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Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate
Accepted Manuscript Title: Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate Author: Lindsay R. Kalan, Deanna M. Pepin, Imran Ul-Haq, Steve B. Miller, Michelle E. Hay, Rod Precht PII: DOI: Reference:
S0924-8579(17)30106-1 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.019 ANTAGE 5068
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
20-10-2016 22-1-2017
Please cite this article as: Lindsay R. Kalan, Deanna M. Pepin, Imran Ul-Haq, Steve B. Miller, Michelle E. Hay, Rod Precht, Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate
Lindsay R. Kalan *, Deanna M. Pepin 1, Imran Ul-Haq, Steve B. Miller, Michelle E. Hay, Rod Precht
Exciton Technologies Inc., Suite 4000, 10230 Jasper Avenue, Edmonton, Alberta, T5J 4P6, Canada
* Corresponding author. Present address: University of Pennsylvania, Perelman School of Medicine, Department of Dermatology, Philadelphia, PA 19104, USA. E-mail address: [email protected] (L.R. Kalan).
1
Present address: University of Alberta, Department of Agriculture, Food and Nutritional
Sciences, Edmonton, AB, Canada T6G 2P5.
ARTICLE INFO Article history: Received 20 October 2016 Accepted 22 January 2017
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Keywords: Antibiotic resistance Biofilm Antimicrobial dressing Wound Infection
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Highlights
Ag Oxysalt releases Ag2+ and Ag3+ ions.
Ag Oxysalt is bactericidal against planktonically growing bacteria.
Ag Oxysalt is bactericidal against established biofilms.
Ag Oxysalt is bactericidal against antibiotic-resistant biofilms.
Ag Oxysalt has a non-toxic safety profile.
ABSTRACT A topical antimicrobial, silver oxynitrate (Ag7NO11), has recently become available that exploits the antimicrobial activity of ionic silver but has enhanced activity because highly oxidised silver atoms are stabilised with oxygen in a unique chemical formulation. The objective of this study was to use a multifaceted approach to characterise the spectrum of antimicrobial and antibiofilm activity of a wound dressing coated with Ag7NO11 at a concentration of 0.4 mg Ag/cm2. Physiochemical properties that influence efficacy were also evaluated, and Ag7NO11 was found to release a high level of Ag ions, including Ag2+ and Ag3+, without influencing the pH of the medium. Time–kill analysis demonstrated that a panel of multidrug-resistant pathogens isolated from wound specimens remained susceptible to Ag7NO11 over a period of 7 days, even with repeated inoculations of 1 106 CFU/mL to the dressing. Furthermore, established 72h-old biofilms of Pseudomonas aeruginosa, Staphylococcus aureus and two carbapenem-resistant Gram-negative bacteria (blaNDM-1-positive Klebsiella pneumoniae and blaVIM-2-positive P. aeruginosa) were disrupted and eradicated by Ag7NO11 in vitro. Ag7NO11 is a proprietary compound that exploits novel Ag chemistry and can be
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considered a new class of topical antimicrobial agent. Biocompatibility testing has concluded Ag7NO11 to be non-toxic for cytotoxicity, acute systemic toxicity, irritation and sensitisation.
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1. Introduction In 1857, Dr J. Marion Sims declared to the President of the New York Academy of Medicine that ‘It is to revolutionize surgical dressings…with silver there is no inflammation, no suppuration’ [1]. Since then, silver (Ag) has held a fundamental place in medicine as one of the earliest antimicrobial agents employed to prevent and treat wound infection. Historically, uses of Ag have been limited to its elemental form [Ag0(s)] or as silver nitrate [AgNO3(s)], but considering the longstanding use of Ag, innovation in Ag technology has been incremental. One major advance in Ag technology was the introduction of nanocrystalline silver (NCS) dressings in the late 1990s [2,3] that demonstrated significantly greater efficacy than AgNO3 [4]. The increased surface area of NCS allows for enhanced surface oxidation and formation of silver oxide [Ag2O(s)], creating a reservoir of available Ag ions [Ag+(aq)] and release of hydroxide [OH–(aq)] upon contact with an aqueous fluid such as a wound bed. The result is a fast and highly efficacious activity against micro-organisms. The novelty in NCS technology lies in the mechanism of action of Ag itself, the Ag+ ion is required for antimicrobial activity [5,6] and the physical form of NCS allows for continued release while creating an unfavourable and basic environment for microbes to thrive. The success of NCS dressings led to an explosive increase in the use of Ag dressings for wound care and the development of subsequent technologies.
Like other transition metals, Ag+ also forms insoluble precipitates [most notably AgCl(s) and Ag2SO4(s)] that are stable and less reactive. It has been demonstrated that Ag chemistry significantly impacts antimicrobial efficacy [7], yet it is these Ag salts that are
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found in the majority of currently available advanced wound care products, resulting in confounding evidence for the supported use of Ag as a topical antimicrobial [8]. Whilst it is established that the solubility and availability of Ag+ is the primary driving factor of antimicrobial efficacy, there have been no major developments in the formulation and delivery of Ag to enhance the applicability and antimicrobial activity since the introduction of NCS nearly 20 years ago.
With a dwindling pipeline of new small-molecule antibiotics and the growing threat of resistance, particularly multidrug-resistant (MDR) Gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae (CRE), the pursuit of novel antimicrobials that can target these types of wound infections is one strategy to preserve the use of antibiotics for systemic infection and to minimise the spread of resistance. Enhancements to Ag formulations and wound dressings, such as the recent application of the silver oxynitrate [Ag7NO11(s) or Ag Oxysalt] compound that contains highly oxidised Ag (Ag2+/3+) stabilised by oxygen atoms, represents a recent major innovation to Ag technology with enhanced antimicrobial efficacy against MDR pathogens [7,9]. Synthesis of Ag Oxysalt requires deliberate oxidation of Ag+ and subsequent capture of the crystalline structure as a pure powder that can then be incorporated into a variety of substrates or matrices for topical delivery [9,10]. Upon interaction with fluid, highly unstable Ag2+ and Ag3+ ions are released, where the difference in the oxidising potential of Ag2+/3+ (E = 1.98 V and 1.8 V, respectively) compared with that of Ag+ (E = 0.8 V) results in access to different chemistry, as electrons are scavenged from microbial metabolic processes. The minimum biocidal concentration of pure Ag Oxysalt required
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for killing a broad spectrum of bacteria has been determined to be significantly lower than all other available Ag compounds. Importantly, the minimum concentrations required both to inhibit and eradicate bacteria growing as biofilms is also significantly lower [7].
Here we describe the antimicrobial and antibiofilm activity of a wound contact layer coated with Ag Oxysalt against a spectrum of Gram-positive and Gram-negative bacteria growing planktonically or in a biofilm. We demonstrate that with a 75% reduction of total silver used, the antimicrobial efficacy is equivalent or surpasses a wound dressing containing NCS. Finally, we show that Ag Oxysalt is efficacious against antibiotic-resistant organisms including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and carbapenem-resistant bacteria expressing the blaNDM-1, blaVIM-2 and blaKPC genes growing both planktonically and in a biofilm.
2. Methods 2.1. Wound dressings Commercially available wound dressings were used in this study as follows: Exsalt ® T7 and SD7 (Ag Oxysalt dressing, 0.4 mg Ag/cm2; Exciton Technologies Inc., Edmonton, Canada); Acticoat 7 (NCS 1.2–1.6 mg Ag/cm2; Smith and Nephew, Hull, UK); Aquacel® Ag and Aquacel® Ag+ ExtraTM (AgCl, 0.083–0.09 mg Ag/cm2; ConvaTec, Greensboro, NC); Mepilex Ag (Ag2SO4, 1.2 mg Ag/cm2; Mölnlycke, Göteburg, Sweden); Silverlon
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(Ag0, 5.46 mg Ag/cm2; Argentum Medical, Geneva, IL); and TheraBond® (Ag0, 15% Ag w/w; Alliqua, Yardley, PA).
2.2. Bacterial strains A panel of Gram-negative (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and Gram-positive (Corynebacterium striatum, vancomycin-sensitive and vancomycin-resistant Enterococcus faecalis, S. aureus and Staphylococcus epidermidis) organisms were supplied by Keystone Labs (Edmonton, Canada). The MRSA isolate tested was isolated from an abscess and was supplied by CanBiocin Inc. (Edmonton, Canada). Pseudomonas aeruginosa ATCC 9027 and S. aureus ATCC 6538 were used for biofilm growth. The antimicrobialresistant strains listed in Table 1 were originally isolated from cutaneous wounds and were kindly supplied by Dr Robert Rennie (Alberta Provincial Laboratory for Public Health, Edmonton, Canada).
