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Antimicrobial Silver in Orthopedic and Wound Care Products

The authors describe the use of silver in orthopedic devices and what you need to know.

Herbert N. Prince and Daniel L. Prince

The use of antibiotics to prevent infections when incorporated onto or into orthopedic devices or wound care products has met with limited success over the years. Limited success has also been seen with non-antibiotic remedies such as chlorohexidine, nitrofurazone, PVP-I, mafenide and mupirocin. Historically, amino-glycoside antibiotics (tobramycin, amikacin, gentamicin) were highly effective, and their original selection was based on the fact that most patients had never been treated with these rather toxic parenterals, as opposed to tetracyclines, semi-synthetic penicillins, cephalosporins, quinolones and macrolides—antibiotics prescribed for practically every patient who ever had either an upper respiratory or urinary tract infection. The thinking was that such little-used antibiotics would not present a threat of the development of drug resistance. This theory was incorrect. As a replacement, one of the remarkable transition elements, silver, has come into wide use—especially for topical treatment. Following are descriptions of some of the theory and mechanisms of action of silver, aspects of clinical deployment and factors relating to resistance. Laboratory methods and results used in the evaluation of silver antimicrobials for submission to regulatory bodies also are discussed.

Theory

Silver was first used clinically in 1881 to prevent eye infections in newborns. The effectiveness of silver revolves around its low propensity to select for resistance, its broad spectrum of activity and its high chemotherapeutic ratio (toxic dose divided by effective dose). Silver is biocidal in the ionic form and, unlike many antibiotics, has at least six mechanisms of action (Table 1). More recently, silver nanoparticles in the range of 2 to 20 nM have been shown to provide a mechanism of closely adhering metallic particles to devices and even environmental surfaces. The deposition on such surfaces may be in the order of 1 to 32 µg/cm2 or otherwise.

The multiple targets suggest that at least six mutations are required for organisms to become resistant. Most antibiotics, on the other hand, have one or two mechanisms of action, usually involving binding to a ribosome or inhibiting some aspect of metabolism. Silver has the following advantages: low induction of resistance compared to antibiotics; “entire spectrum” of activity (bacteria, yeasts and molds); and safety at the proper dose. An entire-spectrum agent surpasses the so-called broad-spectrum agent by killing fungi. No entire-spectrum antibiotics are clinically available.

A second theoretical discussion about silver speaks to its membership in a group of 30 metals known as the Transition Elements. Characteristic of the group is the presence of double energy states for the outer orbits—ie, positive valence may be expressed by loss of an electron from, two energy levels, the outermost or the one beneath (see Figure 1). Of these elements, only silver, mercury and copper have biocidal activity (ppb to ppm). Mercury has the broadest spectrum and kills the most rapidly. Copper is the least active—mainly a fungicide and algacide. Silver is the safest.

Importance of Antimicrobial Attribute

Infections as a result of invasive orthopedic procedures are a major surgical health risk. These infections occur either at the surgical site (dermis, subjacent musculature) or at the implant. Surgical site infections are treated with topical or systemic antibiotics or topical wound care dressings. Infections at the site of implantation (hip, spine, knee, etc.) are more difficult to treat and, in the event that aggressive antibiotic treatment fails, require removal of the implant—an expensive and risky procedure. To counter this problem, manufacturers have developed orthopedic implants with antimicrobial properties. Silver technology has been a part of this development. Antimicrobial devices have been prepared in three ways: (1) direct incorporation of the antimicrobial agent into the physical structure of the implant; (2) surface coating; and (3) incorporation of surface coating of the bone cement employed in the procedure. The major disadvantages of the first and third methods are slow rate of release and unwanted modification of the material’s properties. For surface application, one also sees uncertain rates of release for the organic germicides and antibiotics and poor activity against biofilm. In the area of surface application, silver technology (in the form of nanoparticles, 10 to 20 nM diameter) has come to play an important role by virtue of the fact that such particles can adhere tightly to the device surface and possess antimicrobial properties that potentially mitigate nosocomial infection.

Silver, like most transition elements, is a reducing agent and, upon electron loss, produces an ion with two pathways—one inorganic and the other organic. The inorganic pathway reveals silver ion as an oxidizing agent affecting either electron transport in the cytochrome system or acting as an anionic trap producing insoluble salts from a variety of anions in the cytoplasm. The organic path is depicted in Table 1.

