Burns 30 (2004) 35–41

An in vitro study of the anti-microbial efficacy of a 1% silver

sulphadiazine and 0.2% chlorhexidine digluconate cream夽,
1% silver sulphadiazine cream夽夽 and a silver coated dressing夹
John F. Fraser∗ , Jan Bodman, Ruth Sturgess, Joan Faoagali, Roy M. Kimble
Department of Paediatrics and Child Health, Royal Children’s Hospital, Queensland Health Pathology Service,
University of Queensland, Herston 4029, Brisbane, Qld, Australia

Accepted 19 August 2003

Abstract
Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped
improve the survival in these patients. There are a number of anti-microbials available, one of which, SilvazineTM (1% silver sulphadiazine
(SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared SilvazineTM with
the new dressing ActicoatTM . This study compared the anti-microbial activity of SilvazineTM , ActicoatTM and 1% silver sulphadiazine
(FlamazineTM ) against eight common burn wound pathogens.
Methods: Each organism was prepared as a suspension. A 10 ␮l inoculum of the chosen bacterial isolate (representing approximately
between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were
then incubated and 10 ␮l loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated
for 18 hours and a colony count was performed.
Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (SilvazineTM ) results in the most
effective killing of all bacteria. SSD and ActicoatTM had similar efficacies against a number of isolates, but ActicoatTM seemed only
bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus
mirabilis remained at 24 h.
Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (SilvazineTM ) is a more effective anti-microbial against
a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required.
© 2003 Elsevier Ltd and ISBI. All rights reserved.
Keywords: Anti-microbial; Wound sepsis; Burns; Sulphonamides; Silver

1. Introduction use of more potent systemic antibiotics, infection remains a

The patient suffering major burns is at risk from both
cutaneous and systemic infection. Prior to the routine use
of topical anti-microbial agents (TAAs), burn wound sep-
sis was listed as cause of death in 60% of burns mortal-
ity. The routine application of topical silver nitrate in 1965
[1] rapidly reduced this figure to 28%; a figure further di-
minished by the addition of mafenide acetate [2] and silver
sulphadiazine (SSD or FlamazineTM ) [3] in the late 1960s
to existing therapy [4]. Despite these interventions, and the
夽 SilvazineTM
夽夽 FlamazineTM
夹ActicoatTM
∗Corresponding author.
E-mail address: j.fraser@uq.edu.au (J.F. Fraser).
leading cause of morbidity and mortality in burns patients
[5,6].
The increasing prevalence of multi-resistant and pan-
antibiotic resistant bacteria, has seen a variety of silver
dressings become the mainstay of TAAs [7]. Silver has
been recognised as an effective anti-microbial for over 2000
years [8]. It has broad anti-microbial gram negative and
gram-positive activities as well as anti-fungal properties.
There is also minimal development of bacterial resistance.
There are limited side effects of topical silver therapy; silver
toxicity or argyrosis is uncommon and generally resolves
with cessation of the therapy [9].
The majority of side effects of TAAs are associated with
the co-molecule sulphadiazine. Transient and self-limiting
leukopenia is found in between 3 and 60% of patients
treated with sulphadiazine [7,10,11] and the development

0305-4179/$30.00 © 2003 Elsevier Ltd and ISBI. All rights reserved.

