AAC Accepted Manuscript Posted Online 27 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.05177-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Silver oxynitrate: An Unexplored Silver Compound with Antimicrobial and Antibiofilm Activity Joe A. Lemire1, Lindsay Kalan2, Alexandru Bradu1, and Raymond J. Turner1,# 1. 2.

The Biofilm Research Group. Department of Biological Sciences at the University of Calgary. 2500 University Drive Northwest, Calgary, AB T2N 1N4 Exciton Technologies Inc. 10230 Jasper Ave NW, Edmonton, AB T5J 4P6

# Corresponding Author – Email: [email protected], Phone: 403-220-4308

Running title: Silver oxynitrate has antimicrobial and antibiofilm efficacy Keywords: silver, antimicrobial, biofilm, antibiofilm, wound care, chronic wound treatment

J.Lemire et al. 2015 1

ABSTRACT:

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Historically its been accepted, and recent research has established that silver (Ag) is an

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efficacious antimicrobial agent. A dwindling pipeline of new antibiotics, combined with an increase in

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antibiotic resistant infections, is bringing Ag to the fore as a therapeutic compound to treat infectious

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diseases. Currently, many formulations of Ag are being deployed for commercial and medical

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purposes with varying degrees of effectiveness at killing microbial cells. Here, we evaluated the

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antimicrobial and antibiofilm capacity of our lead compound – silver oxynitrate [Ag(Ag3O4)2NO3 or

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Ag7NO11] – against other metal compounds with documented antimicrobial activity; Ag2SO4, AgNO3,

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silver sulfadiazine (AgSD), AgO, Ag2O, and CuSO4. Our findings reveal that Ag7NO11 eradicates

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biofilm and planktonic populations, of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus

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aureus, uropathogenic Escherichia coli (UPEC), fluoroquinolone-resistant Pseudomonas aeruginosa

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(FQRP), and methicillin-resistant Staphylococcus aureus (MRSA) at lower concentrations than the

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other tested metal salts. Altogether, our results demonstrate that Ag7NO11 has an enhanced efficacy for

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the treatment of biofilm-forming pathogens.

15 16 17 18 19 20 21 22 23 24 25 2

J.Lemire et al. 2015 26

INTRODUCTION

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History has demonstrated that silver (Ag) is an efficacious antimicrobial agent, finding utility as

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an antiquated preservative for food and water (1, 2). Currently, over 100 Ag-containing medical

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devices

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(http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm), and there’s mounting evidence

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that Ag may be effective for preventing the spread of infectious disease (1, 3, 4). Though the

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toxicological profile of orally administered Ag remains to be resolved, topical application for the

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treatment of chronic wounds remains promising (5-8).

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health issue, as chronic wounds represent a substantial – financial and medical – burden to the health

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care system. One of the hallmarks of a chronic wound is the presence of a biofilm; a factor that

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complicates wound healing, and is hypothesized to be the fulcrum between an acute to chronic wound

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transition (6, 9-15). Also, bacterial biofilms contaminate implanted medical devices (16) and exhibit

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high resistance to conventional antibiotics (17-23). New antibiofilm agents are thus desperately needed

39

in medicine.

40

have

been

approved

for

use

by

the

FDA

Accordingly, Ag can address a timely public

Recently, researchers have demonstrated that Ag formulations also have potential as antibiofilm

41

agents; this includes AgSD (24), Ag-nanoparticles [AgNPs] (25, 26), and AgNO3 (27). Currently, the

42

wound care industry is replete with Ag-based ointments and wound dressings with claimed efficacy at

43

reducing bacterial bioburden (7, 28-32). However, only a handful of antibiofilm products exist. There

44

are numerous reasons why this is so. 1) Current standardized, antimicrobial testing methods focus on

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bacteria in their planktonic state – not as biofilms (33, 34). 2) Biofilms are characteristically more

46

tolerant to metal poisoning (35-38), and 3) the detailed mechanism of action for the toxicity of Ag, to

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biofilms, remains to be fully described (1).

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The antimicrobial activity of Ag is intrinsically dependent on the formation of the Ag1+ ion (7,

49

39). Briefly, Ag1+ - a lewis soft acid – poisons the microbial cell by binding to reduced thiols (SH),

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impairing membrane function (1), and disrupting iron-sulfur clusters (40). That being said, the 3

J.Lemire et al. 2015 51

antimicrobial efficacy of higher oxidation states of Ag – Ag2+ and Ag3+ - has not been given adequate

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consideration, mainly due to instability in solution. A promising resolution to the instability of higher

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oxidation states of Ag, are silver oxysalts; for example Ag(Ag3O4)2X -the most stable of which is

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coordinated with nitrate- silver oxynitrate [Ag(Ag3O4)2 NO3 = Ag7NO11], where both the Ag II and III

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oxidation states exist stably at room temperature (41, 42).