2.3. Time–kill curves and log reduction Time–kill log reduction analysis was performed at Keystone Labs. Briefly, from overnight cultures, bacteria inocula were made to 1 106 CFU/mL in maximum recovery diluent (composed of 0.9% NaCl and 0.1% peptone). Dressings were cut to 2.5 cm 2.5 cm and were exposed to 5 mL of each suspension. A single reaction tube in duplicate was set up for each time point. After the specified exposure period (0.5, 1, 2, 3 and 4 h), a sample was removed from the reaction tube and was diluted 1/5 into a
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universal antimicrobial neutralisation fluid [0.4% sodium thioglycolate in saline (STS)] to bind residual silver ions that may carry over from the reaction vessel and interfere with the quantification of viable cells. Serial dilutions were made and were plated onto Mueller–Hinton agar (MHA) to count viable cells and to calculate log reduction from the negative control inoculum. The 4-h log reduction of MRSA followed the same general procedure except 5 cm 5 cm cut dressings were submerged in 10 mL of simulated wound fluid (SWF) (50:50 fetal calf serum:peptone water) inoculated to 1 106 CFU/mL from an overnight culture of the MRSA isolate (in triplicate). Following 4 h of incubation at 37 C, a sample was removed and was diluted 1/10 in 0.4% STS, was serially diluted and was plated for viable cell counts. Log reduction was calculated by subtracting the viable cell counts after exposure to the dressing from the viable cell counts of the inoculum or in the case of longitudinal studies from the negative control vessel, and plotting the log transformed values.
2.4. Biofilm log reduction assay Biofilms were grown on three to five layers of sterile cotton gauze and were placed in SWF in 6-well tissue culture plates for each strain tested. A 1 106 CFU/mL inoculum was added every 24 h up to 72 h and the plates were incubated at 37 C with shaking at 200 rpm. After the incubation period, the gauze was removed from the liquid culture medium, was rinsed three times with sterile water and was placed onto the surface of a MHA plate (Oxoid, Nepean, Canada). The gauze biofilms were overlaid with additional MHA cooled to ca. 50 C such that one-half of the biofilms were embedded in the agar and one-half were exposed. Dressings (5 cm 5 cm) were overlaid onto the biofilms
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and were exposed at 37 C for 4 h or 24 h. After the exposure time, the dressings and biofilms (gauze pieces) were carefully removed from the plates and were placed into 10 mL of 0.4% STS. They were vortexed (3 1 min) to disrupt the biofilm, were serially diluted in saline (0.9% saline) and were spot-plated onto MHA for viable cell counts. Negative controls were made as follows: (i) gauze biofilms were grown and were overlaid with agar as described above; and (ii) blank dressings (porous polyester fibre material that Exsalt® T7 is composed of) but without the addition of Ag Oxysalt were used to cover the biofilms in the same manner as the treatment conditions.
2.5. Scanning electron microscopy (SEM) Biofilm growth and attachment to the sterile cotton gauze was confirmed by SEM. Following growth of biofilms, they were rinsed as above to remove planktonic cells and were fixed with methanol. Biofilms were cut into 5 mm 5 mm squares, were coated (6– 8 nm) with iridium prior to imaging and were imaged with a Hitachi S-4800 highresolution scanning electron microscope (Hitachi Canada, Mississauga, ON, Canada) housed at the National Institute for Nanotechnology (NINT) in Edmonton, Canada.
2.6. Silver release and pH measurements Silver release was measured by two methods. Data in Fig. 1 were measured by potentiometric titration with 0.1 M NaCl titrant. Dressings (5 cm 5 cm) were submerged in 10 mL of reverse osmosis (RO) H2O for the specified time period in triplicate. The fluid was then titrated against NaCl to determine the quantity of free Ag+
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ions in the solution. In Supplementary Fig. S1, silver release in RO H2O or SWF was measured by neutron activation analysis at the University of Alberta SLOWPOKE reactor. Reactions were set up as described above; triplicate samples were pooled prior to analysis. Higher oxidation state silver is detected by exposure of silver dressings to 1:3 HNO3:RO H2O and recording the colour change. As shown in the equations of Fig. 1C, a colour change is only observed for Ag2+ and Ag3+ ions. Furthermore, pH measurements were recorded by setting up vessels as above in triplicate and recording the pH with indicator strips after specified time intervals.
3. Results 3.1. Ag Oxysalt has a broad-spectrum activity To evaluate the speed and biocidal activity of dressings coated with Ag Oxysalt, a 4-h time course was designed, quantifying viable cell counts at 0.5, 1, 2, 3 and 4 h of exposure. A panel of Gram-negative (A. baumannii, E. coli, K. pneumoniae and P. aeruginosa) and Gram-positive (C. striatum, vancomycin-sensitive and vancomycinresistant E. faecalis, S. aureus and S. epidermidis) organisms were evaluated in peptone water. The efficacy was compared with NCS and was found to be equivalent for all organisms tested, with counts below the limit of detection (LOD) within 4 h for all organisms tested (Fig. 1A). We were also interested in assessing the solubility or availability of silver ions released from an Ag Oxysalt or NCS dressing because the ion availability is critical to antimicrobial efficacy. After 4 h, the Ag released from a 5 cm 5 cm dressing was not significantly different for Ag Oxysalt or NCS (0.4 mg Ag/cm2 vs.
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1.2–1.6 mg/cm2). However, the ppm Ag+ released per mg of total Ag on the dressing was significantly higher for the Ag Oxysalt (t-test, P = 0.0057) (Fig. 1B). The oxidation state of Ag released from each dressing was also assessed, with higher oxidation state (Ag2+/3+) released from the Ag Oxysalt dressing and Ag+ from the NCS dressing as observed by a colour change when exposed to a solution of nitric acid (HNO3) (Fig. 1C) [11–14]. Both dressings have been approved for a 7-day wear time so we measured the release of Ag over a 7-day period in RO H2O and a more complex SWF of 1:1 fetal calf serum:peptone water. SWF was tested because it better mimics a wound environment that contains ions (such as chlorides) and proteins (with thiol groups) which are able to bind and inactivate free Ag ions. We found that while both dressings release Ag for this length of time in both types of fluid, the Ag Oxysalt dressing is mostly consumed by Day 7 while the NCS dressing contains >50% of the starting Ag (38% vs. 86% starting Ag remaining), mostly converted to elemental Ag (Supplementary Fig. S1) as determined by X-ray diffraction (data not shown). Solubilising silver oxide releases hydroxide ions, so we assessed changes in the pH of media exposed to the NCS and Ag Oxysalt dressings because drastic pH changes could contribute to antimicrobial kill rates but also cause toxicity. The pH of either saline or SWF (50:50 fetal calf serum:peptone water) exposed to each dressing was monitored over time. In saline, the NCS dressing resulted in a highly alkaline environment at pH 11.5 within the first minute of exposure, eventually stabilising at pH 9 after 4 h. In SWF, the NCS dressing elevated to pH 9.5 within 30 min, whilst the Ag Oxysalt dressing remained below pH 9 and closer to physiological pH in both media types (Supplementary Fig. S1). Previously, it has been reported that the NCS dressing exhibits cytotoxicity to keratinocytes and fibroblasts in
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culture as Ag ions are released into the medium and this ultimately results in inhibition of re-epithelialisation [15]. To fully assess the safety and toxicity profile of the Ag Oxysalt dressing, a full cytotoxicity testing regimen was undertaken in vitro and in vivo (acute system toxicity, in vitro cytotoxicity, minimal essential medium cytotoxicity, irritation and sensitisation) and in all cases the dressing was found to be non-toxic (Supplementary methods; Supplementary Table S1).
3.2. Ag Oxysalt is active against antibiotic-resistant organisms Antibiotic-resistant organisms such as MRSA are a major concern for wound infection. Because we found Ag Oxysalt and NCS dressings were biocidal to methicillin-sensitive S. aureus in 4 h, we wondered whether MRSA would have the same susceptibility. As shown in Fig. 2, a clinical MRSA isolated from an abscess was susceptible both to the Ag Oxysalt and NCS dressing after 4 h of exposure, but not to dressings containing other types of Ag compounds (Ag0, AgCl and Ag2SO4). The Ag Oxysalt dressing is approved for wear up to 7 days so the efficacy against MDR clinical isolates was tested over a 7-day period. Every 24 h the dressings (the same dressing in the same vessel and medium each day) were inoculated with 1 106 CFU/mL and the log reduction values were determined by sampling for quantitative counts every 24 h just prior to the new inoculation. The experimental design represents the extremes where in a clinical setting it is not expected that a wound may be contaminated or inoculated with this level of bioburden each day. The log reduction values over the 7-day period are summarised in Table 1, showing that the Ag Oxysalt dressing was effective in reducing levels of all species tested including MRSA, VRE (VanA) and CRE.
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3.3. Ag Oxysalt kills multidrug-resistant biofilms It has become increasingly evident that the formation of a biofilm in the wound bed contributes to stalled healing, persistent inflammation and a chronic non-healing wound [16–19]. We previously reported that pure Ag Oxysalt powder can both inhibit biofilm formation and eradicate mature biofilms at concentrations significantly lower than other Ag compounds [7]. To expand on this study, the ability of Ag Oxysalt adhered to a substrate (contact layer dressing) to target and reduce biofilms was evaluated. In vitro testing is not always clinically relevant as the environment of a wound is difficult to mimic in the laboratory. To create the most clinically relevant assay, we adapted and enhanced upon a model reported by Seth et al. [20].
Micro-organisms will grow with different biogeography within the three-dimensional (3D) wound bed so it is important to test for antibiofilm activity within a 3D structure. Biofilms were grown over a 72-h period onto multiple layers of cotton gauze and were confirmed by SEM (Fig. 3A,C,D). The gauze biofilms were then embedded into nutrient agar plates before dressings were applied (Fig. 3B). After the specified exposure time, the dressing and gauze were extracted and quantitative counts of viable cells were determined.
