In Comparison Of Dressing And Ointment

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 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 sepsis was listed as cause of death in 60% of burns mortality. The routine application of topical silver nitrate in 1965 [1] rapidly reduced this figure to 28%; a figure further diminished 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: [email protected] (J.F. Fraser).

∗

0305-4179/$30.00 © 2003 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2003.09.008

use of more potent systemic antibiotics, infection remains a leading cause of morbidity and mortality in burns patients [5,6]. The increasing prevalence of multi-resistant and panantibiotic 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

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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 topical 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 successful and the resultant dressing was introduced as SilvazineTM [16]. In a number of clinical and laboratory based studies, this preparation was seen as superior against a number 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 dressing consisting of an absorbent rayon polyester core, with upper and lower layers of nanocrystalline silver coated high-density polyethylene mesh was introduced to Australasia. The nanocrystalline structure was integral in the effect of ActicoatTM , as the inherently unstable structure allowed 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 silver 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 digluconate), FlamazineTM (1% silver sulphadiazine) and ActicoatTM .

2. Methods All tests were carried out in the microbiology laboratory 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 aseptic, sterile manner. SilvazineTM and FlamazineTM samples of 0.1 g were aspirated using sterile syringes. All samples were individually weighed on Delta PC4400 scales (Mettler-Toledo Ltd., Tampa, Fl).

Table 1 Bacterial isolates studied Methicillin-resistant Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Enterococcus faecalis Enterobacter cloacae Proteus mirabilis Acinetobacter baumannii

ATCC 33591 NCTC 10662 ATCC 35218 NCTC 6571 ATCC 29212 ATCC 10347 ATCC 7002 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 patients in the intensive care unit at the hospital where the laboratory 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 microbiologists. Each study was performed three times to ensure reproducibility.

3. Results The results are presented as kill curves for the bacteria studied, (Figs. 1–8). The data demonstrates that the combination 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 number 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

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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 significantly 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 digluconate. 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 efficacy 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 potent 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 immunosuppression 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

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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 Silvazine. 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 patients [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 observation of the wound and application of fresh cream. The scrubbing 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 requires 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 cytotoxicity, 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 effects 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 similar 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 sulphadiazine/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 Pseudomonas 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.

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[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).