorganisms, p<0.05. (Figure 2C and D(Fig. 2)).

Furthermore, we assayed for MDA level in order to evaluate if AgNPs caused lipid peroxidation. Our results showed evidence of lipid peroxidation in both microorganisms following exposure to AgNPs (Figure 3A and B(Fig. 3)). In addition, we determined DNA damage in the bacterial isolates and found that the combination of AgNPs and ascorbic acid caused higher incidence of cellular DNA damage than the effect of AgNPs alone. This was more evident in E. coli, p<0.05 (Figure 3C and D(Fig. 3)). Moreover, the AgNPs exposure led to increased nitric oxide production in the bacteria isolates (Figure 3E and F(Fig. 3)). Taken together, the findings suggest that AgNPs might have caused oxidative stress in the bacterial isolates.

However, and in contrast to its capacity to induce oxidative stress, the AgNPs exposure led to a higher total thiol level in E. coli and S. aureus when compared with the control, p<0.05 (Figure 4A and B(Fig. 4)). In like manner, the SOD activity was elevated in E. coli and S. aureus (Figure 4C and D(Fig. 4)). This may likely be in response to AgNPs-induced oxidative stress noting that SOD is an inducible enzyme. Also, the TAC in the bacterial isolates was increased (p<0.05) after exposure to AgNPs and/or ascorbic acid (Figure 4E and F(Fig. 4)), suggesting probable antioxidant properties.