Letters to the Editor: Polishing the Information on Silver

Dear Editor,
   In the interest of upholding Ostomy/Wound Management's goal to provide accurate, balanced continuing medical education, we respectfully request clarifications regarding the article by Dr. Burrell (A scientific perspective on the use of topical silver preparations. In: Bacteria + Pressure Ulcers: The Role of Silver versus Traditional Antimicrobials. Ostomy/Wound Management. 2003;49[5A supp]:19-24) and suggest that the author's past and present affiliations with commercially involved organizations be properly disclosed. We believe the article shows bias toward nanocrystalline technology and does not address topical silver preparations objectively.

   Table 1 defines the solubility limits for various forms of silver. It infers that nitrates and nanocrystalline forms provide more silver, but the author fails to disclose that these data refer to solubility in pure water. Consequently, these figures bear no relevance to the availability and antimicrobial efficacy of silver in complex media such as wound fluid.

   The only accepted safe and proven antimicrobial species of silver is ionic silver in the form of the monovalent silver cation (Ag⁺).^1,2 Table 2 indicates that nanocrystalline silver provides both Ag⁺ and Ag⁰. Ag⁰ is the chemical symbol for metallic silver for which no evidence of biological or antimicrobial activity exists. Ag⁰ is generally regarded as being insoluble in water, and the only way that it can get into solution and elicit antimicrobial activity is in the form of the silver ion (Ag⁺). Also in Table 2, the water concentrations for AgSD (1%) and AgNO₃ (0.5%) should read 3,000 micrograms/mL and 3,200 micrograms/mL respectively.

   The discussion on the effect of wound fluid on ionic silver levels is limited to dressings containing silver chloride. However, no mention is made of the fact that this same effect will apply to all silver-containing dressings. The concentration of available silver ions is dictated primarily by the solubility limit of silver chloride, which is approximately 1.0micrograms/mL. Ionic silver concentrations in excess of this value (as indicated in Tables 1 and 2) will be rapidly reduced by the formation of inactive silver chloride. Consequently, data showing only solubility and release rates into water are misleading and do not extrapolate to the clinical situation.

   Additional confusion is caused by the presentation of traditional static minimum inhibitory concentration (MIC) data based on the exposure of bacteria to single bolus doses of an ionic silver solution at varying concentrations. With the exception of silver nitrate, all the referenced silver-containing products have been designed to provide some level of controlled silver release into a dynamic wound environment over time. Minimum inhibitory concentration tests are not representative of this situation. This is emphasized in Table 2, which shows that AgCMC is bactericidal (100-fold reduction in 30 minutes) yet produces a concentration of less than 1.0 micrograms/mL of ionic silver, which is a substantially lower value than the referenced MIC values. The term oligodynamic is used, but is not applied to the data presented. This term refers to the ability of bacteria to accumulate substances preferentially; it is the number of silver ions absorbed, not the concentration of silver ions in solution that causes bacterial death.³

   In Table 2, the use of a 30-minute log reduction assay is of questionable clinical relevance, particularly when many of the referenced dressings are considered to demonstrate antimicrobial activity over a period of days. The conditions and time period of this test are deliberately intended to differentiate nanocrystalline silver from other silver-containing dressings.
In the article section titled, "Silver-sodium carboxymethylcellulose dressings" (more correctly known as sodium carboxymethylcellulose with ionic silver), the term limited antimicrobial activity is a gross misinterpretation of the cited reference. On the contrary, this particular dressing (AgCMC) was shown to reduce a bacterial population by approximately 10,000-fold within 24 to 48 hours. Additionally, data presented to, and accepted by, the FDA showed activity for at least 7 days ("Instructions for Use" data).

