The Prevalence of Phenotypic Silver Resistance in Clinical Isolates

  Abstract: Research has shown silver to be an effective antimicrobial agent against bacteria, virus, yeast, and fungi. Due to the increased use of silver-based wound products during the past decade, concerns of widespread silver resistance have been raised.¹ The purpose of this study was to assess the prevalence of phenotypic silver resistance in clinical isolates. Methods. A total of 130 different microorganism strains were collected from patients admitted to a tertiary care hospital. To determine phenotypic silver resistance, a corrected zone of inhibition (CZOI) test was used. The isolate (0.1 mL) was streaked on nutrient agar in 3 directions to form a confluent lawn. A silver dressing and a control gauze dressing were placed on the lawn and incubated for 24 hours. The CZOI was determined by averaging the zone of clearance in both directions across the dressing and then subtracting the dressing size. Corrected zone of inhibition tests were conducted in duplicate. To confirm the silver dressing killed the microorganism and did not simply hinder bacterial growth, a culture was taken from underneath each silver dressing and plated separately. Results. All of the isolates showed no growth when tested against the silver-based dressing. The CZOI values ranged between 0.0 mm and 7.25 mm. No growth was observed in the secondary culture from underneath the dressing, indicating the silver dressing was bactericidal for all 130 isolates tested and not simply bacteriostatic. The control gauze dressing did not show any antimicrobial properties. Conclusion. The threat of widespread silver resistance in clinical isolates remains low. However, continued monitoring for silver resistance should be maintained.

Introduction

  Medical practitioners have utilized silver in advanced wound care for more than 40 years.^2,3 Research has shown silver to be an effective antimicrobial agent against bacteria, virus, yeast, and fungi even at low concentrations.^4-6 Due to silver’s microbicidal properties, minimum toxicity to cells, and therapeutic activity, silver-based treatments have increased considerably over the past decade.^1,4,5 Many silver dressings are commercially available and consistently used for treating wound and burn injuries. The increased use of silver has raised concerns for potential emergence of widespread bacterial silver-resistance in health care.   Silver-resistant bacterial strains that have been isolated include Salmonella,^7,8Enterobacter,^9-12Escherichia coli,^13-15Pseudomonas,¹⁶Acinetobacter,¹⁷Klebsiella,¹⁸ and methicillin-resistant coagulase-negative Staphylococcus aureus (MRSA).¹⁹ Plasmid pMG101, isolated from a Salmonella species, was the initial molecular basis for silver resistance documented by Gupta and colleagues.⁸ The pMG101 180 kilobase (kb) transferable plasmid encodes resistance to multiple antibiotics and metals, including silver. Three transcriptional units encompassing 9 genes (silAB, silC, silE, silP, silSR, ORF105, ORF96, and 2 other open reading frames) account for the genetic basis of silver resistance.²⁰   Although there is clear evidence supporting the molecular basis for silver resistance, prevalence of these genes in clinical isolates appears minimal. Loh et al¹⁹ examined 33 MRSA and 8 methicillin-resistant coagulase-negative Staphylococci isolates for 3 silver-resistant genes. Only 2 MRSA strains contained the silE gene.¹⁹ An in vitro study revealed 2 out of 112 bacterial isolates collected from diabetic foot ulcers contained silver -resistant genes.²¹ The 2 silver-resistant positive isolates were from the Enterobacter cloacae species (an atypical wound pathogen). In a similarly designed study, 6 out of 172 bacteria isolates collected from human and animal clinical origins were positive for silver-resistant genes.²² Again, the 6 isolates positive for silver-resistant genes were E. cloacae strains. Although these studies identified bacteria containing silver-resistant genes, all of the strains were phenotypically sensitive to silver including the few silver-resistant positive isolates.^19,21,22 Due to the widespread use of silver treatments in health care, silver resistance across multiple origins should continue to be monitored for new signs of phenotypic-resistant expression. The purpose of this study was to assess the prevalence for phenotypic silver tolerance in 130 clinical isolates collected from patients admitted to a tertiary care trauma and burn hospital.