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Here, we describe our observations from testing the antimicrobial and antibiofilm activity of

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various metal compounds. Namely, we tested silver oxynitrate (Ag7NO11), silver sulfadiazine (AgSD),

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silver nitrate (AgNO3), silver sulphate (Ag2SO4), silver (I,III) oxide (AgO), silver (I) oxide (Ag2O), and

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copper sulphate (CuSO4) against 6 bacterial strains - Pseudomonas aeruginosa (PAO1),

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Staphylococcus aureus (ATCC 25923), Escherichia coli (JM109), uropathogenic Escherichia coli

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(CFT703) [UPEC], fluoroquinolone-resistant Pseudomonas aeruginosa [FQRP], and methicillin-

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resistant Staphylococcus aureus (USA300) [MRSA] as well as two fungal strains of Candida albicans

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(ATCC 14053) and Candida tropicalis (99916). CuSO4 was included in our analysis because copper

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(Cu) is another thiophilic metal with observed antimicrobial activity (1, 43, 44). Using the minimal

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biofilm eradication concentration (MBEC) assay, we found that Ag7NO11 eradicated planktonic and

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biofilm populations, of the tested strains, at lower concentrations than the other metal compounds

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tested. AgNO3 – the most soluble Ag salt – also had antimicrobial and antibiofilm activity at

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micromolar concentrations. Because AgNO3 has a high solubility, resulting in easily accessible Ag+

69

ions, and it outperformed all other Ag salts, with the exception of Ag7NO11, it was chosen as our

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comparator for testing the efficacy of Ag7NO11 in proceeding experiments. Using the live/dead™

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staining technique as well as crystal violet staining, we demonstrated that Ag7NO11 reduced the

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biomass of the biofilm. In summary, our data demonstrates that Ag7NO11 has greater antimicrobial and

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antibiofilm activity, compared to the other tested metal compounds, and may offer an alternative option

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for the treatment of chronic wounds infected by biofilm-forming microbes.

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J.Lemire et al. 2015 76

MATERIALS AND METHODS

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Bacterial/Fungal Strains and Media

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Bacterial and fungal strains were stored in Microbank ™ vials at -70°C as described by the

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manufacturer (ProLab Diagnostics, Richmond Hill, ON, Canada). Pseudomonas aeruginosa (PA01),

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Staphylococcus aureus (ATCC 25923), Escherichia coli (JM109), Candida albicans (ATCC14053),

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Candida tropicalis (99916), uropathogenic Escherichia coli (CFT703), fluoroquinolone-resistant

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Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (USA300) were streak-

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purified onto tryptic soy agar [TSA] (VWR International, Mississauga, ON, Canada) overnight at 37°C.

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CFT703 and USA300 were generous gifts from Dr. Joe J. Harrison (the University of Calgary), while

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the FQRP isolate was a generous gift from Dr. Michael Parkins (the University of Calgary). To

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simulate a wound environment, simulated wound fluid (SWF) [50% peptone water (0.85% NaCl,

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0.1g/L peptone):50% fetal calf serum (Invitrogen, Life Technologies)] (31, 45) was used as growth

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media and for susceptibility testing.

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Biofilm Formation All biofilms used in this study were cultivated using the Calgary Biofilm Device

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(CBD)/MBEC™ Device as previously described in (38, 46-50) and by the manufacturers guidelines

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(Innovotech, Edmonton, AB, Canada). Briefly, following the overnight growth of the pre-culture, the

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colonies were suspended in SWF to match the density of a 1.0 McFarland standard. The cultures made

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with the optical standard were subsequently diluted 30 times in 150μL of SWF, which served as the

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inoculum for the CBD. The biofilm was formed by placing the lid of the CBD – 96 equivalent pegs –

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into a 96-well microtitre plate (Nunclon, VWR, International) containing the inoculum. The CBD was

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then placed on a gyrorotary shaker operating at 150rpm in a humidified incubator at 37°C for 4h or

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24h. For mature biofilm experiments, the inoculated CBD was incubated for a period of 4d

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J.Lemire et al. 2015 100

(P.aeruginosa), or 6d (S.aureus and E.coli). A period exceeding 4d resulted in overgrowth of PAO1 in

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the MBEC™ device (personal observation)

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Stock Metal Solutions Silver nitrate (AgNO3), silver sulfadiazine (AgSD), silver sulphate (Ag2SO4), silver (I) oxide

