In Vivo Activity of Metal Complexes Containing 1,10-Phenanthroline and 3,6,9-Trioxaundecanedioate Ligands against Pseudomonas aeruginosa Infection in Galleria mellonella Larvae

Abstract

Drug-resistant Pseudomonas aeruginosa is rapidly developing resulting in a serious global threat. Immunocompromised patients are specifically at risk, especially those with cystic fibrosis (CF). Novel metal complexes incorporating 1,10-phenanthroline (phen) ligands have previously demonstrated antibacterial and anti-biofilm effects against resistant P. aeruginosa from CF patients in vitro. Herein, we present the in vivo efficacy of {[Cu(3,6,9-tdda)(phen)₂]·₃H₂O·EtOH}_n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)₂]·₃H₂O·EtOH}_n (Mn-tdda-phen) and [Ag₂(3,6,9-tdda)(phen)₄]·EtOH (Ag-tdda-phen) (tddaH₂ = 3,6,9-trioxaundecanedioic acid). Individual treatments of these metal-tdda-phen complexes and in combination with the established antibiotic gentamicin were evaluated in vivo in larvae of Galleria mellonella infected with clinical isolates and laboratory strains of P. aeruginosa. G. mellonella were able to tolerate all test complexes up to 10 µg/larva. In addition, the immune response was affected by stimulation of immune cells (hemocytes) and genes that encode for immune-related peptides, specifically transferrin and inducible metallo-proteinase inhibitor. The amalgamation of metal-tdda-phen complexes and gentamicin further intensified this response at lower concentrations, clearing a P. aeruginosa infection that were previously resistant to gentamicin alone. Therefore this work highlights the anti-pseudomonal capabilities of metal-tdda-phen complexes alone and combined with gentamicin in an in vivo model.

Keywords: 1,10-phenanthroline; Galleria mellonella; Pseudomonas aeruginosa; antimicrobial resistance; in vivo model; infection; metal complexes.

Figure 1

Kaplan–Meier survival distributions for each P. aeruginosa strain (ATCC 27853, PAO1, CF1, CF2, and CF3) assessed over varying inoculum doses (3 × 10⁰ to 3 × 10⁵ CFU/mL). Significance was assessed through the log-rank (Mantel–Cox) test, and Holm’s correction was applied for multiple comparisons (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Figure 2

Following the inoculation of G. mellonella with P. aeruginosa strains ATCC 27583 (3 × 10⁴ CFU/mL), PAO1 (3 × 10⁰ CFU/mL), CF1 (3 × 10³ CFU/mL), CF2 (3 × 10³ CFU/mL), and CF3 (3 × 10⁰ CFU/mL), the (A) alteration in circulating hemocyte density and (B) bacterial burden, was assessed over a 24 h period. Data are presented as the mean ± SE of the three independent experiments. Statistical analysis was performed by comparing treatments to PBS injected controls at respective time points (A) and to prior time points (B) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Figure 3

G. mellonella representing the different levels of melanization and cuticle discoloration.

Figure 4

Immunomodulation induced by the metal-tdda-phen complexes and gentamicin (2 and 15 µg/larvae) in G. mellonella after 2, 6, and 24 h post-injection. * indicate significant differences in relation to the PBS injected control (p < 0.05).

Figure 5

Relative expression of genes involved in the immune response of G. mellonella when exposed to metal-tdda-phen complexes, gentamicin and phen after 2, 6, and 24 h post-injection. * indicate significant differences to the PBS injected control (p < 0.05).

Figure 6

Immunomodulation induced by the metal-tdda-phen complexes in combination with gentamicin (CN) (2 µg and 2 µg/larvae, and 4 µg and 4 µg/larvae) in G. mellonella after 2, 6, and 24 h post-injection. * indicate significant differences in relation to the PBS injected control (p < 0.05).

Figure 7

Relative expression of genes involved in the immune response of G. mellonella when exposed to metal-tdda-phen complexes in combination with gentamicin (CN) after 2, 6, and 24 h post-injection. * indicate significant differences to the PBS control (p < 0.05).

Figure 8

Survival (%) of G. mellonella inoculated with P. aeruginosa strains, ATCC 27853 (A–D), PAO1 (E–H), CF1 (I–L), CF2 (M–P), CF3 (Q–T), and treated with 2–10 µg/larvae of Mn-tdda-phen (A,E,I,M,Q), Cu-tdda-phen (B,F,J,N,R), Ag-tdda-phen (C,G,K,O,S), and gentamicin (D,H,L,T) over 96 h.

Figure 9

Effect of treatment with Mn-tdda-phen, Cu-tdda-phen, and Ag-tdda-phen alone (1 µg/larvae) and in combination with gentamicin (CN) (1 µg/larvae) infected with ATCC 27853, PAO1, CF1, CF2, and CF3 on survival (left) and larval bacterial burden (right).