Originally Published MDDI August 2005
A new silver nanotechnology chemistry can prevent the formation of life-threatening biofilms on medical devices.
Bruce Gibbins and Lenna Warner
For years, investigators of infection and disease have considered bacteria to be simple, independent, free-floating, single-cell living organisms. Based on this view, many of the strategies for controlling infections in the human body have used laboratory models of free-floating microbes. In the past decade, however, scientists and clinicians have found that bacteria in natural environments often live in highly organized communities and even have the ability to participate in a rudimentary form of communication. These communities are called biofilms.1
Scientists are learning that organisms living in biofilms behave very differently from those living in a free-floating or planktonic state. Their abilities to adhere to a privileged site, to build protective structures, and to communicate by chemical signals enable them to use their strength of numbers and coordinated actions to balance the equation against the host organism's natural defenses. Moreover, a biofilm protects the organism from the therapeutic strategies that have been considered foolproof in treating infections.
Biofilm formation is recognized as the leading culprit in many life-threatening infections in patients who are treated using medical devices. Identifying the role of biofilms has been much easier, however, than discovering a way to control them. Most known pathogenic organisms have the potential of using biofilms. Once established, biofilms are difficult to eliminate from the surface of devices.
Many medical device manufacturers need to prevent biofilm formation. One method under investigation incorporates antimicrobial properties into the devices themselves. The theory is that the antimicrobial treatment might discourage the establishment of biofilms. However, manufacturers are faced with identifying which antimicrobial agent will be effective against a wide range of organisms and yet will be tolerated next to healthy tissue.
Manufacturers are increasingly looking toward silver as the answer. Silver is one of the oldest known antimicrobials. An earlier article examined the benefits and limits of several processes for silver antimicrobial technologies and briefly reviewed ionic plasma deposition.2 This article examines biofilms and how they develop on medical devices. It also describes a new silver nanotechnology chemistry that has proven to be effective against them.
Antimicrobial silver is now used extensively to combat organisms in wounds and burns. It works because pathogens cannot mutate to avoid its antimicrobial effect. In the process of developing burn and wound silver technologies, researchers have studied the ability of silver's antimicrobial properties to remain effective in the face of virulent pathogens.
Silver Kills Microorganisms. When mobilized from its reservoir in aqueous fluids, silver provides an antimicrobial action. The positively charged ionic form is highly toxic for microorganisms but has relatively low toxicity for human tissue cells.
Silver works in a number of ways to disrupt critical functions in a microorganism. For example, it has a high affinity for negatively charged side groups on biological molecules. These include groups such as sulfhydryl, carboxyl, phosphate, and other charged groups distributed throughout microbial cells. This binding reaction alters the molecular structure of the macromolecule, rendering it worthless to the cell.
Silver simultaneously attacks multiple sites within the cell to inactivate critical physiological functions such as cell-wall synthesis, membrane transport, nucleic acid (such as RNA and DNA) synthesis and translation, protein folding and function, and electron transport, which is important in generating energy for the cell. Without these functions, the bacterium is either inhibited from growth or, more commonly, the microorganism is killed.
The development of resistance to antimicrobial silver would be extremely rare because an organism would have to undergo simultaneous mutations in every critical function within a single generation to escape the silver's influence. Spontaneous mutation is rare, occurring in only one per 100,000 divisions, so the probability of multiple dependent mutations occurring in the same generation of microbes is extremely unlikely.
Because silver affects so many different functions of the microbial cell, it is nonselective, resulting in antimicrobial activity against a broad spectrum of medically relevant microorganisms including bacteria, fungi, and yeasts. Silver is also more efficient than traditional antibiotics because it is extremely active in small quantities. For certain bacteria, as little as one part per billion of
silver may be effective in preventing cell growth.3
Biofilms in Nature
Good Biofilms. Not all bio-films are harmful. All animals, including humans, have bacteria growing in various sites in the body. Their presence is actually very useful and important to health. The intestine is an example where mostly anaerobic bacteria help in the digestive process by breaking down complex foods and by producing useful compounds such as vitamins. Their presence even helps in developing resistance against food-borne pathogens.
