Ann-8|oh|m Acnv|ty

Acknowledgments must be made to Glenda Motta Associates who coordinated the clinical case series.
Annm|crob|a| S||ver |n Med|ca| Dev|ces: Compos|non and Lmcacy
Steven M|||er
1
, Ioseph Lem|re
2,3
, A|exandru 8randu
2,3
, Imran U|-naq
1
, Dav|d L|schuk
1
, Car|a Sp|na, 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

ApproxlmaLely $33-43 8llllon uSu/year (Scou. 8.u. 2009) ls spenL on hosplLal
acqulred bacLerlal lnfecuons, many of whlch are mulu- or even LoLally" drug
reslsLanL. ln a wound, presence of bacLerlal blolm ls hypoLheslzed Lo be a key
llnk Lo Lhe chronlc sLaLe and sLalled heallng. Sllver (Ag) ls a meLal used for
cenLurles ln medlclne and ls of renewed lnLeresL for LreaLmenL and prevenuon
of lnfecuon. CurrenLly, many Ag conLalnlng medlcal devlces lncorporaLe elLher
meLalllc Ag or compounds such as AgCl
(s)
and Ag
2
SC
4(s)
. 1he dlerences are
slgnlcanL as lonlc Ag
1+
ls essenual Lo anumlcroblal acuvlLy and Lhe
physlochemlcal properues of Lhese compounds relaLes Lo emcacy.
lurLhermore, research lnLo hlghly oxldlzed Ag (
2+
and
3+
) ls sull ln lLs lnfancy.
1hls sLudy alms Lo dlerenuaLe Lhe "#$%&'()*&"! and "#$*&)+,% emcacy of Ag
specles found ln commerclally avallable wound dresslngs wlLh focus on Ag
oxynlLraLe (Ag
7
nC
11(s)
) conLalnlng Ag
2+
and Ag
3+
.

Introducnon
0
100
200
300
400
300
600
700
800
900
1000
8C waLer
SWl
1) hyslochemlcal properues of sllver compounds lmpacL bloacuvlLy
2) Sllver oxynlLraLe conLalns h|gher ox|danon sLaLes of sllver (Ag
2+
, Ag
3+
)
3) Ag
2+
, Ag
3+
can LargeL muluple phases of Lhe mlcroblal llfesLyle
Ag
7
NO
11

Ag
2
O & Ag
0

Ag
2
SO
4
AgCl
Ag
0

0
0.075
0.15 0.3 0.6
1.22
2.44
4.88 9.7
12.5
19.5 25
39.1 50
78.1
100
156.3
200
312.5
390
625
780
1250
1560
3125 0
1
2
3
4
5
6
7
8
Conc. [M]
L
o
g
[C
F
U
/P
e
g
]
JM109 Established Biofilms 24h - CuSO4, AgNO3, Exsalts
CuSO4
AgNO3
Ag7NO11
0
0.075
0.15 0.3 0.6
1.22
2.44
4.88 9.7
12.5
19.5 25
39.1 50
78.1
100
156.3
200
312.5
390
625
780
1250
1560
3125
6250
12500
25000
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
CuSO4
AgNO3
Ag7NO11
0
0.075
0.15 0.3 0.6
1.22
2.44
4.88 9.7
12.5
19.5 25
39.1 50
78.1
100
156.3
200
312.5
390
625
780
1250
1560
3125
6250
12500
25000
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
CuSO4
AgNO3
Ag7NO11
Wound dresslngs conLalnlng AgCl
(s)
, AgSC
4(s)
, Ag
(s)
, Ag
2
C
(s)
, or Ag
7
nC
11(s)
were
evaluaLed for chemlcal composluon by x-ray dlracuon (x8u) and Ag release
proles lnLo 8C waLer and a slmulaLed wound uld (SWl) by poLenuomeLrlc
uLrauon and neuLron acuvauon analysls (nAA). Log-reducuon assays were
performed for "#$%&'!()())*+ $*-.*+ ln W, challenged wlLh 10
6
cfu/mL and
quanucauon of klll occurred aer 4h exposure. Mlnlmum blolm lnhlbluon
and eradlcauon concenLrauon of Lwo sllver compounds was deLermlned as
descrlbed ln Lhe resulLs.

Methods
S||ver ke|ease, Med|a, and Lmcacy
I|gure 4: 1he eects of med|a type on s||ver re|ease and the M8LC of d|erent s||ver spec|es. A) Sllver release of dlerenL Ag compounds
was measured ln 8C waLer and slmulaLed wound uld (SWl) by neuLron acuvauon analysls (nAA). 8) M8lC assays were performed on Lhe
plankLonlc and blolm populauons of |) /0 $.-*123(+$ (AC1), and ||) "0$*-.*+ (A1CC 23923) grown ln Lhe presence of varylng
concenLrauons of AgnC3, and Ag7nC11 for 4h ln elLher Lhe presence of pepLone waLer (W) or SWl. n > 4 Su.
Silver oxynitrate Ag
7
NO
11

Acknow|edgments
D|scuss|on and Conc|us|ons
Wound dresslngs conLalnlng AgCl
(s)
, AgSC
4(s)
, Ag
(s)
, Ag
2
C
(s)
, or Ag
7
nC
11(s)
have
dlerenL raLes of lonlc Ag dlssoluuon. CuanuLy of sllver does noL LranslaLe lnLo
anumlcroblal emcacy as measured by log reducuon (g. 3).