Methods

  Samples. After approval by the Institutional Review Board, 130 different microorganism strains were randomly collected from patients admitted to a tertiary care unit at Mercy Hospital in Springfield, MO. The samples were collected in sterile collection tubes and transported to the microbiology lab. A microbial identification system (VITEK®2 Microbial Identification System, Biomerieux, Durham, NC) was used to identify species by measuring 47 biochemical substrates including carbon source utilization, enzymatic activities, and resistant patterns. The origins of the isolates include wounds, burns, sputum, and urine.   Microbiology. To determine if silver was microbicidal, a corrected zone of inhibition (CZOI) test was used. This test is similar to the standard zone of inhibition test, except zones are corrected to take into account the differences in shape of the hand-cut testing dressings. This test is a modification of the standard Kirby-Bauer test for antimicrobial sensitivity. Equivalent 0.1 mL of gram-positive bacteria were streaked on tryptic soy agar (Remel, Lenexa, KS) and 0.1 mL of gram negative bacteria were streaked on nutrient agar (Remel, Lenexa, KS) in 3 directions to form a confluent lawn. Silver dressing or control gauze was cut into 0.5 inch x 0.5 inch squares and placed in the center of each lawn. All plates were incubated for 24 hours at 37° C (gram positive) and 26° C (gram negative). The CZOI was then determined by measuring the zone of clearing across 1 direction and subtracting the width of the dressing. This was repeated across the other direction and values were averaged. The CZOI reflects only the width of clearing around the dressing. Corrected zone of inhibition tests were conducted in duplicate. To confirm that silver dressing killed the microorganism under the dressing and did not simply hinder bacterial growth due to pressure, a culture was taken from underneath each silver dressing, plated on appropriate agar plate, and allowed to grow overnight at 37° C.   Descriptive analysis. The data were screened prior to analysis for accuracy and normality. Descriptive and other univariate statistics were investigated and reported.

Results

  The clinical isolates obtained included coagulase-negative Staphylococcus (6 isolates), Escherichia coli (22 isolates), Enterococcus species (9 isolates), Pseudomonas aeruginosa (17 isolates), Staphylococcus aureus (40 isolates), and Staphylococcus epidermidis (4 isolates). In addition, 33 other isolates were collected including Klebsiella pneumonia (10 isolates), Acinetobacter baumannii (3 isolates), Klebsiella oxytoca (5 isolates), Proteus mirabilis (5 isolates), and Proteus vulgaris (5 isolates), Serratia marcescens (4 isolates), and Staphylococcus epidermidis (4 isolates). All of the isolates had no growth to the silver-based dressing showing observable zone of inhibition margins. The mean CZOI across all species was 1.7 mm (SD = 0.93) (See Table 1 for average CZOI summary). No growth was observed in the secondary cultures from underneath the silver dressing. The control gauze dressing did not show any antimicrobial properties. In addition, the multidrug-resistant strains across all species did not grow when exposed to silver with clear zones of inhibition. Again, no growth was observed in the secondary cultures. See the following tables for drug-resistant patterns: Staphylococcus aureus (Table 2), coagulase-negative Staphylococcus (Table 3), Pseudomonas aeruginosa (Table 4), Escherichia coli (Table 5), and Entercoccus spp (Table 6).

Discussion

  Clinical awareness of bacterial silver resistance has increased in the last decade due to the widespread use of silver in medical treatments. Although there continues to be a lack of evidence supporting the development of silver resistance, it remains a topic of concern in wound clinics. In addition, scientific interest in silver resistance has increased due to the potential of cross resistance. Silver-resistant genes are encoded by the same plasmid that encodes traditional antibiotic resistance. Although there are clear molecular basis for silver resistance, phenotypic expression has yet to be observed.   The authors’ results failed to demonstrate any evidence of emerging clinical significance supporting silver’s inability to kill bacteria. All 130 strains tested showed no growth when exposed to the silver dressing, including the multidrug-resistant bacteria. These findings support previous work showing a lack of silver resistance in clinical isolates. An in vitro study showed silver alginate dressings inhibited the growth of 115 clinical samples.²³ In addition, this study showed Enterobacter cloacae to have the highest silver tolerance. This finding is particularly interesting because bacteria strains containing silver-resistant genes identified in previous studies were those of the Enterobacter cloacae species.^21,22 Increases in silver tolerance in this species could potentially be initial observation of mild phenotypic expression. Further testing is needed before conclusions could be made.

Conclusion

  As previously discussed,²⁴ it is important to remember pathogens have been exposed to diminutive concentrations of silver for potentially billions of years without evidence of phenotypic resistance. In addition, the use of silver’s antimicrobial properties is not a novel practice. The threat of widespread silver resistance remains minimal despite ancient civilizations’ use of silver to treat infections and maintain water purity.³ However, due to the genetic potential of cross resistance and the widespread clinical use of silver products, bacterial resistance patterns should continue to be monitored.

Acknowledgments

  This project was funded with an Intramural Mercy Research Project allocation. The authors would like to thank Michael Reidle, BS, MT (ASCP) and the microbiology staff at Mercy Hospital-Springfield for their medical and technical assistance. In addition, the authors would like to thank Cindy Austin, MS, for her continued support throughout this study.

References

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