doi:10.1016/j.burns.2003.09.008
36 J.F. Fraser et al. / Burns 30 (2004) 35–41
of dermatitis and toxic epidermal necrolysis (TEN) has
also been reported [10]. Mafenide acetate can result in the
development of a non-anion gap acidosis, a reflection of its
activity as a carbonic anhydrase inhibitor [9,12–14].
During the late 1960s, SSD was the gold standard topi-
cal anti-microbial used in burns patients around the world.
An outbreak of resistant Staphylococcus aureus in the burns
unit in Royal Melbourne Hospital in 1971 led Dr. M. Clarke
to introduce 0.2% chlorhexidine digluconate to SSD, in an
attempt to control this outbreak [15]. The trial was success-
ful and the resultant dressing was introduced as SilvazineTM
[16]. In a number of clinical and laboratory based stud-
ies, this preparation was seen as superior against a num-
ber of clinically significant bacteria including S. aureus and
methicillin-resistant S. aureus (MRSA) [17,18]. Since that
time, SilvazineTM has been the mainstay of burn therapy in
Australasia.
In September 2001, ActicoatTM , 3-ply gauze dress-
ing consisting of an absorbent rayon polyester core, with
upper and lower layers of nanocrystalline silver coated
high-density polyethylene mesh was introduced to Aus-
tralasia. The nanocrystalline structure was integral in the
effect of ActicoatTM , as the inherently unstable structure al-
lowed for rapid release of both the metallic and ionic silver
to the wound bed. ActicoatTM contains 0.84–1.34 mg/cm2
of silver, which is equivalent to 0.1 ml of SilvazineTM
or FlamazineTM w/w silver. Laboratory tests showed
ActicoatTM to be at least comparable with SSD and sil-
ver nitrate, if not better in terms of anti-microbial efficacy
[19,20]. No studies were conducted comparing ActicoatTM
with SilvazineTM have been conducted to our knowledge,
despite the fact previous data shows SilvazineTM to be a
superior TAA to SSD [17,21].
In an attempt to determine which dressing was the most
effective TAA, our group undertook to perform the first in
vitro study of anti-bacterial efficacy comparing SilvazineTM
(1% silver sulphadiazine and 0.2% chlorhexidine diglu-
conate), FlamazineTM (1% silver sulphadiazine) and
ActicoatTM .
2. Methods
All tests were carried out in the microbiology labora-
tory of a tertiary referral hospital with scientists conducting
the bench work in tandem. Samples of all three dressing
(SilvazineTM , FlamazineTM and ActicoatTM ) were prepared
in a standard, sterile fashion as follows:
2.1. Preparation of dressings
Squares of 1 cm of ActicoatTM were prepared in an asep-
tic, sterile manner. SilvazineTM and FlamazineTM samples
of 0.1 g were aspirated using sterile syringes. All sam-
ples were individually weighed on Delta PC4400 scales
(Mettler-Toledo Ltd., Tampa, Fl).
Table 1
Bacterial isolates studied
Methicillin-resistant Staphylococcus aureus ATCC 33591
Pseudomonas aeruginosa NCTC 10662
Escherichia coli ATCC 35218
Staphylococcus aureus NCTC 6571
Enterococcus faecalis ATCC 29212
Enterobacter cloacae ATCC 10347
Proteus mirabilis ATCC 7002
Acinetobacter baumannii Wild strain
2.2. Preparation of bacterial isolates
Bacteria used in this study were from the American type
culture collection (ATCC) except S. aureus, which was from
the national culture type collection (NCTC). The wild type
Acinetobacter baumannii was a multi-resistant strain which
been responsible for invasive burn sepsis in a number of pa-
tients in the intensive care unit at the hospital where the labo-
ratory work was carried out. A suspension of each organism
was prepared and the turbidity adjusted to a 0.5 McFarland
standard (Table 1).
2.3. Preparation of bacterial broth
Four samples were used for each test; control (no
treatment added), and the three experimental dressings
(ActicoatTM , SilvazineTM and FlamazineTM ). Four vials,
were prepared, each containing 1 ml of Tryptic Soy Broth
(Biomerieux, Australia). A 10 ␮l inoculum of the chosen
bacterial isolate (representing approx between 104 and 105
total bacteria) was added to each of the vials prior to the
introduction of the experimental dressing. The broths were
then incubated with agitation at 35 ◦ C. This method was
repeated for each isolate.
At specified intervals (0, 30 min, 2, 4, 6, 8 and 24 h), a
10 ␮l was removed from the vials and subcultured on Horse
Blood Agar (Biomerieux, Australia). These agar plates were
then incubated at 36 ◦ C for 18 hours, after which time, a
colony count was performed by experienced microbiolo-
gists. Each study was performed three times to ensure re-
producibility.
3. Results
The results are presented as kill curves for the bacteria
studied, (Figs. 1–8). The data demonstrates that the com-
bination of 1% SSD and 0.2% chlorhexidine digluconate
(SilvazineTM ) results in the most effective killing of all
bacteria. There were no detectable bacteria of any isolates
isolated after 30 minutes when SilvazineTM was studied.
SSD and ActicoatTM had similar efficacies against a num-
ber of isolates, but ActicoatTM seemed only bacteriostatic
against E. faecalis and MRSA. Viable S. aureus was also
still present at 24 hours in the ActicoatTM tests. In the
 J.F. Fraser et al. / Burns 30 (2004) 35–41 37

