0

0
.0
7
5
0
.1
5
0
.3
0
.6
1
.2
2
2
.4
4
4
.8
8
9
.7
1
2
.5
1
9
.5
2
5
3
9
.1
5
0
7
8
.1
1
0
0
1
5
6
.3
2
0
0
3
1
2
.5
3
9
0
6
2
5
7
8
0
1
2
5
0
1
5
6
0
3
1
2
5
0
1
2
3
4
5
6
7
8
Conc. [M]
L
o
g
[
C
F
U
/P
e
g
]
JM109 Established Biofilms 24h - CuSO
4
, AgNO
3
, Exsalts
CuSO
4
AgNO
3
Ag7NO
11
0
0
.7
5
0
.1
5
0
.3
0
.6
1
.2
2
2
.4
4
4
.8
8
9
.7
1
9
.5
3
9
.1
7
8
.1
1
5
6
.3
3
1
2
.5
6
2
5
1
2
5
0
-2
0
2
4
6
8
Conc. (uM)
L
o
g

[
C
F
U
/
P
e
g
]
PAOI biofilm PW vs SWF
PW- AgNO
3
SWF - AgNO
3
PW- Ag
7
NO
11
SWF - Ag
7
NO
11
Ann-8|oh|m Acnv|ty
Acknowledgments must be made to Glenda Motta Associates who coordinated the clinical case series.
S||ver and 8|oh|ms: 1he D|erence of Spec|es
Ioseph Lem|re
2,3
, A|exandru 8randu
2,3
, Steven M|||er
1
,kaymond 1urner
2,3
and L|ndsay ka|an
1
*
1LxclLon1echnologleslnc. , LdmonLon, AlberLa, Canada,*lkalan[exclLonLech.com, 780-248-3883
2ueparLmenL of 8lologlcal Sclences, unlverslLy of Calgary, Calgary A8, Canada,
38lolm8esearch Croup, unlverslLy of Calgary, Calgary A8, Canada

ln a wound, presence of bacLerlal blolm ls hypoLheslzed Lo be a key llnk Lo Lhe
chronlc sLaLe and sLalled heallng (g. 2). Sllver (Ag) ls a meLal for LreaLmenL and
prevenuon of lnfecuon and Ag conLalnlng medlcal devlces lncorporaLe elLher
meLalllc Ag or compounds such as AgCl
(s)
and Ag
2
SC
4(s)
. 1he dlerences are
slgnlcanL as lonlc Ag
1+
ls essenual Lo anumlcroblal acuvlLy and Lhe physlochemlcal
properues of Lhese compounds relaLes Lo emcacy. lurLhermore, use of hlghly
oxldlzed Ag (
2+
and
3+
) has recenLly been lnLroduced. 1h|s study a|ms to
d|erennate the "#$%&'()*&"+ and "#$*&),+% emcacy of Ag spec|es found |n
commerc|a||y ava||ab|e wound dress|ngs w|th focus on Ag oxyn|trate (Ag
7
NC
11(s)
)
conta|n|ng Ag
2+
and Ag
3+
.

Introducnon and Cb[ecnves
1) hys|ochem|ca| propernes of s||ver compounds |mpact b|oacnv|ty
2) S||ver oxyn|trate conta|ns h|gher ox|danon states of s||ver (Ag
2+
, Ag
3+
)
3) Ag
2+
, Ag
3+
can target mu|np|e phases of the m|crob|a| ||festy|e
Ag
7
NO
11

