Nanoparticulate silver compounds (AgNP) have widespread use in antibacterial clothing, water filtration, coated medical devices, wound dressings, and cosmetics due to the versatility of nanoparticles and the established activity of silver against bacteria.1–3 Nanoparticles are structures 1–100 nm in diameter, with optical, electrical, and magnetic quantum properties that allow them to adhere to, penetrate, and damage the intracellular structures of bacteria.4–6 Silver ions, released by the nanoparticles, generate free radical oxygen species and bind bacterial components (DNA, enzymes, and cell wall structures) to inhibit their function.7,8 Therefore, AgNP have a greater antibacterial effect than either silver ions or nanoparticles alone and their multi-modal activity may reduce the risk for antibacterial resistance.2 Commercially available preparations of AgNP are composed of 10 nm diameter, spherical, citrate-stabilized nanoparticles.9 Antibiofilm effects against Staphylococcus pseudintermedius have been reported for commercially available preparations of AgNP.10 However, the antibacterial activity of commercial preparations of AgNP has not otherwise been reported.
Silver wound products for topical use are widespread, but none are available that provide sustained local therapy at sites that cannot be left open or removed. Access to products to provide sustained release of silver at these sites may enhance the management of infections while minimizing systemic antimicrobial use. Calcium sulfate hemihydrate (CSH) beads, poloxamer gel, and gelatin sponge are commercially available carrier media with the potential for use in body cavities and wound beds. Elution patterns of AgNP dispersion from calcium sulfate hemihydrate beads, gelatin sponge, and poloxamer 407 were previously studied. A favorable pattern of elution was observed for poloxamer 407 gel, with early and sustained release of silver nanoparticles recorded.11 AgNP variably binds with carrier substrates depending on the substrate characteristics; binding to the substrate may affect the ability of AgNP to engage with bacteria.12,13
The aims of this experiment were (1) to establish the antibacterial activity of AgNP dispersion against E coli and MRSP in vitro, and (2) to evaluate whether this activity was maintained following the incorporation of AgNP into drug carriers for sustained release. We hypothesized that (1) AgNP dispersion would have bactericidal activity against E coli and MRSP in vitro, and (2) AgNP incorporated into drug carriers for sustained release (SR-AgNP) would have similar bactericidal activity in vitro compared to AgNP dispersion alone.
Methods
A pilot study was performed to test the antibacterial effects of 0.02 mg/mL silver nanoparticle dispersion (AgNP) composed of spherical, 10 nm diameter, citrate-stabilized silver nanoparticles (Sigma Aldrich) against a clinical strain of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and a type-strain E coli (ATCC 25922, ATCC) at a range of concentrations before conducting the full study. The pilot study concluded that 0.01 mg/mL AgNP was the lowest dose of AgNP that inhibited the growth of MRSP and E coli bacteria. The full study consisted of 2 aims. In aim 1, bacteria at a range of concentrations were treated with AgNP to determine the highest concentration of bacteria at which growth could be inhibited. In aim 2, AgNP was incorporated into carriers for sustained release and used to treat the concentration of bacteria determined in aim 1. In all experiments, AgNP was filtered with a 0.2 µm filter (Corning) before use to sterilize the suspension following IV medication preparation guidelines.
Aim 1: Antibacterial activity of AgNP
Cation adjusted Mueller-Hinton broth (Thermo Scientific) was added to a 96-well, flat bottom microdilution assay plate (Thermo Fisher Scientific) and inoculated with 10° to 106 colony forming units (CFU)/mL MRSP and 10° to 106 CFU/mL E coli, respectively. Bacteria were incubated overnight on blood agar plates (Thermo Scientific). A few colonies were suspended in 0.85% sterile saline solution. Initial quantification of bacterial concentration was performed by adjusting the optical density of the bacterial suspension to that of 0.5 McFarland Standards (1.5 X 108 CFU/mL) using a DensiCHECK (BioMérieux Inc) instrument to measure the optical density of the solution.14 AgNP 0.02 mg/mL solution (Sigma Aldrich) was added to Mueller-Hinton broth in each well to achieve a concentration of 0.01 mg/mL AgNP in a total volume of 200 µL and incubated for 18–24 hours. Growth controls (MRSP without AgNP and E coli without AgNP) and sterility controls (Mueller-Hinton broth only and AgNP in Mueller-Hinton broth only) were included. From each well, 100 µL was transferred to separate blood agar plates and incubated for 18–24 hours at 36 ± 2 °C in a 5% CO2, ambient O2, 85% to 95% humidity incubator, with humidity provided by a built-in water tray filled with reverse osmosis water (NuAire). The number of bacterial colonies that grew were counted by the unaided eye and recorded. Three trials were performed, with 3 repetitions for each trial.
