Patent Application
20230067150
The present invention relates to a coating that induces an electrochemical/galvanic reaction on coated surfaces when exposed to an electrolyte composed of bodily fluids, resulting in an antimicrobial effect.
Catheters are devices that controllably direct flow of bodily fluids from various passages inside the body to outside the body. A urinary catheter is intended to be deployed into the urinary tract of a patient with urinary incontinence. A Foley's urinary catheter has two separate lumens and ports: one set to flush urine from the bladder, and one set to inflate the balloon at the distal end of the catheter. The balloon is designed to inflate in the bladder and prevent the catheter from inadvertently slipping out from the patient. Aside from biocompatibility, the most important constraints are a smooth surface finish and proper flexibility. In other words, mechanical trauma and shear forces at the biomaterial-tissue interface should be kept to a minimum.
Implants are often associated with infection as they provide an excellent surface for bacterial colonization. During acute infection, bacteria rapidly proliferate and spread as unicellular organisms, however, in chronic infections they predominantly colonize body surfaces and tissues as multicellular aggregates, termed biofilms. In the latter case, planktonic, or free flowing/moving bacteria, adhere to a surface of interest and begin to colonize that surface. Control of infection typically begins with removal of the implant or foreign body followed by a cocktail of antibiotics and anticoagulants. In many cases it is an advantage for bacteria to adhere to surfaces. For example, attachment to horizontal surfaces stimulates growth of bacteria as organic material suspended in liquid settles. Another example would be that of a surface made of nutrient rich material. The disadvantages to adhering to surfaces, is immotility caused by the change in gene expression, which would inhibit the ability to search for nutrients and lead to death by starvation.
The Centers for Disease Control and Prevention, and the Department of Health and Human Resources report that urinary tract infections (UTIs) are the most common type of healthcare-associated infection reported to National Healthcare Safety Network. In fact, UTIs acquired in hospitals are 75% associated with urinary catheters. Between 15-25% of hospitalized patients receive urinary catheters during a regular hospital stay. It is estimated that hospitals spend over $1 billion (US) in managing catheter-associated urinary tract infections (“CAUTI”), which is primarily caused by prolonged catheterization. For a patient, the daily risk of developing a urinary tract infection increases by 3-7% each day that a catheter is present. For decades, the medical community has sought to develop improved antifouling and biocidal materials capable of being incorporated onto the surfaces of medical devices. The ability to maintain the sterility of a surface against the resistive nature of microorganisms would prevent thousands of patients from contracting life-threatening infections.
Bacteria have many strategies to invade the human body. Bacterial adhesion is highly dependent on the physical properties of the media, the surface, and the bacteria membrane. In bacteria/surface interactions, extracellular organelles operate in such a way as to create physical attachments of the cell body to a surface. Cells attach preferentially to hydrophilic materials when the surface energy of the bacterium is larger than the surface energy of the liquid in which they are suspended. Typically, the surface energy of bacteria is smaller than the surface energy of the liquid in which they suspend; a mismatch can allow cells to attach preferentially to hydrophobic materials.
Surface attachment facilitates antibiotic resistance by reducing the net negative surface charge of bacterial cells and enhancing the stability of the membrane. Most bacteria have a net negative surface charge and interact preferentially with positively charged surfaces. The effect can disappear in media with high concentrations of ions due to charge screening. This phenomenon is physiochemical, where water is lost at the interfacial layer as changes to the structure of surface molecules occur during the time the cell body repositions to maximize attachment to that surface.
To complicate matters, bacteria can also attach to surfaces that initially resist the attachment of cells. They do so by depositing a layer of proteins, environmental and innate, that condition the surface and nullify functional groups that would normally reduce adhesion. The dense packing of cells in bacterial “quorums” facilitates an increase in the concentration of secreted small-molecules, forming stronger gradients which transfer information between cells and trigger physiological changes. Quorum sensing and charge screening are abilities of bacteria that allow for modulation of the membrane charge, to better adapt to a surface during initial stages of biofilm formation.
