Harnessing the Dual Antimicrobial Mechanism of Action with Fe(8-Hydroxyquinoline)3 to Develop a Topical Ointment for Mupirocin-Resistant MRSA Infections

Abstract

8-Hydroxyquinoline (8-hq) exhibits potent antimicrobial activity against Staphylococcus aureus (SA) bacteria with MIC = 16.0-32.0 µM owing to its ability to chelate metal ions such as Mn²⁺, Zn^2+, and Cu²⁺ to disrupt metal homeostasis in bacterial cells. We demonstrate that Fe(8-hq)₃, the 1:3 complex formed between Fe(III) and 8-hq, can readily transport Fe(III) across the bacterial cell membrane and deliver iron into the bacterial cell, thus, harnessing a dual antimicrobial mechanism of action that combines the bactericidal activity of iron with the metal chelating effect of 8-hq to kill bacteria. As a result, the antimicrobial potency of Fe(8-hq)₃ is significantly enhanced in comparison with 8-hq. Resistance development by SA toward Fe(8-hq)₃ is considerably delayed as compared with ciprofloxacin and 8-hq. Fe(8-hq)₃ can also overcome the 8-hq and mupirocin resistance developed in the SA mutant and MRSA mutant bacteria, respectively. Fe(8-hq)₃ can stimulate M1-like macrophage polarization of RAW 264.7 cells to kill the SA internalized in such macrophages. Fe(8-hq)₃ exhibits a synergistic effect with both ciprofloxacin and imipenem, showing potential for combination therapies with topical and systemic antibiotics for more serious MRSA infections. The in vivo antimicrobial efficacy of a 2% Fe(8-hq)₃ topical ointment is confirmed by the use of a murine model with skin wound infection by bioluminescent SA with a reduction of the bacterial burden by 99 ± 0.5%, indicating that this non-antibiotic iron complex has therapeutic potential for skin and soft tissue infections (SSTIs).

Keywords: Fe-based antimicrobials; Fenton reaction; bacterial iron metabolism; metal chelation; methicillin-resistant Staphylococcus aureus.

Figure 1

PXRD pattern of the isolated product (a) and X-ray structure of Fe(8-hq)₃ with the stick-and-ball presentation (b) and with the space-filling presentation (c).

Figure 2

Inhibitory effects of Fe(8-hq)₃ in comparison with molar equivalents of 8-hq against MSSA (a), against MRSA^α (b), against MRSA^β (c) and against VISA (d).

Figure 3

Time–kill kinetics of Fe(8-hq)₃ against MRSA^α after 24-h incubation with different concentrations of Fe(8-hq)₃ (a) and cellular uptake of Fe(8-hq)₃ in MRSA^α as represented by the Fe content of the cell lysate (b) (data presented as mean ± s.d, n = 3 replicates; * p < 0.05, ns = not significant).

Figure 4

Relative yields of intracellular ROS generation in MRSA^α bacterial cells treated with Fe(8-hq)₃ in comparison with molar equivalents of 8-hq (a), the intracellular ROS generation in MRSA^α bacterial cells inhibited by TU (b) and by bipy (c) (mean ± s.d, n = 3 replicates; ** p < 0.01, **** p < 0.0001 and ns = not significant).

Figure 5

SEM images of MRSA^α treated with 8-hq and Fe(8-hq)₃ both at the concentration of 32 μM for 2 h (a) and the change in PI fluorescence in MRSA^α treated with 8-hq and Fe(8-hq)₃ (mean ± s.d, n = 3) (b) and microscopic images of PI in MRSA^α treated with 8-hq and Fe(8-hq)₃ (both at 32 μM) with differential interference contrast (DIC) microscopic images (left), fluorescence images (middle) and merged images of MRSA^α (right) (c).

Figure 6

Drug resistance development of Fe(8-hq)₃ vs. ciprofloxacin and 8-hq in MSSA and the overcome of drug resistance the in mutant MSSA^8-hqR (a strain resistant to 8-hq) bacteria by Fe(8-hq)₃ without developing Fe(8-hq)₃ resistance after 50 days of treatment with Fe(8-hq)₃ (a), and resistance development profile of mupirocin and fusidate in MRSA^α and the overcome of high-level mupirocin resistance mutant bacteria (MRSA^mupR) by Fe(8-hq)₃ (b).

Figure 7

Results of growth inhibition measurements (a–d). The average zones of inhibition of PEG-based ointments containing the 2% mupirocin, 2% fusidate and 2% Fe(8-hq)₃ towards MRSA strain with wildtype MRSA^α (a), high-level mupirocin resistant MRSA^α; MRSA ^(mupR) (b), high-level fusidate resistant MRSA^α; MRSA^(fusR) (c) and representative images of antimicrobial zone of growth inhibition of different bacterial strains (d). (mean ± s.d, n = 3 replicates; ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 8

Treatment outcomes of in vivo wound infections by S. aureus (Xen36) in a murine model with the excisional wound. Overview of the in vivo experiments (a), results of in vivo bioluminescent imaging studies of mice with ROI measurements (b), and representative images of CFU enumeration result against S. aureus (c) (mean ± s.d, n = 3 mice per group; ** p < 0.01).