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(Ag2O), silver (I,III) oxide (AgO), and copper sulphate (CuSO4), were all obtained from Sigma-Aldrich

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(St. Louis, MO, USA). Silver oxynitrate (Ag7NO11), was obtained from Exciton Technologies Inc

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(Edmonton, AB, Canada). All stock Ag solutions were made at equivalent molarities of Ag – up to

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5mM – in distilled and deionized (dd)H2O. The CuSO4 stock was made up to 2M in ddH2O. All metal

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solutions were stored in glass vials at 21°C for no longer than one week. Working solutions were made

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in SWF, from stock metal solution, no more than 30 min prior to experimental use. From these, serial-

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dilutions (dilution factor of 2) were made in 96-well plates (the challenge plate). The first row was

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reserved as a sterility control and the second row for a growth control (0mM metal salt). The range of

113

concentrations tested for all Ag compound was 0 – 1250μM, while CuSO4 was tested from 0 –

114

20,000μM.

115 116

Biofilm and Planktonic Culture Susceptibility Testing

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Two scenarios were tested i) the capacity of metal salts to prevent biofilm formation, and ii) the

118

ability of metal salts to eradicate established biofilms. For i) the bacterial cultures were inoculated into

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the challenge plate in the presence of the metal salts and then subsequently placed on a gyrorotary

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shaker at 150rpm, in a humidified incubator at 37°C for 4h. Scenario ii) allowed for the establishment

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of a biofilm for 24h, 4d or 6d on the pegged lid of the CBD. The peg lid containing the established

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biofilm was then rinsed twice with 0.9% NaCl and subsequently placed into the 96-well microtitre plate

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containing the two-fold serial dilutions of the challenge metal (the challenge plate). The plate was then

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incubated for 4h, 24h, or 48h on a gyrorotary shaker at 150rpm, in a humidified incubator at 37°C. 6

J.Lemire et al. 2015 125

To test the metal susceptibility of the planktonic and biofilm populations, the plates from i and

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ii were prepared by removing the peg lid and rinsing it twice with 0.9% NaCl. The biofilms were

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disrupted from the pegs by sonication using a 250HT ultrasonic cleaner (VWR, International), set at

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60Hz for 10min, into 200μL of tryptic soy broth (VWR, International) containing universal neutralizer

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(UN) [0.05 g/L-Histidine (Sigma, USA), 0.05 g/L-Cysteine (Sigma, USA), and 0.l g/L reduced

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glutathione (Sigma, USA) ddH2O] and 0.1% Tween® 20. The minimal biofilm eradication

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concentration (MBEC) of the biofilm populations, was ascertained by performing 8 dilutions -dilution

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factor of 10- of the disrupted biofilms in 0.9% NaCl. The viable cell numbers from the biofilm were

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ascertained by spot plating the diluted sample onto TSA plates, and subsequently inoculating the plates

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overnight at 37°C (38, 51, 52). Similarly, the minimal bactericidal concentration (MBC) of the

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planktonic populations was carried out by serially diluting, 8 times (10-fold dilutions), the neutralized

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(UN; as described above) spent media from the 96-well plate into 0.9% saline. Viable cell counts were

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taken following spot plating of the dilutions onto TSA plates incubated overnight at 37°C (38, 51, 52).

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The MBC and MBEC were determined by monitoring the concentration of metal compound at which

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there were no viable microbial colonies.

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Confocal Microscopy Quantification of live and dead cells in the biofilm was performed using a confocal microscope

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and the Live/Dead® BacLight™ staining kit (Molecular Probes, Burlington, ON, Canada). Briefly,

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following metal exposure, cell viability staining of the bacteria was done by incubating the pegs

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containing the biofilms with the Syto-9 (6.7μM) and propidium iodide (PI) (40μM) stains (provided in

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the BacLight™ kit) at 30°C for 30min as previously described in (46). The pegs were rinsed with 0.9%

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saline twice and monitored using a confocal laser-scanning microscope (Leica DM IRE2 microscope)

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equipped with a 20X objective. Images were processed using Imaris x64 (Bitplane, USA). PI

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excitation wavelength was at 523nm and emission at 617nm (red), while Syto-9 excitation wavelength 7

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was 488nm and emission at 498 (green). Quantification of the compiled confocal images was

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performed using the Fiji software package (http://fiji.sc/Fiji).

