Antimicrobial Activity of Silver-Treated Bacteria against Other Multi-Drug Resistant Pathogens in Their Environment

Silver is a potent antimicrobial agent against a variety of microorganisms and once the element has entered the bacterial cell, it accumulates as silver nanoparticles with large surface area causing cell death. At the same time, the bacterial cell becomes a reservoir for silver. This study aims to test the microcidal effect of silver-killed E. coli O104: H4 and its supernatant against fresh viable cells of the same bacterium and some other species, including E. coli O157: H7, Multidrug Resistant (MDR) Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus (MRSA). Silver-killed bacteria were examined by Transmission Electron Microscopy (TEM). Agar well diffusion assay was used to test the antimicrobial efficacy and durability of both pellet suspension and supernatant of silver-killed E. coli O104:H4 against other bacteria. Both silver-killed bacteria and supernatant showed prolonged antimicrobial activity against the tested strains that extended to 40 days. The presence of adsorbed silver nanoparticles on the bacterial cell and inside the cells was verified by TEM. Silver-killed bacteria serve as an efficient sustained release reservoir for exporting the lethal silver cations. This promotes its use as a powerful disinfectant for polluted water and as an effective antibacterial which can be included in wound and burn dressings to overcome the problem of wound contamination.


Introduction
Increasing bacterial resistance due to inappropriate use of antibiotics is one of the most important problems facing the modern scientific community [1]; moreover, the emergence of new resistant bacterial strains to current antibiotics has become a serious public health issue. This, has raised the need to develop new bactericidal materials [2]. Hence, there is an urgent need for new approaches to overcome antibiotic resistance and to develop alternate antimicrobial agents that can control infectious diseases, the spread of pathogens and which have long-term effectiveness [3]. One area to prolong antimicrobial activity is with the incorporation of antimicrobial agents within sustained-release delivery systems for their continuous use [4][5][6]. The objective of using new sustained-release drug delivery systems is to enhance the therapeutic effectiveness of drugs, by decreasing their side effects and increasing their bioavailability in certain sites. Among the various drug delivery systems, silver nanoparticles have been widely studied, indicating that the slow release of silver cations in trace amounts is toxic to bacteria and achieves prolonged biocidal activity [7,8]. Alternative drug delivery systems include liposomes, alginate based microparticles, and magnetic capsules. Silver nanoparticles

Determination of the Minimum Inhibitory Concentration of AgNO 3
The minimum inhibitory concentration (MIC) of silver nitrate was determined by agar well diffusion assay using AgNO 3 concentrations 40 ppm, 20 ppm, 10 ppm and 5 ppm with an inoculum of 1 × 10 6 cfu/mL. MIC values were evaluated after 24 h incubation at 37 • C by measuring zones of inhibition and averaged. The MIC was calculated by plotting the natural logarithm of the concentrations of AgNO 3 against the square of zones of inhibition. A regression line was drawn through the points. The antilogarithm of the intercept on the logarithm of concentration axis gave the MIC value [23,24].

Evaluating the Antibacterial Effect of Silver-Killed Bacteria
Liquid culture was prepared from E. coli O104:H4 isolate by inoculation into nutrient broth and incubation at 37 • C for 24 h. This overnight culture was centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and pellet was washed in pyrogen free water. The washing process by centrifugation was repeated for three times. Pellet was resuspended in 5 mL pyrogen-free water. The suspension was adjusted to 0.5 McFarland. The bacterial suspension (1.0 mL) was added to sterile centrifuge tubes containing 2 mL solution of AgNO 3 (3.0 mL total volume) at increasing final concentrations of AgNO 3 (1.5, 3, 6, 12, 18 ppm) and the mixture was incubated overnight at 37 • C under dark conditions for 6 h. Silver-treated bacteria were centrifuged at 4000 rpm for 10 min and the pellet was re-suspended in pyrogen free water. The supernatant was filtered using 0.2 µm syringe filter. Then, 2 mL of the supernatant was added to 1 mL of fresh viable E. coli O104:H4 culture and 2 mL of pellet suspension was added to 1 mL of the fresh viable E. coli O104:H4 culture. Both supernatant and pellet suspension containing cultures were incubated overnight at 37 • C. Mixtures were serially diluted (10-fold dilution) in saline and pour-plated. The plates were incubated at 37 • C for 24 h, the bacterial colonies were counted and compared to number of colonies of fresh viable E. coli O104:H4 culture (not treated) [25].

