SUMMARY. Silver sulphadiazine 1 per cent, mupirocin 2 per cent, and fusidic acid 2 per cent were compared to assess the antibacterial effect of once daily application on experimental rat 15 per cent full-skin-thickness burn wounds seeded 24 h earlier with 108 standard strain of methicillin-resistant staphylococci. The quantitative counts of the seeded organism in burn eschar and subjacent muscle were determined on day 7 post-burn, in addition to the cultures of blood and lung biopsies. All tested topical agents were equally effective against methicillin-resistant Staphylococcus aureus in reducing local burn wound bacterial count and preventing systemic infection.
Methicillin-resistant Staphylococcus aureus (MRSA) infected wounds have been a significant cause of morbidity and mortality among thermally injured patients since the 1980s.1,2 The burn wound is particularly susceptible to bacterial colonization and infection because of the physical disruption of the normal skin barrier and the accompanying reduction in cell-mediated immunity.3 Use of topical antimicrobial agents plays an important role in preventing invasive burn wound MRSA infections.4 Numerous topical antibacterial agents are available for clinical use in various concentrations, vehicles, and mixtures. Silver sulphadiazine (SSD), mupirocin, and fusidic acid (FA)
are all topically efficacious against MRSA to variable degrees.5-9 However, the emergence of antibacterial resistance to topical antimicrobial agents is a potential threat for effective control of MRSA in burn patients.3,10-13 Since the antibacterial resistance patterns of MRSA change from one burn centre to another, each centre should monitor the developing resistance profiles and use the proper topical agent.
To our knowledge, there is no report that compares the activities of these agents against MRSA in a burn wound model. This study was designed to compare the topical antibacterial efficacy of SSD, FA, and mupirocin in the treatment of a rat full-skin-thickness burn wound seeded 24 h before with a standard strain of MRSA.
Male Wistar rats (n = 32) weighing 200-220 g were used. The animals were housed under standard conditions at ambient room temperature and given laboratory chow and water ad libitum throughout the study. The experimental protocol was approved by the Marmara University ethical committee prior to commencement of the study.
The weights of the animals were obtained. They were anaesthetized intraperitoneally with 80 mg/kg body weight ketamine hydrochloride, and their backs were shaved. They received a full-skin-thickness dorsal scald burn in boiling water in approximately 15 per cent of the body surface by a standard method.14 The animals were resuscitated with an intraperitoneal injection of 2 ml of lactated Ringer’s solution.
Ten minutes after the burn, each animal was seeded with 0.5 ml of broth containing 1 x 108 c.f.u. MRSA (ATC 38591) using a tuberculin syringe with a 25-gauge needle. Once the inocula had dried, the animals were placed in separate sterilized cages and allowed to recover.
After 24 h, the animals were assigned at random to four groups. Group 1 was the control group (no topical agent applied). Group 2 was the 1% SSD (Silverdin, Deva Holding, Istanbul) treated group. Group 3 was the 2% FA (Fucidin, Abdi Ibrahim, Istanbul) treated group and group 4 was the 2% mupirocin (Bactroban, Smithkline Beecham, Istanbul) treated group. Treatment started 24 h post-burn. The topical agent was applied liberally to the burn wound with a sterile tongue blade once daily. No dressings were applied.
All the animals were sacrificed on day 7 post-burn and their weights were obtained. A loss of more than 15 g (7.5%) was considered indicative of systemic disease. All the cultures were obtained using an aseptic technique. Initially thoracotomy was performed. Blood cultures were obtained from the left ventricle and lung biopsies were obtained. Blood specimens were cultured on 5.0 per cent sheep blood agar and lung specimens were placed in brain heart infusion broth; both were incubated at 35 °C and isolated organisms were identified by standard methods.
A full-skin-thickness 9-mm punch biopsy was obtained from the centre of the burn eschar. Following removal of the eschar and underlying fascia, a separate biopsy of paravertebral muscle deep to the burn eschar was obtained. Separate quantitative cultures of eschar and muscle were performed using a standard method.15
The quantitative count of micro-organisms in burn eschar or muscle was determined. The mean and standard deviations of counts for each treatment group were determined. The variances were too great across groups to permit valid comparison. The logarithm (base 10) of each count was determined. Tissues with no organisms isolated were arbitrarily assigned a value of 10 to permit logarithmic conversion. Means and standard deviations of log-transformed values were determined. The package program SPSS (Statistical Package for Social Sciences for Windows 7.0) was used for statistical analysis. Kruskal-Wallis and Mann-Whitney U tests were used to compare the means and standard deviations of log-transformed values of the groups. The incidence of recovery of the seeded organism from lung and blood and the development of systemic infection were compared by chi square analysis.
No animal death was seen throughout the experimental protocol. The frequency of recovery of the seeded organisms from each culture site is detailed in Table I. There was no statistically significant difference between the treatment groups regarding systemic recovery of MRSA from blood and lung besides development of systemic infection (p > 0.05, chi square analysis), and there was a significant difference between the control group and treatment groups (p < 0.01, chi square analysis). A comparison of quantitative cultures performed on burn eschar and muscle is shown in Table II. Kruskal-Wallis variance analysis of the groups (for burn eschar and muscle columns separately) was significant (p < 0.001). Paired comparison of the groups was performed by the Mann-Whitney U test; there was a statistically significant difference between the treatment groups and the control group (p < 0.01), but no significant difference between the treatment groups (p > 0.05).