Pseudomonas aeruginosa is a prolific biofilm-forming organism, so we first tested the ability of the Ag Oxysalt and NCS dressing to reduce 72-h gauze biofilms. Recently, a hydrofibre (HF) dressing containing AgCl, the divalent cation chelator ethylene diamine tetra-acetic acid (EDTA) and a surfactant benzalkonium chloride (BC) was introduced
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as an antibiofilm dressing, therefore this HF dressing was used as a comparator for the experiment. The Ag Oxysalt dressing resulted in zero counts after a 4-h exposure, significantly lower than the negative control (uncoated substrate) (Tukey’s post-hoc test, P < 0.0001) and the NCS dressing treatment (Tukey’s post-hoc test, P = 0.026) (Fig. 4A). Staphylococcus aureus was also able to form biofilms, so we evaluated the ability of the dressings to reduce viable biofilm counts after a 4-h exposure and noted that the Ag Oxysalt dressing resulted in counts below the LOD (Fig. 4A).
To confirm that the observed biocidal activity is not diminished in MDR isolates, the experiment was repeated with clinical isolates of carbapenem-resistant K. pneumoniae (blaNDM-1) and P. aeruginosa (blaVIM-2) exposed to the Ag Oxysalt-coated wound contact layer or the HF AgCl/EDTA/BC-containing antibiofilm dressing. Both drug-resistant isolates were effectively reduced after 24 h of exposure to the dressings (Fig. 4B). We were curious whether these two dressings were different in their speed of kill because of the considerable differences in dressing construction and composition of antimicrobial agents. The HF dressing has a large absorptive capacity and therefore can reduce cell counts by sequestration in the fibres as they gel and by the combined action of EDTA and BC. The wound contact layer, on the other hand, relies solely on the action and availability of Ag released from the Ag Oxysalt crystals coated onto the surface of polyester fibres with little to no absorptive capacity. The antibiofilm activity against P. aeruginosa biofilms was tested for a 2-h exposure time and the Ag Oxysalt dressing reduced the viable cells counts significantly greater than the HF AgCl/EDTA/BC dressing (log reduction of 3.86 ± 0.7 vs 0.4 ± 0.5; Tukey’s post-hoc test, P = 0.007).
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4. Discussion The formation of microbial biofilms in wounds is a major barrier to healing progression [21], particularly in individuals with a reduced ability to overcome infection. Treatment with antimicrobials is challenging in chronic non-healing wounds of the lower extremities, biofilm formation results in increased tolerance to penetration of active agents, and MDR organisms are becoming increasingly problematic with limited therapeutic options available. Other factors such as poor perfusion can limit the delivery of systemic antibiotics to the wound surface and further increase the complexity in treating these wounds [22,23]. Topical antimicrobials act to control microbial bioburden directly and silver has long been used for this purpose in wound care. Here we describe a new formulation of silver that contains higher oxidation state Ag ions. The current data support previous reports that not all silver is equivalent [24,25] and we show that wound dressings coated with Ag Oxysalt have efficacy against planktonic and biofilm populations at lower overall quantities of silver than commercially available silver wound dressings. Furthermore, the compound maintains efficacy against antibiotic-resistant organisms including MRSA and CRE isolated from cutaneous wounds.
Clinical use of silver dressings has been controversial in relation to their ability to reduce bioburden and contribute to improved wound healing. We argue that this is due in part to an assumption that all silver dressings have the same general mechanism of action, and testing methodologies to evaluate microbial susceptibility have not been standardised in the same manner as pharmaceutical antibiotics [26]. For example, NCS
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has been reported to achieve a 4 log reduction of S. aureus in 30 min [24]; however, as shown in Fig. 1, a 4-h exposure time was required to achieve a 4 log reduction with our method. The methodology employed by Cavanagh et al. [24] involves wetting the dressings with sterile water and inoculating it with 0.06 mL/cm2 of bacterial suspension. In our methodology, a more complex peptone medium was used and the dressings were inoculated with 0.4 mL/cm2 of bacterial suspension, resulting in excess fluid saturating each dressing. The choice of inoculum volume and medium can significantly change the outcome. Silver availability is greatly reduced in a complex SWF owing to binding of free Ag ions by proteins and other ions, notably the complex with chloride ions results in formation of AgCl(s). This is demonstrated in Supplementary Fig. S2. Our method also demonstrates that with the exception of the NCS and Ag Oxysalt dressing, dressings containing other silver formulations do not result in any significant levels of MRSA reduction in this 4-h exposure period (Fig. 2). As a result of NCS chemistry, exposure of an NCS dressing to fluid releases hydroxide ions that elevated the pH and contribute to cellular toxicity. To alleviate discomfort to the wound, the dressing is typically wet with water to dissipate the OH– prior to application. On the other hand, an Ag Oxysalt dressing can utilise much lower silver content and still maintain high antimicrobial efficacy with a non-cytotoxic safety profile. We acknowledge that the dressing construction also influences delivery of silver and thus efficacy is multifactorial.
Biofilms and antibiotic resistance are two serious considerations for wound infection. CRE are a major global health concern [27–29], with mortality rates reaching 50% owing to a deficiency in antibiotics to which the organisms remain susceptible [28]. We
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have demonstrated that the Ag Oxysalt-coated dressing is able to disrupt and kill common wound biofilm pathogens P. aeruginosa and S. aureus more effectively than an NCS dressing containing a four-fold greater silver concentration. CRE clinical wound isolates growing as a biofilm are also susceptible to the Ag Oxysalt dressing (Fig. 4). The efficacy is equivalent to a HF dressing containing a synergistic combination of EDTA, BC and AgCl targeting biofilms. The equivalent HF dressing containing only silver (AgCl) is not active against biofilms [20]. MRSA and VRE also remain susceptible to the Ag Oxysalt dressing.
New therapeutic options for preventing and controlling wound infection are needed as the incidence of diseases such as diabetes increases, where formation of non-healing ulcers contributes to 80% of non-traumatic lower-extremity amputations and 5-year mortality rates exceed that of some cancers [30–32]. Silver remains a viable, non-toxic and effective topical antimicrobial option with exciting innovation and formulation improvements available. Mechanisms of silver resistance have been well described [33–36] but the incidence remains low, and Ag Oxysalt dressings appear to remain active against even the most resistant isolates [37]. The current data support the use of this new, low silver content, Ag Oxysalt dressing for the prevention and management of wound infection.
Acknowledgment: The authors would like to acknowledge Dr Andrew Myles for his support and thoughtful discussion.
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Funding: LRK was supported by Alberta Innovates Technology Futures R&D Associate Fellowship and NSERC Industrial R&D Fellowship. IU-H was supported by an Alberta Innovates NanoWorks Program grant.
Competing interests: All authors were employees of Exciton Technologies (Edmonton, Canada), the manufacturer of silver oxynitrate, at the time the work was completed.
Ethical approval: Not required.
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References [1] Sims JM, Emmet TA. Silver sutures in surgery: the anniversary discourse before the New York Academy of Medicine, delivered in the new building of the Historical Society on November 18, 1857. New York, NY: Samuel S. & William Wood; 1858. [2] Wright JB, Lam K, Burrell RE. Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment. Am J Infect Control 1998;26:572–7. [3] Tredget EE, Shankowsky HA, Groeneveld A, Burrell R. A matched-pair, randomized study evaluating the efficacy and safety of Acticoat silver-coated dressing for the treatment of burn wounds. J Burn Care Rehabil 1998;19:531–7. [4] Yin HQ, Langford R, Burrell RE. Comparative evaluation of the antimicrobial activity of ACTICOAT antimicrobial barrier dressing. J Burn Care Rehabil 1999;20:195–200. [5] Gibbard J. Public health aspects of the treatment of water and beverages with silver. Am J Public Health Nations Health 1937;27:112–9. [6] Xiu Z-M, Zhang Q-B, Puppala HL, Colvin VL, Alvarez PJJ. Negligible particlespecific antibacterial activity of silver nanoparticles. Nano Lett 2012;12:4271–5. [7] Lemire JA, Kalan L, Bradu A, Turner RJ. Silver oxynitrate, an unexplored silver compound with antimicrobial and antibiofilm activity. Antimicrob Agents Chemother 2015;59:4031–9. [8] Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P, Stevenson M. Randomized controlled trial and cost-effectiveness analysis of silver-donating
Page 20 of 28
antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 2009;96:1147–56. [9] Djokić SS. Deposition of silver Oxysalts and their antimicrobial properties. J Electrochem Soc 2004;151:C359–64. [10]
Jack JC, Kennedy T. The thermal analysis of 'argentic oxynitrate' and
silver oxides. J Thermal Analysis 1971;3:25. [11]
Noyes AA, Hoard JL, Pitzer KS. Argentic salts in acid solution. I. The
oxidation and reduction reactions. J Am Chem Soc 1935;57:1221–9. [12]
Noyes AA, Pitzer KS, Dunn CL. Argentic salts in acid solution. II. The
oxidation state of argentic salts. J Am Chem Soc 1935;57:1229–37. [13]
Noyes AA, Kossiakoff A. Argentic salts in acid solution. III. Oxidation
potential of argentous–argentic salts in nitric acid solution. J Am Chem Soc 1935;57:1238–42. [14]
Po HN, Swinehart JH, Allen TL. Kinetics and mechanism of the oxidation
of water by silver(II) in concentrated nitric acid solution. Inorg Chem 1968;7:244– 9. [15]
Burd A, Kwok CH, Hung SC, Chan HS, Gu H, Lam WK, et al. A
comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models. Wound Repair Regen 2007;15:94–104. [16]
Wolcott RD, Dowd S, Kennedy J, Jones CE. Biofilm-based wound care.