Heat-Treated Bacteria Control Test
The effect of E. coli O104: H4 treated with heat towards viable bacteria was performed by autoclaving the bacterial broth at 121 • C for 15 min, and then 2 mL of autoclaved bacterial broth were added to 1 mL of fresh viable E. coli culture for 24 h [25].

Transmission Electron Micrograph
Silver-killed cells of E. coli O104: H4 were centrifuged to separate them from their solution and then re-suspended in 1.0 mL saline. TEM was used to characterize the shape of the silver-killed cells. A drop of bacterial suspension was placed onto carbon-coated copper grid and this was dried in the air to get images for TEM analysis by electron microscope (Hitachi, Japan). All experiments were performed under sterile conditions and in triplicate [26].

Testing Antibacterial Effect of Silver-Killed E.coli O104:H4 Against other Bacterial Species
Agar well diffusion assay was used to test the effect of both silver-killed cells and its supernatant against E. coli O157:H7, MRSA, and MDR Ps. aeruginosa isolates.

In-Vitro Time Kill Assay
Fresh viable E. coli O104:H4 was used to inoculate tubes containing 8 mL of MHB with silver nitrate solution of a concentration of 6 ppm, pellet suspension and supernatant. A tube with MHB alone was inoculated as a control. Tubes were incubated at 37 • C with gentle shaking for defined times (0,1, 15, 30, 60, 90 and 120 min). One ml of bacterial suspension was withdrawn and serially diluted in MHB. Twenty-five microliters of each dilution were spotted on Mueller Hinton agar plates. The test is repeated in triplicate. The number of colony forming units (CFU/mL) was determined and averaged after overnight incubation of the plates at 37 • C [27].

Antimicrobial Efficacy and Durability of Silver Killed Bacteria And Supernatant Against the Tested Strains
Silver-killed E. coli O104:H4 bacteria and supernatant prepared using AgNO 3 at a concentration of 6 ppm were tested for their efficacy and durability against E. coli O104:H4, E. coli O157:H7, MDR Ps. aeruginosa and MRSA. The tested agents were prepared and stored in dark at 4 • C. The test is repeated in triplicate (zones of inhibition are averaged). First, baseline line zone was determined for each of the tested agents after their preparation. Then, the tested agents were stored in dark and tested for their antimicrobial activity after 1, 2, 6, 16, 22, 34, 40 days using agar well diffusion method. Zones of inhibition were measure and recorded as diameters in mm [28].

Statistical Analysis
Each experiment was done in triplicate. Data was represented as mean ± SD. One-Way ANOVA was employed to evaluate any significant difference between values obtained without the tested agents (controls) and those observed in the presence of silver-killed bacteria and supernatant. Differences were done using SPSS, 17 statistical software (SPSS Inc., Chicago, IL, USA).