Testing Methods

The microbiologist employs essentially three test systems in the detection of antimicrobial activity during the screening phase and uses slightly modified procedures for the finished product prior to marketing. The procedures can be a mixture of compendial methods (eg, ASTM, USP, NCCLI—see references at the end of this article), FDA guidelines, in-house methods or a combination. All of these procedures begin with three basic techniques:

1. Zone of inhibition assay—This is an agar diffusion test much like that cited in USP <81 > for antibiotic sensitivity tests and also cited in USP < 87> for cytotoxic tissue culture screening for in-vitro bioreactivity. The procedure is diagrammed in Figure 2.

Agar Diffusion Assay: The size of the zone may or may not be related to the concentration of the agent in the carrier, the physiochemical nature of the carrier and the thickness and chemical concentration of the agar.

Typical carriers are dressings, fabric, putty, gel, tissue, plastic or metal. If one is testing a wound-care product for which the indication is to prevent colonization of purulent organisms into the dressing (preservative effect), an immobilized agent is preferable. If a steady state of diffusion is desirable so as to achieve an antimicrobial or antiseptic affect at the wound, one would look for a medicated sample with a zone of inhibition. Agar diffusion assays frequently do not agree with MIC or log-reduction methods.

2. Minimum Inhibitory Concentration Assay (MIC)—The MIC assay is employed during the developmental phase and can be extremely useful in the clinical phase. One can determine the relative drug resistance of any battery of clinically relevant organisms. During the clinical phase, non-responders can be examined to determine if failure is a function of resistance or delivery. Whether using, for example, a silver salt or a nanoparticle, killing has to occur above the MIC to avoid resistance. Resistance occurs routinely (antibiotics) or rarely (metals) when the delivered dose is at the MIC or below this target value. The MIC values for silver nitrate and Ag+ are presented in a later section (Table 6) expressed in parts per million (μg per mL). Figure 3 shows a diagram of an MIC assay.

3. The Log-Reduction Test—In this procedure, the test organism is inoculated directly into or onto the test article along with placebo controls. The inoculation may be in saline or simulated bodily fluid of various compositions. This is a quantitative time-course assay first promulgated in 1970 with USP 18. It is now promulgated worldwide as the misnamed “Antimicrobial Effectiveness Test,“ originally called the “Preservative Challenge Test.” Quantitative recovery of survivors over time is accomplished by aerobic or anaerobic standard plate count (pour plate or surface streak) or membrane filtration. A log-kill is converted to percent kill as follows:

The D-value is the time to kill one-log. It can be calculated by the equation:

The D-value also can be determined by striking off one-log intercepts on the straight-line portion of the dose-survival curve. Often both can be used and an average value determined. Of course, the lower the D10 value, the more potent the antibacterial event.

A modification of the dynamic log-reduction method has been developed for testing of antibiotic or silver containing PMMA bone cement samples (alt and Bechert “microplate proliferation assay”). The bone cement is surface-contaminated with test organisms, rinsed to release loosely attached cells, followed by a time-course optical density determination as to onset of proliferation of the adherent organisms. The rate of growth relates to the biocidal activity at the bone cement surface.

Results

Tables 2-6 describe some typical results obtained with various device products containing silver employing the test methods described previously. These are in-vitro tests used for guidance in the research process for product development and regulatory approval either as a novel product or as a product put forth for 510K equivalence. In such tests, comparison with an approved product or predicate is included. Data in this respect also are provided on experiments with silver nitrate solution as an example of a pure active ingredient.

Table 2 is informational only and gives some of the silver compounds that have been employed in medicated devices over the past 20 years. It also lists the types of wound care dressings and orthopedic devices that can be associated with infectious disease risk.