The concentration of bacteria against which 0.01 mg/mL AgNP would be expected to cause a major reduction in colony growth was determined based on the microdilution assay, and these concentrations of bacteria were tested against AgNP in a higher-volume environment to ensure that the results of the microdilution assay were repeatable in this setting. Mueller-Hinton broth (10 mL) was added to a 50 mL sterile tube (Thermo Fisher Scientific). AgNP was added to the broth at a concentration of 0.01 mg/mL. MRSP and E coli were added to sterile tubes at a concentration of 102 CFU/mL and 104 CFU/mL, respectively. Tubes were protected from light and incubated for 18–24 hours at 36 ± 2 °C in a 5% CO2, ambient O2, 95% humidity incubator. A growth control using 20 mL Mueller-Hinton broth and bacteria only was prepared for each strain of bacteria. After the initial period of incubation, blood agar plates were streaked with 100 uL from each sample and incubated for 18–24 hours at 36 ± 2 °C in a 5% CO2, ambient O2, 85% to 95% humidity incubator, with humidity provided by a built-in water tray filled with reverse osmosis water to facilitate colony quantification. Plates were visually inspected and the number of colonies that grew was recorded. This was performed in triplicate.
Aim 2: antibacterial activity of sustained release AgNP (SR-AgNP)
SR-AgNP compound preparation—AgNP was filtered using a 0.2 µm filter before preparation of each carrier. Each product was batch-prepared and stored in a sterile carrier, cooled for transport, and protected from light, for short-term storage before use. The 3 carriers used were selected due to their ready availability, inert nature, biocompatibility, and ability to safely degrade in the body over time. Calcium sulfate hemihydrate (CSH) beads (Kerrier) are beads composed of water, calcium, and sulfuric acid. This inert powder can be mixed with liquid to form a paste that will harden as it dries. CSH beads impregnated with antimicrobials have been utilized in the management of infected diabetic foot ulcers where degradation of the CSH over time provided sustained drug release.15 The CAS registry number of this compound is 10034-76-1.16 Poloxamer 407 gel (Pluronic F-127, Sigma Aldrich) is a nonionic triblock copolymer composed of polyethylene glycol and polypropylene glycol. This inert, thermoresponsive, micellar compound is liquid when chilled but forms a gel solid at room temperature. The gel solid state facilitates prolonged drug release as the gel degrades. This compound is approved by the FDA for use as an excipient for a range of pharmaceuticals.17 The CAS registry number of this compound is 9003-11-6.18 Gelatin sponge (Vetspon, Elanco) is a compressed sponge composed of purified porcine skin, gelatin granules, and water. Gelatin sponge is absorbed with little tissue reaction.19 Gelatin sponge has been investigated as a carrier for the controlled delivery of pharmaceuticals including pilocarpine, recombinant peptides, and insulin.20–22 The CAS registry number of this compound is 9000-70-8.23
CSH beads: 15 g CSH powder was added to a sterile jar with 12 mL of filtered 0.02 mg/mL AgNP dispersion and gently stirred until thickened to a consistency that could be aspirated into a syringe using a 16G IV catheter without separation of the components. The product was placed into the 5 mm cavities of the commercial beat mat using the 16G IV catheter. The bead mat was covered with a sterile drape and placed in a Class II biological safety cabinet protected from light for 24 hours to facilitate the hardening of the product. Beads were removed from the bead mat, divided into 3 portions of even weight, and stored in sterile jars. Additional beads were prepared in the same fashion using 0.9% sterile saline for use as controls.
Poloxamer 407 gel: 4 mL of prepared poloxamer 407 (30% w/v) was combined with 4 mL of 0.02 mg/mL AgNP dispersion in a 12 mL syringe and gently agitated for 5 minutes. Poloxamer 407 was expected to achieve a gel state when combined with liquid at a 1:1 ratio.24,25 Poloxamer 407 (30% w/v) without AgNP dispersion was prepared for use as controls.
Gelatin sponge: Two gelatin sponges (each measuring 2 X 6 X 0.7 cm) were placed within a 50 mL sterile tube and each sponge was injected with 2 mL of 0.02 mg/mL AgNP dispersion using a 22G needle to prevent any potential loss of liquid from the sponge during transfer. Pilot data established 2 mL of liquid could be added to each sponge without leakage of liquid from the sponge. Gelatin sponges were prepared in the same fashion with 0.9% sterile saline for use as controls.
Experimental setup—The full study involved the following specimens, prepared in triplicate:
MRSP at a concentration of 10° to 102 CFU/mL and E coli at a concentration of 102 to 104 CFU/mL in Mueller-Hinton broth, combined with each SR-AgNP compound (CSH beads, poloxamer 407 gel, gelatin sponge) in 50 mL sterile tubes. The concentration of bacteria selected for testing corresponded to the susceptible concentration established during aim 1, plus 1 concentration above and below the susceptible concentration.
MRSP at a concentration of 10° CFU/mL and E coli at a concentration of 102 CFU/mL in Mueller-Hinton broth, combined with each carrier prepared without AgNP dispersion (CSH beads, poloxamer 407 gel, gelatin sponge) in 50 mL sterile tubes. These samples were used to assess for any inherent antibacterial activity of the carriers.
MRSP at a concentration of 102 CFU/mL and E coli at a concentration of 104 CFU/mL in Mueller-Hinton broth, combined with 0.01 mg/mL gentamicin in 50 mL sterile tubes. These samples were used as a positive control. A microdilution assay showed an MIC of 0.004 mg/mL for gentamicin against the MRSP isolates used in our study; therefore, susceptibility of this isolate to 0.01 mg/mL gentamicin was expected.