Ag
2
O & Ag
0

Ag
2
SO
4
AgCl
Ag
0

0
0
.0
7
5
0
.1
5
0
.3
0
.6
1
.2
2
2
.4
4
4
.8
8
9
.7
1
2
.5
1
9
.5
2
5
3
9
.1
5
0
7
8
.1
1
0
0
1
5
6
.3
2
0
0
3
1
2
.5
3
9
0
6
2
5
7
8
0
1
2
5
0
1
5
6
0
3
1
2
5
6
2
5
0
1
2
5
0
0
2
5
0
0
0
0
1
2
3
4
5
6
7
8
9
10
Conc. [M]
L
o
g
[C
F
U
/P
e
g
]
PAO1 Established Biofilms - 24h Treatment
CuSO
4
AgNO
3
Ag7NO
11
0
0
.0
7
5
0
.1
5
0
.3
0
.6
1
.2
2
2
.4
4
4
.8
8
9
.7
1
2
.5
1
9
.5
2
5
3
9
.1
5
0
7
8
.1
1
0
0
1
5
6
.3
2
0
0
3
1
2
.5
3
9
0
6
2
5
7
8
0
1
2
5
0
1
5
6
0
3
1
2
5
6
2
5
0
1
2
5
0
0
2
5
0
0
0
0
1
2
3
4
5
6
7
8
Conc. [M]
L
o
g
[
C
F
U
/P
e
g
]
S.aureus Established Biofilms - 24h Treatment
CuSO
4
AgNO
3
Ag7NO
11
.

S||ver ke|ease and Lmcacy
I|gure 4: 1he eecLs of medla Lype on anublolm acuvlLy of dlerenL sllver specles. M8lC assays were
performed on blolm populauons of |) !"#$%&'()*+# (AC1), and ||) -"#&%$&+ (A1CC 23923) grown ln Lhe
presence of varylng concenLrauons of AgnC
3
, and Ag
7
nC
11
for 4h ln elLher pepLone waLer (W) or SWl. n >
4 Su.
Silver oxynitrate Ag
7
NO
11

Acknow|edgments
D|scuss|on and Conc|us|ons
uslng an esLabllshed blolm model,
Lhe mlnlmum blolm eradlcauon
concenLrauon (M8LC) of maLure
blolm ln SWl was evaluaLed.
1) Ag can exlsL wlLh dlerenL oxldauon sLaLes and aqueous avallablllLy
2) Ag
2+/3+
has anumlcroblal and anublolm acuvlLy aL lower concenLrauons Lhan Ag
+

ln all organlsms LhaL were LesLed () .(/%*.
3) 1hese ndlngs supporL evldence for use of hlgh-oxldauon sLaLe Ag
(2+/3+)
as a
Loplcal anumlcroblal LreaLmenL for chronlc lnfecuon caused by blolm.
70
m
Ag7NO11
AgNO3
0.75M4.88M 1250M
0M
80m
AgNO3
0.75M4.88M 1250M
0M
Ag7NO11
70
m
AgNO3
0.75M 4.88M 1250M
0M
Ag7NO11
I|gure 6: 1he M8LC of 24h estab||shed b|oh|ms grow|ng |n the presence of CuSC
4
, AgNC
3
, and Ag
7
NC
11
for 24h.
I) 0"1*2( (!M109),II) !" #$%&'()*+# (AC1), and III) -"#&%$&+ (A1CC 23923) were grown for 24h and Lhen LreaLed
wlLh varylng concenLrauons of CuSC
4
, AgnC
3
, and Ag
7
nC
11
for 24h. n > 4 Su. Confocal mlcroscopy was
performed on Lhe blolms uslng Lhe Llve/uead sLaln [red = propldlumlodlde (dead cells) and green = syLo 9 (llve
cells)] on cells LhaL were LreaLed wlLh varylng concenLrauons of AgnC
3
, and Ag
7
nC
11
for 24h.
I|gure 1: A) Scannlng LlecLron Mlcrograph (SLM) lmage of Ag
7
nC
11
crysLals before and aer exposure Lo
aqueous medla, lnluaung Lhe degradauon process and releaslng oxldlzed sllver from Lhe crysLalllne sLrucLure. 8)
X-Ray Diffraction (XRD) pattern of common silver compounds found in topical antimicrobial medical
devices: Ag
7
NO
11
(s)
,
Ag
2
O(s), Ag
2
SO
4
(s), AgCl(s) and Ag(s) display unique fingerprints due to differing
physiochemical properties.
I|gure 2: 1he mlcroblal llfesLyle ln a blolm can be llnked Lo phases of mlcroblal conLamlnauon Lhrough Lo
lnfecuon and subsequenL lmpalred heallng ln a wound. lnLervenuon LhaL dlsrupLs mlcroblal growLh, lncludlng
blolmcan allow heallng Lo resume.
I|gure 3: A) Sllver release curves of Ag compounds lnLo SWl from a wound dresslng. Analysls was
performed ln LrlpllcaLe, repllcaLes pooled, and measured by nAA. oLenuomeLrlc uLrauon ln 8C waLer
was performed ln LrlpllcaLe as a conLrol (daLa noL shown). 8) 1he same wound dresslngs were assayed for
log reducuon of -" #&%$&+ survlval ln a 4h ume klll assay ln epLone WaLer (M8u).
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
1 3 3 7
C
u
m
u
|
a
n
v
e
A
g
k
e
|
e
a
s
e
m
g
]
m
L