These compounds deployed, for example, in hydrophilic-type wound care pads or orthopedic devices can receive a first pre-clinical in-vitro evaluation in the laboratory by either a passive or dynamic inoculation with a battery of pathogenic microorganisms. The microbial suspension is prepared with simulated tissue fluid as used in the standard cell culture or virology lab or rather with normal sterile saline. Except for the USP “Antimicrobial Effectiveness Test,” we know of no official kill rate for products containing an antimicrobial such as those described in this report. The passive test relies on recovering survivors after inoculation directly onto the device. The dynamic test presents the medicated device to a suspension of bacteria in agitation over a period of time with bacterial counts taken at different intervals as in the above model. In both the passive and dynamic tests, depending if the active agent is diffusible or is immobilized, log reduction calculations are made over periods from a few hours to days, depending on the indication of the product and the clinical directions. A dynamic test, if conducted with a simulated tissue fluid, can usually produce at least a three log kill if the silver ion concentration in the elution fluid is 50 to 100 ppm.

Example of Short-Term Exposure to Silver

It was of interest to determine if silver had a short-exposure (10 minute) rate of kill as is common for many chemical germicides, such as required by Environmental Protection Agency guidelines. The results of this experiment with three pathogenic gram-negative bacilli and the yeast Candida albicans, in which the organisms were inoculated directly into the silver nitrate solution, are shown in Table 3.

As seen, silver nitrate solution 0.5% did not produce a 10-minute kill and, therefore, cannot be ranked amongst common disinfectants as to rate of kill (for example, alcohol and halogens kill in seconds, disinfectants in minutes). However, at least four log kills were obtained at 24 hours, a respectable and obtainable rate of kill with regard to a wound care or orthopedic device. However, variable rates of kill can be seen at 24 hours when formulated products are tested, as shown in Tables 4 and 5. This is a function of the retention of silver ions within the device or inactivation by protein or salts in the inoculating suspension.

Examples of Log-Reduction Comparison of Four Devices Containing Silver

The data in Table 4 show clearly that four of the medicated products would have prevented the growth of the pathogens. These tests are generally conducted with a battery of pathogenic organisms such as the gram-negative rods Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae and others, and the gram-positive cocci Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus (MRSA), Streptococcus pyogenes and the yeast Candida albicans. There are no absolute pass/fail criteria as per current FDA regulations. However, comparisons of sample C with predicate suggest equivalent rapid and sustained kills. Sample A was clearly the least active showing a delayed kill and no more than a two-log decline after seven days. Although Zone-of-Inhibition and MIC tests can be useful in product development, log-reduction tests of the kind depicted in Table 5 afford the best evidence of efficacy.

Examples of Log-Reduction Comparison: Gram-Positive vs. Gram-Negative Bacteria

The data in Table 5 reveal that after 24 hours, gram-negative bacilli were more sensitive to silver than the gram-positive cocci. This frequently is the case with chemical germicides seen in EPA/AOAC–type disinfectant tests. It is the exact opposite of the comparative sensitivities seen with antibiotics, where the gram-negative bacteria frequently are the more difficult to kill than the gram-positive variety.

Comparative MIC Data—Wound and Nosocomial Pathogens

A series of experiments was performed as per the MIC method, in which laboratory strains and clinical isolates were tested for sensitivity to 0.5% silver nitrate solution, as summarized in Table 6.

The MIC results shown in Table 6 demonstrate the “entire-spectrum” effect Ag+ and show that the gram-negative rods were the most sensitive in agreement with log-reduction data. This is important because such pathogens are frequent inhabitants of wound and orthopedic surgical sites. The MIC values in the table help to define the end-use concentration of silver required for clinical efficacy in these indications. It is to be noted that silver, either in the ionic or metallic form, is regarded as an “entire” spectrum agent as opposed to the standard “broad” spectrum designation. Broad spectrum refers to activity against gram-positive and gram-negative bacteria, excluding fungi and viruses. An entire-spectrum agent includes in its range yeast, molds and even algae and protozoa. There is no known entire-spectrum antibiotic of clinical significance.

Some FDA Guidance on Products Containing an Antimicrobial Agent

The FDA has promulgated an FDA draft guidance for medical devices that include antimicrobial agents (available at www.fda.gov). This document speaks to the issues and methods that are the present report. In this document, the FDA distinguished between (a) a product designed to control or prevent infection and to protect the patient (eg, sulfadiazine ointment), an anti-infective product, which is a drug under section 201(g) of the act (CFR 21 (3.2e); and (b) a product such as a wound care treatment or orthopedic or tissue product that makes no chemotherapeutic claim but contains an antimicrobial so as to prevent colonization in or about the product (eg, a hydrocolloid wound dressing) a product containing an antimicrobial. For the first, the primary mode of action is for a drug and for the second, the primary mode of action is that of a device. Appropriate safety and efficacy standards are applied to each indication. It is helpful to list the expectations of the FDA with regards to microbial testing; some of these requirements are shown in Table 7.

