Antibacterial Activity of Colloidal Silver against Gram-Negative and Gram-Positive Bacteria

Due to the emergence of antimicrobial resistance, new alternative therapies are needed. Silver was used to treat bacterial infections since antiquity due to its known antimicrobial properties. Here, we aimed to evaluate the in vitro activity of colloidal silver (CS) against multidrug-resistant (MDR) Gram-negative and Gram-positive bacteria. A total of 270 strains (Acinetobacter baumannii (n = 45), Pseudomonas aeruginosa (n = 25), Escherichia coli (n = 79), Klebsiella pneumoniae (n = 58)], Staphylococcus aureus (n = 34), Staphylococcus epidermidis (n = 14), and Enterococcus species (n = 15)) were used. The minimal inhibitory concentration (MIC) of CS was determined for all strains by using microdilution assay, and time–kill curve assays of representative reference and MDR strains of these bacteria were performed. Membrane permeation and bacterial reactive oxygen species (ROS) production were determined in presence of CS. CS MIC90 was 4–8 mg/L for all strains. CS was bactericidal, during 24 h, at 1× and 2× MIC against Gram-negative bacteria, and at 2× MIC against Gram-positive bacteria, and it did not affect their membrane permeabilization. Furthermore, we found that CS significantly increased the ROS production in Gram-negative with respect to Gram-positive bacteria at 24 h of incubation. Altogether, these results suggest that CS could be an effective treatment for infections caused by MDR Gram-negative and Gram-positive bacteria.


Introduction
Infections caused by multidrug-resistant (MDR) Gram-negative and Gram-positive bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Staphylococcus spp., and Enterococcus spp. represent an increasing worldwide problem [1]. Currently, the appearance of MDR bacteria makes it impossible to find an effective drug to treat certain infectious diseases [2]. Therefore, there is an urgent need to find new therapeutic approaches in order to achieve better success in the bacterial infection treatment.
In this context, colloidal silver gained renewed interest. It was reported that colloidal silver can significantly reduce the duration and severity of many bacterial infections such as septic wounds [3]. This suspension of submicroscopic silver particles does not attack the bacteria directly, but causes a deactivation of enzymes responsible for their respiration, multiplication, and metabolism [4]. One of the main characteristics of silver is its oligodynamic effect, which is defined as the high microbicidal by broth microdilution assay according to the EUCAST (European Committee on Antimicrobial Susceptibility European Committee on Antimicrobial Susceptibility Testing) recommendations [24]. The initial bacterial inoculum 5 × 10 5 Colony Forming Unit (CFU)/mL for each strain was used in a 96-well plate (GreinerBioone, Germany) in the presence of colloidal silver, and incubated for 16-18 h at 37 • C. P. aeruginosa ATCC 27853 was used as a control strain. MIC 50 and MIC 90 , which represent the concentrations shown to be effective for ≥50% and ≥90% of isolates tested, respectively, were determined.

Time-Kill Kinetic Assays
Time-kill curves of susceptible A. baumannii ATCC 17978, P. aeruginosa PAO1, E. coli ATCC 25922, S. aureus Sa24, and E. faecalis VS (vancomycin-susceptible) strains, with an MIC of vancomycin of 0.5 mg/L, and MDR A. baumannii #11, P. aeruginosa Pa238, E. coli Ecmcr1+ (mcr1-producing), S. aureus USA300#1 (clon USA300), and E. faecalis VR (vancomycin-resistant) strains, with an MIC of vancomycin of 128 mg/L, were performed in duplicate as previously described [25]. Initial inoculums of 1 × 10 6 CFU/mL were conducted on Mueller Hinton broth (Sigma, Spain) (in presence of 0.5×, 1×, and 2× MIC of colloidal silver. Drug free broth was evaluated in parallel as a control. Tubes of each condition were incubated at 37 • C with shaking (180 rpm), and viable counts were determined by serial dilution at 0, 2, 4, 8, and 24 h. Viable counts were determined by plating 100 µL of control, test cultures, or the respective dilutions at the indicated times onto sheep blood agar plates (ThermoFisher, Spain). Plates were incubated for 24 h at 37 • C, and, after colony counts, the log 10 of viable cells (CFU/mL) was determined. Bactericidal was defined as a reduction of ≥3 log 10 CFU/mL with the initial inoculum.

