In Vitro Evaluation of Resistance Development to Silver Sulfadiazine and Subsequent Cross-Resistance to Antibiotics.

Treatment of chronic wounds that are at risk of infection, or that are infected, require the use of antimicrobial dressings, most often those that contain silver. Silver exerts its antimicrobial effects by binding to multiple cellular components and, as such, bacterial resistance to it is low; however, molecular silver resistance has been documented and is attributed to the presence of the sil operon or changes in genes encoding porin and efflux pump expression. The aim of this study was to evaluate spontaneous silver resistance development in common opportunistic pathogens, Staphylococcus, Pseudomonas and Enterococcus cloacae, as well as resistance development when exposed to subtherapeutic concentrations over a prolonged period. Furthermore, following silver resistance development, cross-resistance to several classes of antibiotics was evaluated. Following exposure of the strains to silver sulfadiazine (SSD) at two times and four times minimum inhibitory concentration (MIC), the mutation rate was <1010 colony forming unit (CFU)/mL. Serial passage of S. aureus and P. aeruginosa in subinhibitory concentrations of SSD selected for no resistant mutants. The SSD MIC of E. cloacae increased past the solubility limit of SSD at serial passage 17. MIC testing of this isolate showed a >2048-fold increase in MIC to silver in comparison to the parent strain. MIC testing of the serial passage isolates demonstrated no cross-resistance to antibiotics from six different classes. Overall, the results of this study show resistance development to silver is low and, if it does occur, it does not confer resistance to several antibiotic classes. However, as this study was carried out with a small number of strains, a study with a larger panel of strains and sequencing of the strains to determine the exact mechanism of resistance would be needed to investigate the threat of silver resistance further.

Ultrastructural localization and chemical binding of silver ions in human organotypic skin cultures.

Organotypic cultures of human breast skin incubated with silver bandage or treated with silver sulfadiazine accumulated silver in epithelial cells and in macrophages, fibroblasts and collagen fibrils and fibres of underlying connective tissue. Ultrastructurally, the accumulated silver was found in lysosome-like vesicles of the different cells and evenly spread along collagen structures. Apoptotic nuclei were present but few. Autometallographic amplification of 2D-PAGE gels revealed that glutathione S-transferase and glutathion detoxify silver ions in the epidermal cell by binding them in silver-sulphur nanocrystals. Thus, the cytotoxic effect of silver ions seems to be muted by silver ions by being: (1) taken up by undamaged cells, neutralised by glutathione (GSH) and accumulated in lysosomal vesicles, (2) bound extracellularly to SH-groups of the collagen fibres.

Passage of silver ions through membrane-mimetic materials, and its relevance to treatment of burn wounds with silver sulfadiazine cream.

Treatment of acute burn wounds with silver sulfadiazine has raised concern of potential silver toxicity. As the wound heals, a barrier forms between the silver sulfadiazine and the blood, but this membrane is not impenetrable, and so silver absorption is still possible. In this work, we have modeled chemical systems to investigate the transport of silver sulfadiazine and silver chloride through cellulose, chitosan, collagen, and polyethylene membranes into the following media: synthetic serum electrolyte solution (SSES), SSES plus glutathione, and human serum, to simulate some of the chemical processes occurring at a burn wound during healing. Our results clearly indicate that membranes can retard the movement of silver ions, especially those that have silver-binding properties. This suggests that silver absorption at a healing wound will be minimized by entrapment of silver in the growing membrane network, and thus the likelihood of silver toxicity will be reduced.

A simple in vitro model to test the efficacy of antimicrobial agents released from dressings.

A new model for in vitro evaluation of an activity of antibacterial agents released from delivery systems containing antibiotics or antiseptics was developed. The model was composed of two layers of agar gel of two different concentrations in PBS. In the upper gel, the wells were cut and filled with polyurethane sponges saturated with bacterial broth culture. The sponges were then covered with tested dressings containing antibacterial agent. The model was tested by using two bacterial strains, Pseudomonas aeruginosa or Staphylococcus aureus and experimental collagen dressing with doxycyclin, amikacin, or with silver sulfadiazine. The number of colony-forming units (CFU) in the polyurethane sponges was significantly lower under the dressings with antibacterial agents as compared to the control (the same dressing without antimicrobials) during 3 days of observation. The new in vitro model can be recommended for examination of different antibacterial delivery systems instead of experimentally infected wounds in laboratory animals or instead of the other in vitro models.

Serum and urinary silver levels in thermal injury patients.

Although silver sulfadiazine has been used extensively as an effective topical antimicrobial agent in thermal injury patients, little is known about the cutaneous absorption of the silver moiety in these patients. Therefore, we longitudinally evaluated both serum silver concentration and 24-hour urinary excretion of silver in 23 patients with second- and third-degree thermal burns. Mean serum silver concentrations were modestly elevated throughout the patients' hospital course. Urinary excretion of silver was markedly elevated, especially in those patients with more severe burns. Indeed, in patients who had burns covering more than 60% of the total body surface area mean peak silver excretion was 1100 micrograms/24 hr (normal, less than 1 micrograms/24 hr). Thus, silver ion is absorbed across the burn wound in thermal injury patients treated with silver sulfadiazine. The 24-hour urinary excretion of silver appears to be a very sensitive indicator of cutaneous absorption in these patients. Possible implications of this cutaneous silver absorption warrant further evaluation.

Prospective study of side effects associated with the use of silver sulfadiazine in severely burned patients.