Results and Discussion
Silver has long been used as antimicrobial agent in the treatment of infections, dating back to the 19th century. Specifically, silver nitrate has shown different effects against bacteria as at high concentrations, killing bacteria by different mechanisms, which are: binding to the thiol groups of protein and denaturing them, programmed cell death (apoptosis) and causing the DNA to be in the condensed form (not in the relaxed form), which inhibits cell replication. While at low concentrations, bacteria can synthesis silver nanoparticles [29]. With the nanoparticles, the size has been determined by electron microscopy; however, it is recognized that any further study will require additional assessment using a particle size analyzer.
In relation to the experimental methodology, the large inhibition zones obtained for the pellet and the supernatant against E. coli O104:H4 are shown in Figure 1   To demonstrate that the antibacterial activity of the pellet was due to the bacterial residue adsorbing silver and not due to toxins or enzymes produced by the bacterial cells, the effect of heatkilled bacteria (killed by autoclaving) on viable bacteria was examined. Heat-killed bacteria had no effect on the viable bacteria which suggests that silver-killed bacteria are reservoirs of silver ( Figure  2). This supports a study conducted by Wakshlak, et al. [25]. The killing activity of both killed bacteria and its supernatant was shown to increase with an increase in silver concentration. We noticed that at first, supernatant showed a killing activity lower than the effect of killed bacteria at concentrations of 1.5 and 3 ppm. Then, the killing activity of both killed bacteria and its supernatant tend to be close at the concentration of 6 ppm followed by increasing in the activity of the supernatant to a greater extent than the killing activity of killed bacteria at concentrations of 12 and 18 ppm ( Figure 3). These findings were close to that obtained by To demonstrate that the antibacterial activity of the pellet was due to the bacterial residue adsorbing silver and not due to toxins or enzymes produced by the bacterial cells, the effect of heat-killed bacteria (killed by autoclaving) on viable bacteria was examined. Heat-killed bacteria had no effect on the viable bacteria which suggests that silver-killed bacteria are reservoirs of silver ( Figure 2). This supports a study conducted by Wakshlak, et al. [25]. To demonstrate that the antibacterial activity of the pellet was due to the bacterial residue adsorbing silver and not due to toxins or enzymes produced by the bacterial cells, the effect of heatkilled bacteria (killed by autoclaving) on viable bacteria was examined. Heat-killed bacteria had no effect on the viable bacteria which suggests that silver-killed bacteria are reservoirs of silver ( Figure  2). This supports a study conducted by Wakshlak, et al. [25]. The killing activity of both killed bacteria and its supernatant was shown to increase with an increase in silver concentration. We noticed that at first, supernatant showed a killing activity lower than the effect of killed bacteria at concentrations of 1.5 and 3 ppm. Then, the killing activity of both killed bacteria and its supernatant tend to be close at the concentration of 6 ppm followed by increasing in the activity of the supernatant to a greater extent than the killing activity of killed bacteria at concentrations of 12 and 18 ppm (Figure 3). These findings were close to that obtained by The killing activity of both killed bacteria and its supernatant was shown to increase with an increase in silver concentration. We noticed that at first, supernatant showed a killing activity lower than the effect of killed bacteria at concentrations of 1.5 and 3 ppm. Then, the killing activity of both killed bacteria and its supernatant tend to be close at the concentration of 6 ppm followed by increasing in the activity of the supernatant to a greater extent than the killing activity of killed bacteria Antibiotics 2020, 9,181 6 of 14 at concentrations of 12 and 18 ppm (Figure 3). These findings were close to that obtained by Wakshlak and colleagues who explained these activities at low concentrations of silver nitrate as arising from the silver metal being chelated within the bacterial cell [25]. Chelation of silver within bacterial cell has a certain limit; the excess will be available in the supernatant increasing its killing activity against the tested microorganism, as chelation becomes limited and supernatant also shows biocidal activity.