In: Sen CK, editor. Advances in wound care, Vol. 1. New Rochelle, NY: Mary Ann Liebert Inc., 2010. Chapter 54.
Page 21 of 28
[17]
Metcalf DG, Bowler PG. Biofilm delays wound healing: a review of the
evidence. Burns Trauma 2013:1:5–12. [18]
Percival SL, McCarty SM, Lipsky B. Biofilms and wounds: an overview of
the evidence. Adv Wound Care (New Rochelle) 2015;4:373–81. [19]
Zhao G, Usui ML, Lippman SI, James GA, Stewart PS, Fleckman P, et al.
Biofilms and inflammation in chronic wounds. Adv Wound Care (New Rochelle) 2013;2:389–99. [20]
Seth AK, Zhong A, Nguyen KT, Hong SJ, Leung KP, Galiano RD, et al.
Impact of a novel, antimicrobial dressing on in vivo, Pseudomonas aeruginosa wound biofilm: quantitative comparative analysis using a rabbit ear model. Wound Repair Regen 2014;22:712–9. [21]
James GA, Swogger E, Wolcott R, Pulcini ED, Secor P, Sestrich J, et al.
Biofilms in chronic wounds. Wound Repair Regen 2008;16:37–44. [22]
Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res
2010;89:219–29. [23]
Lipsky BA. Medical treatment of diabetic foot infections. Clin Infect Dis
2004;39(Suppl 2):S104–14. [24]
Cavanagh MH, Burrell RE, Nadworny PL. Evaluating antimicrobial efficacy
of new commercially available silver dressings. Int Wound J 2010;7:394–405. [25]
Gallant-Behm CL, Yin HQ, Liu S, Heggers JP, Langford RE, Olson ME, et
al. Comparison of in vitro disc diffusion and time kill-kinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Repair Regen 2005;13:412–21.
Page 22 of 28
[26]
Clinical and Laboratory Standards Institute. Performance standards for
antimicrobial susceptibility testing; sixteenth informational supplement. Document M100-S16. Wayne, PA; CLSI; 2006. [27]
Temkin E, Adler A, Lerner A, Carmeli Y. Carbapenem-resistant
Enterobacteriaceae: biology, epidemiology, and management. Ann N Y Acad Sci 2014;1323:22–42. [28]
Patel G, Huprikar S, Factor SH, Jenkins SG, Calfee DP. Outcomes of
carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol 2008;29:1099–106. [29]
Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL,
Cormican M, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013;13:785–96. [30]
Armstrong DG, Wrobel J, Robbins JM. Guest editorial: are diabetes-
related wounds and amputations worse than cancer? Int Wound J 2007;4:286–7. [31]
Robbins JM, Strauss G, Aron D, Long J, Kuba J, Kaplan Y. Mortality rates
and diabetic foot ulcers. Is it time to communicate mortality risk to patients with diabetic foot ulceration? J Am Podiatr Med Assoc 2008;98:489–93. [32]
Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global
burden of diabetic foot disease. Lancet 2005;366:1719–24. [33]
Silver S. Bacterial silver resistance: molecular biology and uses and
misuses of silver compounds. FEMS Microbiol Rev 2003;27:341–53.
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[34]
Gupta A, Matsui K, Lo JF, Silver S. Molecular basis for resistance to silver
cations in Salmonella. Nat Med 1999;5:183–8. [35]
Asiani KR, Williams H, Bird L, Jenner M, Searle MS, Hobman JL, et al.
SilE is an intrinsically disordered periplasmic 'molecular sponge' involved in bacterial silver resistance. Mol Microbiol 2016;101:731–42. [36]
Woods EJ, Cochrane CA, Percival SL. Prevalence of silver resistance
genes in bacteria isolated from human and horse wounds. Vet Microbiol 2009;138:325–9. [37]
Finley PJ, Norton R, Austin C, Mitchell A, Zank S, Durham P.
Unprecedented silver resistance in clinically isolated Enterobacteriaceae: major implications for burn and wound management. Antimicrob Agents Chemother 2015;59:4734–41.
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Fig. 1. Ag Oxysalt dressings have a 4 log reduction within 4 h. (A) Time–kill curves of common wound-associated Gram-positive and Gram-negative bacteria over a 4-h period. Bacteria were exposed to dressings coated with nanocrystalline silver (NCS) or Ag Oxysalt (n = 2). The control reaction was blank without an antimicrobial. LOD, limit of detection. (B) Silver release into reverse osmosis (RO) H2O after a 4-h period of NCSand Ag Oxysalt-coated dressing (n = 3). Mean ppm Ag was determined by potentiometric titration, and mean ppm Ag/mg Ag was determined by dividing the mean ppm Ag by the total mg of Ag in each dressing. Error bars represent the standard deviation of triplicate measurements. (C) Ag Oxysalt-coated dressings contain higher oxidation states of Ag (Ag2+/3+), but NCS-coated dressings do not. The chemical reaction is represented.
Fig. 2. Ag Oxysalt dressings kill methicillin-resistant Staphylococcus aureus (MRSA). Commercially available silver dressings were exposed to an inoculum of 1 106 CFU/mL of MRSA in simulated wound fluid (SWF) at 37 C for 4 h. The control group represents the inoculum in medium only in the absence of an antimicrobial dressing. Only dressings containing nanocrystalline silver (NCS) (ca. 40 mg Ag) or Ag Oxysalt (10 mg Ag) significantly reduced the viable cell count (P < 0.0001, t-test) to below the limit of detection (LOD) (n = 3).
Fig. 3. Gauze biofilm model. (A) Biofilms were grown on sterile cotton gauze by inoculating with 1 106 CFU/mL at 0, 24 and 48 h. (B) After 72 h at 37 C and 200 rpm, biofilms were embedded in Mueller–Hinton agar and were overlaid with the test
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dressing. (C,E) Biofilms at different magnification were confirmed by scanning electron microscopy (SEM) after rinsing the gauze with sterile water to remove planktonic cells. A biofilm of blaVIM-2-positive Pseudomonas aeruginosa is visualised attached to a fibre of the gauze (C) and individual cells observed within an extracellular matrix. (D) SEM image of sterile cotton gauze fibres not exposed to bacterial culture but incubated in medium.
Fig. 4. Ag Oxysalt dressings kill carbapenem-resistant Enterobacteriaceae (CRE) biofilms. (A) Biofilms of Pseudomonas aeruginosa ATCC 9027 or Staphylococcus aureus ATCC 6538 were exposed to a non-coated control, nanocrystalline silver (NCS), hydrofibre AgCl/EDTA/BC or Ag Oxysalt dressings for 4 h at 37 C and viable cell counts were determined (n = 3). (B) Biofilms of blaNDM-1-positive Klebsiella pneumoniae or blaVIM-2-positive P. aeruginosa were exposed to a non-coated control, hydrofibre AgCl/EDTA/BC or Ag Oxysalt dressing for 24 h at 37 C and viable cell counts were determined (n = 3). EDTA, ethylene diamine tetra-acetic acid; BC, benzalkonium chloride.
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Supplementary Fig. S1. pH changes upon exposure to a silver dressing. Changes to the pH of saline or simulated wound fluid (SWF) (50% fetal calf serum; 50% 0.1% peptone water) was measured after exposure to an Ag Oxysalt or nanocrystalline silver (NCS) dressing. Cut dressings (5 cm 5 cm) were saturated in 10 mL of fluid.
Supplementary Fig. S2. 7-Day silver release. Silver release from a nanocrystalline silver (NCS) or Ag Oxysalt dressing was determined by neutron activation analysis over a period of 7 days with measurements on Days 1, 3, 5 and 7. Triplicate analyses were pooled prior to quantification. Error bars represent standard errors of measurements. RO, reverse osmosis H2O; SWF, simulated wound fluid.
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Table 1. Log reduction values over 7 days (1 106 CFU/mL inoculations occurred every 24 h) of antibiotic-resistant wound isolates exposed to a wound dressing coated with 0.4 mg/cm2 silver oxynitrate Organism
Day
Day
Day
Day
Day
Day
Day
1
2
3
4
5
6
7
Stenotrophomonas maltophilia
4.79
7.45
7.78
7.80
7.53
7.84
7.38
Staphylococcus aureus (mecA)
4.86
3.56
3.95
3.84
3.55
3.14
4.88
Pseudomonas aeruginosa
3.92
7.08
5.98
4.62
5.87
5.21
5.71
Klebsiella pneumoniae (blaKPC)
3.89
6.76
5.94
6.17
5.39
5.46
4.70
K. pneumoniae (blaNDM-1)
3.31
6.70
6.93
6.44
6.35
6.32
6.41
Escherichia coli
4.60
2.76
3.55
3.98
5.02
5.89
5.24
Enterococcus faecalis (vanA)
4.00
5.73
4.93
4.67
4.53
4.82
5.41
(blaVIM-2)
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S0924-8579(17)30106-1 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.019 ANTAGE 5068
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
20-10-2016 22-1-2017
Please cite this article as: Lindsay R. Kalan, Deanna M. Pepin, Imran Ul-Haq, Steve B. Miller, Michelle E. Hay, Rod Precht, Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.01.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Targeting biofilms of multidrug-resistant bacteria with silver oxynitrate
Lindsay R. Kalan *, Deanna M. Pepin 1, Imran Ul-Haq, Steve B. Miller, Michelle E. Hay, Rod Precht
Exciton Technologies Inc., Suite 4000, 10230 Jasper Avenue, Edmonton, Alberta, T5J 4P6, Canada
* Corresponding author. Present address: University of Pennsylvania, Perelman School of Medicine, Department of Dermatology, Philadelphia, PA 19104, USA. E-mail address: [email protected] (L.R. Kalan).