MRSP at a concentration of 10° to 102 CFU/mL and E coli at a concentration of 102 to 104 CFU/mL in Mueller-Hinton broth, combined with 0.0L mg/mL AgNP dispersion in 50 mL sterile tubes. These samples were used as a positive control and to facilitate a direct comparison of the activity of free AgNP dispersion and SR-AgNP for testing performed in parallel.
MRSP at a concentration of 10° CFU/mL and E coli at a concentration of 102 CFU/mL in Mueller-Hinton broth. These samples were used as a growth control.
Each sample was covered in aluminum foil to protect from light and incubated for 72 hours at 36 ± 2 °C in a 5% CO2, ambient O2, 85% to 95% humidity incubator, with humidity provided by a built-in water tray filled with reverse osmosis water. Samples were sub-cultured to blood agar plates at 24, 48, and 72 hours postinoculation and incubated a further 18 to 24 hours at 36 ± 2 °C in a 5% CO2, ambient O2, 85% to 95% humidity incubator, with humidity provided by a built-in water tray filled with reverse osmosis water to facilitate determination of the number of colonies growing on each plate by visual inspection. Plates were categorized as growth (1 or more colonies growing) or no-growth (no colonies growing), as the National Committee for Clinical Laboratory Standards defines a compound as bactericidal if a reduction of bacteria by at least 3-log10 is achieved and bacteria were not assessed at a concentration high enough to facilitate this assessment in this study.26
Statistical methods—Samples were designated “growth” (≥1 colony grew on plate) or “no growth” (0 colonies grew on the plate) based on visual inspection of blood agar plates for bacterial colonies. For aim 1, summary data are presented as a percentage of colonies showing no growth. For aim 2, a mixed-effects linear model was used to compare the binary outcome (growth/no growth) in each group (free AgNP, gentamicin solution, SR-AgNP CSH beads, SR-AgNP poloxamer 407 gel, SR-AgNP gelatin sponge, CSH beads without AgNP, poloxamer 407 gel without AgNP, and gelatin sponge without AgNP) for each concentration of bacteria at each time point. Separate models were used for samples inoculated with each of the bacterial species, MRSP and E coli, respectively. Model assumptions of normality of the residuals and homogeneity of residual variance were assessed by qq-plots, normal curves, and scatter plots; no violations of assumptions were observed. Model fit was evaluated using Akaike information criteria (AIC) with lower values signifying better fit. The Bonferroni method of adjustment of P-values was applied for pairwise comparisons. All statistical analyses were performed using SAS (SAS Institute Inc) and P < .05 was considered significant.
Results
Based on the results of the microdilution assay, the concentration of bacteria against which 0.01 mg/mL AgNP would be expected to cause a major reduction in colony growth was determined to be 101 CFU/mL for MRSP and 103 CFU/mL for E coli.
MRSP
In aim 1, the highest concentration of MRSP against which AgNP exerted a consistent antimicrobial effect was 101 based on a qualitative assessment of colony growth (Table 1). In aim 1, MRSP at 10° CFU/mL had no growth of bacteria in 83% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 66% of samples treated with SR-AgNP CSH beads, 100% samples treated with SR-AgNP poloxamer 407 gel and 0% of samples treated with SR-AgNP gelatin sponge. In aim 1, MRSP at 101 CFU/mL had no growth of bacteria in 55% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 56% samples treated with SR-AgNP CSH beads, 88% of samples treated with SR-AgNP poloxamer 407 gel and 33% of samples treated with SR-AgNP gelatin sponge. In aim 1, MRSP at 102 CFU/mL had no growth of bacteria in 11% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 33% of samples treated with SR-AgNP CSH beads, 100% of samples treated with SR-AgNP poloxamer 407 gel and 0% of samples treated with SR-AgNP gelatin sponge (Table 1).
Percentage of samples of methicillin-resistant Staphylococcus pseudintermedius (MRSP) treated with 0.01 mg/mL nanoparticulate silver (AgNP) showing no growth of bacterial colonies, by concentration.
| No growth (%) | |||
|---|---|---|---|
| Bacterial concentration (CFU/mL) | Trial 1 | Trial 2 | Trial 3 |
| 100 | 100.00 | 66.67 | |
| 101 | 100.00 | 33.33 | 33.33 |
| 102 | 33.33 | 0.00 | 0.00 |
| 103 | 0.00 | 0.00 | 0.00 |
| 104 | 0.00 | 0.00 | |
| 105 | 0.00 | ||
| 106 | 0.00 | ||
MRSP in Mueller-Hinton broth at concentrations of 10° to 106 CFU/mL were treated with 0.01 mg/mL free silver nanoparticle solution. Each inoculum was incubated for 18 to 24 hours at 37 °C, subcultured to blood agar plates, and incubated a further 18 to 24h at 37 °C before visual inspection of each plate for determination of bacterial colony growth. Blank cells indicate concentrations of bacteria not tested.
