P. aeruginosa PAO1 and clinical isolates of P. aeruginosa were used from the previous study [12], with additional clinical isolates obtained from the State University of New York (SUNY) Downstate Medical Center (Brooklyn, NY). To prepare cultures, bacterial strains were grown aerobically on Luria-Bertani (LB; Becton, Dickinson and Company, Sparks, MD) agar plates or in LB broth at 37˚C. All chemical reagents, antibiotics, and bacteriological media were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Hampton, NH).

Four Ag/ZnO-HAp nanocomposites from the previous study [11] were used, which were prepared with the following Ag:ZnO mole ratios: nanocomposite A (0.005:0.005), nanocomposite B (0.01:0.005), nanocomposite C (0.005:0.01), and nanocomposite D (0.01:0.01). Each Ag/ZnO-HAp nanocomposite was dissolved in sterile distilled water to form a colloidal dispersion as previously described [13].

Agar diffusion tests of Ag/ZnO nanocomposites were performed against P. aeruginosa as described previously [14]. Briefly, a bacterial lawn (~5 × 10⁹ cells per milliliter) was prepared on the agar surface, and each Ag/ZnO-HAp nanocomposite (0.1 mg; 5 μL) was applied to the lawn. Each nanocomposite had a specific Ag-to-ZnO mole ratio of A (nanocomposite-A, 0.005:0.005), B (nanocomposite-B, 0.010:0.005), C (nanocomposite-C, 0.005:0.010), D (nanocomposite-D, 0.010:0.010), and Cot (control; unmodified HAp). The agar plates were then incubated overnight (14 to 18 hours), and the results were assessed by measuring the diameter (mm) of the inhibition zones.

A bactericidal assay of P. aeruginosa PAO1 was performed as previously described [14] with a minor modification. Briefly, Ag/ZnO nanocomposites were diluted (3, 2, 1, 0.5, 0.25, and 0.125 mg/mL) in LB broth. Fresh overnight cell cultures were inoculated into the nanocomposite dilutions (3 × 10⁹ cells/mL). The inoculum was then incubated at 37˚C for 18 hours without shaking. After incubation, an aliquot from each dilution was plated on LB agar plates and incubated for 24 hours to count colony-forming units (CFU) per mL. Standard deviations were calculated from three independent experiments. Bactericidal efficacy was quantified as log reductions (log₁₀[N_o] - log₁₀[N_t]), where N_o is the initial inoculum, and N_t is the cell count at different nanocomposite concentrations after 18 hours.

A time-killing assay of P. aeruginosa PAO1 was performed as previously described [15] with a minor modification. Briefly, bacterial cells were diluted in LB broth (5 × 10⁶ cells/mL) with 0.5 mg/mL of each nanocomposite and incubated at 37˚C without shaking. At 30-minute intervals, a 100 μL aliquot was spread on an LB agar plate, and CFU per mL were counted. Experiments were performed in triplicate to determine standard deviations. Survival data at each time point were analyzed by time-kill kinetics to determine the specific death rate (k, min⁻¹) and decimal reduction time (D-value) as previously described [16]. The specific death rate (k) was calculated as k = ln (N_o/N_t)/t, where N_o is the initial cell count per mL, N_t is the cell count at time t, and t is the elapsed time in minutes. The D-value was calculated as D = ln (10)/k.

MIC levels of clinical isolates of P. aeruginosa were determined by the broth dilution method as described previously [14].

MBC levels of Ag/ZnO-HAp nanocomposites were determined as described previously [14]. Briefly, serial two-fold dilutions of each nanocomposite were prepared in LB broth (4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg/mL). Fresh overnight cultures of clinical isolates of P. aeruginosa were diluted in sterile saline, and a portion of the cells (5 μL; 5 × 10⁹ cells/mL) was inoculated into each serial two-fold dilution. The cells were incubated overnight (16 to 18 hours) at 37˚C and then spread on plain LB agar plates. The agar plates were incubated at 37˚C for 24 hours to determine the MBC. MBC values were defined as the lowest nanocomposite concentration required to completely inhibit bacterial growth.

An agar diffusion test was performed to assess the antibacterial activity of four Ag/ZnO-HAp nanocomposites (A, B, C, and D) and their parental HAp (control: Cot) against P. aeruginosa PAO1. As shown in Figure 1, the inhibition zone (diameter) of nanocomposite D was the largest (12 mm), nanocomposite A was the smallest (8 mm), and nanocomposites B and C were similar (11 mm). In contrast, the parental HAp showed no inhibition zone. These results demonstrate that all four Ag/ZnO-HAp nanocomposites exhibit antibacterial activity against P. aeruginosa PAO1, whereas the parental HAp does not. This finding is consistent with the previous report describing antibacterial effects in E. coli and Aggregatibacter actinomycetemcomitans [11]. Although the nanocomposites show clear inhibition zones, the variation in zone size may not be sufficient to differentiate their antibacterial efficacy.

