Patent Application
20130183435
The present invention relates to applications of low temperature plasma coatings for implantable medical and other devices with enhanced inhibition against biofilm formation, more specifically, to applications of organo-silicon low temperature plasma coatings on implantable medical devices to prevent biofilm formation and reduce the risk of nosocomial infections.
Bacteria can develop biofilm on a submerged surface. One common place for biofilm formation is on or within indwelling medical devices such as catheters, respirators, and artificial cardiovascular implants, prosthetic joints, and contact lenses, which pose a critical problem for medical care. Approximately 1 million nosocomial infections are associated with indwelling devices annually in the United States, which incurs enormous healthcare cost.
Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections, and promotes local progressive tissue destruction. In particular, Staphylococcus epidermidis (S. epidermidis) infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which, in combination with the increasing prevalence of antibiotic resistance among human pathogens, is further complicating treatment of biofilm-related device infections. Therefore, it would be beneficial to coat the surfaces of a device with anti-biofilm plasma coating that inhibits the formation of biofilm on such devices.
Bacteria in biofilm on medical devices behave differently from planktonic bacteria, demonstrating high level of resistance to antibiotic and host immunity. The inherent resistance of biofilms to antibiotics and host immune responses makes eradication of biofilm from medical device difficult. Biofilm infections are treated in clinic by removal or replacement of the infected medical devices, combined with systemic antibiotic therapy. However, biofilm's resistance to antibiotics makes antibiotic treatment less effective. In addition, antibiotic therapy could also exacerbate the antibiotic resistance problems among human pathogens. As a result, much effort has been made to prevent biofilm formation on medical devices.
The development of biofilm could be roughly divided into two steps. Bacterial cells attach and colonize the implanted medical devices in the initial attachment step, following by a maturation step in which bacterial cells would proliferate and accumulate on the device surfaces. Once the biofilm was established, it would form a persistent source for bacterial dissemination and infections. Multiple surface-modified methods have been developed to prevent or decrease biofilm formation on medical devices. The most commonly used materials for surface coating are antibiotics. However, long term usage of antibiotics risks selecting for resistance among pathogens. Aminoglycoside antibiotics can even induce biofilm formation at subinhibitory concentrations. Other materials, which also demonstrated anti-biofilm function when being coated on biomaterial surfaces, include heavy metal ions, furanone, hydrophilic polyethylene glycol (PEG) derivatives and Zwitterionic polymer.
S. epidermidis is one of the most prevalent pathogens involved in biofilm formation on medical devices. S. epidermidis is part of the normal human epithelial bacterial flora, but can cause infection when skin or mucous membrane is injured. S. epidermidis could develop into biofilm and become a persistent source for device associated infections. Antibiotic resistance is wide spread in S. epidermidis and further limits the treatment options.
Another common place for biofilm's formation is the submerging surfaces of water pipelines and reservoirs/containers in a water plant or a residential water system. Multiple bacteria can attach to pipe wall and form biofilm which protect them from disinfection. Pathogens that can form biofilm in water distribution system include Campylobacter jejuni, pathogenic Escherichia coli, Yersinia enterocolitica, Legionella spp., Aeromonas spp., Mycobacterium spp. and Pseudomonas aeruginosa. Disinfectants are typically used to combat biofilm formation. Biofilm formation in water distribution system could cause taste and odor problems and corrosion of pipe materials. Pathogens could be shed into the water and cause infectious diseases.
Low temperature plasma coating deposition is a thin film forming process in a vacuum chamber, where thin films deposit on the surface of substrates under plasma conditions. In a plasma deposition process, monomers are introduced into a plasma reactor and get activated to produce a gaseous complex composed of highly energetic electrons, ions, free radicals and excited monomer molecules, known as the plasma state. In recent years, plasma process has been widely used in the preparation of biomedical materials with unique performance and in the manufacturing of medical devices.
Therefore, there is a need to provide a new and improved device with a modified plasma coating on its contacting/submerging surfaces (which contact with fluids containing bacterial cells), whereas the modified plasma coating having the beneficial properties of inhibiting bacterial attachment and preventing biofilm formation. There is also a need to provide a new and improved implantable medical device with modified plasma coating on its contacting surfaces with the beneficial properties of inhibiting bacterial attachment and preventing biofilm formation and a new and improved methods and process for coating such a device.