Upon aggregating at a surface, adhered bacteria initiate expression of new phenotypes, the result of which changes them into biofilm. Biofilms are aggregates of bacteria that communicate and maintain proliferation via the secretion of an extracellular polymeric substance (EPS) in which they encompass themselves. This matrix-like network allows a bacterial colony to be able to respond to environmental stress that would normally inhibit proliferation. Stresses include but are not limited to external attack, physical conditions, and nutrient limitation. For example, EPS secretion provides protection from mechanical damage and shear stress caused by fluid flow. The EPS assists bacterial colonies in many ways that include improved adhesion, communication, and nutrient supply.
Numerous studies reveal that rather than focusing solely on planktonic bacteria, it is important to also focus on the prevention of the biofilm; slow growth of the biofilms can confer resistance. These studies have led to the definition of two main approaches to antimicrobial agents: antifouling and biocidal.
Antifouling materials are organized into two categories: hydrophilic materials and polyzwitterions. The former group inhibits fouling by forming a barrier of hydration on the surface that induces a type of steric repulsion. The latter group employs electrostatic and low surface energy. Both antifouling categories represent coatings that do not kill bacteria, but thermodynamically prevent attachment of bacteria and/or protein to the surfaces. On the other hand, biocidal materials are designed to kill microbes instead of minimizing their deposition. These materials are seen as more important because they protect the patients from infection and encrustation development. Clinically tested catheters use silver, a well-known biocide, or antibiotic coatings. While the coatings are most commonly used, other biocidal materials are currently being researched.
An alternative approach to surface coatings involves the use of electrical fields and currents. In 2009, Del Pozo et al. conducted a study with the aim to determine the effect of prolonged exposure to low intensity direct electrical current (DC) on Pseudomonas aeruginosa, S. aureus, and S. epidermidis biofilms. These infectious species are well known to be the causes of most UTIs. The investigators tested biofilm-covered TEFLON® coupons submerged in a slightly ionic media exposed to DC electrical current. The study's conclusion was that higher amperage currents correlated with increased inhibition of biofilm at every time point during the study. These results also demonstrated that electrical current can substantially reduce the viability of biofilm on a TEFLON® surface. The mechanism, referred to as the “electricidal effect”, resulted from one or a combination of disruption of the integrity of the bacterial membrane and generation of chlorine, oxygen, and/or hydrogen peroxide as a result of electrolysis.
Catheter-associated urinary tract infection is still prevalent in hospitals around the world. Bacteria are the ultimate survivors, able to groom surfaces to their liking and pass on genes that enable resistance to harsh environments. The need exists for materials that can be used to passively mitigate the transmission of infection. The design and analysis of materials that are able to synergistically work to inhibit the growth and proliferation of bacteria on medical devices is the key to infection control.
According to embodiments of the invention, a surface film or coating applied to the surfaces of urinary catheters inhibits the growth and proliferation of biofilm on the surfaces of catheters and the urinary tract by inducing an electrochemical/galvanic reaction when exposed to an electrolyte composed of bodily fluids. These reactions produce small scale electric fields and potentials capable of generating an ambipolar diffusion of ions, resulting in passive micro-current and formation of hydrogen peroxide. Testing confirms that these reactions are harmful to microorganisms, particularly bacteria and the biofilms they can form. In some embodiments, the reaction reagents, i.e., active materials, are heavy metals, metal alloys, and/or metal oxides that occur naturally in the human body or are non-toxic to humans in low doses. Examples include magnesium, calcium, manganese, zinc, silver, and others. In in vitro studies, the films are effective in inhibiting growth of planktonic E. coli in synthetic urine. After exposure of the films to inoculated urine for six days, the biofilm was found to be reduced in concentration by five orders of magnitude relative to a control (p<0.05)—a clinically significant improvement.
The inventive composite films induce reactions between the solution and the active materials in the films. These reactions are similar to those that occur within a battery. Stored electrochemical potential is released during the reaction with the urine, producing low level electric fields and potentials capable of inducing an ambipolar diffusion of ions, resulting in passively generated micro-current and formation of hydrogen peroxide. The precise mechanism is not known. Without intending to be bound by a particular theory, it is believed that the film generates an entourage effect that involves different antimicrobial mechanisms at the interface between the solution and the surface, making it difficult for microorganisms to survive. These mechanisms may include one or more of micro-currents, electric fields, metal toxicity, changes in pH, and generation of hydrogen peroxide.