Biofilms create a hostile environment so that the bacteria fail to attach to and invade the tissues. Organisms such as Escherichia, Bacteroides, Enterococcus, Lactobacillus, Clostridium, Streptococcus, and Proteus are just a few that are found in high numbers in feces. These unique biofilm communities actually guarantee their retention within the gut during bowel elimination, thus maintaining the population. Also, biofilms are important as the natural cleansers of water. Waste-water treatment plants are designed to encourage the biofilm formation responsible for the removal of dissolved solids from sewage.4
Bad Biofilms. Not all biofilm formation is desirable, however. For example, dental plaque is one undesired biofilm. Artificial joints made of robust biocompatible materials are commonly used to replace joints surfaces damaged by injury or disease. Pacemakers implanted in soft tissue provide the electrical signal needed to regularize abnormal heart beats. Artificial heart valves and Teflon sleeves for vessel repair are commonplace. Implanted monitors will soon be used to drive the delivery of insulin from indwelling pumps to control blood sugar levels in diabetic patients. In all cases, the nonliving surfaces of such medical devices are typically biocompatible and, therefore, are highly prone to supporting biofilms. This gives harmful organisms an advantage. They can establish a foothold that may cause a life-threatening infection.
Biofilms can form on the surfaces of artificial hearts, stents, urinary catheters and central lines, contact lenses, intrauterine devices, joint implants, and dental implants. Because of the rapidly growing implant market, the concern over controlling biofilms on these surfaces has greatly increased. Infections related to indwelling medical devices are a national public health issue and rank as the fifth-leading cause of hospital patient deaths in the United States.5
Harmful Biofilms at Work. Unfortunately, infection associated with indwelling devices is common. It has been reported that 2–4% of all patients who are placed on an IV or central line in the hospital will develop an infection. One in 20 patients gets an infection while hospitalized. A single hospital infection is estimated to add $38,600 to the cost of medical care and as much as $58,000 for a serious bout with post-operative sepsis.6
A Closer Look at Biofilms
Biofilms are organisms that establish a layer typically attached to a surface. The slime on rocks in a babbling brook is a classic example of a natural biofilm. Biofilms are made up of microorganisms that transition from a free-living planktonic existence to one where they adhere to surfaces. This transition begins by switching on genes that make adhesion possible. Once an
organism adheres to the surface, it continues to multiply to form a colony in which all members have this adherence property.
One interesting note about biofilms is their ability to communicate by chemical signals with other microbes.7 Their chemical signals have special meaning. For example, an attached organism can signal the free-swimming forms to turn on genes that cause them to also adhere to a surface. A different signal may cause all the organisms in the biofilm to begin secreting the sticky carbohydrate that surrounds the organisms with a thick polysaccharide layer. This layer shields the organisms from the protective infection-fighting cells and humoral (fluid-borne) factors of a patient's resistance system that sequester and eliminate pathogens.
In 1979, it was discovered that yet another signal causes all cells in the community to begin production of toxins simultaneously. This coordinated signaling is called quorum sensing. Organisms hiding in the biofilm are essentially invisible to the host's resistance system until sufficient numbers develop to cause migration to deeper tissues. The biofilm is an effective way of staging in preparation for an organism to cause an infection. It enables the organisms to increase to sufficient numbers to overwhelm the host's defense mechanisms by essentially appearing invisible to the immune system during the biofilm formation stage.8
The problem in the clinical environment is that once a biofilm has formed, it is extremely difficult to eliminate. Antibiotics designed to fight the free-living microbes may reduce the clinical signs of infection. However, once antibiotic therapy is withdrawn, the organisms in the biofilm mount a new attack to cause the infection to recur. Recurrence can be expensive to address, and, for many compromised patients, it may lead to either the removal of the implanted devices or, worse, death.
