In Vivo Antimicrobial Activity of Nisin Z Against S. aureus and Polyurea Pharmadendrimer PUREG4OEI48 Against P. aeruginosa from Diabetic Foot Infections

Abstract

Background/objectives: Diabetic foot infections (DFIs) are commonly associated with frequent hospitalizations, limb amputations, and premature death due to the profile of the bacteria infecting foot ulcers. DFIs are generally colonized by a polymicrobial net of bacteria that grows in biofilms, developing an increased antimicrobial resistance to multiple antibiotics. DFI treatment is a hurdle, and the need to develop new therapies that do not promote resistance is urgent. Therefore, the antibacterial efficacy of Nisin Z (antimicrobial peptide), a core-shell polycationic polyurea pharmadendrimer (PURE_G4OEI₄₈) (antimicrobial polymer), and amlodipine (antihypertensive drug) was evaluated against S. aureus and P. aeruginosa isolated from a DFI and previously characterized.

Methods: The antibacterial activity was analyzed in vitro by determining the minimal inhibitory concentration (MIC) and in vivo in a Galleria mellonella model by assessing the larvae survival and health index.

Results: The results indicate that Nisin Z exhibited antibacterial activity against S. aureus in vivo, allowing larvae full survival, and no antibacterial activity against P. aeruginosa. Nisin Z may have reduced the antibacterial effectiveness of both PURE_G4OEI₄₈ and amlodipine. PURE_G4OEI₄₈ significantly increased the survival of the larvae infected with P. aeruginosa, while amlodipine showed no activity against both bacteria in vivo.

Conclusions: These findings suggest that both Nisin Z and PURE_G4OEI₄₈ could potentially be used individually as adjunct treatments for mild DFIs. However, further studies are needed to confirm these findings and assess the potential toxicity and efficacy of PURE_G4OEI₄₈ in more complex models.

Keywords: Amlodipine; Galleria mellonella; Nisin Z; Pseudomonas aeruginosa; Staphylococcus aureus; core–shell polycationic polyurea pharmadendrimer; diabetic foot infection.

Figure 1

Health index of larvae infected with S. aureus Z25.2 (a) and with P. aeruginosa Z25.1 (b) treated with Nisin Z (0.2 mg/mL), PURE_G4OEI₄₈ (2.07 mg/mL) with and without supplementation with Nisin Z, and amlodipine (0.16 mg/mL) with and without supplementation with Nisin Z at 24, 48, 72, and 96 h post-infection. Positive control: infected larvae + NaCl 0.9% (grey bar); negative control: uninfected larvae + NaCl 0.9%/each treatment. Data are shown as the mean ± standard error (larvae activity, cocoon formation, melanization, and survival) of three independent determinations for 10 animals per treatment. ns, not significant; ****, p < 0.0001 from the control at 96 h (one-way ANOVA, Tukey’s multiple comparison test). PURE, PURE_G4OEI₄₈; AML, amlodipine.

Figure 2

Degrees of larvae melanization: (a) no melanization; (b) dark spots on cream larvae; (c) dark spots on brown larvae; (d) full melanization. Larvae melanization at 24 h: (e) negative control (saline plus saline) showing no melanization (cream larvae); (f) larvae challenged with S. aureus showing partial melanization (brown dead larvae, inside the circle) and no melanization (cream live larvae); (g) larvae challenged with P. aeruginosa showing full melanization (dark dead larvae). Cocoon development at 96 h: (h) negative control (saline plus saline) with a full development of the cocoon; (i) caterpillars following P. aeruginosa infection and PURE_G4OEI₄₈ treatment with a partial and altered cocoon; (j) negative control (saline plus PURE_G4OEI₄₈) with a partial and altered cocoon. As shown, PURE_G4OEI₄₈ delayed and affected the normal development of G. mellonella cocoons (medium larval length: 2 cm).

Figure 3

Kaplan–Meier survival curves for G. mellonella larvae after infection with S. aureus (1 × 10⁶ CFU) and P. aeruginosa (100 CFU), either with (full line) or without (dashed line) administration of the compounds under evaluation at 1 h post-infection. (a,c) Nisin Z (0.2 mg/mL), PURE_G4OEI₄₈ (2.07 mg/mL), and amlodipine (0.16 mg/mL); (b,d) PURE_G4OEI₄₈ supplemented with Nisin Z and amlodipine supplemented with Nisin Z. Positive control: infected larvae + NaCl 0.9% (dashed line); negative control: uninfected larvae + NaCl 0.9%/treatment. In subfigures (c,d), the dotted line is present but may not be visible due to overlap with other lines in the figure. The results represent the mean of three independent determinations for 10 animals per treatment. The bars represent the standard error. The Mantel–Cox test significance between survival curves of treated larvae and nontreated larvae: ns, not significant; *, p = 0.01; **, p = 0.005; ***, p = 0.0001; ****, p < 0.0001. PURE, PURE_G4OEI₄₈; AML, amlodipine.

Figure 4

Larvae survival rates (%) at 96 h after challenging them with S. aureus Z25.2 (a) and with P. aeruginosa Z25.1 (b) and treating them with PURE_G4OEI₄₈ (2.07 mg/mL) with and without Nisin Z, amlodipine (0.16 mg/mL) with and without Nisin Z, and Nisin Z (0.2 mg/mL). Positive control: infected larvae + NaCl 0.9% (grey bar); negative control: uninfected larvae + NaCl 0.9%/each treatment. The P. aeruginosa control corresponded to zero larvae alive at 96 h. Data are shown as the mean ± standard error of three independent determinations for 10 animals per treatment. ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA, Tukey’s multiple comparison test). NZ, Nisin Z; PURE, PURE_G4OEI₄₈; AML, amlodipine.

Figure 5

Chemical structures of Nisin Z, amlodipine, and the core–shell polycationic polyurea pharmadendrimer PURE_G4OEI₄₈ (cartoon showing only the surface structure of one branch).