10000000
Control
1000000
Acticoat
Colony Count

100000
Silvazine
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 1. Methicillin-resistant S. aureus viability curve induced by 1% silver sulphadiazine combind with 0.2% chlorhexidine digluconate (SilvazineTM ),
1% silver sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 2. Acinectobacter baumannii viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1%
silver sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 3. Pseudomonas aeruginosa viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1%
silver sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.
38 J.F. Fraser et al. / Burns 30 (2004) 35–41

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Lo g10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 4. Escherichia Coli viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1% silver
sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 5. Enterobacter cloacae viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1% silver
sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 6. Staphylococcus aureus viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1% silver
sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.
 J.F. Fraser et al. / Burns 30 (2004) 35–41 39

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Log10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 7. Enterococcus faecalis viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1% silver
sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

10000000
Control
1000000 Acticoat
Silvazine
Colony Count

100000
Flamazine
Lo g10

10000

1000

100

10

1
0 4 8 12 16 20 24
Time (Hrs)

Fig. 8. Proteus mirabilis viability curve induced by 1% silver sulphadiazine combined with 0.2% chlorhexidine digluconate (SilvazineTM ), 1% silver
sulphadiazine (FlamazineTM ) and ActicoatTM dressing. Each point is the mean from three replicate experiments ± standard deviation.