How Mutating Pathogens Defeat Antibiotics
A microorganism goes through radical changes when it transitions from the free-living form to living in a biofilm. It accomplishes this adaptation by activating genes that code for proteins needed for the formation of biofilm. The expression of these genes makes the organism look entirely different, so much so that it could be misperceived as being an entirely different species from its planktonic form. Such extensive change makes it difficult to tailor drug therapies to fight infection. Up to 40% of the proteins in the cell walls of the biofilms differ from the original planktonic version. The constant changes make the biofilms extremely difficult to kill. In recent research, as many as five stages have been identified in the profile of biofilm proteins throughout the various stages of development.9
The picture that emerges of microorganisms is one in which there is a tenacious, heterogeneous form that is capable of orchestrating changes within its population to cope with the environment. The planktonic form enables rapid, widespread dissemination, whereas the biofilm form withstands resistance factors.The mutating nature of the pathogens as well as the high antibiotic resistance of biofilms make dependency on antibiotic treatment ineffective against recurrent infections associated with indwelling medical devices. The rule of thumb is that 1500 times more of an antibiotic agent is needed to kill a biofilm than to kill planktonic bacteria.10
Fighting Biofilms with Antimicrobial Silver
Biofilm formation is extremely difficult to eliminate once it has begun. Preventing biofilms on medical devices and implants is key to controlling their contribution to establishing infection. Because biofilm formation is dependent upon a surface, one strategy is to modify the surface to make it hostile to microorganisms. Ionic silver is becoming a favored substance for surface modification for a number of reasons, including the following:
• It has broad-spectrum antimicrobial action.
• It is well tolerated by tissues.
• It is compatible with most materials used in making medical devices.
• It can be compounded into the submatrix or applied on the surface, and resistance to it is largely nonexistent.
Surface-application chemistries vary, but they are usually designed to deposit either metallic silver or an ionic salt of silver to the medical device surface. Both forms are activated when placed in the presence of moisture. The limitation of ionic salts is that they are only active for a short period of time—often only for a few days. By contrast, metallic silver nano-particles persist in delivering antimicrobial silver for as long as 100–200 days.
Nanosilver particles (as small as 1000th the diameter of a bacterium) constitute the reservoir for the antimicrobial effect. This reservoir effect results when metallic silver, which has no antimicrobial properties, undergoes a chemical change called oxidation, which results in the release of the ionic form. This chemical reaction occurs at the surface of the particle when it is exposed to moisture such as body fluids. Silver metal oxidizes very slowly, however, so it persists on the device to extend its usefulness.
Surface Area. Because silver doesn't readily oxidize, nanoparticles are critical to achieving the reservoir effect. However, the smaller the particle size, the greater the ratio of surface area to volume, and the greater the area available for oxidation. For example, a gram of pure, solid silver in the form of a sphere has a surface area of 10.6 cm2, whereas a gram of silver nanoparticles averaging 10 nm in diameter has a surface area of 0.6 million cm2. This huge increase provides the surface area necessary to allow a continuous release of silver ions.
Silver Nanotechnologies. Several methods are used to enable silver antimicrobial nanotechnologies to adhere to the surface of a medical device. Typically, vacuum-sputter coating and plasma-arc deposition technologies direct vaporized silver at the device surface. These technologies require special equipment.
Ionic Plasma Deposition (IPD). IPD technology is a surface-engineering process that deposits ordered layers of silver oxide molecules. These molecules can be adjusted for silver release rate or for a total release period. Recent test results show that IPD silver antimicrobial nanotechnology is effective against pathogens associated with biofilms including Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, and Asperigus niger.11
Silver Nanoparticles in Solution. Silver nanoparticles in solution is a recently developed technology that originated from silver antimicrobial applications for burns and wound dressings. These dressings are now used worldwide. This new platform for antimicrobial silver has proven to prevent biofilms on medical devices while it eliminates most of the problems of current commercial technologies. The technology uses either an aqueous or a solvent-based process, depending on the needs or characteristics of the medical device to be treated. During the process, the outer layer of each silver nanoparticle oxidizes upon exposure to air or body fluids. This step forms a monolayer of silver oxide (Ag2O) on the outside of each nanoparticle. The silver oxide then dissolves in the body fluid to produce the Ag+ form of antimicrobial silver that fights microorganisms. Because the vast number of nanoparticles on the medical device surface range from 5 to 15 nm in size, a large reservoir and large surface area of antimicrobial silver form on the device. The duration of activity depends on the level of treatment. Elution studies using radioactive silver have predicted that it is possible to achieve antimicrobial treatment levels that last for more than a year.