It is hoped that the type of data presented in this report with products containing an antimicrobial agent will assist investigators and manufacturers in developing unique or comparative medical products and to be better aware of appropriate methodology, as required by the FDA.

Summary

• It is noted that the silver compounds and particles, in many respects, have replaced antibiotics and certain chemical germicides in wound and burn care treatment. They have proven useful when deployed into or onto carriers such as orthopedic devices, catheters, implants, fabrics, plastics and a variety of hard surfaces and polymers, among others.
• There is an established relationship of the atomic structure of silver to its antimicrobial activity as well as the theoretical basis for its low level of selectivity for microbial resistance.
• Laboratory methods often required by the FDA for use in the study of silver and other antimicrobial agents were described, and representative examples of the pre-clinical data as shown in the tables can be used for comparison with predicate materials and submission to regulatory bodies, as required.
• Experimental results obtained with silver nitrate solutions are presented so as to determine rate of kill. In addition, data describing the activity of silver nitrate solutions against a variety of gram-positive and gram-negative pathogens show the good relationship between log-reduction and MIC methodologies. A combination of log-reduction and MIC tests can be useful in determining the concentration of silver required for effectiveness in these indications.

Dr. Herbert N. Prince is the scientific director and founder of Gibraltar Laboratories, Inc. As a national expert on pharmaceutical microbiology and virology emphasizing the control of pathogens in vivo and in vitro, he is a recipient of the Selman Waksman Award and has published more than 50 papers. He has lectured extensively on microbiological control and held numerous teaching appointments at Fairleigh Dickinson University and Montclair State University. He is a longstanding member of many microbiology organizations including ASM, SIM and TSS.

Dr. Daniel Prince is president of Gibraltar Laboratories, Inc. He has served on numerous USP, ASTM, A2LA committees and published more than 20 papers. He has worked as electron microscopist at Osborne Aquarium and later at Gibraltar in many capacities including virologist, endotoxin analyst, molecular biologist, assistant scientific director, and associate scientific director. For other companies, he has led scientific efforts in HIV-1 and human HBV testing and contributed to OSHA’s Bloodborne Pathogen rule. He recently led the expansion of Gibraltar Laboratories’ building additional state-of-the-art laboratories for the microbiology program and creating additional lab space for the chemistry department.

References

1. Yamanaka, M. et al 2005, Bactericidal Activity of Silver ion solutions on E.Coli, Applied Environmental Microbiology 71 (11) 75-83.
2. Chopra, I. 2007, The Increasing use of Silver based products as Antimicrobial agents: A Useful development or a cause for concern? Journal Antimicrobial Chemotherapy 59:584-587
3. CLSI Methods 2006, Performance Standards for Anti-microbial susceptibility tests, 9th edition, NCCL Document M2-Ag Disc methods; CLSI document M7-A7 Dilution methods; NCCLS 940 W. Valley Road, Suite 1400, Wayne, PA.
4. USP 24, 2007 Chapter <51> Antimicrobial Effectiveness Test; Disinfectants and Antiseptics; Chapter <1072>
5. ASTM-2149 (2001) Standard Test Method for Determining Antimicrobial Activity of Immobilized Agents under Dynamic Contact conditions.
6. FDA, July 19, 2007, Draft Guidance for Industry and FDA staff – Pre-Market Notification (510K) Submissions for Medical Devices that include Antimicrobial agents – US Department of Health and Human Services, Center for Device and Radiological Health.
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14.    Prince, H.N. 1983, Disinfectant Activity Againest Bacteria and Viruses: A Hospital Guide, Particulate and Microbial Control, Canon Publications.
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Alt, Volker, Bechert, T. In Vitro testing of Antimicrobial Activity of Bone Cement. Antimicrobial Agents and Chemother, Nov. 2004, 48, No. 11, 4084-4088.