In an exemplary implementation, the interaction with zinc and silver in solution creates an interfacial environment that is potentially too chaotic for bacteria to thrive. At the surface of the urinary catheter, bacteria encounter changes in pH, micro-currents generated from the ions caught in electric fields, toxic metal compounds, and peroxides. The selected active materials are thermodynamically capable of coupling with an ensemble of atomic and molecular ions that exist in urine. These ions can react with active materials to produce other chemical species or potentially produce a passivation layer on the surface of the active materials that inactivates them. A key goal of the inventive approach is to effectively combine these active materials with commercial urinary catheter materials to produce a new material with surfaces capable of perpetuating the electrochemical reactions upon exposure to bodily fluids. Urologists have suggested that it would be a major accomplishment to design a catheter with surfaces that are able to maintain sterility for 30 days. This is in contrast to the observation that most catheter patients develop a bacterial infection over that period.
In one aspect of the invention, an antimicrobial coating is formed from a biocompatible flexible polymer having incorporated therein an active material having a reducible form of silver, wherein at least a portion of the active material is exposed at the surface of the polymer. In some embodiments, the flexible polymer is polydimethylsiloxane (PDMS). The active material may include silver oxide or silver chloride, and may further include one or more of zinc and silver. The active material may be incorporated into the polymer by forming a paste from a monomer, a cross-linker and a powder of the active material and thermally curing the paste. In some embodiments, the paste is filled into a mold configured to form a lumen.
In another aspect of the invention, a method for inhibiting growth and proliferation of biofilm on a medical device includes applying an antimicrobial coating formed from a biocompatible flexible polymer having incorporated therein an active material having a reducible form of silver to surfaces of the medical device, wherein the coating is configured to induce an electrochemical/galvanic reaction on the device surfaces when exposed to an electrolyte comprising bodily fluids.
In another aspect of the invention, a catheter lumen includes a molded biocompatible flexible polymer having incorporated therein an active material comprising a reducible form of silver, wherein at least a portion of the active material is exposed at the surface of the polymer, wherein the active material is configured to induce an electrochemical/galvanic reaction when exposed to an electrolyte comprising bodily fluids. In some embodiments, the flexible polymer is polydimethylsiloxane (PDMS). The active material may include silver oxide or silver chloride, and may further include one or more of zinc and silver. The active material may be incorporated into the polymer by forming a paste from a monomer, a cross-linker and a powder of the active material and thermally curing the paste. In some embodiments, the paste is filled into a mold configured to form the lumen.
The description of the materials and procedures used for preparation and testing, employs a number of acronyms and abbreviations that are generally well known among those in the art. To avoid ambiguity, Table 1 below provides a list of acronyms/symbols and their meanings as used herein.
The embodiments disclosed herein relate to a surface film or coating applied to the surfaces of a urinary catheter for the purpose of inhibiting the growth and proliferation of biofilm on the surfaces of catheters and the urinary tract. The inventive coating induces an electrochemical/galvanic reaction when exposed to an electrolyte composed of bodily fluids. The unique interaction of zinc and silver in solution creates an interfacial environment that is potentially too chaotic for bacteria to manage. At the surface of the urinary catheter, bacteria are exposed to changes in pH, micro-currents generated from the ions caught in electric fields, toxic metal compounds, and peroxides. The selected active materials are thermodynamically capable of coupling with an ensemble of atomic and molecular ions that exist in urine. These ions can react with active materials, referred to herein as “AMs”, to produce other chemical species or potentially produce a passivation layer on the surface of the active materials that inactivates them. The inventive approach is directed to an effective combination of these active materials (AMs), with commercial urinary catheter materials to produce a new material with surfaces capable of perpetuating the electrochemical reactions when exposed to bodily fluids. Urologists have suggested that it would be a major accomplishment to design a catheter with surfaces that maintain sterility for up to 30 days based on the observation that nearly every catheter recipient tends to develop a bacterial infection by that time.