This approach is a surface treatment rather than a coating. Unlike many surface coatings, the method does not change the dimensions of even minuscule medical devices. This solution-based approach also creates a surface comprising a conformal deposition of tightly adhering particles of silver. This deposition occurs on any surface brought in contact with the fluid vehicle. It is difficult to get silver flow to turn corners. This limitation can be overcome by silver nanoparticles in solution, which uses a fluid delivery vehicle rather than sputter coating or plasma to deliver the silver to the desired surface area. For example, it is possible to treat both the surface and the lumen of a catheter simultaneously.
It's important to note that masking can be used on the areas where treatment is not desired. This is currently beyond the capability of other commercial silver microbial technologies that are applied either by vacuum deposition or by electroplating with this process.
The antimicrobial silver nanoparticles are designed to be deposited onto the surfaces of medical devices after they are manufactured. Most medical device manufacturers prefer surface application because it prevents having to requalify the device's base material resulting from compositional changes caused by adding something to the mix.
|Figure 1. Optical density of various microorganisms treated with antimicrobial silver compared with untreated controls. The silver prevents the formation of biofilms (click to enlarge).|
Depending on the application and duration requirements, studies show that depositing from 100 to 3000 parts per million of silver on medical device surfaces can prevent biofilm formation across a broad spectrum of pathogens (see Figure 1). In one study, biofilm reduction was evaluated by exposing treated and untreated materials to microorganisms under culture conditions that encourage biofilm formation. This was done by placing test materials into a medium containing 0.1% neopeptone with 0.25% glucose and 1% adult bovine serum. The medium was then inoculated with 104–105 organisms from fresh O/N cultures of clinical isolates (E. coli, methicillin-resistant Staph aureus, Pseudomonas aeruginosa, and Candida albicans). The cultures were incubated for 72 hours and then rinsed exhaustively to remove nonbiofilm organisms.
Biofilm was quantitatively assayed by adding the metabolic dye XTT for 4 hours. The color formation was measured spectrophotometrically. The results showed that treated materials had no more conversion of XTT dye than material that had never seen organisms. This finding was interpreted to mean that the silver treatment completely resists the formation of biofilm.
|Figure 2. Silver particles deposited on glass and imaged by electron transmission microscopy. The size distribution of the particles varies between 2 and 20 nm with the average size being 11.6 nm (see arrow). No silver was detected on untreated materials (see insets) (click to enlarge).|
Although silver is slowly released from a treated surface, its main function is likely to be close to the surface, where it kills organisms in close contact. Figure 2 shows silver particles deposited on glass and imaged by electron transmission microscopy. The size distribution of the particles varies between 2 and 20 nm with the average size being 11.6 nm. In separate analyses, treated and untreated support material was analyzed for elemental composition of the nanoparticles using energy-dispersion spectral analysis. Particles on the treated materials were analyzed for silver. No silver was detected on untreated materials (see inserts on Figure 2). The actual amount released is well below the level that would harm healthy human cells.
|Figure 3. Sustained release of silver from a treated matrix. The elution shows three phases of release. Approximately 20% of total silver is lost within the first 72 hours, a further 9% is
eluted over the next 7 days, and then a steady-state release of approximately 0.7% per day occurs thereafter (click to enlarge).
