Herbert N.Prince, PhD

GibraltarLaboratories,

Inc.

Daniel L.Prince, PhD

GibraltarLaboratories,

lnc.

Antimicrobial Silver in Orthopedicand Wound Gare Products

The authors describe the useof silver in orthopedic devicesand what you need to know.

he use of antibioticsto prevent infectionswhen incorporated

onto or into orthopedicdevices or wound care prod-ucts has met with limitedsuccess over the years.Limited success has also been seen withnon-antibiotic remedies such as chlorohex-idine, nitrofurazone, PVP-I, mafenide andmupirocin. Historically amino-glycosideantibiotics (tobramycin, amikacin, gen-tamicin) were highly effective, and theiroriginal selection was based on the factthat most patients had never been treatedwith these rather todc parenterals, asopposed to tetracyclines, semi-syntheticpenicillins, cephalosporins, quinolones andmacrolides-antibiotics prescribed forpractically every patient who ever hadeither an upper respiratory or urinarytract infection. The thinkingwas that suchlittle-used antibiotics would not present athreat ofthe development ofdrug resist-ance. This theory was incorrect. As areplacement, one of the remarkable tran-sition elements, silver, has come into wideuse-especially for topical treatment.Following are descriptions of some of thetheory and mechanisms of action of silver,aspects ofclinical deplol.rnent and factorsrelating to resistance. Laboratory methodsand results used in the evaluation ofsilverantimicrobials for submission to regulato-ry bodies also are discussed.

TheorySilver was first used clinically in1881 to prevent eye infections innewborns. The effectiveness of silverrevolves around its low propensity to

select for resistance, its broad spec-trum of activity and its highchemotherapeutic ratio (toxic dosedivided by effective dose). Silver isbiocidal in the ionic form and,unlike many antibiotics, has atleast six mechanisms of action (Table1). More recently, silver nanoparticlesin the range of 2 to 20 nM havebeen shown to provide a mechanismof closely adhering metallic particlesto devices and even environmentalsurfaces. The deposition on suchsurfaces may be in the order of 1 to32 1tg/cm2 or otherwise.

The multiple targets suggest thatat least six mutations are requiredfor organisms to become resistant.Most antibiotics, on the other hand,have one or two mechanisms ofaction, usually involving binding to aribosome or inhibiting some aspect ofmetabolism. Silver has the followingadvantages: low induction of resist-ance compared to antibiotics; "entirespectrum" of activity (bacteria,yeasts and molds); and safety at theproper dose. An entire-spectrumagent surpasses the so-called broad-spectrum agent by killing fungi. Noentire-spectrum antibiotics are clin-ically available.

A second theoretical discussionabout silver speaks to member-

May/June 2oo8 6 l

Table 1

1. Ribosome (m-RNA) (t-RNA) - Silver binds disallowingpolypeptide init iation

2. Sulfhydryl compounds (Oxidation) - sulfur amino acidis inactivated to the disulfide form

3. Protein denaturation (Ag-salts) - coagulation ofenzymatic and other proteins

4. Nucleic acid interactions - interaction with DNA,disrupting mRNA synthesis

5. Gytoplasmic membrane - disruption and leaching ofmetabolites

6. Interference with electron transport in cytochrome system

ship in a group of 30 metals known as the TransitionElements. Characteristic of the group is the presence ofdouble energy states for the outer orbits-ie, positivevalence may be expressed by loss of an electron from twoenergy levels, the outermost or the one beneath (seeFigure 1). Of these elements, only silver, mercury and cop-per have biocidal activity (ppb to ppm). Mercury has thebroadest spectrum and kills the most rapidly. Copper isthe least active-mainly a fungicide and algacide. Silver isthe safest.

Figure I

OXIDATION STATES[and electronic toxicity]

DermalToxicity

Ag+ (Ag2O) Tolerated

Ag++ (AgO) lrritant [divalent]

lmpoftance of Antimicrobial AttributeInfections as a result of invasive orthopedic proceduresare a major surgical health risk. These infections occureither at the surgical site (dermis, subjacent muscula-

ture) or at the implant. Surgical site infections aretreated with topical or systemic antibiotics or topicalwound care dressings. Infections at the site of implanta-tion (hip, spine, knee, etc.) are more difficult to treatand, in the event that aggressive antibiotic treatmentfails, require removal of the implant-an expensive andrisky procedure. To counter this problem, manufacturershave developed orthopedic implants with antimicrobialproperties. Silver technology has been a part of thisdevelopment. Antimicrobial devices have been preparedin three ways: (1) direct incorporation of the antimicro-bial agent into the physical structure of the implant; (2)

surface coating; and (3) incorporation of surface coatingof the bone cement employed in the procedure. Themajor disadvantages of the first and third methods areslow rate of release and unwanted modification of thematerial's properties, For surface application, one alsosees uncertain rates of release for the organic gerrni-

cides 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) hascome to play an important role by virtue of the fact thatsuch particles can adhere tightly to the device surfaceand possess antimicrobial properties that potentiallymitigate nosocomial infection.