<% createTable "Table I ","The frequency of recovery of seeded MRSA from the groups and the development of systemic infection",";;Animals seeded;Eschar;Muscle;Blood;Lung;Systemic infection (weight loss > 15 g) @;Control;8;8;8;7;6;6@;Silver sulphadiazine;8;8;6;0;1;1@;Fusidic acid;8;8;6;1;1;0@;Mupirocin;8;8;6;0;0;0","",4,300,true %> <% createTable "Table II ","Concentration of seeded MRSA (log of c.f.u./gram tissue) recovered from burn eschar and muscle",";;Animals studied;Eschar (mean ± s.d.);Muscle (mean ± s.d.)@;Control;8; 8.60 ± 0.26; 6.63 ± 0.12@;Silver sulphadiazine;8; 5.94 ± 1.79; 3.41 ± 2.74@;Fusidic acid;8; 6.72 ± 1.39; 3.85 ± 2.54@;Mupirocin;8; 5.71 ± 0.88; 3.12 ± 1.94","",4,300,true %>SSD 1 per cent is one of the most commonly used topical antibacterial creams for the prevention of burn wound infection. It is most effective against Pseudomonas aeruginosa and the enterics, and equally as effective as any antifungal drug against Candida albicans. Staphylococcus aureus and some strains of the Klebsiella species have been less effectively controlled. Despite its wide spectrum, SSD treatment fails with continued use in large burns (> 50% TBSA) and resistance has been reported. Because of its poor burn-eschar penetration, it is not so effective in the treatment of established burn wound infections.5,16,17
Fusidic acid is an antibiotic isolated from culture media of the fungus Fusidium coccineum. It has a narrow bacterial spectrum, mainly against gram-positive bacteria; there is exceptionally high in vitro activity against Staphylococcus aureus. Its topical form has been effectively used in primary and secondary skin infections.18 Although staphylococcal resistance to fusidic acid has been documented,19,20 in vitro evidence suggests that resistance to Staphylococcus aureus is less likely to occur after exposure to high concentrations of fusidic acid, reflecting the situation with topical use.5
Mupirocin is a topical broad-spectrum antibiotic produced by fermentation of Pseudomonas fluorescens. It is highly active in vitro against MRSA, staphylococcal strains resistant to other antibacterials, and streptococci that are associated with primary and secondary skin infections.21 Successful treatment of MRSA-infected burn wounds with topical mupirocin has been reported.6,22 Mupirocin has also been used with variable success for the elimination of chronic nasal MRSA colonization but the development of mupirocin resistance in MRSA has become a problem after widespread use of nasal mupirocin.23,24
Although mupirocin seemed to be more effective than SSD and FA against MRSA in our study, there was no statistically significant difference between the treatment groups. They were equally effective in reducing burn eschar colonization and subjacent muscle invasion with MRSA. They also prevented systemic MRSA infection.
In comparative trials of fusidic acid for the therapy of primary and secondary skin infections, fusidic acid has been found to be equivalent or inferior to mupirocin.25,26 Deng27 and Vizcaino-Alcaide28 reported that the effect of mupirocin on a burn wound with Staphylococcus aureus infection was superior to that of SSD and recommended that mupirocin should be the first choice of topical antibacterial agents in MRSA-infected burn wounds. However, mupirocin has been found to be costly for treatment of MRSA.7,29
The true topical antibiotics such as FA and mupirocin should not be used as a prophylactic agent for long periods in burns. The emergence of resistant strains constitutes a major risk, besides significant absorption of antibiotic and systemic toxicity. They should be used cautiously, for as brief a period as possible, and preferably in a limited surface area.16 Rode et al. recommended that mupirocin should be used only on a selective basis when current prophylactic topical therapy has failed to control MRSA infection in burns of less than 20 per cent of the total body surface area, and that it should be applied only for a limited period of five days. The safety and the efficacy of mupirocin in burns exceeding 20 per cent of the total body surface area need to be established.6 Fusidic acid alone has not been recommended in the treatment of MRSA-infected burn wounds. However, mixtures of topical antibacterial agents can also be used: this leads to synergistic effects and may delay the selection of resistant micro-organisms. Fusidic acid should be kept in mind for selected cases as a part of topical combination therapy for limited periods.5,30,31
RESUME. La sulphadiazine argentée à 1 pour cent, la mupirocine à 2 pour cent et l’acide fusidique à 2 pour cent ont été confrontés pour évaluer l’effet antibactérien, dans le rat, de l’application une fois par jour sur des brûlures expérimentales à toute épaisseur (15% de la surface corporelle) ensemencées 24 h avant avec une souche standard (108) de staphylococci résistants à la méticilline. Les comptes quantitatifs de l’organisme ensemencé dans l’escarre et le muscle subjacent ont été déterminés le jour 7 après la brûlure, comme aussi les cultures des biopsies du sang et du poumon. Tous les agents topiques testés se sont démontrés également efficaces contre le Staphylococcus aureus résistant à la méticilline pour réduire le compte local bactérien des brûlures et pour prévenir l’infection systémique.