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Crystal Violet Assay

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Crystal violet staining was used to quantify the amount of biomass in the biofilm. Following

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metal treatment, the peg lids from the CBD were rinsed twice in 200μL of 0.9% saline. Following a

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procedure similar to O’Toole GA, 2011 (53), the biofilms were stained with 200μL of 0.1% crystal

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violet solution for 10min. Following the staining, the pegs were washed with 200μL ddH2O thrice, and

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blotted on paper towel to remove excess dye. The stain was left to set overnight at room temperature.

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Quantification of the biofilm was performed by solubilizing the crystal violet stain with 200μL of 30%

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acetic acid for 15min, into a microtitre plate, and reading the absorbance at 550nm using acetic acid as

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the blank.

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Statistical Analysis Statistical significance of the results was performed using a non-parametric, one-way analysis

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of variance (ANOVA) for the MBC and MBEC assays, a two-way ANOVA for the crystal violet

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assays, or a students T-test for Supplementary Figure 3 and 4. All experiments were performed, at

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minimum (stated otherwise in the figure captions), with two biological replicates and in duplicate.

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RESULTS

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Silver oxynitrate (Ag7NO11) prevents biofilm formation. In order to assess the capacity of various

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metal compounds to prevent biofilm formation in our selected pathogenic strains (E.coli, P.aeruginosa,

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and S.aureus), we employed the minimal biofilm eradication concentration (MBEC) assay (Fig. 1).

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Concurrently, the minimal bactericidal concentration (MBC) for the metal compounds was also

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established. Compared to the other metal compounds tested, significantly lower concentrations of 8

J.Lemire et al. 2015 175

Ag7NO11 were needed to prevent the formation of biofilms (Fig. 1A) and –with the exception of

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AgNO3- eliminate planktonic populations of our tested strains (Fig. 1B), following a 4h incubation

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period.

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Silver oxynitrate (Ag7NO11) eradicated established biofilm populations. Our next aim was to

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assess the capacity of various metal compounds to eradicate an established biofilm. Biofilms from

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E.coli, P.aeruginosa, and S.aureus were established following a 24h incubation period in the MBEC™

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device. The established biofilms were then exposed to serial dilutions (two-fold) of Ag2O, AgO,

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Ag2SO4, AgNO3, Ag7NO11, AgSD, and CuSO4 for 24h (Fig. 2). Exposing established biofilms for 4h

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to AgNO3, Ag7NO11, and CuSO4 was insufficient time to eradicate them (Suppl. Fig. 1) – with the

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exception of E.coli biofilms. Thus, a 24h exposure time to the metal compounds was evaluated as a

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potentially efficacious treatment period. Following a 24h incubation period with the various metal

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compounds, we observed that Ag7NO11 significantly reduced biofilm populations of E. coli, P.

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aeruginosa, and S. aureus at lower concentrations than the other tested metal compounds – with the

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exception of AgNO3 in P. aeruginosa (Fig. 2). When mature biofilms were established in the

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MBEC™ device, following a 4d (P.aeruginosa) or 6d (E.coli and S.aureus), higher concentrations of

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AgNO3, CuSO4, and Ag7NO11 were required to reach a MBEC with the exception of CuSO4 in E.coli

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(Fig. 3). Nonetheless, Ag7NO11 eradicated the mature biofilms of S.aureus and E.coli at lower

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concentrations than AgNO3 and CuSO4 (Fig. 3). Additionally, at the highest concentrations –of

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equivalent Ag- of AgNO3 and Ag7NO11 tested (1250μM), Ag7NO11 significantly outperformed AgNO3

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and CuSO4 at eradicating C.albicans and C.tropicallis biofilms (Suppl. Fig 2).

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Silver oxynitrate (Ag7NO11) reduces the biomass of established biofilms. Since AgNO3 and

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Ag7NO11 were the most efficacious at eliminating planktonic populations, preventing biofilm

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formation, and eradicating established biofilms (Fig 1. and Fig. 2), these two metal compounds were 9

J.Lemire et al. 2015 200

chosen as lead compounds to study the capacity of Ag to abolish established biofilms. We employed

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Live/Dead® staining followed by confocal microscopy analysis to visualize the effects of a 4h and 24h

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treatment with 5μM and 12.5μM of AgNO3 and Ag7NO11 (at equivalent Ag concentrations) on

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established biofilms of E. coli, P. aeruginosa, and S. aureus (Suppl. Fig. 3 and Fig. 4). Though, a 4h

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exposure to AgNO3 and Ag7NO11 was not adequate to eradicate the biofilm populations, an increase in

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the quantity of dead cells was observed in the 12.5μM AgNO3 and Ag7NO11 – exposed biofilms

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(Suppl. Fig.3). Following a 24h exposure to AgNO3 and Ag7NO11 we observed an increase in dead

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cells in the 12.5μM AgNO3-exposed biofilms and reduction in biofilm quantity in the Ag7NO11-

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exposed biofilms (Fig. 4 and Suppl. Fig. 4). To confirm that Ag7NO11 bestowed the capacity to reduce

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the quantity of the biomass in the biofilms, we performed a crystal violet staining assay –following the

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O’Toole method (53) - on the established E .coli, P. aeruginosa, and S .aureus biofilms following a

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24h exposure to AgNO3, Ag7NO11, and CuSO4 (Fig. 5). We observed that Ag7NO11 had significantly

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greater efficacy than AgNO3 and CuSO4 at reducing the quantity of biomass in E. coli and P.