1
Present address: University of Alberta, Department of Agriculture, Food and Nutritional
Sciences, Edmonton, AB, Canada T6G 2P5.
ARTICLE INFO Article history: Received 20 October 2016 Accepted 22 January 2017
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Keywords: Antibiotic resistance Biofilm Antimicrobial dressing Wound Infection
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Highlights
Ag Oxysalt releases Ag2+ and Ag3+ ions.
Ag Oxysalt is bactericidal against planktonically growing bacteria.
Ag Oxysalt is bactericidal against established biofilms.
Ag Oxysalt is bactericidal against antibiotic-resistant biofilms.
Ag Oxysalt has a non-toxic safety profile.
ABSTRACT A topical antimicrobial, silver oxynitrate (Ag7NO11), has recently become available that exploits the antimicrobial activity of ionic silver but has enhanced activity because highly oxidised silver atoms are stabilised with oxygen in a unique chemical formulation. The objective of this study was to use a multifaceted approach to characterise the spectrum of antimicrobial and antibiofilm activity of a wound dressing coated with Ag7NO11 at a concentration of 0.4 mg Ag/cm2. Physiochemical properties that influence efficacy were also evaluated, and Ag7NO11 was found to release a high level of Ag ions, including Ag2+ and Ag3+, without influencing the pH of the medium. Time–kill analysis demonstrated that a panel of multidrug-resistant pathogens isolated from wound specimens remained susceptible to Ag7NO11 over a period of 7 days, even with repeated inoculations of 1 106 CFU/mL to the dressing. Furthermore, established 72h-old biofilms of Pseudomonas aeruginosa, Staphylococcus aureus and two carbapenem-resistant Gram-negative bacteria (blaNDM-1-positive Klebsiella pneumoniae and blaVIM-2-positive P. aeruginosa) were disrupted and eradicated by Ag7NO11 in vitro. Ag7NO11 is a proprietary compound that exploits novel Ag chemistry and can be
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considered a new class of topical antimicrobial agent. Biocompatibility testing has concluded Ag7NO11 to be non-toxic for cytotoxicity, acute systemic toxicity, irritation and sensitisation.
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1. Introduction In 1857, Dr J. Marion Sims declared to the President of the New York Academy of Medicine that ‘It is to revolutionize surgical dressings…with silver there is no inflammation, no suppuration’ [1]. Since then, silver (Ag) has held a fundamental place in medicine as one of the earliest antimicrobial agents employed to prevent and treat wound infection. Historically, uses of Ag have been limited to its elemental form [Ag0(s)] or as silver nitrate [AgNO3(s)], but considering the longstanding use of Ag, innovation in Ag technology has been incremental. One major advance in Ag technology was the introduction of nanocrystalline silver (NCS) dressings in the late 1990s [2,3] that demonstrated significantly greater efficacy than AgNO3 [4]. The increased surface area of NCS allows for enhanced surface oxidation and formation of silver oxide [Ag2O(s)], creating a reservoir of available Ag ions [Ag+(aq)] and release of hydroxide [OH–(aq)] upon contact with an aqueous fluid such as a wound bed. The result is a fast and highly efficacious activity against micro-organisms. The novelty in NCS technology lies in the mechanism of action of Ag itself, the Ag+ ion is required for antimicrobial activity [5,6] and the physical form of NCS allows for continued release while creating an unfavourable and basic environment for microbes to thrive. The success of NCS dressings led to an explosive increase in the use of Ag dressings for wound care and the development of subsequent technologies.
Like other transition metals, Ag+ also forms insoluble precipitates [most notably AgCl(s) and Ag2SO4(s)] that are stable and less reactive. It has been demonstrated that Ag chemistry significantly impacts antimicrobial efficacy [7], yet it is these Ag salts that are
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found in the majority of currently available advanced wound care products, resulting in confounding evidence for the supported use of Ag as a topical antimicrobial [8]. Whilst it is established that the solubility and availability of Ag+ is the primary driving factor of antimicrobial efficacy, there have been no major developments in the formulation and delivery of Ag to enhance the applicability and antimicrobial activity since the introduction of NCS nearly 20 years ago.
With a dwindling pipeline of new small-molecule antibiotics and the growing threat of resistance, particularly multidrug-resistant (MDR) Gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae (CRE), the pursuit of novel antimicrobials that can target these types of wound infections is one strategy to preserve the use of antibiotics for systemic infection and to minimise the spread of resistance. Enhancements to Ag formulations and wound dressings, such as the recent application of the silver oxynitrate [Ag7NO11(s) or Ag Oxysalt] compound that contains highly oxidised Ag (Ag2+/3+) stabilised by oxygen atoms, represents a recent major innovation to Ag technology with enhanced antimicrobial efficacy against MDR pathogens [7,9]. Synthesis of Ag Oxysalt requires deliberate oxidation of Ag+ and subsequent capture of the crystalline structure as a pure powder that can then be incorporated into a variety of substrates or matrices for topical delivery [9,10]. Upon interaction with fluid, highly unstable Ag2+ and Ag3+ ions are released, where the difference in the oxidising potential of Ag2+/3+ (E = 1.98 V and 1.8 V, respectively) compared with that of Ag+ (E = 0.8 V) results in access to different chemistry, as electrons are scavenged from microbial metabolic processes. The minimum biocidal concentration of pure Ag Oxysalt required
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for killing a broad spectrum of bacteria has been determined to be significantly lower than all other available Ag compounds. Importantly, the minimum concentrations required both to inhibit and eradicate bacteria growing as biofilms is also significantly lower [7].
Here we describe the antimicrobial and antibiofilm activity of a wound contact layer coated with Ag Oxysalt against a spectrum of Gram-positive and Gram-negative bacteria growing planktonically or in a biofilm. We demonstrate that with a 75% reduction of total silver used, the antimicrobial efficacy is equivalent or surpasses a wound dressing containing NCS. Finally, we show that Ag Oxysalt is efficacious against antibiotic-resistant organisms including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and carbapenem-resistant bacteria expressing the blaNDM-1, blaVIM-2 and blaKPC genes growing both planktonically and in a biofilm.
2. Methods 2.1. Wound dressings Commercially available wound dressings were used in this study as follows: Exsalt ® T7 and SD7 (Ag Oxysalt dressing, 0.4 mg Ag/cm2; Exciton Technologies Inc., Edmonton, Canada); Acticoat 7 (NCS 1.2–1.6 mg Ag/cm2; Smith and Nephew, Hull, UK); Aquacel® Ag and Aquacel® Ag+ ExtraTM (AgCl, 0.083–0.09 mg Ag/cm2; ConvaTec, Greensboro, NC); Mepilex Ag (Ag2SO4, 1.2 mg Ag/cm2; Mölnlycke, Göteburg, Sweden); Silverlon
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(Ag0, 5.46 mg Ag/cm2; Argentum Medical, Geneva, IL); and TheraBond® (Ag0, 15% Ag w/w; Alliqua, Yardley, PA).
2.2. Bacterial strains A panel of Gram-negative (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and Gram-positive (Corynebacterium striatum, vancomycin-sensitive and vancomycin-resistant Enterococcus faecalis, S. aureus and Staphylococcus epidermidis) organisms were supplied by Keystone Labs (Edmonton, Canada). The MRSA isolate tested was isolated from an abscess and was supplied by CanBiocin Inc. (Edmonton, Canada). Pseudomonas aeruginosa ATCC 9027 and S. aureus ATCC 6538 were used for biofilm growth. The antimicrobialresistant strains listed in Table 1 were originally isolated from cutaneous wounds and were kindly supplied by Dr Robert Rennie (Alberta Provincial Laboratory for Public Health, Edmonton, Canada).