Antibiotics 2020, 9, x FOR PEER REVIEW 6 of 14 Wakshlak and colleagues who explained these activities at low concentrations of silver nitrate as arising from the silver metal being chelated within the bacterial cell [25]. Chelation of silver within bacterial cell has a certain limit; the excess will be available in the supernatant increasing its killing activity against the tested microorganism, as chelation becomes limited and supernatant also shows biocidal activity. We used killed bacteria and supernatant, where their killing activities were approximately equal when tested against E. coli O157:H7, MDR Ps. aeruginosa and MRSA. It was found that a pellet suspended in deionized water and supernatant showed inhibition zones diameters that ranged from 16± 0.4 to 30 ± 0.23 mm. Also, we noticed that there are no significant differences in inhibition zone diameters obtained from neither killed bacteria nor its supernatant towards any of the tested organisms p > 0.05 (Supplementary file Table S1 & Figure 4). The activity of both killed bacteria and supernatant towards other microorganisms may be attributed to the fact that AgNps generate reactive oxygen species which have cytotoxic effect on the treated bacteria [30,31]. In addition, the presence of fresh viable bacteria acts as new, unoccupied site for silver, and so silver is shifted, according to Le-Chattelier principle, from the dead bacteria to the fresh ones [25] . In addition, when silver comes into contact with bacteria, AgNPs tends to accumulate at the bacterial membrane and form aggregates [32,33]. Other studies had supported this finding and found that the amount of adsorbed Ag+ ions and their aggregation as silver nanoparticles affects the antibacterial efficacy. As nanoparticles release ions, unless they consist of fully insoluble compounds, [34] and as a result, silver is released from nanoparticles by slow oxidation [35]. The release of silver depends on the size of the nanoparticles, their surface area, the temperature, and the composition of the surrounding medium [36,37]. In keeping with this, Xiu, et al. [38] found that in absence of dissolved molecular oxygen, there is no dissolution of silver nanoparticles. We used killed bacteria and supernatant, where their killing activities were approximately equal when tested against E. coli O157:H7, MDR Ps. aeruginosa and MRSA. It was found that a pellet suspended in deionized water and supernatant showed inhibition zones diameters that ranged from 16± 0.4 to 30 ± 0.23 mm. Also, we noticed that there are no significant differences in inhibition zone diameters obtained from neither killed bacteria nor its supernatant towards any of the tested organisms p > 0.05 (Supplementary file Table S1 & Figure 4). The activity of both killed bacteria and supernatant towards other microorganisms may be attributed to the fact that AgNps generate reactive oxygen species which have cytotoxic effect on the treated bacteria [30,31]. In addition, the presence of fresh viable bacteria acts as new, unoccupied site for silver, and so silver is shifted, according to Le-Chattelier principle, from the dead bacteria to the fresh ones [25]. In addition, when silver comes into contact with bacteria, AgNPs tends to accumulate at the bacterial membrane and form aggregates [32,33]. Other studies had supported this finding and found that the amount of adsorbed Ag+ ions and their aggregation as silver nanoparticles affects the antibacterial efficacy. As nanoparticles release ions, unless they consist of fully insoluble compounds, [34] and as a result, silver is released from nanoparticles by slow oxidation [35]. The release of silver depends on the size of the nanoparticles, their surface area, the temperature, and the composition of the surrounding medium [36,37]. In keeping with this, Xiu, et al. [38] found that in absence of dissolved molecular oxygen, there is no dissolution of silver nanoparticles.  TEM images of bacteria treated with silver nitrate clearly showed the accumulated silver as small nanoparticles having larger surface area distributed throughout the bacterium's cross section and released outside the cell. Silver nanoparticles not only adhered at the surface of cell membrane but also penetrated inside the bacterial cell ( Figure 5). Kooti, et al. [39] reported that silver had a significant effect on bacterial cell wall, changing the shape of cells from rods to cocci or irregular shapes, causing loss of cell wall integrity, releasing of cytoplasmic content, triggering swelling of bacterial cell, and finally cell lysis. Le Ouay and Stellacci [40] reported that nanoparticles that get close to a bacterium can release several tens of thousands of silver atoms in the organism's vicinity, producing a locally high concentration of antibacterial ions. This is known as the Trojan horse effect; and here adhesion and bioactivity of positively charged Ag + towards the negatively charged bacterial cell is due to electrostatic forces [41]. Silver nanoparticles, when penetrating the bacteria, interact with its DNA [2], inactivate its enzymes, generate hydrogen peroxide, causing bacterial cell death. In addition, the particles bind to functional groups of proteins, resulting in protein denaturation [10]. TEM images of bacteria treated with silver nitrate clearly showed the accumulated silver as small nanoparticles having larger surface area distributed throughout the bacterium's cross section and released outside the cell. Silver nanoparticles not only adhered at the surface of