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aeruginosa biofilms – albeit, much higher concentrations were needed to reduce the P. aeruginosa

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biofilm-biomass (Fig. 5). Meanwhile, Ag7NO11 only outperformed CuSO4 for reducing the biomass of

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S.aureus biofilms (Fig. 5).

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Silver oxynitrate (Ag7NO11) is more efficacious against select clinical isolates and antibiotic-

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resistant strains of E.coli [UPEC (CFT703)], P.aeruginosa [FQRP], and S.aureus [MRSA

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(USA300)]. To demonstrate the potential of using Ag7NO11 as an antimicrobial beyond lab strains of

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bacterial pathogens, we performed MBC and MBEC assays on UPEC, FQRP, and MRSA planktonic

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and biofilm populations, respectively, with serial dilutions of Ag7NO11, AgNO3, and CuSO4 (Fig. 6, 7,

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and 8; respectively). Our observations suggest that greater concentrations of AgNO3, Ag7NO11, and

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CuSO4 are required to eradicate planktonic and biofilm populations of UPEC, FQRP, and MRSA (Fig.

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6,7, and 8), with the one exception of Ag7NO11 in FQRP; which has a similar effectiveness at 10

J.Lemire et al. 2015 225

eradicating planktonic populations, as well as preventing and eradicating biofilm populations as it does

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in the PAO1 strain (Fig. 7 and 1; respectively). Nonetheless, we observed that Ag7NO11 eliminated

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planktonic populations, prevented biofilm formation, and eradicated established biofilms of UPEC,

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FQRP, and MRSA at lower concentrations than AgNO3, and CuSO4; with the exception of established

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UPEC biofilms which had no considerable log-reduction values at the concentrations and treatment

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times tested (Fig. 6,7,8).

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DISCUSSION Biofilms represent an earnest concern in medicine, as they contribute to chronic infections,

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complicate wound healing, contaminate medical devices, and exhibit resistance to conventional

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antimicrobials (54-63). Ionic silver (Ag+) has a demonstrated antimicrobial outcome at low

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concentrations (64-66) (67) [and references within] and may offer an alternative option for treating

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antibiotic-resistant infections. Here, we demonstrated how silver oxynitrate (Ag7NO11) – a high

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oxidation state Ag compound - carries an enhanced antimicrobial and antibiofilm capacity.

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Additionally, our present study adds to the body of literature supporting the antimicrobial and

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antibiofilm potential of Ag.

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In this study we confirmed that Ag is effective at eliminating planktonic populations of E. coli,

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P. aeruginosa, and S. aureus. Additionally, we established that Ag is effective at preventing biofilm

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formation (Fig. 1) and eradicating established biofilms (Fig. 2). Indeed, we also observed that Cu is

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effective at killing planktonic and biofilm populations of lab pathogens, albeit at millimolar

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concentrations. Mechanistically, both Cu and Ag are thiophilic metals that have similar targets in the

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microbial cell (1). One key distinction between the two metals, is that Cu is an essential metal, while

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Ag is non-essential and has no known function in the bacterial cell (1). However, it’s important to

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recognize that the precise manner with which Ag poisons the microbial cell remains to be fully

249

elaborated. 11

J.Lemire et al. 2015 250

One novel highlight of this study was that it demonstrated, for the first time, that Ag7NO11 –a

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source of higher oxidation states of Ag- had a MBC and MBEC that was much lower (2 fold or lower)

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than the other metal compounds tested. The advantage of having a lower MBC and MBEC is that less

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of the compound would be required to have the desired therapeutic effect. Interestingly, Ag7NO11 was

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not significantly better than AgNO3 at preventing E. coli and P. aeruginosa biofilm formation, or at

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eradicating established P. aeruginosa biofilms (Fig. 1a and 2). But, Ag7NO11 was significantly better

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than AgNO3 at eradicating planktonic populations of all tested bacterial strains (Fig. 1b). This

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observation is not surprising as biofilms are notoriously more difficult to eliminate than their