Membrane Permeabilization Assay
Bacterial cells were grown in Luria Bertani Broth (Sigma, Spain) and incubated in the absence or presence of 0.25× MIC of colloidal silver for 24 h as previously described [26]. The pellet was harvested by ultracentrifugation at 4600× g for 15 min. Bacterial cells were washed with phosphate-buffered saline (PBS) 1×, and, after centrifugation in the same conditions described before, the pellet was resuspended in 100 µL of PBS 1× containing 10 µL of ethidium homodimer-1 (EthD-1) (ThermoFisher, Spain). After 10 min of incubation, 100 µL of mixture was placed into a 96-well plate to measure fluorescence at 0, 5, 10, 20, 30, 60, 90, 120, 240, and 300 min using a Typhoon FLA 9000 laser scanner (GE Healthcare Life Sciences, USA) and quantified by ImageQuant TL software (GE Healthcare Life Sciences, USA).

Statistical Analysis
Group data are presented as means ± standard errors of the means (SEM). The Student t-test was used to determine differences between means. A p-value < 0.05 was considered significant. The SPSS software, version 23.0 (IBM Corporation, Somers, New York, NY, USA) was used.

Antimicrobial Activity of Colloidal Silver
Colloidal silver was tested against reference and clinical strains of A. baumannii, P. aeruginosa, E. coli, K. pneumoniae, S. aureus, S. epidermidis, and Enterococcus spp. The MIC 50 and MIC 90 concentrations, which were shown to be effective for ≥50% and ≥90% of isolates tested , are presented in Table 1. The MICs for the Gram-negative bacteria strains ranged from 0.5 to >16 mg/L, while those for the Gram-positive strains ranged from 1 to >16 mg/L. The MIC 50 and MIC 90 for Gram-negative and Gram-positive strains ranged from 2 to 8 mg/L and 4 to 8 mg/L, respectively. These data show the antibacterial activity of colloidal silver.

Time-Kill Curves
Using time course assays, we examined the bactericidal activity of colloidal silver against susceptible and MDR strains of A. baumannii (ATCC 17978 and #11), P. aeruginosa (PAO1 and Pa238), E. coli (ATCC 25922 and Ecmcr1+), S. aureus (Sa24 and USA300#1), and E. faecalis (VS and VR). Figure 1A shows that 0.5×, 1×, and 2× MIC of colloidal silver presented bactericidal effects against susceptible and MDR A. baumannii strains at 8 h decreasing the bacterial count by >3 log 10 CFU/mL compared to the initial inoculum. These reductions persisted at 24 h at 0.5×, 1×, and 2× MIC of colloidal silver for the susceptible strain and at 2× MIC for the MDR strain. In the case of P. aeruginosa, 1× and 2× MIC of colloidal silver presented bactericidal effects against susceptible and MDR strains at 24 h ( Figure 1B). Regarding E. coli, colloidal silver at 1× and 2× MIC were bactericidal against susceptible and MDR strains at 8 h. These bactericidal activities persisted at 24 h for 2× MIC of colloidal silver against the susceptible strain, and for 1× and 2× MIC against the MDR strain ( Figure 1C). In the case of S. aureus and E. faecalis, only 2× MIC of colloidal silver presented bactericidal activity against susceptible and MDR strains at 24 h, although 1× MIC of colloidal silver was bactericidal against the susceptible E. faecalis strain at 24 h ( Figure 1D,E).

Colloidal Silver Effect on ROS Production
To assess whether colloidal silver caused an increase in cellular stress in the bacteria, the quantification of ROS was carried out by determining relative fluorescence units. The incubation of susceptible and MDR strains of A. baumannii, P. aeruginosa, and E. coli with 0.25×, 0.5×, and 1× MIC of colloidal silver increased progressively and significantly affected the production of ROS during 24 h when compared with untreated strains (Figure 1A-C). The positive controls (ampicillin and ciprofloxacin) also increased the production of ROS.

Colloidal Silver Effect on ROS Production
To assess whether colloidal silver caused an increase in cellular stress in the bacteria, the quantification of ROS was carried out by determining relative fluorescence units. The incubation of susceptible and MDR strains of A. baumannii, P. aeruginosa, and E. coli with 0.25×, 0.5×, and 1× MIC of colloidal silver increased progressively and significantly affected the production of ROS during 24 h when compared with untreated strains (Figure 1A-C). The positive controls (ampicillin and ciprofloxacin) also increased the production of ROS.
Regarding S. aureus and E. faecalis, lower but significant changes in the production of ROS were observed under different concentrations of colloidal silver during 24 h, except for the vancomycin-susceptible E. faecalis ( Figure 2D,E). The positive control (vancomycin) also increased the production of ROS.
Regarding S. aureus and E. faecalis, lower but significant changes in the production of ROS were observed under different concentrations of colloidal silver during 24 h, except for the vancomycinsusceptible E. faecalis ( Figure 2D,E). The positive control (vancomycin) also increased the production of ROS.