2.3. Time–kill curves and log reduction Time–kill log reduction analysis was performed at Keystone Labs. Briefly, from overnight cultures, bacteria inocula were made to 1 106 CFU/mL in maximum recovery diluent (composed of 0.9% NaCl and 0.1% peptone). Dressings were cut to 2.5 cm 2.5 cm and were exposed to 5 mL of each suspension. A single reaction tube in duplicate was set up for each time point. After the specified exposure period (0.5, 1, 2, 3 and 4 h), a sample was removed from the reaction tube and was diluted 1/5 into a
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universal antimicrobial neutralisation fluid [0.4% sodium thioglycolate in saline (STS)] to bind residual silver ions that may carry over from the reaction vessel and interfere with the quantification of viable cells. Serial dilutions were made and were plated onto Mueller–Hinton agar (MHA) to count viable cells and to calculate log reduction from the negative control inoculum. The 4-h log reduction of MRSA followed the same general procedure except 5 cm 5 cm cut dressings were submerged in 10 mL of simulated wound fluid (SWF) (50:50 fetal calf serum:peptone water) inoculated to 1 106 CFU/mL from an overnight culture of the MRSA isolate (in triplicate). Following 4 h of incubation at 37 C, a sample was removed and was diluted 1/10 in 0.4% STS, was serially diluted and was plated for viable cell counts. Log reduction was calculated by subtracting the viable cell counts after exposure to the dressing from the viable cell counts of the inoculum or in the case of longitudinal studies from the negative control vessel, and plotting the log transformed values.
2.4. Biofilm log reduction assay Biofilms were grown on three to five layers of sterile cotton gauze and were placed in SWF in 6-well tissue culture plates for each strain tested. A 1 106 CFU/mL inoculum was added every 24 h up to 72 h and the plates were incubated at 37 C with shaking at 200 rpm. After the incubation period, the gauze was removed from the liquid culture medium, was rinsed three times with sterile water and was placed onto the surface of a MHA plate (Oxoid, Nepean, Canada). The gauze biofilms were overlaid with additional MHA cooled to ca. 50 C such that one-half of the biofilms were embedded in the agar and one-half were exposed. Dressings (5 cm 5 cm) were overlaid onto the biofilms
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and were exposed at 37 C for 4 h or 24 h. After the exposure time, the dressings and biofilms (gauze pieces) were carefully removed from the plates and were placed into 10 mL of 0.4% STS. They were vortexed (3 1 min) to disrupt the biofilm, were serially diluted in saline (0.9% saline) and were spot-plated onto MHA for viable cell counts. Negative controls were made as follows: (i) gauze biofilms were grown and were overlaid with agar as described above; and (ii) blank dressings (porous polyester fibre material that Exsalt® T7 is composed of) but without the addition of Ag Oxysalt were used to cover the biofilms in the same manner as the treatment conditions.
2.5. Scanning electron microscopy (SEM) Biofilm growth and attachment to the sterile cotton gauze was confirmed by SEM. Following growth of biofilms, they were rinsed as above to remove planktonic cells and were fixed with methanol. Biofilms were cut into 5 mm 5 mm squares, were coated (6– 8 nm) with iridium prior to imaging and were imaged with a Hitachi S-4800 highresolution scanning electron microscope (Hitachi Canada, Mississauga, ON, Canada) housed at the National Institute for Nanotechnology (NINT) in Edmonton, Canada.
2.6. Silver release and pH measurements Silver release was measured by two methods. Data in Fig. 1 were measured by potentiometric titration with 0.1 M NaCl titrant. Dressings (5 cm 5 cm) were submerged in 10 mL of reverse osmosis (RO) H2O for the specified time period in triplicate. The fluid was then titrated against NaCl to determine the quantity of free Ag+
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ions in the solution. In Supplementary Fig. S1, silver release in RO H2O or SWF was measured by neutron activation analysis at the University of Alberta SLOWPOKE reactor. Reactions were set up as described above; triplicate samples were pooled prior to analysis. Higher oxidation state silver is detected by exposure of silver dressings to 1:3 HNO3:RO H2O and recording the colour change. As shown in the equations of Fig. 1C, a colour change is only observed for Ag2+ and Ag3+ ions. Furthermore, pH measurements were recorded by setting up vessels as above in triplicate and recording the pH with indicator strips after specified time intervals.
3. Results 3.1. Ag Oxysalt has a broad-spectrum activity To evaluate the speed and biocidal activity of dressings coated with Ag Oxysalt, a 4-h time course was designed, quantifying viable cell counts at 0.5, 1, 2, 3 and 4 h of exposure. A panel of Gram-negative (A. baumannii, E. coli, K. pneumoniae and P. aeruginosa) and Gram-positive (C. striatum, vancomycin-sensitive and vancomycinresistant E. faecalis, S. aureus and S. epidermidis) organisms were evaluated in peptone water. The efficacy was compared with NCS and was found to be equivalent for all organisms tested, with counts below the limit of detection (LOD) within 4 h for all organisms tested (Fig. 1A). We were also interested in assessing the solubility or availability of silver ions released from an Ag Oxysalt or NCS dressing because the ion availability is critical to antimicrobial efficacy. After 4 h, the Ag released from a 5 cm 5 cm dressing was not significantly different for Ag Oxysalt or NCS (0.4 mg Ag/cm2 vs.
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1.2–1.6 mg/cm2). However, the ppm Ag+ released per mg of total Ag on the dressing was significantly higher for the Ag Oxysalt (t-test, P = 0.0057) (Fig. 1B). The oxidation state of Ag released from each dressing was also assessed, with higher oxidation state (Ag2+/3+) released from the Ag Oxysalt dressing and Ag+ from the NCS dressing as observed by a colour change when exposed to a solution of nitric acid (HNO3) (Fig. 1C) [11–14]. Both dressings have been approved for a 7-day wear time so we measured the release of Ag over a 7-day period in RO H2O and a more complex SWF of 1:1 fetal calf serum:peptone water. SWF was tested because it better mimics a wound environment that contains ions (such as chlorides) and proteins (with thiol groups) which are able to bind and inactivate free Ag ions. We found that while both dressings release Ag for this length of time in both types of fluid, the Ag Oxysalt dressing is mostly consumed by Day 7 while the NCS dressing contains >50% of the starting Ag (38% vs. 86% starting Ag remaining), mostly converted to elemental Ag (Supplementary Fig. S1) as determined by X-ray diffraction (data not shown). Solubilising silver oxide releases hydroxide ions, so we assessed changes in the pH of media exposed to the NCS and Ag Oxysalt dressings because drastic pH changes could contribute to antimicrobial kill rates but also cause toxicity. The pH of either saline or SWF (50:50 fetal calf serum:peptone water) exposed to each dressing was monitored over time. In saline, the NCS dressing resulted in a highly alkaline environment at pH 11.5 within the first minute of exposure, eventually stabilising at pH 9 after 4 h. In SWF, the NCS dressing elevated to pH 9.5 within 30 min, whilst the Ag Oxysalt dressing remained below pH 9 and closer to physiological pH in both media types (Supplementary Fig. S1). Previously, it has been reported that the NCS dressing exhibits cytotoxicity to keratinocytes and fibroblasts in
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culture as Ag ions are released into the medium and this ultimately results in inhibition of re-epithelialisation [15]. To fully assess the safety and toxicity profile of the Ag Oxysalt dressing, a full cytotoxicity testing regimen was undertaken in vitro and in vivo (acute system toxicity, in vitro cytotoxicity, minimal essential medium cytotoxicity, irritation and sensitisation) and in all cases the dressing was found to be non-toxic (Supplementary methods; Supplementary Table S1).
3.2. Ag Oxysalt is active against antibiotic-resistant organisms Antibiotic-resistant organisms such as MRSA are a major concern for wound infection. Because we found Ag Oxysalt and NCS dressings were biocidal to methicillin-sensitive S. aureus in 4 h, we wondered whether MRSA would have the same susceptibility. As shown in Fig. 2, a clinical MRSA isolated from an abscess was susceptible both to the Ag Oxysalt and NCS dressing after 4 h of exposure, but not to dressings containing other types of Ag compounds (Ag0, AgCl and Ag2SO4). The Ag Oxysalt dressing is approved for wear up to 7 days so the efficacy against MDR clinical isolates was tested over a 7-day period. Every 24 h the dressings (the same dressing in the same vessel and medium each day) were inoculated with 1 106 CFU/mL and the log reduction values were determined by sampling for quantitative counts every 24 h just prior to the new inoculation. The experimental design represents the extremes where in a clinical setting it is not expected that a wound may be contaminated or inoculated with this level of bioburden each day. The log reduction values over the 7-day period are summarised in Table 1, showing that the Ag Oxysalt dressing was effective in reducing levels of all species tested including MRSA, VRE (VanA) and CRE.
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3.3. Ag Oxysalt kills multidrug-resistant biofilms It has become increasingly evident that the formation of a biofilm in the wound bed contributes to stalled healing, persistent inflammation and a chronic non-healing wound [16–19]. We previously reported that pure Ag Oxysalt powder can both inhibit biofilm formation and eradicate mature biofilms at concentrations significantly lower than other Ag compounds [7]. To expand on this study, the ability of Ag Oxysalt adhered to a substrate (contact layer dressing) to target and reduce biofilms was evaluated. In vitro testing is not always clinically relevant as the environment of a wound is difficult to mimic in the laboratory. To create the most clinically relevant assay, we adapted and enhanced upon a model reported by Seth et al. [20].
Micro-organisms will grow with different biogeography within the three-dimensional (3D) wound bed so it is important to test for antibiofilm activity within a 3D structure. Biofilms were grown over a 72-h period onto multiple layers of cotton gauze and were confirmed by SEM (Fig. 3A,C,D). The gauze biofilms were then embedded into nutrient agar plates before dressings were applied (Fig. 3B). After the specified exposure time, the dressing and gauze were extracted and quantitative counts of viable cells were determined.