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planktonic counterparts (51, 52, 68, 69) – an observation confirmed by this study (Fig. 1, 2, and

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Suppl. Fig. 1). In terms of the MBC and MBEC of the Ag salts, the trend in efficacy appeared to

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follow the molar solubility of the salts in water; AgNO3 > Ag2SO4 > AgO > Ag2O (Ksp = 51.6M,

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1.4x10-5M, 4.8x10-8M, and 3.6x10-11M; respectively). However, we did note strain specific

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sensitivities. For example, E. coli planktonic populations were more sensitive to Ag2SO4 in

263

comparison to P. aeruginosa and S. aureus. We were surprised at the poor efficacy of AgSD –the

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silver/sulfonamide synergistic biocidal compound- as it is part of the current standard-of-care for the

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treatment of burn wounds (70-72). In some cases, CuSO4 outperformed AgSD as a bactericidal

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compound (Fig. 1B and 2). Not surprisingly, much higher concentrations and treatment periods with

267

Ag and Cu were needed to reduce the viable cells in mature biofilms (4d; P.aeruginosa, or 6d; E.coli

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and S.aureus) [Fig. 3]. The enhanced metal resistance of mature biofilms has been reported previously

269

(73). However, making comparisons across studies is difficult due to differing methodologies used for

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growing the biofilms. Regardless, this study demonstrated that Ag7NO11 outperformed AgNO3 and

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CuSO4 for eradicating mature E.coli and S.aurues biofilms.

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A universally recognized challenge in treating biofilm-associated infections is reducing the

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biomass of the biofilm. Without reduction of the biofilm-biomass, many antimicrobials will kill the

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outermost bacteria in the biofilm, but will fail to penetrate and kill the innermost bacterial cells – 12

J.Lemire et al. 2015 275

resulting in recurring or persistent infections (9, 74-76). Performing live/dead™ staining, following a

276

24h AgNO3 and Ag7NO11 exposure to established biofilms, revealed that Ag7NO11 has the capacity, to

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reduce the biomass E. coli, P. aeruginosa, and S. aureus biofilms. Both, AgNO3 and Ag7NO11

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increased the amount of dead cells within the biofilm, but Ag7NO11 was superior for reducing the

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biomass of the biofilm (Fig. 4 and Suppl. Fig 4). Monitoring the quantity of biomass in established

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biofilms of E. coli, P. aeruginosa, and S. aureus following a 24h exposure to CuSO4, AgNO3, and

281

Ag7NO11, revealed that Ag7NO11 reduces the amount of biofilm-biomass significantly more than

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AgNO3 and CuSO4 in E. coli, and P. aeruginosa. Meanwhile, Ag7NO11 was only significantly better

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than CuSO4 in reducing the biomass of S. aureus biofilms (Fig 4). Interestingly, while the biomass-

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reducing effects of the metal compounds on E. coli biofilms were concentration dependent, this was not

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the case with P. aeruginosa or S. aureus. This phenomenon raises interesting insights into the potential

286

necessity to target therapies toward particular infections. However, no conclusions should be drawn

287

from these limited observations.

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Our overarching goal was to demonstrate the universal antimicrobial and antibiofilm potential

289

of Ag7NO11. Accordingly, we performed an MBC and MBEC assays on a three clinically-relevant

290

pathogenic strains – uropathogenic E.coli, fluoroquinolone-resistant P.aeruginosa, and methicillin-

291

resistant S.aureus - (Fig. 6, 7, and 8) as well as MBEC assays on two laboratory strains of fungi – C.

292

albicans and C. tropicalis (Suppl. Fig. 2). Again, Ag7NO11 had a lower MBC against planktonic

293

populations, prevented biofilm formation, and eradicated established biofilms at lower concentrations

294

when compared to AgNO3 and CuSO4. Certainly, Ag has been explored as a strategy to control

295

infection of uropathogens (77), as well as combatting and controlling antibiotic-resistant pathogens,

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including MRSA (78-82). The results from this study demonstrated variable sensitivities of the

297

planktonic and biofilm populations to CuSO4, AgNO3, and Ag7NO11. In general, all of the planktonic

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and biofilm populations of the clinically-relevant strains were capable of tolerating higher

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concentrations of the tested metals than their laboratory counterparts (Fig. 1, 2, 6, 7, and 8). The single 13

J.Lemire et al. 2015 300

exception to this trend was observed with FQRP, where Ag7NO11 was exceptionally efficacious in the

301

low micromollar range; tempting speculation that Ag7NO11 can be considered as an antimicrobial

302

coating for endotracheal tubes. It is also noteworthy that Ag7NO11 eliminated planktonic populations,

303

prevented biofilm formation, and eradicated established biofilms at lower concentrations than AgNO3

304

and CuSO4, with the exception of established UPEC biofilms, which could not be reduced to a

305

significant level by Ag under the conditions tested. Again, confirming the challenge involved in

306

eradicating an established biofilm. Though only limited analyses were performed in this study, we

307

observed that Candida sp. biofilms are also difficult to eradicate (Suppl. Fig 4). The enhanced

308

tolerance of Candida sp. has been observed previously within our research group (38, 83, 84).