Figure 1.Agar diffusion test of Ag/ZnO-HAp nanocomposites against P. aeruginosa PAO1. A bacterial lawn was prepared on the agar surface, and each Ag/ZnO-HAp nanocomposite was applied to the lawn. Each nanocomposite had a specific mole ratio (Ag:ZnO): A (nanocomposite A, 0.005:0.005), B (nanocomposite B, 0.010:0.005), C (nanocomposite C, 0.005:0.010), D (nanocomposite D, 0.010:0.010), and Cot (control), as described in the Materials and Methods.

The agar (or disk) diffusion test is the most common method used in clinical microbiology for initial antimicrobial susceptibility screening because of its cost-effectiveness, simplicity, and high versatility [17]. Despite these advantages, the test method has notable limitations in precisely quantifying antimicrobial susceptibility or resistance. For example, high-molecular-weight or poorly soluble antimicrobial agents are unsuitable because their diffusion in agar matrices may be impeded, causing inaccurate inhibition zones [17][18]. The Ag/ZnO-HAp nanocomposites used in this study have relatively high molecular weights (>1000 g/mol) and poor solubility in pure water. Consequently, the antibacterial activity of the nanocomposites, as measured by the size of the inhibition zones, may be underestimated.

Bactericidal activity was evaluated by measuring bacterial killing patterns at different concentrations of Ag/ZnO-HAp nanocomposites. Results showed that nanocomposite D killed cells 100% at 0.5 mg/mL over 18 hours, whereas nanocomposites A, B, and C killed cells 100% at 1 or 2 mg/mL over the same period (Figure 2). However, at 0.125 mg/mL, the initial inoculum (3 × 10⁹ cells/mL) decreased to ~2 × 10⁴ cells/mL for nanocomposites B and D, while to ~1.5 × 10⁸ cells/mL for nanocomposites A and C. In parallel, bacterial colonies in the nanocomposite-untreated growth and in HAp-only controls were uncountable. This corresponds to log reductions of 1.34, 5.07, 1.67, and 5.69 for nanocomposites A, B, C, and D, respectively. Notably, the Ag content in nanocomposites B and D (0.01 mole) is twice that in nanocomposites A and C (0.005 mole) but does not correlate with the ZnO content. Thus, these results suggest that the enhanced bactericidal activity of nanocomposites B and D may be associated with a 2-fold increase in Ag. However, although Ag content is the primary factor in bactericidal activity rather than ZnO content, the bactericidal assay conditions, such as bacterial culture conditions, media, and cell concentrations, may alter the relative roles of Ag and ZnO in bactericidal activity.

Figure 2. Bactericidal patterns of Ag/ZnO-HAp nanocomposites at different concentrations. Bactericidal patterns were determined by bacterial killing at varying concentrations (0.125, 0.25, 0.5, 1, 2, 3 mg/mL) of Ag/ZnO-HAp nanocomposites against P. aeruginosa PAO1, as described in the Materials and Methods. Standard deviations represent the results of three independent experiments.

A time-killing assay of P. aeruginosa PAO1 was performed in the presence of 0.5 mg/mL of each nanocomposite, including the parental HAp (control). Results showed that the initial inoculum (5 × 10⁶ cells/mL) decreased to ~3 × 10⁴ cells/mL over the first 30 minutes for all nanocomposites as well as the control (Figure 3(A)). Over the next 120 minutes, cell concentrations for nanocomposites A, B, C, and D decreased to an average of 253, 4, 57, and 0 cells/mL, respectively, while cell concentrations in the control increased (Figure 3(A)). These results indicate that nanocomposites B and D have significantly higher killing rates than A and C, and that C and D have slightly higher killing rates than A and B. The differences in killing rate correspond to the amounts of Ag and ZnO in the nanocomposites. A 2-fold increase in Ag (from 0.005 to 0.01 mole) leads to significantly higher killing rates (A to B and C to D), and a 2-fold increase in ZnO (from 0.005 to 0.01 mole) results in slightly higher killing rates (A to C and B to D) (Figure 3(A)). Therefore, both Ag and ZnO increase bactericidal activity. However, Ag likely plays a primary role, with ZnO as a secondary contributor.

