Silver Nanoparticles Templated by the M13 Phage Exhibit High Antibacterial Activity against Gram-Negative Pathogens and a Reduced Rate of Bacterial Resistance In Vitro

Abstract

Silver and silver nanoparticles (AgNPs) are well-known for their antibacterial properties. However, their low potency and the emergence of resistance are major barriers to the use of AgNPs in systemic therapy. Biological products can be used to reduce and cap AgNPs during synthesis from silver salts, but the structure and properties of biotemplated AgNPs are not understood well. We observed that AgNPs templated by the Escherichia coli phage M13 showed unusually high potency as well as activity against multiple Gram-negative pathogens, including E. coli, Pseudomonas aeruginosa, and Vibrio cholerae. The increased antimicrobial activity was attributable to the structural properties of the AgNPs rather than to the contributions from the phage. Furthermore, M13-templated AgNPs elicited bacterial resistance >10-fold more slowly than commercially purchased AgNPs and exhibited good cytocompatibility above the concentrations needed for bacterial inhibition. The improvements in antimicrobial properties obtained through biotemplating move AgNPs toward becoming a viable candidate for future systemic applications.

1

M13-templated silver nanoparticles. (a) M13 is a filamentous coliphage whose major coat protein, g8p, has a surface-exposed N-terminal domain. Solvent-accessible electron-rich residues include Lys8 and Tyr21. (b) M13-seeded silver reduction results in AgNP synthesis, as shown by the LSPR absorbance spectrum of AgM13_2h (blue) and AgM13_1d (red) compared to a AgNO₃ solution (yellow) and M13 alone (gray). AgNP spectra were normalized by the maximum value for comparison of the peak shape. The inset shows photographs of representative synthesis products. (c) TEM micrographs show AgNPs associated with phages for AgM13_1d (top row), which are larger than AgNPs from AgM13_2h (bottom row), consistent with the shift in the LSPR peak. Scale bar of 50 nm. (d) AgM13_2h synthesis was conducted with controls and various M13 phage mutants. Nanoparticle formation was quantified by calculating the area under the curve (AUC) of UV–vis absorbance spectra from 400 to 800 nm using Simpson’s rule implemented via the SciPy library in Python. AUC values represent the mean of three independent replicates, and error bars denote the standard error of the mean (SEM). Data for control samples are colored red and blue, those of wild-type (WT) M13 green, and those of M13 mutants purple, orange, and yellow.

2

High-resolution TEM (HR-TEM) and selected area electron diffraction (SAED) pattern of silver–M13 nanoparticles. (a) Bright field HR-TEM image of a single AgM13_1d nanoparticle showing lattice fringes with interplanar spacings of 2.3 and 2.9 Å, corresponding to the (111) and (110) crystallographic planes, respectively. (b) SAED of the AgM13_1d nanoparticle showing a single-crystal diffraction pattern with 6-fold symmetry, characteristic of spherical face-centered cubic (fcc) nanocrystals predominantly exposing low-energy {111} facets. (c) Bright field HR-TEM image of a single AgM13_2h nanoparticle displaying lattice fringes with an interplanar distance of 1.67 Å, attributed to the (211) plane. (d) SAED pattern of the AgM13_2h nanoparticle that reveals preferential exposure of the {211} facets within the fcc crystal structure.

3

Effect of AgM13_1d, AgM13_2h, and AgNP_comm nanoparticles on the growth of various bacterial strains: (a) E. coli ER2738, (c) E. coli DH5α, (e) P. aeruginosa, and (g) V. cholerae following treatment with AgNPs at Ag concentrations of 0.04, 0.1, and 0.2 μg mL^–1 (Ag concentration determined by ICP-MS). Data are presented as means ± SEM (n = 3) biologically independent replicates. Areas under the curve (AUC), representing bacterial growth potential, are shown for (b) E. coli ER2738, (d) E. coli DH5α, (f) P. aeruginosa, and (h) V. cholerae. AUC values are expressed as the mean ± SD. Statistical significance was assessed using one-way analysis of variance (ANOVA) for all groups of the same species, followed by Tukey’s test with false discovery rate (FDR) post hoc correction (n = 3); p values compared to the control are indicated in the corresponding graphs.