309

However, Ag7NO11 still has an MBEC in the millimolar range – much within a potentially therapeutic

310

range. This is ever important as Candida sp. are biofilm-forming microorganisms with potential to be

311

infectious and can cause deleterious health outcomes (85-88).

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To conclude, here we have demonstrated that thiophilic metals – such as Cu and Ag – are

313

efficacious antimicrobial and antibiofilm agents. Furthermore, we’ve demonstrated that silver

314

oxynitrate (Ag7NO11) – a compound that delivers higher oxidations states of Ag – has a greater

315

antimicrobial and antibiofilm capacity, at equal concentrations of Ag, than AgNO3, Ag2SO4, AgO,

316

Ag2O, and CuSO4. Additionally, we demonstrated that Ag7NO11 reduces the biomass of the biofilm –

317

an important strategy for combatting biofilm-associated infections.

318

Recent realizations have focused on biofilms as the causative agent of many infectious diseases,

319

but we are at a loss for adequate therapies to eradicate them. This includes chronic wounds, where

320

many wound care products exist, but very few have an antibiofilm capacity. Fortunately, wound care

321

products offer the greatest potential for implementing metal-based antimicrobials, due to decreased

322

internalization of the therapeutic by the patient. This is the venue with which we envision the best

323

utility for Ag7NO11 as a strategy for preventing and controlling infectious disease. Thus, Ag7NO11 will

14

J.Lemire et al. 2015 324

add to our arsenal for combatting infection, an arsenal that is desperately needed in the face of

325

increasing antibiotic resistant infections and a dwindling pipeline of new antibiotics.

326 327 328

ACKNOWLEDGEMENTS AND CONFLICTS OF INTEREST We would like to extend a special thanks to Dr. Joe Harrison at the University of Calgary for

329

providing the MRSA strain (USA 300) and the uropathogenic E.coli strain (CFT703). We would also

330

like to extend our gratitude to Dr. Michael Parkins at the Cummings School of Medicine for providing

331

us with the fluoroquinolone-resistant P.aeruginosa isolate. J.L. is currently funded by the Natural

332

Sciences and Engineering Council of Canada - Banting Postdoctoral Fellowship Program, as well as

333

Alberta Innovates Health Solutions. A.B. received summer internship support from NSERC. This

334

work was supported by an NSERC-Engage grant and by a MITACS Accelerate fellowship for J.L.;

335

both of which required financial and in-kind contributions from Exciton Technologies Inc. (ETI). L.K

336

was funded by the NSERC Industrial R&D Fellowship (IRDF) and Alberta Innovates Technology

337

Futures (AITF) R&D Associates Fellowship for this work. ETI manufactured and supplied the silver

338

oxynitrate. In addition, ETI provided technical assistance in performing some of the experiments.