   Although nanocrystalline silver is an efficacious topical antimicrobial agent, this article promotes one technology to the detriment of others that have equal merit from an antimicrobial perspective and, in some cases, superior properties from a wound management perspective.
   D. Parsons, PhD
   P.G. Bowler, MPhil
   M. Walker, PhD
   ConvaTec Global Development Centre
   Deeside, Flintshire, UK

Reply
   With regard to a full disclosure of past and present affiliations with commercially involved organizations, I have worked on silver-based technologies for more than 18 years in commercial and university settings. I am a named inventor on numerous patents, many of which pertain to silver technology. A former Vice President of Science and Technology and Chief Scientific Officer of Nucryst Pharmaceuticals, I am now, and have been for more than a year, a Professor of Chemical and Materials Engineering at the University of Alberta, Edmonton, Alberta, and an Adjunct Professor in the Biofilms Research Group, Department of Biology at the University of Calgary, Calgary, Alberta. I do not have consulting agreements related to silver technology with any of the commercial entities involved in manufacturing, marketing or selling silver based products. I speak on an invitational basis at educational symposia on silver technology and receive honoraria from companies such as HMP Communications and Smith and Nephew for these engagements. I have no financial interests in Smith and Nephew (the company that markets and sells nanocrystalline silver-based products).

   Table 1 clearly shows that silver nitrate and nanocrystalline silver release more silver into solution than do other silver complexes. I respectfully disagree with Parsons et al's opinion that these figures bear no relevance to the availability and antimicrobial efficacy of silver in complex media such as a wound fluid. The efficacy of silver is determined, in part, by the concentration of silver added, the concentration of available silver, the stability of complexes formed in the media, and the frequency of new additions of silver (exposure time). This is in close agreement with McManus¹ who notes that while the MIC is the standard way of thinking about antimicrobial activity, the observed activity is more likely to be a compound function of the MIC, concentration achieved, and the time of exposure above an effective concentration. The relationship between dose of silver, available silver, and antimicrobial efficacy was demonstrated when Ricketts et al² looked at the antimicrobial activity of silver nitrate and silver chloride solutions. Silver nitrate (0.5 mg/L) solutions generated greater than 5 log reductions in 60 minutes while the supernatant from the silver chloride solutions was not bactericidal in the same time period. They also examined free silver in solution as a function of added silver in a bacterial growth medium with variable levels of sodium chloride. In these experiments, they found a direct relationship between added silver and free silver, as would be expected. They also showed that while a 0.5 mg/L solution of silver nitrate reduced a Pseudomonas population by 5 logs in 1 hour, the minimum inhibitory concentration (MIC) of silver nitrate in bacterial growth media was up to 80 times higher. They concluded that the efficacy of silver nitrate dressings was a result of the presence of high levels of unbound silver immediately after silver replenishment and that using poorly soluble salts would be problematic because at no time is a high level of unbound silver present. This was exemplified by the low bactericidal activity of the silver chloride supernatant that contrasts greatly with the effectiveness of silver nitrate, even when in contact with a complex media containing high levels of chloride.

   The importance of using sufficiently high concentrations of silver was more recently demonstrated by Spacciapoli et al.³ They showed that silver nitrate was bactericidal (>3 log reduction) to a population of Porphyromonas gingivalis at concentrations as low as 0.05 mg/L in phosphate buffered saline. However, when the test was performed in human serum, the required concentration jumped to 25 mg/L - an approximately 500-fold increase in required silver. Scheirholz et al⁴ demonstrated a 100-fold increase in required silver when albumin and NaCl were added to a microbial growth medium (Mueller-Hinton Broth). These results show that adding more silver to the system can compensate when the introduction of organics and other silver-consuming entities reduces the concentration of available silver. These studies also show that silver concentrations between 25 mg/L and 100 mg/L are required in complex media.
Several of the silver dressings in the marketplace today instruct users to activate the dressing using sterile water. This activation process releases silver into the dressing and determines the initial dose. By using water, the initial dose of available silver is much higher than that which can be obtained by moistening with chloride-containing media. Based upon this information, testing silver release in water provides useful comparative data that may be linked to clinical efficacy.