cell membrane but also penetrated inside the bacterial cell ( Figure 5). Kooti, et al. [39] reported that silver had a significant effect on bacterial cell wall, changing the shape of cells from rods to cocci or irregular shapes, causing loss of cell wall integrity, releasing of cytoplasmic content, triggering swelling of bacterial cell, and finally cell lysis. Le Ouay and Stellacci [40] reported that nanoparticles that get close to a bacterium can release several tens of thousands of silver atoms in the organism's vicinity, producing a locally high concentration of antibacterial ions. This is known as the Trojan horse effect; and here adhesion and bioactivity of positively charged Ag + towards the negatively charged bacterial cell is due to electrostatic forces [41]. Silver nanoparticles, when penetrating the bacteria, interact with its DNA [2], inactivate its enzymes, generate hydrogen peroxide, causing bacterial cell death. In addition, the particles bind to functional groups of proteins, resulting in protein denaturation [10]. A time kill assay was performed to test the effect of the tested agents on bacterial viability and to assess the time necessary to reach the bactericidal activity threshold. Figure 6 showed that pellet suspension and the supernatant caused a sudden drop in the average number of viable cells showing no colonies or growth after one minute for E. coli O104:H4 and after 15 min for Ps. aeruginosa and MRSA. On the other hand, the bactericidal action of silver nitrate solution at a concentration of 6 ppm was observed after 30 min. For E. coli O157:H7, pellet suspension and the supernatant showed bactericidal activity after 30 min with no detectable CFUs; while silver nitrate solution achieved the bactericidal effect against E. coli O157:H7 after 1 h. Das, et al. [42] found that bactericidal activity of AgNO3 was achieved after 8 h at MBC concentration against E. coli and S. aureus. Also, the researchers A time kill assay was performed to test the effect of the tested agents on bacterial viability and to assess the time necessary to reach the bactericidal activity threshold. Figure 6 showed that pellet suspension and the supernatant caused a sudden drop in the average number of viable cells showing no colonies or growth after one minute for E. coli O104:H4 and after 15 min for Ps. aeruginosa and MRSA. On the other hand, the bactericidal action of silver nitrate solution at a concentration of 6 ppm was observed after 30 min. For E. coli O157:H7, pellet suspension and the supernatant showed bactericidal activity after 30 min with no detectable CFUs; while silver nitrate solution achieved the bactericidal effect against E. coli O157:H7 after 1 h. Das, et al. [42] found that bactericidal activity of AgNO 3 was achieved after 8 h at MBC concentration against E. coli and S. aureus. Also, the researchers reported that silver nanoparticles were effective in inhibiting bacterial growth and reproduction in a dose and time dependent manner, which agrees with our study. In another study conducted by Yamanaka, et al. [43], AgNO 3 was shown to exhibit the bactericidal activity after 14 h. Pal, et al. [44] reported that the effect of silver nanoparticles was similar to that of the silver ions; in contrast, our results showed that pellet suspension containing the formed silver nanoparticle had higher activity than the tested silver nitrate.
Antibiotics 2020, 9, x FOR PEER REVIEW 9 of 14 reported that silver nanoparticles were effective in inhibiting bacterial growth and reproduction in a dose and time dependent manner, which agrees with our study. In another study conducted by Yamanaka, et al. [43], AgNO3 was shown to exhibit the bactericidal activity after 14 h. Pal, et al. [44] reported that the effect of silver nanoparticles was similar to that of the silver ions; in contrast, our results showed that pellet suspension containing the formed silver nanoparticle had higher activity than the tested silver nitrate.
(B) Ps. aeruginosa  Figure 7A,B showed the result of the efficacy and the durability of pellet (killed bacteria) suspension and the supernatant, against the tested bacteria. The baseline zones of inhibition diameters were determined after the preparation of the tested agents. Then, pellet suspension and supernatant were stored at 4 °C in dark. We tested the agent for its antibacterial activity at 1, 2, 6, 16, 22, 34 and 40 days. Results showed that pellet suspension and the supernatant continued to exhibit antibacterial activity until 40 days. Furthermore, the antibacterial activity of silver-killed E.coli O104:H4 cells and its supernatant was nearly the same against all the tested strains (no significant difference between the average zone of inhibition diameters produced by pellet suspension and the supernatant against each strain p > 0.05) after 24 h, 48 h, 6 days and 16 days against E.coli O157:H7, MRSA and MDR P. aeruginosa isolates, indicating the sustained release of silver ions from the silverkilled cells and its supernatant up to 40 days. While nanotoxicology research remain on-going, current data suggests there is not an on-going risk to human health once this time period has elapsed [14,34,45].  