Silver, like most transition elements, is a reducingagent and, upon electron loss, produces an ion with twopathways-one inorganic and the other organic. The inor-ganic pathway reveals silver ion as an oxidizing agentaffecting either electron transport in the cytochrome oxi-dase system or acting as an anionic trap producing insol-uble salts from a variety of anions in the cy'toplasm. Theorganic path is depicted in Table 1.

Testing MethodsThe microbiologist employs essentially three test sys-tems in the detection of antimicrobial activity duringthe screening phase and uses slightly modified proceduresfor the frnished product prior to marketing. The proce-dures can be a mixture of compendial methods (eg, ASTM,USR NCCLI-see references at the end of this article),FDA guidelines, in-house methods or a combination. All ofthese procedures begin with three basic techniques:

l. Zone of inhibition assay-This is an agar diffusiontest much like that cited in USP <81 > for antibiotic sensi-tivity tests and also cited in USP < 87> for cytotoxic tissueculture screening for in-uitro bioreactivity. The procedureis diagrammed in Figure 2.

62 May/June zoog

Figure 2

Disc

Cylinder

Well

Agar Diffusion Assay: The size of the zone may or maynot be related to the concentration ofthe agent in the car-rier, the physiochemical nature of the carrier and thethickness and chemical concentration ofthe agar.

Typical carriers are dressings, fabric, putty, gel, tis-sue, plastic or metal. If one is testing a wound-care prod-uct for which the indication is to prevent colonization ofpurulent organisms into the dressing (preservativeeffect), an immobilized agent is preferable. If a steadystate of diffusion is desirable so as to achieve an antimi-crobial or antiseptic affect at the wound, one would lookfor a medicated sample with a zone of inhibition. Agardiffusion assays frequently do not agree with MIC orlog-reduction methods.

2. Minimum Inhibitory Concentration Assay (MIC)-The MIC assay is employed during the developmentalphase and can be extremely useful in the clinical phase.One can determine the relative drug resistance of anybattery of clinically relevant organisms. During the clini-cal phase, non-responders can be examined to determineif failure is a function of resistance or delivery. Whetherusing, for example, a silver salt or a nanoparticle, killinghas to occur above the MIC to avoid resistance.Resistance occurs routinely (antibiotics) or rarely (met-als) when the delivered dose is at the MIC or below thistarget value. The MIC values for silver nitrate and Ag+are presented in a later section (Table 6) expressed inparts per million (Irg per mL). Figure 3 shows a diagramof an MIC assay.

Figure 3

| -rl-l-l-r -f-r -f-i--\ u,,,",

r_r r_r r_r u u .i_100 ppm 50 25 12.5 Control

3. The Log-Reduction Test-In this procedure, the testorganism is inoculated directly into or onto the test articlealong with placebo controls. The inoculation may be insaline or simulated bodily fluid of various compositions.This is a quantitative time-course assay first promulgatedin 1970 with USP 18. It is now promulgated worldwide asthe misnamed "Antimicrobial Effectiveness Test," original-ly called the "Preservative Challenge Test." Quantitativerecovery of survivors over time is accomplished by aerobicor anaerobic standard plate count (pour plate or surfacbstreak) or membrane filtration. A loe-kill is converted topercent kill as follows:

The D-value is the time to kill one-loe. It can be calcu-lated by the equation:

Time lnterval or DoseDro = Log,oN umberSurvivorsTime(0) - Log,oNumberSurvivorsTime(t)

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

A modification of the dynamic log-reduction methodhas been developed for testing of antibiotic or silvercontaining PMMA bone cement samples (Alt andBechert "microplate proliferation assay"). The bonecement is surface-contaminated with test organisms,rinsed to release loosely attached cells, followed by atime-course optical density determination as to onset ofproliferation of the adherent organisms. The rate of

1 . 0

2.0

J .U

90o/o

99%

99.9%

Counts are conve(ed to% kill by the expression:% kill = (B-A)/B x 100whe reB= thebeg inn ingcount and A is the end count

May/June 2oog 63

growth relates to the biocidal activity at the bone

cement surface.