Medla componenLs do lndeed lnuence
Lhe emcacy of Ag, Lherefore a cllnlcally
relevanL medlum should be uullzed for
assays evaluaung Lhe anumlcroblal and
anublolm acuvlLy of meLal specles.
SWl mlmlcs a wound envlronmenL
where Loplcal agenLs would be placed.
uslng an esLabllshed blolm model, Lhe
ablllLy Lo eradlcaLe maLure blolm ln
SWl was evaluaLed.
1) Ag can exlsL ln many compounds wlLh dlerenL oxldauon sLaLes and aqueous
avallablllLy - 1hese properues lnuence anumlcroblal emcacy.
2) Plgher oxldauon sLaLe Ag has anumlcroblal and anublolm acuvlLy aL lower
concenLrauons Lhan AgnC
3
(Ag
+
) and CuSC
4
(Cu
2+
) ln all organlsms LhaL were
LesLed 23 42#-(.
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.
70m Ag7NO11
AgNO3
0.75M 4.88M 1250M
0M
80m
AgNO3
0.75M 4.88M 1250M
0M
Ag7NO11
70m
AgNO3
0.75M 4.88M 1250M
0M
Ag7NO11
III) S.aureus (ATCC 25923)
II) P. aeruginosa (PAO1)
I) E.coli (JM109)
I|gure 6: 1he M8LC of 24h estab||shed b|oh|ms from var|ous mode| organ|sms grow|ng |n the presence of CuSC4, AgNC3, and Ag7NC11
for 24h . M8LC assays were performed on blolm populauons of I) 50)(!2 (!M109),II) /0 $.-*123(+$ (AC1), and III) "0$*-.*+ (A1CC 23923)
LhaL were grown for 24h and Lhen LreaLed wlLh varylng concenLrauons of CuSC4, AgnC3, and Ag7NO11 for 24h. n > 4 Su. Confocal
mlcroscopy was performed on Lhe blolms uslng Lhe Llve/uead sLaln [red = propldlum lodlde (dead cells) and green = syLo 9 (llve cells)]
on cells LhaL were LreaLed wlLh varylng concenLrauons of AgnC3, and Ag7nC11 for 24h.
I|gure 1: A) Scannlng LlecLron Mlcrograph (SLM) lmage of Ag7nC11 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: Ag7NO11(s), Ag2O(s), Ag2SO4(s), AgCl(s) and Ag(s) display unique
fingerprints due to differing physiochemical properties.

I|gure 2: 1he mlcroblal llfesLyle ln a blolmcan 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.
[
A
g
]
io
n

g
/m
L

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 "0 $*-.*+ 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
anuml crobl al emcacy agal nsL boLh
plankLonlc cells and Lhose growlng as a
blolm. Charged specles and proLelns ln
c ompl ex medl um bl nd l onl c 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
0.75
0.15
0.3
0.6
1.22
2.44
4.88
9.7
19.5
39.1
78.1
156.3
312.5
625
1250
0
2
4
6
8
10
Conc. [M]
L
o
g
[C
F
U
/m
L
]
PAO1 Planktonic PWvs SWF
PW-AgNO3
SWF - AgNO3
PW- Ag7NO11
SWF - Ag7NO11
0
0.75
0.15
0.3
0.6
1.22
2.44
4.88
9.7
19.5
39.1
78.1
156.3
312.5
625
1250
0
2
4
6
Conc. [M]
L
o
g
[C
F
U
/P
e
g
]
S.aureus BiofilmPWvs SWF
PW- AgNO3
SWF - AgNO3
PW- Ag7NO11
SWF - Ag7NO11
0
0.75
0.15
0.3
0.6
1.22
2.44
4.88
9.7
19.5
39.1
78.1
156.3
312.5
625
1250
0
2
4
6
8
10
Conc. [M]
L
o
g
[C
F
U
/m
L
]
S.aureus Planktonic PWvs SWF
PW- AgNO3
SWF - AgNO3
PW- Ag7NO11
SWF - Ag7NO11
0
0.75
0.15
0.3
0.6
1.22
2.44
4.88
9.7
19.5
39.1
78.1
156.3
312.5
625
1250
-2
0
2
4
6
8
Conc. (uM)
L
o
g
[C
F
U
/P
e
g
]
PAOI biofilmPWvs SWF
PW- AgNO3
SWF - AgNO3
PW- Ag7NO11
SWF - Ag7NO11
A)
B)
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
I
C
M
B
I
C

MBEC
*
*
* Maintained for 7 days
with daily 106 cfu/mL
challenge
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 measure th|s and assess d|erent |ntervennons?
Week 1
Week 4