studies with Proteus mirabilis and Enterobacter cloacae,
increasing bacterial survival was seen in the ActicoatTM
assays between 8 and 24 hours, consistent with increased
bacterial survival in the broth mixture.
4. Discussion
Silver in its numerous forms has been used for over 200
years in the treatment of burn injury [22]. The alteration of
the physical form and the addition of co-molecules signifi-
cantly alters the anti-microbial activity of silver. This is the
first in vitro comparison between the most commonly used
TAA in Australasia and ActicoatTM . The data is very clear in
the superior killing effect of the SilvazineTM over the other
dressings. The only difference between SilvazineTM and
FlamazineTM is the addition of 0.2% chlorhexidine diglu-
conate. This water-soluble biguanide is a potent bactericidal
agent in its own right. Its activity is concentration and pH
dependant, and peak effect occurs within 20 seconds result-
ing in damage to cell walls, and at higher concentrations, to
the intracellular contents. Damage occurs to the cell walls,
and at higher concentrations, to the intracellular contents.
The majority of bacteria and yeast, with the exception of
mycobacteria, are sensitive to chlorhexidine. Hence, it is to
be expected that the addition of chlorhexidine substantially
improves the potency of the SSD.
A number of previous studies have examined TAA ef-
ficacy using zone of inhibition. Clinically, however, any
skin that is susceptible to infection is covered with TAA,
rendering the zone distal to the TAA irrelevant in a clinical
context. The question that our study addressed is, ‘how po-
tent an anti-microbial action does the TAA have in optimal
growing conditions for bacteria?’ The burn wound, with
its high protein content and limited immune system due
to local reduction of blood supply and systemic immuno-
suppression represents such an area. The broth inoculation
method allows for optimal bacterial growth. In agitating
the broths, which contain the TAA, the surface area of the
creams (SilvazineTM and FlamazineTM ) may be larger as
40 J.F. Fraser et al. / Burns 30 (2004) 35–41
they are broken into smaller particles. However, SSD is
relatively water insoluble. As has been mentioned earlier,
chlorhexidine is water soluble, and this may partly explain
the very rapid kill curve obtained consistently with Sil-
vazine. Our group also investigated these agents, using a
zone of inhibition method, and chlorhexidine leached out
of the cream, resulting in similar results.
Of interest is the relative efficacy of ActicoatTM and
FlamazineTM . FlamazineTM was included in this study
to identify the relative efficacy of SilvazineTM and
FlamazineTM , and hence demonstrate the importance of
the chlorhexidine digluconate. However, the comparison of
ActicoatTM and FlamazineTM show significantly different
results to the published literature. This may be due to the
differing study methods. Whilst there are pros and cons
of all study methods, none truly reflect clinical practice,
and it is an important finding of this study that ActicoatTM
does not perform so well under these laboratory conditions.
However, it must be born in mind that the graphs represent
log10 scale, so differences that appear very large may not
reflect an important clinical difference in efficacy. Both
dressings performed well against Pseudomonas, the most
common cause of invasive gram negative sepsis in burn pa-
tients [4] and coliforms, but ActicoatTM was less effective
against Staphylococcus, Proteus and Enterobacter. We have
used ActicoatTM in over 500 patients clinically, and have
seen Proteus infection on several occasions, but there has
been minimal problem encountered with Staphylococcus or
Enterobacteria.
Along with anti-microbial efficacy, other aspects must
be considered in prescribing a TAA. Topical anti-microbial
creams must be removed on a daily basis to allow observa-
tion of the wound and application of fresh cream. The scrub-
bing action associated with this action is painful, particularly
in the paediatric patient, and may be associated with loss of
newly formed and poorly adherent fibroblasts in the wound,
thus delaying closure of the wound. A dressing, which re-
quires less abrasive removal and reduced trauma to the new
epithelial layer, is an important addition to the treatment of
burns patients.
An in vitro test does not prove clinical potency. There are
also many other important factors, such as relative cytotoxic-
ity, and ease of application and removal of dressings that play
an important role in the decision of which dressing is best
