A combination therapy strategy for treating antibiotic resistant biofilm infection using a guanidinium derivative and nanoparticulate Ag(0) derived hybrid gel conjugate

Abstract

Bacteria organized in biofilms show significant tolerance to conventional antibiotics compared to their planktonic counterparts and form the basis for chronic infections. Biofilms are composites of different types of extracellular polymeric substances that help in resisting several host-defense measures, including phagocytosis. These are increasingly being recognized as a passive virulence factor that enables many infectious diseases to proliferate and an essential contributing facet to anti-microbial resistance. Thus, inhibition and dispersion of biofilms are linked to addressing the issues associated with therapeutic challenges imposed by biofilms. This report is to address this complex issue using a self-assembled guanidinium-Ag(0) nanoparticle (AD-L@Ag(0)) hybrid gel composite for executing a combination therapy strategy for six difficult to treat biofilm-forming and multidrug-resistant bacteria. Improved efficacy was achieved primarily through effective biofilm inhibition and dispersion by the cationic guanidinium ion derivative, while Ag(0) contributes to the subsequent bactericidal activity on planktonic bacteria. Minimum Inhibitory Concentration (MIC) of the AD-L@Ag(0) formulation was tested against Acinetobacter baumannii (25 μg mL^-1), Pseudomonas aeruginosa (0.78 μg mL^-1), Staphylococcus aureus (0.19 μg mL^-1), Klebsiella pneumoniae (0.78 μg mL^-1), Escherichia coli (clinical isolate (6.25 μg mL^-1)), Klebsiella pneumoniae (clinical isolate (50 μg mL^-1)), Shigella flexneri (clinical isolate (0.39 μg mL^-1)) and Streptococcus pneumoniae (6.25 μg mL^-1). Minimum bactericidal concentration, and MBIC₅₀ and MBIC₉₀ (Minimum Biofilm Inhibitory Concentration at 50% and 90% reduction, respectively) were evaluated for these pathogens. All these results confirmed the efficacy of the formulation AD-L@Ag(0). Minimum Biofilm Eradication Concentration (MBEC) for the respective pathogens was examined by following the exopolysaccharide quantification method to establish its potency in inhibition of biofilm formation, as well as eradication of mature biofilms. These effects were attributed to the bactericidal effect of AD-L@Ag(0) on biofilm mass-associated bacteria. The observed efficacy of this non-cytotoxic therapeutic combination (AD-L@Ag(0)) was found to be better than that reported in the existing literature for treating extremely drug-resistant bacterial strains, as well as for reducing the bacterial infection load at a surgical site in a small animal BALB/c model. Thus, AD-L@Ag(0) could be a promising candidate for anti-microbial coatings on surgical instruments, wound dressing, tissue engineering, and medical implants.

Scheme 1. The methodology adopted for the synthesis of AD-L.

Fig. 1. The schematic representation of the plan of analysis with AD-L@Ag(0).

Fig. 2. Physico-chemical characterization of AD-L@Ag(0) using UV-Vis absorbance spectra and emission spectra of the corresponding systems in methanol–water (1 : 1, v/v): (a′) and (a′′) (blue traces) show the absorbance and emission spectra of the pure ligand AD-L, and (b′) and (b′′) (red trace) show the absorbance and emission spectra of the AD-L@Ag(0) material.

Fig. 3. XPS analysis: XPS survey spectra of (a) AD-L and (b) AD-L@Ag(0), respectively. (c, e and g) High resolution deconvolution spectra for C, N and O of AD-L, respectively; (d, f and h) high resolution deconvolution spectra for C, N and O of AD-L@Ag(0); and (i) the high resolution Ag 3d region for AD-L@Ag(0).

Fig. 4. Oscillatory rheology measurements of the ligand (AD-L) and metallogel (AD-L@Ag(0)) were obtained in methanol–water (1 : 1, v/v). Strain sweeps (at a frequency of 1 Hz) of the storage modulus G′ (■) and loss modulus G′′ (●) for (a) AD-L and (c) metallogel AD-L@Ag(0). Frequency sweeps (at 0.1% strain amplitude) for (b) AD-L, and (d) metallogel AD-L@Ag(0).