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FIGURE CAPTIONS Figure 1: Silver oxynitrate (Ag7NO11) prevents biofilm formation at lower concentrations versus other metal compounds. The MBEC™ device was inoculated with E.coli (JM109), P.aeruginosa (PAO1), and S.aureus (ATCC 25923) concurrently with serial dilutions (two-fold) of Ag2O, AgO, Ag2SO4, Ag7NO11, AgSD, and CuSO4. The A) MBEC and B) MBC were then determined after a 4h incubation with the various metal compounds. Values are represented as the median ± the range, n=4. * Indicates a significant difference between Ag7NO11 and the indicated metal compound p≤0.05. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 2: Silver oxynitrate (Ag7NO11) eradicates established biofilms at lower concentrations versus other metal compounds. The MBEC™ device was inoculated with E.coli (JM109), P.aeruginosa (PAO1), and S.aureus (ATCC 25923). Biofilms were established following a 24h incubation. The established biofilms were then treated with serial dilutions (two-fold) of Ag2O, AgO, Ag2SO4, Ag7NO11, AgSD, and CuSO4 for 24h. The MBEC was then determined for the various metal compounds. Values are represented as the median ± the range, n=4. * Indicates a significant difference between Ag7NO11 and the indicated metal compound p≤0.05. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 3: Silver oxynitrate (Ag7NO11) is more efficacious for eradicating mature biofilms of E.coli and S.aureus. E.coli (JM109), P.aeruginosa (PAO1), and S.aureus (ATCC 25923) biofilms were cultivated using the MBEC™ device. Established biofilms were formed following an incubation of 4d for P.aeruginosa, and 6d for E.coli as well as S.aureus. The biofilms were then treated with various concentrations of  CuSO4 (closed line),  AgNO3 (broken line), and Ag7NO11 (dotted line) for 48h. The MBEC was then determined for the various metal compounds. Values are represented as the mean ± the SD, n=3. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 4: Silver oxynitrate (Ag7NO11) reduces biofilm quantity. Biofilms of E.coli (JM109), P.aeruginosa (PAO1), and S.aureus (ATCC 25923) were established in the MBEC™ device for 24h. The established biofilms were then exposed to A) 0 μM, B) 5μM and C) 12.5μM AgNO3 or Ag7NO11 (at equivalent Ag concentrations) for 24h. Live/dead™ staining was then performed on the treated biofilms and the biofilm was quantified using a confocal microscope operating at 20X objective magnification. Green (Syto-9; Ex: 488nm, Em: 498nm) = live cells. Red (propidium iodide; Ex:523nm, Em: 617nm) = dead cells. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 5: Silver oxynitrate (Ag7NO11) reduces biofilm-biomass. Biofilms of E.coli (JM109), P.aeruginosa (PAO1), and S.aureus (ATCC 25923) were established in the MBEC™ device for 24h. The biofilms were then exposed to various concentrations of A) CuSO4, B) AgNO3 and C) Ag7NO11. Pursuant, a crystal violet staining protocol –using the O’Toole method (53) – was performed on the biofilms. The biomass was quantified by measuring the absorbance at 550 nm. Values are represented as the mean ± the SD, n=15. * Indicates a significant difference between CuSO4 and Ag7NO11, where 21

J.Lemire et al. 2015 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668

* = p≤0.05, ** = p≤0.01, *** is p≤0.001, and **** is p≤0.0001. + Indicates a significant difference between AgNO3 and Ag7NO11, where + = p≤0.05, ++ = p≤0.01, +++ is p≤0.001, and ++++ is p≤0.0001. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 6: Silver oxynitrate (Ag7NO11) has antimicrobial and antibiofilm activity against uropathogenic E.coli (UPEC) [CFT703]. UPEC biofilms were cultivated using the MBEC™ device. The A) MBC, on the planktonic population, and B) MBEC, on the biofilm population, were determined after a 4h incubation in the presence of serial dilutions (two-fold) of various metal compounds. C) Established biofilms were cultivated in the MBEC™ device for 24h. The MBEC was then determined following a 24h treatment with serial dilutions (two-fold) of various metal compounds.  CuSO4 (closed line),  AgNO3 (broken line), and Ag7NO11 (dotted line). Values are represented as the mean ± the SD, n=4. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. . Figure 7: Silver oxynitrate (Ag7NO11) has antimicrobial and antibiofilm activity against a fluoroquinolone-resistant P.aeruginosa (FQRP) isolate. FQRP biofilms were cultivated using the MBEC™ device. The A) MBC, on the planktonic population, and B) MBEC, on the biofilm population, were determined after a 4h incubation in the presence of serial dilutions (two-fold) of various metal compounds. C) Established biofilms were cultivated in the MBEC™ device for 24h. The MBEC was then determined following a 24h treatment with serial dilutions (two-fold) of various metal compounds.  CuSO4 (closed line),  AgNO3 (broken line), and Ag7NO11 (dotted line). Values are represented as the mean ± the SD, n=4. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself. Figure 8: Silver oxynitrate (Ag7NO11) has antimicrobial and antibiofilm activity against a methicillin-resistant S.aureus (MRSA) strain, [USA300]. MRSA biofilms were cultivated using the MBEC™ device. The A) MBC, on the planktonic population, and B) MBEC, on the biofilm population, were determined after a 4h incubation in the presence of serial dilutions (two-fold) of various metal compounds. C) Established biofilms were cultivated in the MBEC™ device for 24h. The MBEC was then determined following a 24h treatment with serial dilutions (two-fold) of various metal compounds.  CuSO4 (closed line),  AgNO3 (broken line), and Ag7NO11 (dotted line). Values are represented as the mean ± the SD, n=4. #Note: all Ag stock solutions were prepared at equal molar equivalents of Ag molecules. Hence, concentrations found in this figure are reflective of the concentration of Ag and not the silver containing compound itself.

22