Pseudomonas aeruginosa is a prolific biofilm-forming organism, so we first tested the ability of the Ag Oxysalt and NCS dressing to reduce 72-h gauze biofilms. Recently, a hydrofibre (HF) dressing containing AgCl, the divalent cation chelator ethylene diamine tetra-acetic acid (EDTA) and a surfactant benzalkonium chloride (BC) was introduced
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as an antibiofilm dressing, therefore this HF dressing was used as a comparator for the experiment. The Ag Oxysalt dressing resulted in zero counts after a 4-h exposure, significantly lower than the negative control (uncoated substrate) (Tukey’s post-hoc test, P < 0.0001) and the NCS dressing treatment (Tukey’s post-hoc test, P = 0.026) (Fig. 4A). Staphylococcus aureus was also able to form biofilms, so we evaluated the ability of the dressings to reduce viable biofilm counts after a 4-h exposure and noted that the Ag Oxysalt dressing resulted in counts below the LOD (Fig. 4A).
To confirm that the observed biocidal activity is not diminished in MDR isolates, the experiment was repeated with clinical isolates of carbapenem-resistant K. pneumoniae (blaNDM-1) and P. aeruginosa (blaVIM-2) exposed to the Ag Oxysalt-coated wound contact layer or the HF AgCl/EDTA/BC-containing antibiofilm dressing. Both drug-resistant isolates were effectively reduced after 24 h of exposure to the dressings (Fig. 4B). We were curious whether these two dressings were different in their speed of kill because of the considerable differences in dressing construction and composition of antimicrobial agents. The HF dressing has a large absorptive capacity and therefore can reduce cell counts by sequestration in the fibres as they gel and by the combined action of EDTA and BC. The wound contact layer, on the other hand, relies solely on the action and availability of Ag released from the Ag Oxysalt crystals coated onto the surface of polyester fibres with little to no absorptive capacity. The antibiofilm activity against P. aeruginosa biofilms was tested for a 2-h exposure time and the Ag Oxysalt dressing reduced the viable cells counts significantly greater than the HF AgCl/EDTA/BC dressing (log reduction of 3.86 ± 0.7 vs 0.4 ± 0.5; Tukey’s post-hoc test, P = 0.007).
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4. Discussion The formation of microbial biofilms in wounds is a major barrier to healing progression [21], particularly in individuals with a reduced ability to overcome infection. Treatment with antimicrobials is challenging in chronic non-healing wounds of the lower extremities, biofilm formation results in increased tolerance to penetration of active agents, and MDR organisms are becoming increasingly problematic with limited therapeutic options available. Other factors such as poor perfusion can limit the delivery of systemic antibiotics to the wound surface and further increase the complexity in treating these wounds [22,23]. Topical antimicrobials act to control microbial bioburden directly and silver has long been used for this purpose in wound care. Here we describe a new formulation of silver that contains higher oxidation state Ag ions. The current data support previous reports that not all silver is equivalent [24,25] and we show that wound dressings coated with Ag Oxysalt have efficacy against planktonic and biofilm populations at lower overall quantities of silver than commercially available silver wound dressings. Furthermore, the compound maintains efficacy against antibiotic-resistant organisms including MRSA and CRE isolated from cutaneous wounds.
Clinical use of silver dressings has been controversial in relation to their ability to reduce bioburden and contribute to improved wound healing. We argue that this is due in part to an assumption that all silver dressings have the same general mechanism of action, and testing methodologies to evaluate microbial susceptibility have not been standardised in the same manner as pharmaceutical antibiotics [26]. For example, NCS
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has been reported to achieve a 4 log reduction of S. aureus in 30 min [24]; however, as shown in Fig. 1, a 4-h exposure time was required to achieve a 4 log reduction with our method. The methodology employed by Cavanagh et al. [24] involves wetting the dressings with sterile water and inoculating it with 0.06 mL/cm2 of bacterial suspension. In our methodology, a more complex peptone medium was used and the dressings were inoculated with 0.4 mL/cm2 of bacterial suspension, resulting in excess fluid saturating each dressing. The choice of inoculum volume and medium can significantly change the outcome. Silver availability is greatly reduced in a complex SWF owing to binding of free Ag ions by proteins and other ions, notably the complex with chloride ions results in formation of AgCl(s). This is demonstrated in Supplementary Fig. S2. Our method also demonstrates that with the exception of the NCS and Ag Oxysalt dressing, dressings containing other silver formulations do not result in any significant levels of MRSA reduction in this 4-h exposure period (Fig. 2). As a result of NCS chemistry, exposure of an NCS dressing to fluid releases hydroxide ions that elevated the pH and contribute to cellular toxicity. To alleviate discomfort to the wound, the dressing is typically wet with water to dissipate the OH– prior to application. On the other hand, an Ag Oxysalt dressing can utilise much lower silver content and still maintain high antimicrobial efficacy with a non-cytotoxic safety profile. We acknowledge that the dressing construction also influences delivery of silver and thus efficacy is multifactorial.
Biofilms and antibiotic resistance are two serious considerations for wound infection. CRE are a major global health concern [27–29], with mortality rates reaching 50% owing to a deficiency in antibiotics to which the organisms remain susceptible [28]. We
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have demonstrated that the Ag Oxysalt-coated dressing is able to disrupt and kill common wound biofilm pathogens P. aeruginosa and S. aureus more effectively than an NCS dressing containing a four-fold greater silver concentration. CRE clinical wound isolates growing as a biofilm are also susceptible to the Ag Oxysalt dressing (Fig. 4). The efficacy is equivalent to a HF dressing containing a synergistic combination of EDTA, BC and AgCl targeting biofilms. The equivalent HF dressing containing only silver (AgCl) is not active against biofilms [20]. MRSA and VRE also remain susceptible to the Ag Oxysalt dressing.
New therapeutic options for preventing and controlling wound infection are needed as the incidence of diseases such as diabetes increases, where formation of non-healing ulcers contributes to 80% of non-traumatic lower-extremity amputations and 5-year mortality rates exceed that of some cancers [30–32]. Silver remains a viable, non-toxic and effective topical antimicrobial option with exciting innovation and formulation improvements available. Mechanisms of silver resistance have been well described [33–36] but the incidence remains low, and Ag Oxysalt dressings appear to remain active against even the most resistant isolates [37]. The current data support the use of this new, low silver content, Ag Oxysalt dressing for the prevention and management of wound infection.
Acknowledgment: The authors would like to acknowledge Dr Andrew Myles for his support and thoughtful discussion.
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Funding: LRK was supported by Alberta Innovates Technology Futures R&D Associate Fellowship and NSERC Industrial R&D Fellowship. IU-H was supported by an Alberta Innovates NanoWorks Program grant.
Competing interests: All authors were employees of Exciton Technologies (Edmonton, Canada), the manufacturer of silver oxynitrate, at the time the work was completed.
Ethical approval: Not required.
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References [1] Sims JM, Emmet TA. Silver sutures in surgery: the anniversary discourse before the New York Academy of Medicine, delivered in the new building of the Historical Society on November 18, 1857. New York, NY: Samuel S. & William Wood; 1858. [2] Wright JB, Lam K, Burrell RE. Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment. Am J Infect Control 1998;26:572–7. [3] Tredget EE, Shankowsky HA, Groeneveld A, Burrell R. A matched-pair, randomized study evaluating the efficacy and safety of Acticoat silver-coated dressing for the treatment of burn wounds. J Burn Care Rehabil 1998;19:531–7. [4] Yin HQ, Langford R, Burrell RE. Comparative evaluation of the antimicrobial activity of ACTICOAT antimicrobial barrier dressing. J Burn Care Rehabil 1999;20:195–200. [5] Gibbard J. Public health aspects of the treatment of water and beverages with silver. Am J Public Health Nations Health 1937;27:112–9. [6] Xiu Z-M, Zhang Q-B, Puppala HL, Colvin VL, Alvarez PJJ. Negligible particlespecific antibacterial activity of silver nanoparticles. Nano Lett 2012;12:4271–5. [7] Lemire JA, Kalan L, Bradu A, Turner RJ. Silver oxynitrate, an unexplored silver compound with antimicrobial and antibiofilm activity. Antimicrob Agents Chemother 2015;59:4031–9. [8] Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P, Stevenson M. Randomized controlled trial and cost-effectiveness analysis of silver-donating
Page 20 of 28
antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 2009;96:1147–56. [9] Djokić SS. Deposition of silver Oxysalts and their antimicrobial properties. J Electrochem Soc 2004;151:C359–64. [10]
Jack JC, Kennedy T. The thermal analysis of 'argentic oxynitrate' and
silver oxides. J Thermal Analysis 1971;3:25. [11]
Noyes AA, Hoard JL, Pitzer KS. Argentic salts in acid solution. I. The
oxidation and reduction reactions. J Am Chem Soc 1935;57:1221–9. [12]
Noyes AA, Pitzer KS, Dunn CL. Argentic salts in acid solution. II. The
oxidation state of argentic salts. J Am Chem Soc 1935;57:1229–37. [13]
Noyes AA, Kossiakoff A. Argentic salts in acid solution. III. Oxidation
potential of argentous–argentic salts in nitric acid solution. J Am Chem Soc 1935;57:1238–42. [14]
Po HN, Swinehart JH, Allen TL. Kinetics and mechanism of the oxidation
of water by silver(II) in concentrated nitric acid solution. Inorg Chem 1968;7:244– 9. [15]
Burd A, Kwok CH, Hung SC, Chan HS, Gu H, Lam WK, et al. A
comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models. Wound Repair Regen 2007;15:94–104. [16]
Wolcott RD, Dowd S, Kennedy J, Jones CE. Biofilm-based wound care.