To assess bactericidal efficacy, we determined the time-kill kinetics of each nanocomposite by calculating the specific death rate (k) and decimal reduction time (D-value), as described in Materials and Methods, over a 120-minute period starting at 30 minutes (cell concentration was 5.5 × 10⁴ per mL), with measurements taken every 30 minutes. As shown in Figure 3(B), nanocomposite D had the most potent bactericidal activity, with the highest specific death rate (0.082 min⁻¹) and the lowest D-value (28.08 minutes), implying that approximately 8.2% of surviving cells are killed each minute and that it takes 28.08 minutes to kill 90% of the cells. Conversely, nanocomposite A was the least potent, with the lowest specific death rate (0.034 min⁻¹) and the highest D-value (67.72 min). However, the control HAp showed a negative specific death rate and negative D-values, indicating that it promoted cell proliferation rather than inducing cell death (Figure 3(A) and Figure 3(B)). Overall, the results suggest that bactericidal efficacy follows the order D > B > C > A, as also supported by the log reductions in bacterial killing at 0.125 mg/mL for each nanocomposite, as shown in Figure 2.

Figure 3. Time-killing and time-kill kinetics of P. aeruginosa PAO1. (A) Time-killing in the presence of Ag/ZnO-HAp nanocomposites (0.5 mg/mL), performed as described in Materials and Methods. Nanocomposite A: square; Nanocomposite B: diamond; Nanocomposite C: triangle; Nanocomposite D: closed circle; and Cot (control): open circle. Three independent experiments were used for standard deviation. (B) Time-kill kinetics were calculated using the specific death rate (k) and D-value for each nanocomposite, as described in the Materials and Methods.

The time-kill kinetics also demonstrated that doubling the Ag concentration from 0.005 to 0.01 mole (A to B) led to more than a two-fold increase in the specific death rate (0.07/0.034 = 2.1) when combined with 0.005 mole ZnO. In contrast, doubling Ag (C to D) resulted in less than a two-fold increase (0.082/0.054 = 1.5) when combined with 0.01 mole ZnO. The same D-value trends were also seen from A to B and C to D (Figure 3(B)). Likewise, doubling the ZnO concentration from 0.005 to 0.01 mole produced less than a two-fold increase in specific death rate, both A to C (0.054/0.034 = 1.6) and B to D (0.082/0.07 = 1.2) with the same as D-values when combined with either 0.005 or 0.01 mole Ag. These results suggest that Ag’s bactericidal activity may be higher at lower ZnO concentrations, but that ZnO’s bactericidal activity may not be significantly affected by Ag. Taken together, high ZnO levels may hinder Ag’s bactericidal activity, and ZnO’s effect may be insignificant on overall bactericidal activity. Thus, optimizing the Ag:ZnO ratio in HAp nanocomposites may be crucial for maximizing bactericidal activity.

HAp-based nanocomposites have been proposed for various biomedical applications, including bone tissue engineering, orthopedic implant coatings, and dental applications. These HAp-based nanocomposites are expected to exhibit high antimicrobial activity while maintaining biocompatibility and material stability [4][8][19]. To evaluate the Ag/ZnO-HAp nanocomposites in biomedical applications, eight multidrug-resistant clinical isolates of P. aeruginosa were used to determine MBC. Results showed that MBC levels for the four Ag/ZnO-HAp nanocomposites varied widely, ranging from < 0.06 to >4 mg/mL, regardless of susceptibility or resistance to traditional antibiotics (Table 1). Although clinical isolates showed different MBC levels, nanocomposite D consistently had the lowest MBCs. Nanocomposite B had lower MBCs than the other two nanocomposites; nanocomposite C likely had lower MBCs than nanocomposite A, but, exceptionally, in isolate NY215, nanocomposite A showed lower MBCs than nanocomposites B and C (Table 1). These results indicate that the bactericidal activity of Ag/ZnO-HAp nanocomposites may be strain-specific, reflecting heterogeneity in cellular targets among isolates. Clinical isolates of P. aeruginosa have marked heterogeneity in both core genome sequences and accessory gene profiles [20]. Therefore, biomedical applications of Ag/ZnO-HAp nanocomposites may need to be customized to each clinical isolate.

Table 1. Bactericidal effect of Ag/ZnO-HAp nanocomposites against clinical isolates of P. aeruginosa.

MBC: minimum bactericidal concentration; MIC: minimum inhibitory concentration; *CAR: Carbenicillin (R: MIC: > 128 μg/mL, S: MIC: <64 μg/mL); CHL: Chloramphenicol (R: MIC: > 128 μg/mL, S: MIC: <64 μg/mL); CIP Ciprofloxacin (R: MIC: > 2 μg/mL, S: MIC: <1 μg/mL); KAN: Kanamycin (R: MIC: > 50 μg/mL, S: MIC: <25 μg/mL); TET: Tetracycline (R: MIC: > 8 μg/mL, S: MIC: <4 μg/mL).