Catheters are typically formed from polymers based on their desirable physical and chemical properties as well as ease of manufacture. Unlike vulcanized natural rubber latex, polydimethylsiloxane (PDMS) is inert, non-cytotoxic, and flexible. PDMS is one of the most widely used silicon-based organic polymers due to its versatility, with mechanical properties that can be varied depending on how much cross-linker is used for the polymerization reaction. For this reason, PDMS was selected as the preferred substrate to combine with our active materials. As will be readily apparent to those in the art, other polymers known to be appropriate for medical applications, particularly for implants, may be substituted. Polymer additives, as are known in the art, may include fillers, reinforcements, release agents, internal lubricants, catalysts, impact and toughness modifiers, thermal and radiation stabilizers, plasticizers, pigments, coupling agents, and antistats. A commercial-off-the-shelf silver coated Foley catheter was used as a benchmark for the study.
Different methods to fabricate thin-film and flexible zinc-silver batteries were evaluated. Methods that can be used to form a composite of the active materials with PDMS include chemical adhesion, chemical-free adhesion, and in situ synthesis. Covalent bonding of silicone elastomers on metallic substrates is difficult due to an incompatible surface chemistry. Adhesion can be promoted by the use of chemical coupling agents such as: organotitanates, amide/imides, zircoaluminates, organosilanes, etc. Such approaches involve the use of toxic reagents and may not be optimal. Nandkumar et al. described use of a chemical solvent and dissolved silver to impregnate latex Foley catheters. The described method used only silver as an active ingredient, used a different flexible substrate (Latex), and still involved the use of strong reagents. Thus, the chemical-free adhesion and in situ synthesis method would generally be a more attractive method for making the zinc-silver PDMS composites. Anubha Goyal et al. prepared a homogenous mixture of metal salt, silicone elastomer and the curing agent to produce metal nanoparticle embedded PDMS films. During the curing process, the hardener simultaneously crosslinks the elastomer and reduces the metal salt to form nanoparticles in situ. Although the authors reported reduced bacteria concentrations in solution following exposure to the material, it appears that silver ions that were not reduced and have not agglomerated into nanoparticles are able to escape the surface of the polymer matrix and therefore are unable to sustain the desired electrochemical behavior. To expand on the phenomena, the bacteria are most likely being affected by residual silver ions, not by nanoparticle interaction. A suspension of particles, flakes or fibers in a thermoset polymer is considered to be a composite, especially when the mechanical properties are improved.
It is a common practice to fully embed fiber in resin when making a composite for mechanical loads. However, to achieve the results required of the inventive coating, the active material must be exposed at the surface of the polymer matrix rather than being fully embedded in it. This led to an approach that employed mechanical adhesion. In this case, a powder or metal thin film can be partially encased in PDMS and thermally bonded, leaving partial surfaces of the metal exposed. Authors Domin Koh et al. used a lithography-based method where a thin film of metal was evaporated onto chromium coated glass, etched into an electrode, and then coated in PDMS; the cured PDMS film could be separated from the chromium with the metal electrode imbedded at the surface. This process, described below with reference to
Referring to
The method used to make the final prototype biomaterial for testing combined a couple of the methods described above. PDMS monomer, cross-linker and a powdered form of the active materials were combined and thermally cured. To ensure that sufficient amounts of the active materials were exposed at the surface, a percolation threshold was employed. At some ratio, the mass of the powdered metals will be high enough to conduct through the PDMS. If this conduction can be measured at the surface, it is probable that the active materials are available at the surface. This approach can be taken with zinc and silver, however, because silver oxide is not good conductor, the proper ratio was found iteratively through AMR testing, using one or more of known test methods for detecting antimicrobial resistance. A few examples of commonly-used antimicrobial sensitivity testing methods include dilution (broth and agar dilution), disk-diffusion, E-test, mechanism-specific tests such as beta-lactamase detection test and chromogenic cephalosporin test, and genotypic methods such as PCR and DNA hybridization methods.