4

Influence of AgM13_1d, AgM13_2h, and AgNP_comm nanoparticles on E. coli biofilms. (a–c) Quantification of biofilm biomass by the crystal violet assay for three different E. coli strains. The absorbance at 520 nm reflects the extent of biofilm formation following exposure to different AgNP formulations. Data are expressed as the mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA for groups of the same species, followed by Tukey’s test with false discovery rate (FDR) post hoc correction (n = 3); p values for comparison to the control are indicated in the corresponding graphs. (d) Confocal laser scanning microscopy (CLSM) maximum projection images of E. coli ER2738 biofilms treated with various silver nanoparticles, stained with SYTO 9 and propidium iodide (PI). SYTO 9 (green fluorescence) indicates viable cells, while PI (red fluorescence) indicates compromised membranes, suggesting dead cells. (e) Three-dimensional reconstructions of corresponding biofilms showing the spatial distribution of live (green) and dead (red) cells following nanoparticle treatments as labeled.

5

Bactericidal activity and bacterial resistance to AgM13_1d. (a) E. coli culture was grown to midexponential phase, and then AgM13_1d nanomaterial was added. Growth without AgM13_1d (black curve) or with different concentrations of AgM13_1d (red curves) was monitored by OD₆₀₀. The concentration of AgM13_1d is shown on the right side in micrograms per milliliter. (b) End point samples were plated for the control (no AgM13_1d), 0.2 μg mL^–1 AgM13_1d, or 0.6 μg mL^–1 AgM13_1d, to count colony-forming units (CFUs). No CFUs were observed at 0.6 μg mL^–1 AgM13_1d, consistent with the observed decrease in OD₆₀₀. (c) The E. coli culture was propagated over 15 rounds of serial passage in the presence of AgM13_1d (red), AgM13_2h (blue), or commercially purchased AgNPs (yellow). The minimum inhibitory concentration (MIC) was determined after each round, and the development of resistance was monitored by an increase in MIC. Commercial AgNPs show a high starting MIC and a 2-fold increase in MIC every two or three rounds. Black dots indicate the MIC above the detection range (>40 μg mL^–1) for AgNP_comm; the MIC after round 15 was determined to be 120 μg mL^–1. In contrast, AgM13_1d and AgM13_2h exhibit low starting MICs and a 2-fold increase in MIC after eight rounds.

6

AgNP_comm-M13 and acid-digested AgM13_1d. (a) TEM micrographs showing the networked structure of nanomaterial (AgNP_comm-M13) made from commercially purchased AgNPs and M13. (b) AgNP_comm-M13 did not exhibit substantial antibacterial activity compared to AgM13_1d and AgM13_2h at the same concentration (0.2 μg mL^–1 Ag) on E. coli ER2738. (c) TEM micrographs of acid-digested AgM13_1d indicate a loss of phage structures and the presence of aggregates of silver nanoparticles and amorphous organic matter. (d) Acid-digested AgM13_1d showed antibacterial activity with a modest decrease in potency on E. coli ER2738, as digestion caused a 4–6 h decrease in lag time compared to undigested AgM13_1d at the same concentration. No nanomaterial was added to the control.

7

Cytotoxicity assessment of AgM13_1d and AgM13_2h nanoparticles. (A) Metabolic activity was measured in human embryonic kidney (HEK293) cells using the MTT assay. Cell activity is expressed as the mean ± standard deviation (SD) from three biologically independent replicates (n = 3). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s test and false discovery rate (FDR) post hoc correction. Corresponding p values for pairwise comparisons are indicated in the graphs. No significant decreases were noted compared to those of the control. (B) Hemolytic activity was measured in sheep red blood cells (n = 3). Less than 2% hemolysis was observed.