In aim 2, growth of bacterial colonies after incubation was associated with the treatment group (P < .001) (Table 2; Figure 1) but not bacterial concentration (P = .292) nor time (P = .289) (Figure 2). Gelatin sponges were observed to dissolve rapidly (within 12 hours) when exposed to MRSP while sponges remained intact in samples where AgNP prevented growth of MRSP. In pairwise comparisons, the following groups were not different from each other: free AgNP, gentamicin solution, SR-AgNP poloxamer 407 gel, and poloxamer 407 gel without AgNP (P > .800). However, they were different from the following groups: SR-AgNP CSH beads, SR-AgNP gelatin sponge, CSH beads without AgNP, and gelatin sponge without AgNP (P < .011). The treatment groups were rank ordered from best (highest percentage of samples with no growth) to worst (highest percentage of samples with growth) as follows: free AgNP, gentamicin solution, poloxamer 407 gel without AgNP, SR-AgNP poloxamer 407 gel, SR-AgNP CSH beads, CSH beads without AgNP, SR-AgNP gelatin sponge, and gelatin sponge without AgNP.
Percentage of samples inoculated with methicillin-resistant Staphylococcus pseudintermedius (MRSP) showing no growth of bacterial colonies, by treatment group.
| Treatment group | No growth (%) |
|---|---|
| Free AgNP | 100.00 |
| Gentamicin solution | 100.00 |
| SR-AgNP CSH beads | 51.80 |
| SR-AgNP poloxamer 407 gel | 96.30 |
| SR-AgNP gelatin sponge | 11.11 |
| CSH beads without AgNP | 33.33 |
| Poloxamer 407 gel without AgNP | 100.00 |
| Gelatin sponge without AgNP | 0.00 |
MRSP in Mueller-Hinton broth at concentrations of 10° to 102 CFU/mL were treated with 0.01 mg/mL free silver nanoparticle (AgNP) solution, 0.01 mg/mL gentamicin solution, 0.01 mg/mL AgNP bound to carriers for sustained release (SR-AgNP) and each of the carriers without incorporation of AgNP. The carriers used were calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel and gelatin sponge. Each inoculum was incubated for 18 to 24 hours at 37 °C, subcultured to blood agar plates, and incubated a further 18 to 24 hours at 37 °C before visual inspection of each plate for determination of bacterial colony growth.
Percentage of samples with growth of methicillin-resistant Staphylococcus pseudintermedius (MRSP) (A) and Escherichia coli (E coli) (B) after treatment with gentamicin, free silver nanoparticulate (AgNP), and AgNP combined with 3 carriers for sustained release. (A) MRSP in Mueller-Hinton broth at concentrations of 10° to 102 CFU/mL and B) E coli in Mueller-Hinton broth at concentrations of 102 to 104 CFU/mL were treated with 0.01 mg/mL free AgNP solution, 0.01 mg/mL gentamicin solution, 0.01 mg/mL AgNP bound to carriers for sustained release (SR-AgNP) and each of the carriers without incorporation of AgNP. The carriers used were calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel and gelatin sponge. Each inoculum was incubated for at 37 °C and sub-cultured to blood agar plates at 24, 48, and 72 hour postinoculation. Plates were incubated a further 18 to 24 hours at 37 °C before visual inspection for determination of bacterial colony growth. For MRSP, the percentage of samples where bacterial growth was suppressed by free AgNP solution did not differ when compared to SR-AgNP poloxamer 407 gel (P = 1.00) but differed from SR-AgNP bead (P < .001) and SR-AgNP gelatin sponge (P < .001). For E coli, the percentage of samples where bacterial growth was suppressed by free AgNP solution did not differ when compared to SR-AgNP poloxamer 407 gel (P = .837) but differed from SR-AgNP bead (P < .006) and SR-AgNP gelatin sponge (P < .001). Y-error bars indicate the 95% binomial confidence interval for the proportion.
Citation: American Journal of Veterinary Research 85, 3; 10.2460/ajvr.23.10.0229
Percentage of samples with bacterial growth after treatment with gentamicin, free AgNP, AgNP combined with 3 carriers for sustained release and carriers for sustained release without AgNP. (A) MRSP in Mueller-Hinton broth at concentrations of 10° to 102 CFU/mL and (B) Escherichia coli (E coli) in Mueller-Hinton broth at concentrations of 102 to 104 CFU/mL were treated with 0.01 mg/mL free silver nanoparticle (AgNP) solution, 0.01 mg/mL gentamicin solution, 0.01 mg/mL AgNP bound to carriers for sustained release (SR-AgNP) and each of the carriers without incorporation of AgNP. The carriers used were calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel and gelatin sponge. Each inoculum was incubated for at 37 °C and sub-cultured to blood agar plates at 24, 48, and 72 hours postinoculation. Plates were incubated a further 18 to 24 hour at 37 °C before visual inspection for determination of bacterial colony growth. The rate of bacterial suppression did not change over time for E coli (P = .095) nor MRSP (P = .289).