An important design constraint for urinary catheter coatings is wettability or the ability of a liquid to contact a solid surface due to the intermolecular forces between them. Depending on the ratio of AM:PDMS, changes in surface wettability can also be expected. The wettability of the catheter both influences the biofilm growth on the surface and the comfort of a patient using the catheter. Studies have suggested that interactions between bacterial cell walls and other surfaces are affected by electrostatic interactions and Van der Waals forces. Bacteria tend to have a surface energy smaller than the liquids in which they are suspended and, therefore, attach preferentially to hydrophobic surfaces. Studies regarding the adhesion of different strains of E. coli have also found that they are highly attracted to hydrophobic surfaces, while being slightly repelled by hydrophilic ones. Furthermore, catheters coated with hydrophilic materials also reduce trauma to the urethral surfaces and enable easy and comfortable catheterization for patients, as compared to more hydrophobic conventional catheters. As such, measuring the wettability of the inventive catheter coatings is both important for the prevention of biofilm formation and improvement of patient comfort. One method used to measure wettability is Sessile drop goniometry. The goal is to measure wettability of a surface by measuring the angle that a droplet of liquid forms with a given surface. A low contact angle would indicate a high wettability and a hydrophilic surface, whereas a high contact angle indicates a low wettability and a hydrophobic surface.
The following examples describe the materials and methods used in preparing embodiments of the inventive coating, forming a catheter with the coating and the testing procedures and results for evaluating the coating and devices.
Preparing a complete Foley catheter adds technical challenges. A mold must be prepared to form the main body of the catheter including two lumens/ports for flushing urine and inflating the balloon. One-way valves must be added to the proximal balloon port using a medical grade adhesive. Finally, a balloon must be adhered to the distal end of the catheter. The ratio of active materials to polymer could lead to the material becoming so costly that would not be economical to manufacture, and could degrade its mechanical toughness. To address these issues, a high ratio paste can be made and applied as a thin coating to the surface of a mold over which pure PDMS can be poured/injected. This approach, which is illustrated diagrammatically in
The sequence illustrated in
Standard commercially-available silicone catheters without antimicrobial properties were cut to have an outside surface area of 168 mm2. The catheters were cleaned using an ultrasonic bath to remove any debris from the surface and secured to a rod set up to be slowly rotated about its axis. A uniform coating of the selected AM:PDMS paste was applied to the outer surface as the rod was rotated. The catheter was heated using a heat gun for about 1 minute while to rod continued to rotate. The rod with the catheter was then placed in an oven at 125° C. for 10 minutes to complete the curing process. The result was a small piece of silicone catheter with a uniform coating thickness of approximately 500 μm.
The foregoing examples describe two possible approaches for catheter preparation. The described options are provided as examples only and are not intended to be limiting. The key is to form a biocompatible flexible polymer coating that has an active material incorporated in a way that at least a portion of the active material is exposed at the polymer surface. Accordingly, variations or combinations of the described examples as well as other methods for forming and applying the inventive antimicrobial coating, will be readily apparent to those of skill in the art.
Synthetic urine was prepared by combining reagents listed in Table 3, and filtering through a 0.2 μm filter.
In order to explore the quantity of hydrogen peroxide produced by the samples containing the active materials being studied, replicates (n=3) of Zn:PDMS, Ag:PDMS, Ag2O:PDMS, Ag/Ag2O:PDMS, Zn/Ag2O:PDMS, and Zn:PDMS+Ag2O:PDMS striped pattern specimens were tested over a 13 day period. Samples were sterilized with 100% ethanol and allowed to dry in the bottom of glass containers overnight. While working under a flame, 1.5 mL of synthetic urine was injected into the containers and onto the surface of the sample using a 1 mL syringe. 20 μL of synthetic urine were removed from each sample container and the extracted solution was placed directly on the tile of either Quantofix 25 or Quantofix 100 peroxide strips (Quantofix; Macherey-Nagel, Dueren,
Germany) to reveal a color change used to quantify hydrogen peroxide concentration via comparison to a color scale. Quantofix 25 concentration scale measures: 0, 0.5, 2, 5, 10, and 25 mg/L hydrogen peroxide. Quantofix 100 concentration scale measures: 0, 1, 3, 10, 30, and 100 mg/L hydrogen peroxide. The pH of the urine was measured with litmus paper before being separated into the glass containers, and again for each specimen at the conclusion of the 13 day test.