   Parsons et al say the only accepted safe and proven antimicrobial species of silver is ionic silver in the form of the monovalent silver cation (Ag⁺). Lansdown,⁵ an author cited by Parsons et al, states, "Microbiological studies illustrate that the 'activated' silver ion (Ag⁺ or other species) can exert its lethality through action on the bacterial cell membrane ...." Clearly, he contemplates the presence of other silver species. Silver is available in many different forms, including ionic complexes (AgNO₃, Ag sulfadiazine, AgCl, and AgCMC), metals (silver metal, silver alloys, nanocrystalline silver) and adsorbed or trapped materials (silver carbon and silver zeolites). These materials all release some Ag⁺ that has well demonstrated antimicrobial properties. The FDA and many other regulatory bodies around the world have accepted these materials. Nanocrystalline silver, unlike all of the other products, also releases Ag⁰ that is thought to be present as a cluster structure.⁶ Numerous papers in the literature attest to the rapid kill associated with the nanocrystalline silver technology, including bacteria,^7-9 antibiotic-resistant bacteria,¹⁰ and fungi.^9,11 This rapid kill must be related to the dissolution products of the nanocrystalline silver, so far identified as Ag⁺ and Ag⁰,⁶ since direct comparison with other sources of Ag⁺ has shown that the nanocrystalline silver is a faster-acting antimicrobial agent. Further, several articles examine the efficacy and safety of the nanocrystalline silver in vivo^12,13 and clinically.¹⁴ More recently, several papers and a conference presentation have looked at the anti-inflammatory activity associated with the nanocrystalline silver.^15-17 Again, these properties are not associated with other silver delivery systems that only provide Ag⁺. Thus, ample evidence appears to link the nanocrystalline silver and its dissolution products with improved antimicrobial efficacy and other desirable biological activities. While the mechanisms of action for nanocrystalline silver are unclear, it may be as simple as an improved way to deliver Ag⁺ from a less reactive remote source - ie, the Ag⁰ moiety.

   In Table 2, the footnote indicating that the silver nitrate and silver sulfadiazine silver release numbers were calculated based upon dilution of the original materials was inadvertently omitted. The numbers are correct as shown based upon a 1 in 10 dilution of the starting materials.
While chloride will interact with silver ions from all silver dressings, the assumption that the effect will be the same for all dressings is incorrect. Simply, the chemistry of silver and chloride ions is as follows:
   Ag⁺ + Cl- U AgCl where the Ksp is 1.8 x 10^-10 M

   Thus,
   [Ag⁺] [Cl-] = 1.8 x 10^-10 M
   and
   [Ag⁺] = 1.34 x 10^-5 M
   [Ag⁺] = 1.25 x 10^-3 g/L
   [Ag⁺] = 1.25 mg/L

   The above only applies at conditions where the molar concentrations of silver and chloride ions are equal. If the chloride ion levels are higher than the silver ion levels, the equilibrium is pushed in favor of the precipitation of silver chloride. In the wound environment, the chloride levels are about 5,000 mg/L or about 0.14 molar. If a dressing that releases 1 mg/L or 9.3 x 10^-6 molar silver ions is used, an approximately 15,000-times difference exists in molar concentrations between the two ions. This will significantly reduce the free silver ion in solution. These losses of silver ions as AgCl can be compensated for in two ways. The quantity of silver can be increased and/or the frequency of application can be increased with controlled release being the best case. Historically, silver nitrate has been used at a high concentration (3,176 mg/L) and a high frequency of application (12 times per day). Silver sulfadiazine was an improvement over silver nitrate - although the concentration of silver was high (3,025 mg/L), the frequency of application was lower (twice per day) because the sulfadiazine moiety controlled the release of silver. If Parsons et al's theory was correct and all silver above 1 mg/L was precipitated as the inactive chloride, all MIC experiments in chloride containing media would yield the same result - an MIC of greater than or equal to 1 mg/L - since silver additions greater than this would apparently not alter available silver concentrations. Clearly, this is not the case, as has been shown by Spacciapoli et al,³ Schierholz et al,⁴ and Ricketts et al.² Because some dressings' instructions for use call for pre-moistening in water, and it is clear that different amounts of silver will give different results in tests, the collection of silver release data in water is useful in that an initial dose of silver can be determined.