Fig. 5. (a and b) TEM images of the metallogel from AD-L@Ag(0) showing the gel fibres with in situ formed silver nanoparticles. (c) Representative HR-TEM image of nanoparticles showing lattice fringes. (d) Particle size distribution of Ag(0) nanoparticles based on the TEM image analysis. (e and f) SAED pattern of Ag nanoparticles. (g) FE-SEM image of the AD-L@Ag(0) gel. (h and i) Photographs of gels developed from AD-L and AD-L@Ag(0).

Fig. 6. Graphical representation of a broth microdilution method to determine the MIC and MBC of the AD-L@Ag(0) nano-formulation and ligand (guanidine alone) against (a) Acinetobacter baumannii, (b) Pseudomonas aeruginosa, (c) Staphylococcus aureus, (d) Klebsiella pneumoniae, (e) E. coli, (f) Klebsiella pneumoniae (clinical), (g) Shigella flexneri, and (h) Streptococcus pneumoniae. The minimum inhibitory concentration (MIC) was estimated as the lowest concentration of AD-L@Ag(0) that will suppress visible growth of microorganisms after a certain incubation period. As per the Clinical and Laboratory Standards Institute (CLSI) guidelines, the minimum bactericidal concentration (MBC) determination assay was used for evaluating this new antimicrobial agent/drug for predicting its efficacy towards eradication (killing 99.9% of a specific bacterial species) of the species used in this study. (i) MBC in the form of RLU as a measure of bacterial viability. The encircled values on the X-axis are the MICs of AD-L@Ag for each species. RLU – relative light unit, AB – Acinetobacter baumannii, PA – Pseudomonas aeruginosa, SA – Staphylococcus aureus, KP(a) – Klebsiella pneumoniae (ATCC), EC – E. coli, KP(b) – Klebsiella pneumoniae (clinical), SF – Shigella flexneri, and SP – Streptococcus pneumoniae.

Fig. 7. Time–kill curves of AD-L@Ag(0) for two different strains, (a) Klebsiella pneumoniae (clinical) and (b) Staphylococcus aureus, are shown. Four doubling dilutions are plotted, the highest concentration corresponds to 0.5× to 4× MIC. The antimicrobial agent was added at timepoint 0 and monitored until 24 h.

Fig. 8. Graphical representation of the Minimum Biofilm Inhibitory Concentration (MBIC) of AD-L@Ag(0) in (a) Acinetobacter baumannii, (b) Pseudomonas aeruginosa, (c) Staphylococcus aureus, (d) Klebsiella pneumoniae, (e) Escherichia coli, (f) Klebsiella pneumoniae (clinical), (g) Shigella flexneri, and (h) Streptococcus pneumoniae.

Fig. 9. Graphical representation of the Minimum Biofilm Eradication Concentration (MBEC) of AD-L@Ag(0) in (a) Acinetobacter baumannii, (b) Staphylococcus aureus, (c) Klebsiella pneumoniae, (d) Klebsiella pneumoniae (clinical), and (e) Shigella flexneri.

Fig. 10. Confocal images (60× magnification) illustrating the (a) Pseudomonas aeruginosa biofilm grown for 24 hours onto the surface of a glass coverslip (control), and (b) residual biofilm after 24 h of AD-L@Ag(0) formulation treatment.

Fig. 11. Graphical representation of the cytotoxicity assay. Grouped graph from 4 hour and 24 hour data plotted with stranded error mean. Vero E6 – cells only, 100–100 μg ml⁻¹, 50–50 μg ml⁻¹, 25–25 μg ml⁻¹, 12.5–12.5 μg ml⁻¹, and 6.26–6.26 μg ml⁻¹ concentration of the AD-L@Ag(0) formulation.

Fig. 12. Day-wise reduction of bacterial CFUs with AD-L@Ag treatment (a), with representative images of surgical wound generation and topical application of the AD-L@Ag(0) formulation in BALB/c mice. (b) Bacterial strains used, PA – Pseudomonas aeruginosa (ATCC 27853), SA – Staphylococcus aureus (ATCC 33592), KP – Klebsiella pneumoniae (ATCC 19050). SC – control uninfected.