Figure 4

6

Ol

i 4i3A2

1

0

Time or Dose Exposure

c=control P=predicate x=test

-P

ResultsTables 2-6 describe some typical results obtained with var-ious device products containing silver employing the testmethods described previously. These are in-uitro tests usedfor guidance in the research process for product develop-ment and regulatory approval either as a novel product oras 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 experi-ments with silver nitrate solution as an example of a pure

active ingredient.Table 2 is informational only and gives some of the sil-

ver compounds that have been employed in medicateddevices over the past 20 years. It also lists the types of

wound care dressings and orthopedic devices that can beassociated with infectious disease risk.

These compounds deployed, for example, in hydrophilic-type wound care pads or orthopedic devices can receive afirst pre-clinical in-uitro evaluation in the laboratory byeither a passiue or dynamic inoculation with a battery ofpathogenic microorganisms. The microbial suspension isprepared with simulated tissue fluid as used in thestandard cell culture or virology lab or rather with normalsterile saline. Except for the USP 'Antimicrobial

Effectiveness Test," we know of no official kill rate for prod-ucts containing an antimicrobial such as those described inthis report. The passiue /esf relies on recovering survivorsafter inoculation directly onto the device. The dynarnic testpresents the medicated device to a suspension ofbacteria inagitation over a period of time with bacterial counts taken atdifferent intervals as in the above model. In both the passive

and dynamic tests, depending if the active agent is diffusibleor is immobilized, log reduction calculations are made overperiods from a few hours to days, depending on the indica-

tion of the product and the clinical directions. A dpramic

test, if conducted with a simulated tissue fluid, can usuallyproduce at least a three log kill ifthe silver ion concentrationin the elution fluid is 50 to 100 ppm (organism dependent).

Example of Shoft-Term Exposure to SilverIt was of interest to determine if silver had a short-expo-sure (10 minute) rate of kill as is common for many chem-ical germicides, such as required by EnvironmentalProtection Agency guidelines. The results of this experi-ment with three pathogenic gram-negative bacilli and the

Product Types AssociatedWith Lowering the Risk

of lnfection

Woven and non'wovenfabrics (3)

Other solid or semisolid carriers

Catheters, venous lines,drains, pins

Metals, nylon, latex, steel,Teflon. titanium. others

Particles of silver 2 to 100 nMare claimed to provide

efficient and rapid penetrationinto the bacterial cell

IllThe combination of ionic silver with CMC insteod of saltalone ollows for o mote effective moisture-,etentive

I2lAllows Zeolite ion exchange for steody state delivery.

I3lNanoparticle deposition to foce masks, environmentolsurfaces, device surfoces, etc, for contamination control orinfection control, Concentrations on a weight/area basis conbe 3O to 65 mg/square inch or centimeter, or other.

64 May/June 2oo8

yeast Cand.id,a albicans,inwhich the organisms were inoculat-ed directly into the silver nitrate solution, are shown in Table 3.

As seen, silver nitrate solution 0.5Vo did not produce a10-minute kill and, therefore, cannot be ranked amongstcommon disinfectants as to rate of kill (for example, alco-hol 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 awound care or orthopedic device. However, variable rates ofkill can be seen at 24 hours when formulated products aretested, as shown in Tables 4 and 5. This is a function of theretention ofsilver ions within the device or inactivation bvprotein or salts in the inoculating suspension.

Examples of Log-Reduction Comparisonof Four Devices Containing SilverThe data in Table 4 show clearly that four of themedicated products would have prevented thegrowth of the pathogens. These tests are generallyconducted with a battery of pathogenic organismssuch as the gram-negative rods Escherichia coli,Pseudomonas aeruginosa, , Proteus mirabi l is ,Klebsie l la pneurnoniae and others, and the gram-pos-it ive cocci Staphylococcus aureus, Staphylococcusepidermidis , Staphylococcus aureus (MRSA),Streptococcus pyogenes and the yeast Candida albi-cans. There are no absolute pass/fail criteria as percurrent FDA regulations. However, comparisons ofsample C with predicate suggest equivalent rapidand sustained kil ls. Sample A was clearly the leastactive showing a delayed kil l and no more than atwo-log decline after seven days. Although Zone-of-Inhibit ion and MIC tests can be useful in productdevelopment, log-reduction tests of the kind depictedin Table 5 afford the best evidence of efficacy.

Examples of Log-Reduction Comparison:Gram-Positive vs. Gram-Negative BacteriaThe data in Table 5 reveal that after 24 hours, gram-neg-ative bacilli were more sensitive to silver than the gram-positive cocci. This frequently is the case with chemicalgermicides seen in EPA/AOAC-type disinfectant tests. Itis the exact opposite of the comparative sensitivitiesseen with antibiotics, where the gram-negative bacteriafrequently are more difficult to kill than the gram-posi-tive variety.