   It is confusing to argue that traditional static MIC data, based on the exposure of bacteria to a single bolus, is not applicable to controlled release devices if you also argue that all silver above 1 mg/L is precipitated as inactive silver chloride. As previously noted, if this were the case, all MIC values would be greater than or equal to 1mg/L. The fact that MIC values in the literature range from 0.005 mg/L to > 100 mg/L suggests that increasing the concentration of silver in complex media increases the available silver which, in turn, increases the kill of bacteria. This was shown by Ricketts et al² Spacciapoli et al.³ correlated the results of a clinical trial of a controlled release silver wafer to standard MIC and MBC data. They concluded that an in vitro positive bactericidal result (MBC) in serum may well represent the threshold level for clinical applications. From this information, it appears that standard MIC and MBC data may be useful in predicting clinical outcomes for controlled release devices.

   Using Table 2 data to show a bactericidal effect is not supported by the literature. A bactericidal effect is defined as a three-log reduction in a population over the time of the test.¹⁸ A 100-fold or two-log reduction does not meet that criteria and the dressing would at best be considered bacteriostatic based on the data presented. As a result, you cannot say that this organism is any different than the referenced MIC values. A further difficulty with the argument is that many different isolates of Pseudomonas aeruginosa have different MIC values, which makes comparisons difficult.

   The term oligodynamic is defined in the article as, "the high level of antibacterial activity that was generated from relatively small amounts of silver and other heavy metals," and applies to the antimicrobial effects of silver described in the article. A dictionary definition would include words such as "active in very small quantities." These definitions focus on activity, not accumulation or absorption because activity is the critical element of the term - ie, silver is oligodynamic because of its antibacterial effect, not because it is accumulated. Some bacteria are able to accumulate silver without toxic effects; in these cases, silver would not exert an oligodynamic effect even though it is accumulated.

   Parsons et al state emphatically that, "it is the number of silver ions absorbed and not the concentration of silver ions in solution that causes bacterial death." This is an interesting concept in that it calls into question the use of MIC and MBC test methods for evaluating antimicrobial efficacy of silver. Both techniques have been used extensively to generate data that are presented as a concentration to either inhibit or kill bacteria. While the number of ions absorbed is related to the death of a bacterium, concentration is a measure of the number of atoms/ions per unit volume. The concentration dictates the likelihood of an atom/ion being absorbed since it must come within a specific distance of the cell to be absorbed. It is for this reason that many efficacy tests are based upon concentration. It is interesting to note that Shierholz et al⁴ (a reference used by Parsons et al) state, "The Ag⁺ kill rate is directly proportional to Ag⁺ concentrations, typically acting at multiple targets. The higher the silver-ion concentration, the higher the antimicrobial efficacy."

   Parsons et al state that a 30-minute log reduction assay is of questionable clinical relevance and that it is deliberately used to differentiate nanocrystalline silver from other silver-containing dressings. The test clearly differentiates the nanocrystalline silver dressing from all other silver-containing dressings. This type of test, a log-reduction or time-kill kinetic assay, is often used to compare new therapeutic agents.¹⁹ It is used more frequently, now that speed of kill is being recognized as an important factor in the treatment of infections. The original theories of antimicrobial treatment state that the active agent only needed to have bacteriostatic properties because preventing the organisms' growth for a period of time would allow the patient's immune system to function and remove the infection. This no longer holds true for certain types of infections (eg, meningitis and endocarditis) and patients (eg, immunocompromised individuals including those with autoimmune conditions, transplant recipients, the elderly, burn victims, and the like) where a bactericidal effect is required.
McCracken's studies on infants with meningitis led Barry¹⁹ to conclude that 1) rapidity of sterilization was important, and 2) time-kill assays have better correlations with endocarditis cures than do MBCs, suggesting the rate of kill was important. Time-kill analyses are used to determine positive and negative interactions between therapeutic agents. In these types of studies, rate-of-kill data are considered by some to be more important than MBCs.¹⁹ Stratton and Cooksey,¹⁸ based upon Jackson and Riff's work,²⁰ concluded that the time-kill kinetic assay was more sensitive than MIC-MBC techniques for evaluating antimicrobial activity - they noted that the best clinical results followed more closely the maximal killing rates than the MICs. As previously noted, McManus¹ thinks time is an important factor. He believes, "In the future, the area under the concentration and time curve above the MIC of the agent is likely to become a more meaningful predictor of expected agent activity."