We report the results of a prospective study of 45 burn patients in whom serum and urine levels of cream constituents (sulfadiazine and propylene glycol) were determined and correlated with clinical parameters and the presence of an immune response to sulfadiazine. Propylene glycol, measured by gas chromatography, was found in the serum of 53% of study patients. Sulfa, determined by spectroscopy, was found in the serum of all patients, with values as high as 9.1 mg/dl within 24 hours of topical application. Ninety percent of tested patients had circulating sulfadiazine antibodies, predominantly IgG. Fourteen patients died, 11 of whom had interpretable renal biopsies. Five of these biopsies showed positive immunofluorescent linear or granular staining for IgG, IgM, IgA, or C'3; none of these 5 patients had clinical evidence for the nephrotic syndrome. The association between antisulfadiazine antibodies, circulating immune complexes, immunoglobulin disposition on glomerular basement membranes, and topical application of silver sulfadiazine provides evidence for sulfadiazine sensitization in patients with severe burns.

Tissue deposition of silver following topical use of silver sulphadiazine in extensive burns.

Silver sulphadiazine has been applied to the burn wounds of 509 patients during the past 10 years. Eleven patients with burns covering more than 20 per cent of the body surface showed silver deposits in the mucosa of the lips, gingiva and cheeks. The colour of the burn wound was also slightly darker than in patients not treated with silver compounds. This darker colour spontaneously disappeared during the year following discharge from hospital. The pathogenesis of silver deposition has been discussed in relation to other published studies.

Silver sulfadiazine: an antibacterial agent for topical use in burns. A review of the literature.

Topical antibacterial treatment is of major importance in the burn patient. Silver sulfadiazine is an effective agent with low toxicity and few side effects. Deposition of silver in tissues, and absorption of sulfadiazine are both minimal. Present and future problems are represented by the emergence of resistant Gram negative bacilli, including Pseudomonas aeruginosa. The development of related metal sulfadiazines to be used against resistant bacteria is on an investigational stage, and clinical trials are few. Silver sulfadiazine may be used in a variety of other conditions than burns.

Topical therapy and the development of silver sulfadiazine.

Topical therapy is a selective approach to the prevention and treatment of infection in burns and other surgical wounds. Effective compounds possess certain chemical and physical properties. Prerequisites include low solubility, slow absorption, nonreactivity with wound exudates, proteins and ions and ability to achieve prolonged antimicrobial activity in the wound. These compounds yield much higher levels in the zone of infection than can be achieved by diffusion into the wound after systemic administration. The wide spectrum of antibacterial activity, the low toxicity, minimal tissue reaction, ease of application suggest that topical silver sulfadiazine therapy can safely be extended to other wound infections, wound covers and certain transplant materials.

Electron-microscopic observation on silver deposition in burn wounds treated with silver sulphadiazine cream.

The absorption and distribution of silver has been studied in 8 burned patients treated with silver sulphadiazine cream by electron-microscopic examination. Biopsies were taken from the burn wounds and from epithelized areas surrounding wounds. Only 1 to 2 patients treated for 12 days was observed to have silver deposits in the cytoplasm of both the epidermal cells and sweat glands. The silver deposits appeared as roughly round and oval bodies of varied size. The results indicated that very little of the silver moiety in silver sulphadiazine cream penetrated the body through the wound.

Absorption, excretion and tissue distribution of silver sulphadiazine.

It was clarified that the absorption of silver sulphadiazine cream, a widely-used topical agent against Pseudomonas infection after burns, through both superficial and deep dermal burn surfaces as well as the normal skin, was negligible, but that its absorption increased when the blister was removed. After absorption, the silver sulphadiazine was dissociated into two portions. The sulphadiazine portion was excreted into the urine quickly and the silver portion, on the contrary, remained at a certain level in the body for a long time. Our results given some suggestions about the indications for silver sulphadiazine cream. The cream seems to be inadequate for treating second-degree burns but may be useful for the treatment of third- or fourth-degree burns.

Pharmacology of sulfadiazine silver. Its attachment to burned human and rat skin and studies of gastrointestinal absorption and extension.

The attachment of Ag110-labeled sulfadiazine silver (AgSU) to the burn wound of humans and full and partial thickness scald burns of rats was studied over time. The duration of Ag adherence to burned skin and the absorption and organ distribution of ingested AgSU was studied. Peak attachment to human burns was 1% of the administered dose in 24 hours. Rat wounds showed greater attachment. Dissections of the wounds showed 81% to 98.7% of this attachment to be in the most superficial layers of cells and no silver was observed in organs of surface-treated animals. Duration of attachment after one application was until wound slough with percent attachment dropping from 5% to 1.7% over that time. Oral ingestion resulted in substantial silver deposition, particularly in liver and lungs. Clearance occurs in three weeks. The basic function of AgSU may be through the slow release of silver into the superficial wound environment.

Mechanism of silver sulfadiazine action on burn wound infections.

The role of silver and sulfadiazine in the mechanism of action of silver sulfadiazine on burn wound infections was investigated. Silver, but not sulfadiazine, was bound by bacteria. Sulfadiazine did not act as an antibacterial agent in low concentrations, but exhibited specific synergism in combination with subinhibitory levels of silver sulfadiazine. The efficacy of silver sulfadiazine is thought to result from its slow and steady reactions with serum and other sodium chloride-containing body fluids, which permits the slow and sustained delivery of silver ions into the wound environs. In this circumstance, a relatively minute amount of sulfadiazine appears active.