Table 4

Hours or Days After Inoculation

Product 6 24 48 72 7 Days

UnmedicatedControl 0 0 +

Sample A 0 0 z z z

Sample B 1 2 z z

Sample C 2 4 4 >4 >4

PredicateDevice 4 4 >4 >4

Legenct: eocn numDer rndtcates decltne meosured dscfu/sample plote count; + = indicates growth beyond.O = time inoculotion. lnoculations were occomplished withsimuloted wound/tissue fluid carrier supplemented with serumto final concentration of 5o/o to simulate exudates.

24HourLog

ReductionBacillus anthracis (spores) cP

Staphylococcus aureus cpMSSA

GP = Gram Positive IV1SSA = l\,,lethicillin SensitiveGN = Gram Negative MRSA= lvlethicillin Resistant

C = Communitv/Hosoital Associated

May/June 2oos 65

Comparative MIC Data-Woundand Nosocomial PathogensA series of experiments was performed as per the MICmethod, in which laboratory strains and clinical isolateswere tested for sensitivity Lo 0.57o silver nitrate solution,as summarized in Table 6.

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 gtam-negative bacteria, exclud-

ing fungi and viruses. An entire-spectrum agent includes in

Table 6

Legend: + = Growth (turbidity) 0 = No Growth (no turbidity)

1. Staphylococcus aureusstandard test strain MSSA

2. Staphylococcus aureusMRSAATTC strain

3. Staphylococcu s au reu sMRSA (CA)(cl inical isolate)

4. Staphylococcus aureusMRSA (HA) (cl inical isolate)

5. Escherichia coli USP strain

6. Escherichia coll EPA strain

7. Escherichia coli(cl inical isolate)

9. Pseudomonas aeruginosaUSP strain

1 0. Pse udomon as ae ruginosaEPA strain

The MIC results shown in Table 6 demonstrate the

"entire-spectrum" effect of Ag+ and show that the gram-

negative rods were the most sensitive, in agreement withlog-reduction data. This is important because suchpathogens are frequent inhabitants ofwound and orthope-

dic surgical sites. The MIC values in the table help to defrnethe end-use concentration of silver required for clinical

its range yeast, molds and even algae and protozoa. There is

no known entire-spectrum antibiotic of clinical significance.

Some FDA Guidance on ProductsContaining an Antimicrobial AgentThe FDA has promulgated an FDA draft guidance for med-

ical devices that include antimicrobial agents (available at

66 May/June 2oog

www.fda.gov). This document speaks to the issues andmethods that are in the present report. In this document,the FDA distinguished between (a) a product designed tocontrol or prevent infection and to protect the patient (eg,

sulfadiazine ointment), an anti-infectiue ptoduct, which isa drug under section 201(g) ofthe act (CFR 21 (3.2e); and(b) a product such as a wound care treatment or orthopedicor tissue product that makes no chemotherapeutic claimbut contains an antimicrobial so as to prevent colonizationin or about the product (eg, a hydrocolloid wound dressing)a product containing an antimicrobial. For the frrst, theprimary mode of action is for a drug and for the second, theprimary mode of action is that of a device. Appropriatesafety and efficacy standards are applied to each indica-tion. It is helpful to list the expectations of the FDA withregards to microbial testing; some of these requirementsare shown in Table 7.

Table 7

ldentity of the aqent and basic properties

Concentration in the deviceMethod used to deploy the agent to the product

Release Kinetics for the aoentAntimicrobial soectrumMechanism of action

Minimum effective concentration (MEC) (MlC)

Biocompatibil i tyMicrobial test methods: organisms,

inoculation and recovery procedures

IDRH Guidance Draft Document. FDA. 19 Julv 2007

It is hoped that the type of data presented in this reportwith products containing an antimicrobial agent will assistinvestigators and manufacturers in developing unique orcomparative medical products and to be better aware ofappropriate methodology, as required by the FDA.