Citation: American Journal of Veterinary Research 85, 3; 10.2460/ajvr.23.10.0229
E coli
In aim 1, the highest concentration of E coli against which AgNP exerted a consistent antimicrobial effect was 103 based on the qualitative assessment of colony growth (Table 3). In aim 1, E coli at 102 CFU/mL had no growth of bacteria in 100% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 66% of samples treated with SR-AgNP CSH beads, 100% of samples treated with SR-AgNP poloxamer 407 gel and 33% of samples treated with SR-AgNP gelatin sponge. In aim 1, E coli at 103 CFU/mL had no growth of bacteria in 88% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 44% of samples treated with SR-AgNP CSH beads, 88% of samples treated with SR-AgNP poloxamer 407 gel and 0% of samples treated with SR-AgNP gelatin sponge. In aim 1, E coli at 104 CFU/mL had no growth of bacteria in 100% of samples treated with 0.01 mg/mL AgNP; in aim 2 at this concentration, no growth of bacteria was seen in 33% of samples treated with SR-AgNP CSH beads, 100% of samples treated with SR-AgNP poloxamer 407 gel and 0% of samples treated with SR-AgNP gelatin sponge (Table 3 and Table 4). In aim 2, the growth of bacterial colonies after incubation was associated with group (P < .001) (Table 4) and bacterial concentration (P = .029) but not time (P = .095) (Figure 2). In pairwise comparisons, the following groups were not different from each other: free AgNP, gentamicin solution, SR-AgNP poloxamer 407 gel, and poloxamer 407 gel without AgNP (P = .053). However, they were different from the following groups: SR-AgNP CSH beads, SR-AgNP gelatin sponge, CSH beads without AgNP, and gelatin sponge without AgNP (P = .015). The treatment groups were ranked ordered from best (highest percentage of samples with no growth) to worst (highest percentage of samples with growth) as follows: gentamicin solution, poloxamer 407 gel without AgNP, SR-AgNP poloxamer 407 gel, free AgNP, SR-AgNP CSH beads, SR-AgNP gelatin sponge, gelatin sponge without AgNP, and CSH beads without AgNP.
Percentage of samples of Escherichia coli (E coli) treated with 0.01 mg/mL nanoparticulate silver (AgNP) showing no growth of bacterial colonies, by concentration.
| No growth (%) | |||
|---|---|---|---|
| Bacterial concentration (CFU/mL) | Trial 1 | Trial 2 | Trial 3 |
| 100 | 100.00 | ||
| 101 | 100.00 | 100.00 | 100.00 |
| 102 | 100.00 | 100.00 | 100.00 |
| 103 | 66.67 | 100.00 | 100.00 |
| 104 | 100.00 | 100.00 | |
| 105 | 66.67 | 33.33 | |
| 106 | 0.00 | ||
E coli in Mueller-Hinton broth at concentrations of 10° to 106 CFU/mL were treated with 0.01 mg/mL free silver nanoparticle solution. Each inoculum was incubated for at 37 °C and subcultured to blood agar plates at 24, 48, and 72 hours postinoculation. Plates were incubated a further 18 to 24 hours at 37 °C before visual inspection for determination of bacterial colony growth. Blank cells indicate concentrations of bacteria not tested.
Percentage of samples inoculated with Escherichia coli (E coli) at 3 concentrations showing no growth of bacterial colonies, by treatment group.
| No growth (%) | |||
|---|---|---|---|
| Treatment group | 102 CFU/mL | 103 CFU/mL | 104 CFU/mL |
| Free AgNP | 96.3 | 100.00 | 44.44 |
| Gentamicin solution | 100.00 | ||
| SR-AgNP CSH beads | 66.67 | 44.44 | 33.33 |
| SR-AgNP poloxamer 407 gel | 100.00 | 88.89 | 100.00 |
| SR-AgNP gelatin sponge | 33.33 | 0.00 | 0.00 |
| CSH beads without AgNP | 0.00 | ||
| Poloxamer 407 gel without AgNP | 100.00 | ||
| Gelatin sponge without AgNP | 0.00 | ||
E coli in Mueller-Hinton broth at concentrations of 102 to 104 CFU/mL were treated with 0.01 mg/mL free silver nanoparticle (AgNP) solution, 0.01 mg/mL gentamicin solution, 0.01 mg/mL AgNP bound to carriers for sustained release (SR-AgNP) and each of the carriers without incorporation of AgNP. The carriers used were calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel and gelatin sponge. Each inoculum was incubated for at 37 °C and subcultured to blood agar plates at 24, 48, and 72 hours postinoculation. Plates were incubated a further 18 to 24 hours at 37 °C before visual inspection for determination of bacterial colony growth. Blank cells indicate concentrations of bacteria not tested.
Discussion
Although bactericidal activity of AgNP has been widely reported against a range of bacteria, current literature does not provide a precise concentration of AgNP at which bactericidal activity can be expected: the MIC of AgNP against Staphylococcus aureus has been reported from 1.35 μg/mL to 100 μg/mL27–30 and the MIC of AgNP against E coli has been reported from 3.38 μg/mL to 100 μg/mL.27,29–31 The only report on S pseudintermedius states AgNP disrupts biofilm formation at a concentration of 0.01 mg/mL but did not report the concentration of AgNP at which inhibition of bacterial growth was seen.10 The range in MICs reported for AgNP is due to the variety of test conditions used across studies, with many researchers compounding nanoparticles with particular characteristics.6,7,31–35 Ipe et al (2020) synthesized spherical AgNP with an average size of 26 nm. Bacterial species grown in Brain Heart Infusion were challenged with AgNP and antibacterial effect was assessed using a broth microdilution method and disk diffusion method. Martinez-Castanon et al (2008) synthesized pseudospherical AgNP with sizes ranging between 7 to 89 nm. Bacterial species grown in Mueller-Hinton broth were challenged with AgNP and antibacterial effect was assessed using a broth microdilution method. Agnihotri et al (2014) synthesized AgNP with sizes ranging between 5 to 100 nm. Bacterial species cultured on agar plates were challenged with AgNP were impregnated on filter paper disks and the zone of inhibition was recorded. Pal et al (2007) synthesized triangular, rod, and spherical AgNP. Bacterial species were challenged with each respective AgNP preparation using a broth microdilution method and bacterial assessment on transmission electron microscopy. There are few reports on the antibacterial properties of commercially available AgNP.