1|me (days)
Ag7NO11 (s) - 10 mg
Ag2O/Ag (s) 37.5 mg
Ag2SO4 (s)- 30 mg
Ag(s) 62.5 mg
AgCl(s) 3 mg
A)
1.00L+00
1.00L+01
1.00L+02
1.00L+03
1.00L+04
1.00L+03
1.00L+06
1.00L+07
L
o
g
C
I
U
]
m
L

8)
Medla lnuences boLh Ag release and anumlcroblal emcacy agalnsL boLh
plankLonlc cells and Lhose growlng as a blolm. Charged specles and proLelns ln
complex medlum blnd lonlc Ag dramaucally reduclng bloavallablllLy. Ag
2+/3+

specles are able Lo malnLaln greaLer anumlcroblal emcacy regardless of medla
Lype due Lo hlgher oxldauve power (g. 4)

0
0
.7
5
0
.1
5
0
.3
0
.6
1
.2
2
2
.4
4
4
.8
8
9
.7
1
9
.5
3
9
.1
7
8
.1
1
5
6
.3
3
1
2
.5
6
2
5
1
2
5
0
0
2
4
6
Conc. [M]
L
o
g
[
C
F
U
/P
e
g
]
S.aureus Biofilm PW vs SWF
PW- AgNO
3
SWF - AgNO
3
PW- Ag
7
NO
11
SWF - Ag
7
NO
11
i)
ii)
Ag
2+]3+
|s s|gn|hcant|y beuer than Ag
1+

at k||||ng b|oh|ms. I|gure S: Schemauc of M8LC
assay.
Lxc|ton 1echno|og|es Inc.: SupporL of Lhe presenLed research
Natura| Sc|ence and Lng|neer|ng kesearch Counc||: l8ul Lo L.k, ul Lo !.L and Lngage granL Lo 8.1
M|tacs: AcceleraLe granL for an lndusLry-parLnered fellowshlp for !.L
A|berta Innovates 1echno|ogy Iutures: r&u AssoclaLes lellowshlp Lo L.k
Nanona| Insntute of Nanotechno|ogy (NIN1): Access Lo SLM and x8u lnsLrumenLauon
M
B
I
C

*
*
* Ma|nta|ned for 7 days
w|th da||y 106 cfu]mL
cha||enge
A) 8)
Colonization
Contamination
Critical Colonization
Infection
2 ppm
16.8 ppm
2 ppm
Harrison et al. 2008. Nature Rev Micro
Phillips et al. 2010. Wounds International

Intervennon
now can you measure and assess d|erent annm|crob|a| |ntervennons?
Week 1
Week 4
Sllver release proles of wound dresslngs conLalnlng AgCl
(s)
, AgSC
4(s)
, Ag
(s)
,
Ag
2
C
(s)
, or Ag
7
nC
11(s)
were evaluaLed ln a slmulaLed wound uld (SWl) by
neuLron acuvauon analysls (nAA). Log-reducuon assays were performed for
-/#3452*1*11&+ #&%$&+ ln pepLone waLer (W), challenged wlLh 10
6
cfu/mL and
quanucauon of klll occurred aer 4h exposure. CuanuLy of sllver does noL
LranslaLe lnLo anumlcroblal emcacy as measured by log reducuon (g. 3).
Ag
2+]3+
|s beuer than Ag
1+
at
|nh|b|nng b|oh|m formanon