Summarye It is noted that the silver compounds and particles, inmany respects, have replaced antibiotics and certain chemi-cal germicides in wound and burn care treatment. Theyhave proven useful when deployed into or onto carriers suchas orthopedic devices, catheters, implants, fabrics, plasticsand a variety of hard surfaces and pol;rmers, among others.o There is an established relationship ofthe atomic struc-ture of silver to its antimicrobial activitv as well as the

theoretical basis for its low level of selectivity for micro-bial resistance.o Laboratory methods often required by the FDA for use inthe study of silver and other antimicrobial agents weredescribed, and representative examples of the pre-clinicaldata as shown in the tables can be used for comparisonwith predicate materials and submission to regulatorybodies, as required.. Experimental results obtained with silver nitrate solutionsare presented so as to determine rate ofkill. In addition, datadescribing the activity ofsilver nitrate solutions againsta variety of gram-positive and gram-negative pathogensshow the good relationship between log-reduction andMIC methodologies. A combination of log-reduction andMIC tests can be useful in determining the concentration ofsilver reouired for effectiveness in these indications. *

Dn Herbert N. Prince is the scientific director and founderof Gibraltar Laboratories, Inc. As a national expert on phaf-maceutical microbiology and uirology emphasizing the con-trol of pathogerus in vivo and in vitro, he is a recipient of theSelman Wahsman Award and has published more than50 papers, He has lectured extensiuely on microbiologicalcontrol and held numerotls teaching appointm.ents atFairleigh Dichinson Uniuersity and Montclair StateUniuersity. He is a longstanding member of many microbi-ology organizations including ASM, SIM and TSS.

Dn Daniel Prince is president of Gibraltar Laboratories,Inc. He has serued on numerous USnASTM,A2LA commit-tees and published more than 20 papers. He has worked aselectron microscopist at Osborne Aquarium and later atGibraltar in many capacities including uirologist, endotox-in analyst, molecular biologist, assistant scientific director;and associate scientific director. For other companies, hehas led scientific efforts in HIV-I and human HBV testingand contributed to OSH.Ns Bloodborne Pathogen rule. Herecently led the expansion of Gibraltar Laboratories' build-ing additional state-of-the-art laboratories for the microbi-ology program and creating additional lab space for thechemistry department.

Referencesl.Yamanaka, M. et al2005, BactericidalActivity of Silver ion solu-tions onE Coll,Applied Environmental Mcrobiologz 71 (11) 75-83.2. Chopra, I.2007,T)he Increasing Use of Silver Based Productsas Antimicrobial agents: A Usefirl Development or a Cause forConcern? Journal Antimicrobial Chemotherapy 59:584-587.3. CISI Methods 2006, Performance Standards for Anti-misobialSusceptibility Tbsts, 9th Edition, NCCL Document M2-fu DiscMethods; CI"SI document M7-A7 Dlution Method$ NCCIS 940 WValleyRoad, Suite 1400,Wayrre, PA4. USP 24,2007 Chapter <51> Antimicrobial EffectivenessTest; Chaptet <7072> Disinfectants and Antiseptics.

May/June 2oog 67

5. ASTM-2149 (2001) Standard Test Method for DeterminingAntimicrobial Activity of Immobilized Agents Under Dynarnic

Contact conditions.6. FDA, July 19, 2007, Draft Guidance for Industry and FDAstaff--Pre-Market Notification (510K) Submissions for MedicalDevices That Include Antimicrobial Aagents-US Department

of Health and Human Services, Center for Devices and

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in keventing Infecbions in Health Care, In Bloc[ S, Chapter 19'

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ftir G;.nekologie.9. Gibbins, B.L. 2006, The Orthopedic "Silvef' Lining Orthopedic

Design and Technolog', May/June 2006, 52.10. Gibbins, L. 2005, Antimicrobial Silver Nano-TechnologyTreatment for Medical Devices, Technical Summary Bulletin,Acny Med Inc., Beaverton, OR.11. Mc Donnell, G. and Rr:sse[AD.,Antiseptics and Dsinfectants:Activity Action and Resistance, Clinical Mi<robiolory Reviews, June79pp.,147-r53.12. Noronh4 C. and Almeida A, 2000, Local Bum Tt'eabnent -

Topical Antimicrobial Agents, Burn Unit, St. Joseph Hospital,

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of Microbiology and Society of Industrial Microbiolog5', Cook

College, Rutgers University, New Brunswick, N'J

14. Prince, H.N. 1983, DsinfectantAchivityfuainest Bacteria and

Viruses: A Hospital Guide, Particulate and Microbial Control'

Canon Publications.1,5. Prince, D.L. and Prince, H.N. and Geering, G. 7994;

Methodological Approaches to Topical Virucidal Materials and

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Compounds, Proceedings of the First Workshop on Antiviral

Claims for Topical Antiseptics, U.S. FDA, CDER, U'S'

Government Printing Offi ce.16. Smith, L. 2008 MRSA Antibiotic Patterns, Personal

Communications, St. Michael's Medical Center, Newark, NJ'

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