Characteristics such as shape, state, and size affect the efficacy of AgNP against bacteria. Truncated triangular nanoparticles have greater antibacterial activity than spherical and rod-shaped nanoparticles, but are not readily commercially available.36 Colloidal AgNP has enhanced antibacterial activity compared to AgNP alone; AgNP in liquid systems have low colloidal stability.31 Nanoparticles of decreasing size have increased antibacterial activity due to providing a relatively higher surface area: volume ratio for bacterial interaction and silver ion release, and due to direct bacterial damage caused by particles 10 nm and less.4,8,27 This may account for the poorer suppression of bacterial growth in this study compared to others: In the present study, 0.01 mg/mL AgNP inhibited the growth of E coli up to 103 CFU/mL when spherical 10 nm particles were used, while under similar test conditions, 0.01 mg/mL AgNP inhibited growth of E coli up to 107 when spherical 5 nm particles were used.4
AgNP exerts dose-dependent antibacterial activity.8 Therefore, increasing the concentration of AgNP used in this study may have facilitated the use of higher bacterial concentrations to improve the clinical relevance of the results. However, the concentration was limited to 0.01 mg/mL AgNP by the concentration of commercially available AgNP dispersion available to the authors and the supposition that only 4 mL fluid could be used in the formulation of the CSH beads, per manufacturer recommendations.37 Despite this, it was possible to establish the lowest effective dose of AgNP that could suppress the growth of E coli and S pseudintermedius, and determine the effect on the activity of AgNP after incorporation into carriers for sustained release. During the study, it was determined that 12 mL fluid could be added to the CSH powder although 24 hours was required for the beads to set.
Only free nanoparticles can interact with bacteria: in wound dressings, only nanoparticles that left the dressing matrix exerted an antimicrobial effect.7 Additionally, using stabilizers to prevent particle aggregation in liquid systems has a greater impact on the antibacterial potential of AgNP than the net charge of the solution (citrate stabilizers are negatively charged, which could theoretically reduce interaction between positively charged AgNP and negatively charged bacterial components).32 Therefore, nanoparticles that irreversibly bind to a drug carrier for sustained release will similarly fail to interact with bacteria. In the present study, sufficient AgNP was added to each carrier to achieve a concentration of 0.01 mg/mL AgNP in the solution assuming 100% elution of AgNP dispersion from each carrier. However, a previous study showed that this degree of elution could not be expected from the drug carriers used in this study.11 Therefore, using a higher dose of AgNP in the formulation of carriers for sustained release may have provided better resolution in the data to compare the antibacterial activity of SR-AgNP and free AgNP. A superior elution pattern was reported for SR-gelatin sponge compared to SR-CSH beads.11 However, although not statistically significant, a trend toward greater antibacterial activity was seen for SR-AgNP CSH beads compared to SR-AgNP gelatin sponge for both MRSP and E coli (Figure 1). The technique used to formulate SR-AgNP CSH beads in the present study differed from that in the reported elution study which may have affected the elution of AgNP from the CSH beads. Macroscopic dissolution of gelatin sponges was observed in specimens with growth of MRSP. Coagulase-positive Staphylococcus spp, such as MRSP, produce gelatinase which liquifies gelatin.38 Therefore, other factors inherent to each material may affect the ability of AgNP to interact with bacteria, revealing the importance of assessing the performance of AgNP with a range of drug carriers for sustained release despite reported elution patterns.
Poloxamer 407 gel showed rapid and sustained elution of AgNP.11 SR-AgNP poloxamer 407 gel was the only sustained-release formulation of AgNP to show comparable antibacterial activity to free AgNP. However, this was confounded by the antibacterial activity exerted by the poloxamer 407 gel alone. Poloxamer 407, also known as PF-127, is a micellar compound that forms aggregates at high concentrations and with increasing temperature to achieve a gel consistency. Previous studies using 20% to 40% poloxamer 407 gel against S aureus and E coli reported that the compound did not possess intrinsic antibacterial activity against these species.39 However, poloxamer 407 gel can alter cell membranes due to their micellar composition and has been shown to prevent bacterial adhesion to surfaces.40 It is feasible that poloxamer 407 gel works synergistically with AgNP, preventing bacterial adhesion to improve exposure of bacteria to AgNP. Exploration of the antibacterial properties of poloxamer 407 gel may enhance the use of this versatile compound in wound management.