The treatment has been shown to be effective on a variety of materials, including glass, stainless steel, titanium, polyvinyl chloride, polyethylene, polypropylene, silicone, nylon, and even Teflon. The adhesion is such that this silver treatment cannot be removed by scouring or ultrasonic cleaning. Electron microscopy shows that the treatment consists of discrete particles of silver confined to the surface of the substrate material. The efficacy period can be varied by the density of silver applied during treatment. Figure 3 shows the sustained release of silver from a treated matrix. For this analysis, nylon support material was treated with the silver to deposit ~650 mg of 110-m Ag per kilogram to the surface. The material was then serially transferred to fresh eluate (water) daily. At each change, the total remaining silver was determined by counting the residual silver using a gamma counter. The elution showed three phases of release. Approximately 20% of total silver was lost within the first 72 hours. A further 9% was eluted over the next 7 days, and then a steady-state release of approximately 0.7% per day occurred thereafter. This finding suggests that biological activity may last for more than 150 days on this application.
Its flexibility enables the process to be customized depending on the requirements of the device. For example, a device that resides in tissues for only a few days would need a lower load of microbial silver than a permanently implanted device. The silver can be prepared relatively easily without special equipment and has been shown to produce effective sustained-release activity.
Identifying the role of biofilms has been much easier than finding ways to control them. Once established, biofilms are particularly difficult to eliminate from the surfaces of medical devices. Biofilm formation is responsible for many life-threatening infections in patients who are treated using medical devices, and so many medical device manufacturers need to take steps to prevent biofilm formation. Devices such as artificial joints, pacemakers, artificial heart valves, and implanted monitors are all typically biocompatible and, therefore, are highly prone to supporting biofilms.
One method under investigation incorporates antimicrobial properties into the devices themselves. The theory is that the antimicrobial treatment might discourage the establishment of biofilms. However, manufacturers are faced with identifying which antimicrobial agent will be effective against such a wide range of organisms and yet will be tolerated next to healthy tissue.
Silver is one of the oldest known antimicrobials, and manufacturers are increasingly looking to silver as the solution. Defeating biofilm formation is the only way to outsmart the microbes. Broad-spectrum silver offers an efficient way to achieve this objective. In the future, providing only the duration of protection needed will allow manufacturers to better conform their products to a particular medical application.
The authors would like to thank Balu Karandikar, PhD, senior research scientist at AcryMed (Portland, OR), for his expertise and advice.
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2. D Tobler and L Warner, “Nanotech Silver Fights Microbes in Medical Devices,” Medical Device & Diagnostic Industry 27, no. 5 (2005): 164–169.
3. B Gibbins, “The Antimicrobial Benefits of Silver and the Relevance of Microlattice Technology,” Ostomy Wound Management 49, no. 6 (2003): 5–6.
4. J Deacon et al., “The Microbial World: Biofilms,” [online] (Edinburgh, Scotland: Institute of Cell and Molecular Biology, 2003) [cited July 2005]; available from Internet: http://helios.bto.ed.ac.uk/bto/microbes/biofilm.htm.
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7. K Sauer, “Biofilm Phenotype and Signaling Mechanisms in Pseudomonas aeruginosa” (paper presented at the American Society of Microbiology General Meeting, Orlando, FL, May 23, 2001).
8. E Peter Greenberg, “Bacterial Communication and Group Behavior,” Journal of Clinical Investigation 112, no. 9 (2003): 1288–1290.
9. D Davis et al., “Studying Slime,” Environmental Health Perspectives 106, no. 12 (1998): 106–112.
10. W Costerton, “Innovations: Studying Slime” [online] (Bethesda, MD: National Institutes of Health, 2005); available from Internet: http://ehp.niehs.nih.gov/docs/1998/106-12/innovations.html.
11. JM Ryan III, “Silver Antimicrobial Nanotech: An Alternative to Antibiotic Use” [online] (Longmont, CO: Ionic Fusion Corp., 2005, cited April 15, 2005); available from Internet: www.ionicfusion.com/silveroxidestudy.doc.
Bruce Gibbins is the founder of AcryMed Inc. (Portland, OR). Lenna Warner is vice president and a principal at Mamalu Partners Inc. (Palm Beach, FL).
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