   Clearly, time and rates of kill are considered important factors by many authors. In addition, time-kill kinetic assays have a stronger link to clinical outcomes than do other in vitro test results. Therefore, using this type of data to compare dressings is warranted and should be utilized by healthcare providers.

   Parsons et al say the term limited antimicrobial activity is a gross misinterpretation of the cited reference. Again, I respectfully disagree. Antimicrobial efficacy data can be presented numerous ways. Two common ways to present data are bacterial log reduction and bacterial survival. Bacterial survival data are collected, typically using a tube dilution and plate count technique after exposing the organisms to an antimicrobial agent, converted to log numbers and plotted against linear time. A dressing that contains an effective bactericidal agent will reduce the number of survivors quickly; thus, when the data are plotted, the graph will start high at time 0 and fall at least 3 logs in a short span of time. Bacterial log reduction data are the inverse of bacterial survival data. Bacterial log reductions are determined by first calculating the log survival number as above and subtracting that from the log of the initial number of cells. The difference between the log number of survivors and the log number of initial cells is the log reduction number.

   The cited reference is an advertising brochure entitled, "Gentle" on the outside and "Ferocious" on the inside (AQUACEL Ag, ConvaTec, a Bristol-Myers Squibb company, Princeton, NJ). The brochure contains a graph that shows the antimicrobial activity of the dressing against Pseudomonas aeruginosa and Staphylococcus aureus. The x - axis is labeled "Time (days)" and covers 0 to 14 days. The y-axis is labeled "Bacteria reduction (log value)" and covers log values from 0 -7. The curves start high on the y - axis (log values close to 6) and fall over 24 to 48 hours to 1. This clearly indicates that the material will initially cause a large log reduction in the bacterial population, but that after 24 - 48 hours, the log reduction drops to 1, indicating no bactericidal efficacy as the log reduction was less than three.¹⁸ No misinterpretation of this data occurred. As presented, the data clearly show that beyond 24 to 48 hours, this material generates 1-log reductions and, therefore, has limited antimicrobial activity. This is of concern in light of Schierholz et al,²¹ who believe that devices coated with antimicrobial agents should be bactericidal. Whether bacteriostatic devices will lead to severe infections through the colonization of the surface with potentially resistant organisms is a concern.⁴

   The difficulty in writing this type of educational article lies in the lack of peer-reviewed published data for many of the devices. A reasonable number of papers has been published on silver nitrate, silver sulfadiazine, and nanocrystalline silver, but few on the other types of material. To provide the readers with as much information as possible, corporate marketing information was included in the references where limited peer-reviewed data were available. In the future, more articles may appear that will add to the healthcare providers' database of knowledge.

   Robert E. Burrell, PhD

Letter
1. Russell AD, Hugo WB. Antimicrobial activity and action of Silver. In: Ellis GP, Luscombe DK (eds). Progress in Medicinal Chemistry, Vol 31. St. Louis, Mo.:Elsevier Science;1994:351-370.

2. Schierholz JM, Wachol-Drewek Z, Lucas LJ, Pulverer G. Activity of silver ions in different media. Zentralblatt fur Bakteriologie. 1998;287:411-420.

3. Lansdown ABG. The role of silver. European Tissue Repair Society: Concepts in Wound Healing. 2002;9:108-111.

Reply
1. McManus AT. 2002. Precautions regarding acquired resistance. Wounds. 2002;14(7 suppl):11S-13S.