Colloidal Silver Effect on Bacterial Membrane Permeability
To evaluate whether colloidal silver is capable of causing any damage to the bacterial membrane, membrane permeability tests were performed. Susceptible and MDR strains of A. baumannii, P. aeruginosa, E. coli, K. pneumoniae, S. aureus, and E. faecalis were treated with 0.25× MIC colloidal silver and incubated with EthD-1. Five hours of fluorescence monitoring using a Typhoon scanner did not show an increase in the membrane permeability (data not shown).

Discussion
The emergence of MDR Gram-negative bacteria prompted the use of colistin, as the last resort in the treatment of severe infections by these pathogens. Although uncommon, colistin resistance is increasing and its spread is being considered a global health threat.
Due to the disruptive action of silver on bacteria reviewed by Barras et al. and its ability to bind to the sulfur atoms present in sulfhydryl groups of proteins and enzymes located on the bacterial cell surface [4], we hypothesized that colloidal silver may present antibacterial activity against Gram-negative and Gram-positive bacteria. In this study, we showed that colloidal silver presented bactericidal activity with MIC 90 values between 4 and 8 mg/L against a collection of 270 isolates of A. baumannii, P. aeruginosa, E. coli, S. aureus, S. epidermidis, and Enterococcus spp. These data are consistent with previous work reporting that colloidal silver was active against three reference strains of A. baumannii, P. aeruginosa, and S. aureus [27]. In other work, using Kirby-Bauer disc diffusion test, colloidal silver at 30 ppm was shown to be active against S. aureus, S. epidermidis, and Bacillus subtilis but not against E. coli [28].
It is noteworthy to mention that sub-MICs of colloidal silver did not significantly affect the membrane permeability of both Gram-negative and Gram-positive bacteria, in accordance with previously published data by Fenq et al. which showed that S. aureus is less permeable to silver ions when compared with E. coli [8]. In contrast, other studies showed that silver enhanced the cell permeability of a reference E. coli strain [7]. This difference in the membrane permeability results between both studies could be due to the fact that, in our study, we used MDR clinical isolates of E. coli that may have reduced membrane permeability.
Although, in our study, the colloidal silver at sub-MIC against the studied strain could not alter the bacterial membrane permeability, colloidal silver may increase the production of ROS such as ampicillin [29], ciprofloxacin [30,31], and vancomycin [32]. Barras et al. reported in their review that silver is a non-redox active metal that cannot directly produce ROS [4]. The production of ROS by silver occurred through the perturbation of the respiratory electron transfer chain [9], Fenton chemistry following destabilization of Fe-S clusters, or displacement of iron [7], and inhibition of anti-ROS defenses by thiol-silver bond formation [33]. We observed in our study that colloidal silver at lower and higher concentration produced ROS in Gram-negative bacteria and to a much lesser extent in Gram-positive bacteria, especially in E. feacalis, which may explain the lower bactericidal activity of colloidal silver against Gram-positive bacteria in time-kill curves assays. Similar results were observed by Kim et al., who reported that the differences in structure, thickness, and composition of cells between Gram-negative and Gram-positive can explain why E. coli shows substantial inhibition by silver nanoparticles, whereas S. aureus is less inhibited [34]. The antimicrobial potential of silver ions is influenced by the thickness and composition of the cell wall of the microorganisms, and the difference in the organization of the peptidoglycan layer [35]. Gram-negative bacteria contain lipopolysaccharides (LPS) in the cell membrane, which contributes to structural integrity of the membrane, in addition to protecting the membrane from chemical attacks. However, the negative charge of LPS promotes the adhesion of silver and renders the bacteria more susceptible to antimicrobial therapy [35]. Several studies showed the pronounced adhesion and deposition of silver onto the cell surface of Gram-negative bacteria in particular, due to the presence of LPS in their cell membrane [36]. In Gram-positive bacteria, the cell wall is composed of a negatively charged peptidoglycan layer, and the amount of peptidoglycan is comparatively higher in Gram-positive bacteria than Gram-negative bacteria [35]. The lower susceptibility of Gram-positive bacteria to antibiotic therapy can be explained on the basis of the fact that their cell wall is comparatively much thicker than that of Gram-negative bacteria [37]. The thicker cell wall of Gram-positive bacteria, as well as the negative charge of the peptidoglycan layer, allows the adhesion of silver ions. For this reason, S. aureus, which possesses a thick cell wall and more peptidoglycan molecules, prevents the action of the silver ions and renders the bacterium comparatively more resistant to silver [8].

Conclusions
The results of this study provide new insights into the use of colloidal silver against MDR Gram-negative and Gram-positive bacteria, where the therapeutic options are reduced. Nevertheless, further studies are needed in order to elucidate the action of colloidal silver in vivo, as well as to determine the optimal dosage to achieve, in terms of efficacy and safety, clinical efficacy in the treatment of infections by MDR Gram-negative and Gram-positive bacteria.