Time-dependent antibacterial effects were reported for Klebsiella pneumoniae treated with variably-shaped AgNP with diameters 20–90 nm, measured by disk diffusion method.3,41 Time-dependent antibacterial effects were reported for multi-drug resistant strains of E coli and S aureus treated with green-synthesized triangular AgNP with a mean diameter of 18 nm, measured by killing kinetic assay in which bacterial cell viability was assessed spectrophotometrically.42 The potential for sustained activity of AgNP has been reported. A 0.02% AgNP gel was reported to disrupt biofilm more effectively than irrigation with 0.1% AgNP solution.43 In the present study, it was expected that as more AgNP eluted from the carriers for sustained release, increased antibacterial activity would be seen over time. However, there was no change in the efficacy of free AgNP nor SR-AgNP over time. This may be due to the greater initial concentration gradient between the carrier for sustained release and broth promoting greater elution of AgNP initially, with a reduced rate of elution and lower effective dose of AgNP in the broth over time. Additionally, aggregation of nanoparticles in the broth over time may have similarly reduced the effective dose of AgNP in the broth over time. Given the complex nature of AgNP elution and behavior in solutions, greater exploration of its potential to provide sustained activity is required.
The efficacy of AgNP against MRSP and E coli seen in the microdilution study was confirmed by testing AgNP against the concentration of bacteria expected to be inhibited by 0.01 mg/mL AgNP in a higher volume (20 mL) environment. Nanoparticles become entangled with bacteria in an aqueous environment, favoring strong activity even in a large-volume setting such as an exudative wound.43 However, in 1 study the MIC of AgNP against E coli changed by up to 2 orders of magnitude depending on the culture medium used, as some components of the culture medium can impair silver ion release and promote aggregation of nanoparticles.44 For example, blood interferes with the activity of silver.32 Mueller-Hinton broth was selected for use in this study as bacterial growth could not be inhibited, even with high doses of free AgNP, when thioglycolate broth was used. Similarly, silver dressings had higher MIC values in simulated wound fluid, and under anaerobic conditions, when compared to traditional broths in aerobic conditions.45 An acidic environment favors the oxidation of AgNP to release silver ions.43 Therefore, it is essential to test antimicrobials in the environment they will be used in; further research including the use of simulated wound fluid and in vivo studies should be performed.
Silver has been used in wound care for a considerable time with scant reports of antimicrobial resistance. Resistance to AgNP has been reported in only 2 cases.46 While resistance to silver was induced in Enterobacteriaceae spp by increasing protein production to cause nanoparticle aggregation, resistance was not maintained when silver-selective pressure was removed.34 Drug-resistant and drug-susceptible strains of bacteria, including Pseudomonas aeruginosa, E coli, and Streptococcus pyogenes, were affected by AgNP in the same manner.34 Furthermore, there is potential synergy between AgNP and antibiotics; when tested against P aeruginosa, E coli, and Klebsiella pneumoniae several antibiotics were effective at concentrations below their reported MIC when combined with AgNP.47 Therefore, there is the potential for the use of AgNP against multi-drug resistant bacteria either with or without concomitant antibiotic administration.
MRSP was selected for use in the present study as MRSP are reported to be an increasingly common isolate in surgical site infections.48–51 E coli is amongst the most commonly cultured gram-negative isolates in surgical site infections, particularly those occurring after gastrointestinal surgery.49 Gram-positive bacteria are relatively resistant to AgNP compared to gram-negative bacteria. This has been observed for many bacterial species under a range of test conditions.2,8,27,30 The cell wall of gram-positive bacteria contains a thick, negatively charged peptidoglycan layer that may limit penetration of the cell by nanoparticles.3 Furthermore, hydroxyl and amido groups on gram-positive bacteria may chelate silver.46 Therefore, the difference in susceptibility between MRSP and E coli to AgNP in this study is expected to have resulted from inherent differences between gram-positive and gram-negative bacteria rather than use for a MRSP. However, testing both MRSP and non-MRSP may have provided greater clarity on the greater susceptibility of E coli to AgNP when compared to MRSP.
In this study, microdilution assay was selected to establish the expected antibacterial activity of AgNP to facilitate efficient testing of a range of concentrations for each trial. Interpretation of cloudiness of the microdilution assay wells to assess inhibition of bacterial growth was not possible as the AgNP solution affected the color of the media; thus, each well was sub-cultured to blood agar plates to determination of CFU by visual inspection. This study design meant that traditional methods to determine MIC and MBC could not be used which limits comparison to other studies on AgNP. Classification of the sub-cultured plates as growth or no-growth may have reduced the sensitivity of data. Other studies used a cutoff of <30 colonies to define no-growth, allowing a distinction between concentrations of AgNP that inhibited bacterial growth to some degree and those that provided no inhibition at all.43
The relationship between silver and wound healing is unclear, however, as with any antimicrobial agent the potential for cytotoxicity must be considered. In 1 study, fibroblast viability and collagen synthesis were reduced when cells were cultured with silver.52 Similarly, the percentage of viable fibroblasts was reduced when exposed to increasing concentrations of AgNP.53 However, other studies state the addition of silver to wound dressings promoted re-epithelialization and accelerated wound healing.52 Silver was reported to promote fibroblast differentiation and proliferation and migration of keratinocytes.1 An in vivo study utilizing mice supported the use of AgNP in wounds infected with S pseudintermedius.35 Despite conflicting literature, the greatest utility for AgNP remains wounds that are infected or at high risk for infection. Caution should be exercised in un-infected wounds, particularly those undergoing granulation and re-epithelialization.