In: Sen CK, editor. Advances in wound care, Vol. 1. New Rochelle, NY: Mary Ann Liebert Inc., 2010. Chapter 54.
Page 21 of 28
[17]
Metcalf DG, Bowler PG. Biofilm delays wound healing: a review of the
evidence. Burns Trauma 2013:1:5–12. [18]
Percival SL, McCarty SM, Lipsky B. Biofilms and wounds: an overview of
the evidence. Adv Wound Care (New Rochelle) 2015;4:373–81. [19]
Zhao G, Usui ML, Lippman SI, James GA, Stewart PS, Fleckman P, et al.
Biofilms and inflammation in chronic wounds. Adv Wound Care (New Rochelle) 2013;2:389–99. [20]
Seth AK, Zhong A, Nguyen KT, Hong SJ, Leung KP, Galiano RD, et al.
Impact of a novel, antimicrobial dressing on in vivo, Pseudomonas aeruginosa wound biofilm: quantitative comparative analysis using a rabbit ear model. Wound Repair Regen 2014;22:712–9. [21]
James GA, Swogger E, Wolcott R, Pulcini ED, Secor P, Sestrich J, et al.
Biofilms in chronic wounds. Wound Repair Regen 2008;16:37–44. [22]
Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res
2010;89:219–29. [23]
Lipsky BA. Medical treatment of diabetic foot infections. Clin Infect Dis
2004;39(Suppl 2):S104–14. [24]
Cavanagh MH, Burrell RE, Nadworny PL. Evaluating antimicrobial efficacy
of new commercially available silver dressings. Int Wound J 2010;7:394–405. [25]
Gallant-Behm CL, Yin HQ, Liu S, Heggers JP, Langford RE, Olson ME, et
al. Comparison of in vitro disc diffusion and time kill-kinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Repair Regen 2005;13:412–21.
Page 22 of 28
[26]
Clinical and Laboratory Standards Institute. Performance standards for
antimicrobial susceptibility testing; sixteenth informational supplement. Document M100-S16. Wayne, PA; CLSI; 2006. [27]
Temkin E, Adler A, Lerner A, Carmeli Y. Carbapenem-resistant
Enterobacteriaceae: biology, epidemiology, and management. Ann N Y Acad Sci 2014;1323:22–42. [28]
Patel G, Huprikar S, Factor SH, Jenkins SG, Calfee DP. Outcomes of
carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol 2008;29:1099–106. [29]
Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL,
Cormican M, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013;13:785–96. [30]
Armstrong DG, Wrobel J, Robbins JM. Guest editorial: are diabetes-
related wounds and amputations worse than cancer? Int Wound J 2007;4:286–7. [31]
Robbins JM, Strauss G, Aron D, Long J, Kuba J, Kaplan Y. Mortality rates
and diabetic foot ulcers. Is it time to communicate mortality risk to patients with diabetic foot ulceration? J Am Podiatr Med Assoc 2008;98:489–93. [32]
Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global
burden of diabetic foot disease. Lancet 2005;366:1719–24. [33]
Silver S. Bacterial silver resistance: molecular biology and uses and
misuses of silver compounds. FEMS Microbiol Rev 2003;27:341–53.
Page 23 of 28
[34]
Gupta A, Matsui K, Lo JF, Silver S. Molecular basis for resistance to silver
cations in Salmonella. Nat Med 1999;5:183–8. [35]
Asiani KR, Williams H, Bird L, Jenner M, Searle MS, Hobman JL, et al.
SilE is an intrinsically disordered periplasmic 'molecular sponge' involved in bacterial silver resistance. Mol Microbiol 2016;101:731–42. [36]
Woods EJ, Cochrane CA, Percival SL. Prevalence of silver resistance
genes in bacteria isolated from human and horse wounds. Vet Microbiol 2009;138:325–9. [37]
Finley PJ, Norton R, Austin C, Mitchell A, Zank S, Durham P.
Unprecedented silver resistance in clinically isolated Enterobacteriaceae: major implications for burn and wound management. Antimicrob Agents Chemother 2015;59:4734–41.
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Fig. 1. Ag Oxysalt dressings have a 4 log reduction within 4 h. (A) Time–kill curves of common wound-associated Gram-positive and Gram-negative bacteria over a 4-h period. Bacteria were exposed to dressings coated with nanocrystalline silver (NCS) or Ag Oxysalt (n = 2). The control reaction was blank without an antimicrobial. LOD, limit of detection. (B) Silver release into reverse osmosis (RO) H2O after a 4-h period of NCSand Ag Oxysalt-coated dressing (n = 3). Mean ppm Ag was determined by potentiometric titration, and mean ppm Ag/mg Ag was determined by dividing the mean ppm Ag by the total mg of Ag in each dressing. Error bars represent the standard deviation of triplicate measurements. (C) Ag Oxysalt-coated dressings contain higher oxidation states of Ag (Ag2+/3+), but NCS-coated dressings do not. The chemical reaction is represented.
Fig. 2. Ag Oxysalt dressings kill methicillin-resistant Staphylococcus aureus (MRSA). Commercially available silver dressings were exposed to an inoculum of 1 106 CFU/mL of MRSA in simulated wound fluid (SWF) at 37 C for 4 h. The control group represents the inoculum in medium only in the absence of an antimicrobial dressing. Only dressings containing nanocrystalline silver (NCS) (ca. 40 mg Ag) or Ag Oxysalt (10 mg Ag) significantly reduced the viable cell count (P < 0.0001, t-test) to below the limit of detection (LOD) (n = 3).
Fig. 3. Gauze biofilm model. (A) Biofilms were grown on sterile cotton gauze by inoculating with 1 106 CFU/mL at 0, 24 and 48 h. (B) After 72 h at 37 C and 200 rpm, biofilms were embedded in Mueller–Hinton agar and were overlaid with the test
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dressing. (C,E) Biofilms at different magnification were confirmed by scanning electron microscopy (SEM) after rinsing the gauze with sterile water to remove planktonic cells. A biofilm of blaVIM-2-positive Pseudomonas aeruginosa is visualised attached to a fibre of the gauze (C) and individual cells observed within an extracellular matrix. (D) SEM image of sterile cotton gauze fibres not exposed to bacterial culture but incubated in medium.
Fig. 4. Ag Oxysalt dressings kill carbapenem-resistant Enterobacteriaceae (CRE) biofilms. (A) Biofilms of Pseudomonas aeruginosa ATCC 9027 or Staphylococcus aureus ATCC 6538 were exposed to a non-coated control, nanocrystalline silver (NCS), hydrofibre AgCl/EDTA/BC or Ag Oxysalt dressings for 4 h at 37 C and viable cell counts were determined (n = 3). (B) Biofilms of blaNDM-1-positive Klebsiella pneumoniae or blaVIM-2-positive P. aeruginosa were exposed to a non-coated control, hydrofibre AgCl/EDTA/BC or Ag Oxysalt dressing for 24 h at 37 C and viable cell counts were determined (n = 3). EDTA, ethylene diamine tetra-acetic acid; BC, benzalkonium chloride.
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Supplementary Fig. S1. pH changes upon exposure to a silver dressing. Changes to the pH of saline or simulated wound fluid (SWF) (50% fetal calf serum; 50% 0.1% peptone water) was measured after exposure to an Ag Oxysalt or nanocrystalline silver (NCS) dressing. Cut dressings (5 cm 5 cm) were saturated in 10 mL of fluid.
Supplementary Fig. S2. 7-Day silver release. Silver release from a nanocrystalline silver (NCS) or Ag Oxysalt dressing was determined by neutron activation analysis over a period of 7 days with measurements on Days 1, 3, 5 and 7. Triplicate analyses were pooled prior to quantification. Error bars represent standard errors of measurements. RO, reverse osmosis H2O; SWF, simulated wound fluid.
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Table 1. Log reduction values over 7 days (1 106 CFU/mL inoculations occurred every 24 h) of antibiotic-resistant wound isolates exposed to a wound dressing coated with 0.4 mg/cm2 silver oxynitrate Organism
Day
Day
Day
Day
Day
Day
Day
1
2
3
4
5
6
7
Stenotrophomonas maltophilia
4.79
7.45
7.78
7.80
7.53
7.84
7.38
Staphylococcus aureus (mecA)
4.86
3.56
3.95
3.84
3.55
3.14
4.88
Pseudomonas aeruginosa
3.92
7.08
5.98
4.62
5.87
5.21
5.71
Klebsiella pneumoniae (blaKPC)
3.89
6.76
5.94
6.17
5.39
5.46
4.70
K. pneumoniae (blaNDM-1)
3.31
6.70
6.93
6.44
6.35
6.32
6.41
Escherichia coli
4.60
2.76
3.55
3.98
5.02
5.89
5.24
Enterococcus faecalis (vanA)
4.00
5.73
4.93
4.67
4.53
4.82
5.41
(blaVIM-2)
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