2. Ricketts CR, Lowbury EJL, Lawrence JC, Hall M, Wilkins MD. Mechanism of prophylaxis by silver compounds against infection of burns. British Medical Journal. 1970;2:444-446.

3. Spacciapoli P, Buxton D, Rothstein D, Friden P. Antimicrobial activity of silver nitrate against periodontal pathogens. Journal of Periodontal Research. 2001;36:108-113.

4. Schierholz JM, Wachol-Drewe, Z, Lucas LJ, Pulverer G. Activity of silver ions in different media. Zentralblatt fur.Bakteriologie. 1998;287:411-420.

5. Lansdown ABG. The role of silver. European Tissue Repair Society: Concepts in Wound Healing. 2002;9:108-111.

6. Fan FF-R, Bard AJ. Chemical and electrochemical, gravimetric, and microscopic studies on antimicrobial silver films. Journal of Physical Chemistry B. 2002;106(2):279-287.

7. Yin HQ, Langford R, Burrell RE. Comparative evaluation of the antimicrobial activity of Acticoat Antimicrobial Barrier Dressing. J Burn Care Rehabil. 1999;20:195-200.

8. Wright JB, Hansen DL, Burrell RE. The comparative efficacy of two antimicrobial barrier dressings: in vitro examination of two controlled release silver dressings. Wounds. 1998;10(6):179-188.

9. Thomas S, McCubbin P. A comparison of the antimicrobial effects of four silver containing dressings on three organisms. Journal of Wound Care. 2003;12(3):101-106.

10. Wright JB, Lam K, Hansen DL, Burrell RE. Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment. Am J Infect Control. 1998;26:572-577.

11. Wright JB, Lam K, Burrell E. Efficacy of topical silver against fungal burn wound pathogens. Am J Infect Control. 1999;27:344-350.

12. Burrell RE, Wright JB, Heggers JP, Davis GJ. Efficacy of silver coated dressings as barriers in a rodent burn sepsis model. Wounds. 1999;11(4):64-71.

13. Olson ME, Wright JB, Lam K, Burrell RE. 2000. Healing of donor sites covered with silver coated dressings. Eur J Surg. 2000;166:486-489.

14. Tredget EE, Shankowsky HA, Groeneveld A, Burrell RE. A matched-pair, randomized study evaluating the efficacy and safety of Acticoat Silver-Coated Dressing for the treatment of burn wounds. J Burn Care Rehabil. 1998;19:531-537.

15. Wright JB, Lam K, Buret AG, Olson ME, Burrell RE. Early healing events in a porcine model of contaminated wounds: impact of nanocrystalline silver on matrix metalloproteinases, cellular apoptosis and wound healing. Wound Repair and Regeneration. 2002;10:141-151.

16. Kirsner R, Orsted H, Wright JB. Matrix metalloproteinases in normal and impaired wound healing: a potential role of nanocrystalline silver. Wounds. 2001;13:4S-12S.

17. Paddock HN, Schultz GS, Perrin KJ, Moldawer LL, Wright B, Mozingo DW. Clinical assessment of silver-coated antimicrobial dressing on MMPs and cytokine levels in non-healing wounds. Annual Meeting Wound Healing Society. Baltimore, Md. May 28 to June 1, 2002.

18. Stratton CW, Cooksey RC. Susceptibility Tests: Special Tests. In: Manual of Clinical Microbiology, 5th ed. ASM, Washington, DC;1991.

19. Barry AL, Craig WA, Nadler H, Reller LB, Sanders CC, Swenson JM. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Guideline. NCCLS document M26-A (ISBN 1-56238-384-1);1999.

20. Jackson GG, Riff LJ. Pseudomonas bacteremia: pharmacologic and other bases for failure of treatment with gentamicin. J Infect Dis. 1971;124:S185-S191.

21. Schierholz JM, Rump AFE, Pulverer G. Drug delivery concepts for efficacious prevention of foreign body infections. Zentralblatt fur Bakteriologie. 1996;284:390-401.