for each individual patient. In particular, the well-recognised
cytotoxicity of chlorhexidine with respect to newly formed
keratinocytes is of concern, and is the subject of a further
study by our group [23].
5. Conclusions
This series of in vitro assays of anti-bacterial efficacy,
comparing ActicoatTM , SilvazineTM and FlamazineTM ,
demonstrated all preparations exhibiting anti-bacterial ef-
fects against a wide range of clinical pathogens The data
show that SilvazineTM is a much more potent TAA against
all isolates tested. FlamazineTM and ActicoatTM have sim-
ilar efficacy in a number of isolates, but FlamazineTM is
superior to ActicoatTM in others, in particular S. aureus. The
most potent agent was a combination of 1% silver sulpha-
diazine/0.2% chlorhexidine digluconate. A clinical study
comparing the efficacy of ActicoatTM and SilvazineTM in
the prevention of burn wound sepsis is required.
Acknowledgements
The authors acknowledge Miss Leila Cuttle (B.Sc.) and
Ms. Margit Kempf for their comments on the manuscript.
Smith and Nephew (Australia) provided the dressings free
of charge.
References
[1] Moyer CA. Some effects of 0.5% silver nitrate and high humidity
upon the illness associated with large burns. J Natl Med Assoc
1965;57:95–100.
[2] Dressler DP, Rozin RR, Skornik W, Bellas AE, Soroff HS. An
evaluation of mafenide acetate in experimental burn wound sepsis.
Arch Surg 1967;95:1000–8.
[3] Fox Jr CL. Silver sulfadiazine: a new topical therapy for Pseudo-
monas in burns. Therapy of Pseudomonas infection in burns. Arch
Surg 1968;96:184–8.
[4] Pruitt Jr BA, McManus AT, Kim SH, Goodwin CW. Burn wound
infections: current status. World J Surg 1998;22:135–45.
[5] Nguyen TT, Gilpin DA, Meyer NA, Herndon DN. Current treatment
of severely burned patients. Ann Surg 1996;223:14–25.
[6] Muller MJ, Herndon DN. The challenge of burns. Lancet
1994;343:216–20.
[7] Monafo WW, West MA. Current treatment recommendations for
topical burn therapy. Drugs 1990;40:364–73.
[8] Russell AD, Hugo WB. Anti-microbial activity and action of silver.
Prog Med Chem 1994;31:351–70.
[9] Hollinger MA. Toxicological aspects of topical silver
pharmaceuticals. Crit Rev Toxicol 1996;26:255–60.
[10] Lockhart SP, Rushworth A, Azmy AA, Raine PA. Topical silver
sulphadiazine: side effects and urinary excretion. Burns Incl Therm
Inj 1983;10:9–12.
[11] Monafo WW, Freedman B. Topical therapy for burns. Surg Clin
North Am 1987;67:133–45.
[12] Lee JJ, Marvin JA, Heimbach DM, Grube BJ. Use of 5% sulfamylon
(mafenide) solution after excision and grafting of burns. J Burn Care
Rehab 1988;9:602–5.
[13] Liebman PR, Kennelly MM, Hirsch EF. Hypercarbia and acidosis
associated with carbonic anhydrase inhibition: a hazard of topical
mafenide acetate use in renal failure. Burns Incl Therm Inj
1982;8:395–8.
[14] Shuck JM, Moncrief JA. Safeguards in the use of topical mafenide
(Sulfamylon) in burned patients. Am J Surg 1969;118:864–70.
[15] Clarke AM, Solomon J, Keogh J, Nixon M, Burchett J. Chlorhexidine
with silver-sulphadiazine in the treatment of burns. Med J Aust
1971;2:446.
[16] Clarke AM. Topical use of silver sulphadiazine and chlorhexidine
in the prevention of infection in thermal injuries. Med J Aust
1975;1:413–5.
[17] Inman RJ, Snelling CF, Roberts FJ, Shaw K, Boyle JC. Prospective
comparison of 1% silver sulfadiazine plus 0.2% chlorhexidine
 J.F. Fraser et al. / Burns 30 (2004) 35–41
digluconate (Silvazine) and 1% silver sulfadiazine (Flamazine) as
prophylaxis against burn wound infection. Burns Incl Therm Inj
1984;11:35–40.
[18] George N, Faoagali J, Muller M. Silvazine (silver sulfadiazine
and chlorhexidine) activity against 200 clinical isolates. Burns
1997;23:493–5.
[19] Yin HQ, Langford R, Burrell RE. Comparative evaluation of the
anti-microbial activity of Acticoat anti-microbial barrier dressing. J
Burn Care Rehab 1999;20:195–200.
[20] Wright JB, Lam K, Hansen D, Burrell RE. Efficacy of topical
silver against fungal burn wound pathogens. Am J Infect Control
1999;27:344–50.
41
[21] Gray JH, Henry DA, Forbes M, Germann E, Roberts FJ, Snelling
CF. Comparison of 1% silver-sulphadiazine, 1% silver sulphadiazine
plus 0.2% chlorhexidine digluconate and 8.5% mafenide acetate for
topical antibacterial effect in infected full skin thickness rat burn
wounds. Burns 1991;17:37–40.
[22] Klasen HJ. Historical review of the use of silver in the treatment of
burns. I. Early uses. Burns 2000;26:117–30.
[23] Fraser JF, Cuttle L, Kempf M, Kimble RM, Cytotoxicity of topical
antimicrobial agents used in burn wounds in Australasia. ANZJS
(in press).