Figure 7A,B showed the result of the efficacy and the durability of pellet (killed bacteria) suspension and the supernatant, against the tested bacteria. The baseline zones of inhibition diameters were determined after the preparation of the tested agents. Then, pellet suspension and supernatant were stored at 4 • C in dark. We tested the agent for its antibacterial activity at 1, 2, 6, 16, 22, 34 and 40 days. Results showed that pellet suspension and the supernatant continued to exhibit antibacterial activity until 40 days. Furthermore, the antibacterial activity of silver-killed E.coli O104:H4 cells and its supernatant was nearly the same against all the tested strains (no significant difference between the average zone of inhibition diameters produced by pellet suspension and the supernatant against each strain p > 0.05) after 24 h, 48 h, 6 days and 16 days against E.coli O157:H7, MRSA and MDR P. aeruginosa isolates, indicating the sustained release of silver ions from the silver-killed cells and its supernatant up to 40 days. While nanotoxicology research remain on-going, current data suggests there is not an on-going risk to human health once this time period has elapsed [14,34,45].
Thus, small dose of silver is enough to kill high number of bacteria and it was observed that this element is released in a gradual and controlled manner to provide an adequate amount of the antimicrobial activity for an extended period of time. Testing both as soluble silver salts release silver ions when they come in contact with water and these silver ions are the biochemically active agent [46] which suggests its use in water disinfection. Guo, et al. [45] reported that the slower release of silver ions helps to prevent rapid depletion of the film. Such a slow and continuous release of Ag + ions was due to the adhesion forces between coatings and the substrates ensuring a lasting antibacterial performance. These researchers also investigated the different bond strength between anchored Ag + and electron donor (e.g., O, N and S), which resulted in different Ag + release rate. Another study showed a high release of silver from Ag NPs at the first day (0.35 μg/mL), inferring that Ag NPs can quickly prevent bacterial invasion when covering a wound site. Furthermore, constant silver ion release could still be observed at the seventh day, indicating that Ag NPs had prolonged and steady antibacterial activity, which could protect Thus, small dose of silver is enough to kill high number of bacteria and it was observed that this element is released in a gradual and controlled manner to provide an adequate amount of the antimicrobial activity for an extended period of time. Testing both as soluble silver salts release silver ions when they come in contact with water and these silver ions are the biochemically active agent [46] which suggests its use in water disinfection.
Guo, et al. [45] reported that the slower release of silver ions helps to prevent rapid depletion of the film. Such a slow and continuous release of Ag + ions was due to the adhesion forces between coatings and the substrates ensuring a lasting antibacterial performance. These researchers also investigated the different bond strength between anchored Ag + and electron donor (e.g., O, N and S), which resulted in different Ag + release rate. Another study showed a high release of silver from Ag NPs at the first day (0.35 µg/mL), inferring that Ag NPs can quickly prevent bacterial invasion when covering a wound site. Furthermore, constant silver ion release could still be observed at the seventh day, indicating that Ag NPs had prolonged and steady antibacterial activity, which could protect cutaneous wounds from infection [47]. In addition, a further study showed that a sustained-release silver dressing had faster wound healing and lower levels of pain for the patient, facilitating earlier hospital discharge. Moreover, this helped to reduce expenses for a burns-treating medical facility [48]. Therefore, modern silver dressings are set to become very important in coming years for burn treatment; thus, understanding the clinical efficacy coupled with the cost effectiveness will become a very important subject in wound management.

Conclusions
Silver-killed bacteria can act as effective weapons against viable bacteria which are present in their environment or media. This is, as literature suggests, by acting as a reservoir for adsorbed silver nanoparticles. This phenomenon may play an important role in the management of wound infections using dressings containing silver and for the disinfection of water using silver.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/4/181/s1. Table S1: Antimicrobial activity of Silver treated bacteria (killed bacteria and the supernatant) on Ps. aeruginosa, E. coli O157:H7 and MRSA using well agar diffusion method. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.