A standard electrochemical setup with a high impedance electrometer was used to determine the potential difference between silver (Alpha Aesar 99.99%) and zinc (Alpha Aesar 99.9%) foils in air-saturated aqueous solution at room temperature (21° C.) under atmospheric conditions. Synthetic urine was used as the electrolyte for one experiment and DI water for the other. Potential differences were measured using a Solartron 1286 Electrochemical Interface and a two-electrode cell configuration with a 10 mm separation between electrode surfaces. Open circuit potential measurements were obtained at 1 second intervals over a 6 hour period starting immediately after placing the fluid media in the cell.
To determine the wettability of the samples, Sessile drop, contact angle goniometry was performed. The samples were placed under a Keyence VHX 1000 video microscope, positioned at 90 degrees. A 2 μL drop of pure DI water was pipetted onto the separate edges of each sample. The angle is measured using the line and angle measurement tools. Testing was performed on pure PDMS as well as samples made with PDMS mixed with Ag2O, Ag, Ag/Ag2O, and Zn/Ag2O.
48 hour (n=2) and 144 hour (n=5) antimicrobial resistance studies were performed to compare the short-term and long-term effectiveness of selected antimicrobial samples. The samples for the 48 hour short-term study included: PDMS, Ag:PDMS, Zn:PDMS, Ag/Ag2O:PDMS, Zn/Ag2O:PDMS, Zn:PDMS+Ag2O:PDMS striped pattern, and sections of a commercially available “silver-coated” catheter. The samples for the 144 hour long term study included: PDMS, Ag:PDMS, Zn:PDMS, Ag/Ag2O:PDMS, Zn/Ag2O:PDMS, Ag2O:PDMS, and a bioelectric dressing (BED) (Procellera®, Vomaris Innovations, Inc., Tempe, Ariz.), and sections of a commercially available “silver-coated” catheter. A polyester woven cloth served as the control for the BED, and pure PDMS was used as control for the AM:PDMS samples. There was no control for the “silver-coated” catheter. Positive controls for absorbance measurements were test tubes containing only urine and the inoculum. Negative controls for absorbance measurements were test tubes containing only urine without inoculum.
All samples were cut into squares with 168 mm2 (12 mm×14 mm) surface area. All samples, apart from the BED and the BED control, were washed with 100% ethanol and placed at the bottom of a test tube to dry overnight. Procellera® and its control were sterilized under UV light for 8 hours on each side, and placed into individual test tubes. The sample-containing test tubes were filled with 3 mL of synthetic urine and inoculated with E. coli (BW 25113) to a known optical density (OD600 =0.01).
The tubes were placed in an incubator at 37° C. without shaking to facilitate growth. The absorbance of each sample was measured using a spectrophotometer (BIORAD SmartSpec™ 3000), at regular intervals. Samples for the 144-hour study were placed into new test tubes containing 3 mL fresh synthetic urine every 48 hours and re-inoculated with bacteria to OD600=0.01 in order to simulate the environmentthat would be present in a urethra.
Biofilm formation was monitored at the end of the short term and long term tests. Sample were placed into a separate sterile glass test tube containing 3 mL distilled water to stunt further growth. The test tubes were then placed in an ultrasonic bath (Branson Ultrasonics CPX Series M 1800) at maximum frequency for three, thirty second intervals, ten seconds apart to shed biofilm from the samples into solution for quantification. A serial dilution was performed until colonies are quantifiable on LB agar plates incubated at 37° C. for 24 hours.
Zone of inhibition studies are a common practice for researchers and pathology labs for testing resistance to antibiotics. The AM:PDMS thin film composites were cut into 8 mm diameter discs using a biopsy tissue punching device. Bacteria were cultured for 24 hours in synthetic urine and coated onto LB 1.5% agar plates. Disks were placed in the center of agar, and plates are incubated overnight at 37° C. Radii of zones of inhibition are measured from center of disk to the edge of bacterial lawn.
Data are plotted using means and standard deviations. The Welch's t-test was used to test the hypothesis that the AM:PDMS coatings are more effective at inhibiting biofilm than just PDMS (placebo) using a confidence interval of 95%.
The results shown in
The mean and standard deviation for contact angles of water droplets on each composite and a control are shown as bars in
Compared to the pure PDMS surface, all composite samples were more hydrophilic (n=5, p<0.05). Based on the standard deviations, it is difficult to say which composite was more or less wettable, only that they were less than the control. This hydrophilicity is welcomed as it has been shown to resist bacteria adhesion. However, because all samples form water drop contact angles that were greater than 90 degrees, they were all still considered to be slightly hydrophobic or have low wettability.