Only 2% to 4% AgNP is retained in tissues after absorption by the body.34 However, extended exposure to silver can lead to argyria and argyrosis. In vitro cell culture studies have indicated the toxic effects of AgNP in bronchial epithelial cells, keratinocytes, erythrocytes, neuroblastoma cells, embryonic kidney cells, liver cells, and colon cells.3 In vivo studies have found AgNP accumulates in the liver, spleen, and lung of rodents, and can cross the blood-brain barrier.3 Furthermore, AgNP induced dose-dependent hematologic changes including red blood cell hemolysis at 30 μg/mL.54 Similar to the antibacterial effects of AgNP, particular nanoparticle characteristics such as polygonal shapes and smaller particle size were associated with greater cytotoxicity.36,55 Given the low rates of tissue retention reported for AgNP, used at appropriate doses, AgNP is considered safe for use in wounds.
There is a growing concern for the environmental impact of AgNP due to the marked increase in the production and use of these particles across a range of industries.32 AgNP oxidize to silver ions when they enter aquatic environments, which are toxic to aquatic organisms. They also form salts that can accumulate within marine food chains and be ingested by humans and other organisms.3 Nanoparticle synthesis through environmentally friendly means, known as green synthesis, has become increasingly popular to reduce the environmental footprint of nanoparticle fabrication.53 The potential risk to the environment should be established to develop protocols for safe use.
Limitations
This study did not assess the effect of the wound microenvironment on the efficacy and availability of AgNP, nor the ability of AgNP bound to a carrier to disperse throughout a wound bed. However, AgNP dispersion was assessed in a large volume of fluid (20 mL) which is expected to replicate the volume of fluid that may be found in a wound bed. The concentration of AgNP that could be used in this study was limited by the concentration of commercial dispersion selected for use and the maximum volume of liquid that could be added to the carriers. The range of bacterial concentrations tested varied in each trial due to miscalculations detected during data transcription for analysis. However, the results had utility due to the overlap in ranges of bacterial concentrations creating a set of concentrations that were repeatably tested. The unaltered poloxamer 407 gel exhibited antibacterial activity that confounded some results. Finally, the combination of poloxamer 407 gel and AgNP dispersion at a 1:1 ratio did not form a stable gel which may have influenced the elution and dispersion of AgNP throughout the broth. However, when incubated to 37 °C, this ratio of poloxamer 407 gel and AgNP dispersion did completely gel and it is possible that the compound formed a gel during bacterial culture incubation. A few plates were suspected to have shown growth due to contamination during plate preparation as subsequent plates from the same broth did not grow bacteria. In statistical analysis, these plates were treated as “growth” as their contamination could not be proven. Performing speciation on plates that grew bacteria but were suspected to have been the result of contamination during the study could have allowed these to be interpreted more accurately during statistical analysis. A small sample size was used due to financial limitations; therefore, statistical analysis may have been susceptible to type II error. Similarly, only 2 bacterial species were assessed. Evaluation of a broader range of bacterial species would be valuable to establish the lowest effective dose of commercially available AgNP against a range of bacteria; this could be investigated in future studies. MICs were not reported within this study; investigation and reporting of breakpoints is needed and would be valuable to establish the concentration of AgNP necessary to be used against these microbes, clinically.
Future directions
Assessment of the antibacterial activity of AgNP dispersion in an in vivo wound environment and assessment of the local and systemic toxicity of AgNP in animal models should be performed. Further assessment of the poloxamer 407 gel’s antibacterial activity should be assessed, including evaluation of any synergistic antibacterial activity between AgNP dispersion and poloxamer 407 gel. Future studies could be performed with higher concentrations of AgNP by obtaining dispersions of higher concentration or utilizing the novel mixing practice described to generate beads of higher AgNP concentration. This would facilitate testing against higher concentrations of bacteria, improving utility of this substance in a clinical setting. Finally, testing the activity of AgNP dispersion against a wider range of bacterial species should be performed to increase the scope of its utility.
Conclusion
Free AgNP at a concentration of 0.01 mg/mL inhibited growth of 101 MRSP and 103 E coli. Poloxamer 407 gel alone and combined with AgNP exerted the same degree of antibacterial activity against E coli and MRSP as free AgNP. AgNP combined with CSH beads or gelatin sponge had reduced activity against MRSP and E coli when compared to free AgNP. The degree of antibacterial activity shown by each test group was not different over time.
Clinical significance
AgNP dispersion has antibacterial activity against MRSP and E coli. The activity of AgNP against these bacterial species was diminished by incorporation with CSH beads and gelatin sponge, but not poloxamer 407 gel, which also had antibacterial activity against these species when used alone. Further studies are required to assess the utility of SR-AgNP in the clinical setting.
Acknowledgments
Dr. Bates’ current address is Arizona Regional Intensive Care, Specialty and Emergency Veterinary Center, Queen Creek, AZ.
Presented in part at the American College of Veterinary Surgeons Surgery Summit, October 13, 2022.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors have nothing to disclose.
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