Synergistic Antimicrobial Effects of Silver/Transition-metal Combinatorial Treatments

Abstract

Due to the emergence of multi-drug resistant strains, development of novel antibiotics has become a critical issue. One promising approach is the use of transition metals, since they exhibit rapid and significant toxicity, at low concentrations, in prokaryotic cells. Nevertheless, one main drawback of transition metals is their toxicity in eukaryotic cells. Here, we show that the barriers to use them as therapeutic agents could be mitigated by combining them with silver. We demonstrate that synergism of combinatorial treatments (Silver/transition metals, including Zn, Co, Cd, Ni, and Cu) increases up to 8-fold their antimicrobial effect, when compared to their individual effects, against E. coli and B. subtilis. We find that most combinatorial treatments exhibit synergistic antimicrobial effects at low/non-toxic concentrations to human keratinocyte cells, blast and melanoma rat cell lines. Moreover, we show that silver/(Cu, Ni, and Zn) increase prokaryotic cell permeability at sub-inhibitory concentrations, demonstrating this to be a possible mechanism of the synergistic behavior. Together, these results suggest that these combinatorial treatments will play an important role in the future development of antimicrobial agents and treatments against infections. In specific, the cytotoxicity experiments show that the combinations have great potential in the treatment of topical infections.

Figure 1

Inhibitory Effect of the MIC dilutions of Ag potentiated by transition metals against E. coli. Inhibitory percentage by sub-inhibitory combinations of Ag-transition metals in Escherichia coli ATCC 11229. Experiments with combinatorial treatments of Ag with (A) Ni, (B) Cu, (C) Zn and (D) Cd. Each combinatorial treatment with inhibition >80% is significantly different to the effects of the individual treatments. Each checkerboard treatment was performed in triplicates.

Figure 2

Inhibitory Effect of the MIC dilutions of Ag potentiated by transition metals against Bacillus subtilis. Inhibitory percentage by sub-inhibitory combinations of Ag-transition metals in Bacillus subtilis ATCC 23857. Experiments with combinatorial treatments of Ag with (A) Ni, (B) Cu, (C) Zn and (D) Co. Each combinatorial treatment with inhibition >80% is significantly different to the effects of the individual treatments. Each checkerboard treatment was done in triplicates.

Figure 3

Analysis of Ag-Transition Metal Interactions. Classification of the different interactions between Ag-transition metal in Escherichia coli ATCC 11229. The interactions of Ag with (A) Ni, (B) Cu, (C) Zn and (D) Cd, are classified as synergistic, additive or antagonistic, value >0, =0 and <0, respectively. Each checkerboard treatment was done in triplicates.

Figure 4

Analysis of Ag-Transition Metal Interactions. Classification of the different interaction between Ag-transition metal in Bacillus subtilis ATCC 23857. The interactions of Ag with (A) Ni, (B) Cu, (C) Zn and (D) Co, are classified as synergistic, additive or antagonistic, value >0, =0 and <0, respectively. Each checkerboard treatment was done in triplicates.

Figure 5

Bactericidal Effect of Ag Potentiated by Transition Metals. Log change in CFUs/mL with respect to time zero, in Escherichia coli ATCC 11229 after 1 hour treatment with: LB (control), Ag, specific transition metal and their combination. As nominal concentrations: (A) Ag 30 µM, Ni 0.5 mM and the combination; (B) Ag 30 µM, Cd 1 mM and the combination; (C) Ag 15 µM, Cu 1 mM and 2 mM, and the respective combinations; (D) Ag 30 µM, Zn 0.5 mM and the combination. ***Corresponds to a p < 0.05, tested with an ANOVA, that there is a difference with respect to the control and each of the individual treatments. Error bars correspond to the standard deviation from experiments performed in triplicates.

Figure 6

Viability and cell permeability assay of B. subtilis and E. coli treated with STMCs. Nominal concentrations of Ag/Cu Ag/Ni and Ag/Zn (15 μM/0.5 mM) by 24 h (37 °C). (A) SyBR-G B. subtilis positive cells represented as a measure of % of viability; whereas (B) B. subtilis PI positive cells are represented as a measure of % of permeability. (C) SyBR-G E. coli positive cells represented as a measure of % of viability; whereas (D) E. coli PI positive cells are represented as a measure of % of permeability Values represent mean ± SD (n = 3 experiments for each treatment). P values *P < 0.05 vs. Control, **P < 0.01 vs. Control.

Figure 7

Cytotoxicity effects of Ag and transition metals on H9c2 and B16F10 cells. (A) H9c2 and B16F10 cells were treated with Ag at the indicated concentrations for 24 h and viability was determined. (B) Effect of transition metals on the viability of cells H9c2. Cells were treated with each transition metal at a concentration of 1 mM for 48 h and viability was determined. Bars represent means of three independent experiments and their respective standard deviations. (C) Dose-response effect of transition metals on the viability of H9c2 cells. Cells were treated with transition metals at the stated concentration for 48 h before viability was determined. Indicator points represent means of three independent experiments and their respective standard deviations. Values are mean ± SD (n = 3 experiments at least for each treatment).

Figure 8

Cytotoxicity of Individual Transition Metals on HaCat cells. Effect of transition metals on the viability of HaCat cells. Cells were treated with individual treatments of transition metals at different concentrations for 24 h and viability was determined. Data are expressed as percent of control mean ± SEM and analyzed by one-way ANOVA, with a Tukey post hoc test. **p < 0.01 vs. control; N = 3.

Figure 9

Cytotoxicity of STMCs on HaCat cells. Effect of STMCs on the viability of HaCat cells. Cells were treated with STMCs at different concentrations for 24 h and viability was determined. Data are expressed as percent of control mean ± SEM and analyzed by one-way ANOVA, with a Tukey post hoc test. **p < 0.01 vs. control; N = 3.

Figure 10

Cytotoxicity effects of Ag and transition metals at concentrations adjusted for speciation on melanocytes and myoblast cells. Effect of transition metals, alone or in combination, with Ag on the viability of (A) B16F10 and (B) H9c2 cells. Cells were treated with each transition metal (in nominal µM: Cu 80; Co 400; Zn 31.2) alone or in the combination with Ag (15 µM), for 24 h. Values are mean ± SD. *p < 0.05 vs. Control; (a) vs. Ag. (n = 3 experiments at least for each treatment).