The growth of planktonic bacteria in solution can be tracked using spectrophotometry. The absorbance of light (600 nm) emitted by the machine was measured when passing through a cuvette containing an aliquot of the media containing the inoculum. The optical density (OD600) increases as more light is absorbed by colloid in solution. The sigmoidal curves plotted in
The biofilm shed from samples via ultrasonic bath can be quantified by counting the number of colony forming units (CFUs) that grow from a known volume of solution onto LB agar plates. The number of colonies that form on the plates were counted and used to calculate a concentration using units, CFU/mL. A greater number of colony forming units per milliliter corresponds to a higher number of bacteria growing on the surfaces of the sample. It should be noted that the vertical axis scale for CFU/mL is logarithmic. As shown in
The long term study was initially designed to last as long as necessary to observe clear distinctions. Following a 48-hour interval, bacteria in the synthetic urine reach a lag cycle. At this point in time, most of the nutrients available to the remaining bacteria in solution are gone and the bacteria begin to be affected by increasing concentrations of their own metabolic byproducts, which can force gene mutations and cell death. For this reason, the synthetic urine was refreshed at 48-hour intervals during the six-day study. This also better mimics in vivo conditions in the bladder as urine is constantly being produced and flushed out of the bladder. Physiologically, the refreshed urine in a urinary tract is expected to still have small concentrations of bacteria present at the time the patient is catheterized. As mentioned previously, the mechanisms by which bacteria are inhibited are complex and can affect growth in solution and on surfaces independently. The study was terminated following 144 hours, when the absorbance of well performing samples approached the magnitude of the controls.
The growth curves following each 48-hour cycle in
Biofilm was quantified from all samples following repeated exposure to fresh urine and E. coli over six days. The results are shown in
Zone of inhibition results in
Samples containing silver (I) oxide were able to inhibit E. coli up to 9 mm away from the edge of the disk. This suggests that these particular films have an improved delivery of a diffusive antimicrobial mechanism.
Comparing the test results, it appears that samples containing reducible forms of silver perform the best in inhibiting planktonic E. coli. Samples made with zinc seemed to lessen the strength on the surface and in solution. This suggests that the e-fields and microcurrents formed between zinc and silver are not significant enough to inhibit biofilm. This also suggests that at the current concentration, zinc could be a nutrient for the E. coli. While this may be the case for E. coli, many other infectious bacteria and fungi are more sensitive to zinc. It is possible that the amount of zinc used to make the Zn/Ag2O:PDMS samples may have been too high. The goal was to initially load enough zinc to make the coating surface conductive. This proportion of zinc and PDMS was used, including when mixing with silver. To promote a better electrochemical reaction, the amounts of zinc and silver could be varied or patterned in way that improves the electricidal performance. To conclude, these findings may suggest these samples cannot be described as electricidal.
As for the effect of hydrogen peroxide on bacteria, it is apparent that E. coli are indifferent to the concentration of hydrogen peroxide being generated by the samples. The highest concentration of hydrogen peroxide measured was 100 mg/mL or 2.94 millimolar using only zinc. Not only was zinc ineffective at inhibiting the growth of E. coli, so too were high concentrations of hydrogen peroxide. This led to a conclusion that even non-virulent strains of bacteria are capable of protecting themselves from hydrogen peroxide and peroxide anions.
The samples containing silver performed well enough to consider using silver as the main active material for a biocidal catheter. More important than elemental silver is a reducible form of silver, such as silver oxide or silver chloride. These have the ability to participate in reactions with oxygen, hydrogen, hydroxide, and other ions in solution to free silver ions into solution. As is known, silver ions are extremely toxic to bacteria. The elemental silver used to make the composites may have had a thin layer of oxide, similar to the zinc, that initially released silver ions in solution. However, it appears that AMs containing silver oxide are preferred to permit reduction reactions to increase in number or to perpetuate for longer durations of time.
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This application claims the benefit of the priority of U.S. Provisional Application No. 62/968,096, filed Jan. 30, 2020, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/015919 | 1/29/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62968098 | Jan 2020 | US |