In one embodiment of the present inveniton, a process for surface treatment of a fluid-contacting device where a plasma coating is applied to at least one contacting surface of the device. The plasma coating inhibits bacterial attachment to the device and prevents biofilm formation on said device. The plasma coating is generally made up of at least one organo-silicon monomer. In one aspect of the invention the monomer from the silane group and is selected from the following monomers: dimethylsilane, trimethylsilane, vinyltrichlorosilane, tetraethoxysilane, vinyltriethoxysilane, hexamethyldisilazane, tetramethylsilane, vinyldimethylethoxysilane, vinyltrimethoxysilane, tetravinylsilane, vinyltriacetoxysilane, methyltrimethoxysilane, or combinations thereof.
In the process of the present invention the coating is applied to the device at a low temperature by a plasma deposition technique to form a continuous layer on one or more surfaces of the devices. The coating preferably has a thickness from about 1 nm to about 100 nm, more preferably from about 20 nm to about 30 nm.
In another emobidment of the present invention, the coating also includes a gas where the gas is selected from oxygen, O3, or CO2. The coating can comprise trimethylsilane and oxygen gas mixed in an approximate ratio of 1 to 4.
In one aspect of the invention, a series of new and improved fluid-contacting devices having at least one contacting/submerging surface with modified plasma coating, whereas said coating has the beneficial properties of inhibiting bacterial attachment and preventing biofilm formation, is described. The inventive fluid-contacting devices can be implantable medical devices, such as catheters, respirators, and artificial cardiovascular implants, prosthetic joints, and contact lenses, whereas one or multiple surfaces of a particular device can contact bacteria containing body fluid of a subject (human or animal). The inventive fluid-contacting devices can also be devices employed in a water system, such as water pipes and water reservoirs/containers, or machineries, where one or multiple surfaces of a particular device can be submerged in the bacteria containing fluid (such as water).
According to one embodiment of the invention, the modified plasma coating with properties of inhibiting bacterial attachment and preventing biofilm formation comprises at least one continuous layer of organo-silicon monomers at a preferable thickness between 1 to 100 nm, with 20 to 30 nm as more preferred. Various organo-silicon monomers can be employed in the invention, including but not limited to a trimethylsilane (TMS) monomer.
According to another embodiment of the invention, the modified plasma coating with properties of inhibiting bacterial attachment and preventing biofilm formation can also comprise at least one continuous layer or organo-silicon monomers and oxygen, or other gases, such as O3 or CO2. The modified plasma coating comprises TMS and oxygen, where the ratio of TMS to oxygen is about 1 to 4.
In another aspect of the invention, a new and improved method for reducing and preventing biofilm formation on the contacting/submerging surfaces of a fluid-contacting device is described. The inventive method for reducing and preventing biofilm formation on contacting/submerging surfaces of a fluid-contacting device comprises the step of depositing at least one layer of bacterial-inhibiting plasma coating on surfaces of the device via a low temperature plasma deposition technique, in which the process environment is nearly room temperature.
According to one embodiment of the invention, the inventive method comprises the step of depositing at least one layer of organo-silicon monomer coating on surfaces of said device via low temperature plasma deposition technique, whereas the thickness of said organo-silicon monomer coating preferably ranges from about 1 to 100 nm and more preferably from about 20 to 30 nm, where the organo-silicon monomer is TMS.
According to another embodiment of the invention, the inventive method comprises the step of depositing at least one layer of organo-silicon and oxygen, O3 or CO2 plasma coating on surfaces of said device via low temperature plasma deposition technique, where the coating comprises TMS and oxygen with the ratio of about 1 to 4, respectively.
In the accompanying drawings that form a part of the specification and that are to be read in conjunction therewith:
The invention provides a series of new and improved fluid-contacting devices having at least one contacting/submerging surface with modified plasma coating, whereas said coating has the beneficial properties of inhibiting bacterial attachment and preventing biofilm formation. The inventive devices can be implantable medical devices, such as catheters, respirators, and artificial cardiovascular implants, prosthetic joints, and contact lenses, whereas one or multiple surfaces of a particular device can contact bacteria containing body fluid of a subject (human or animal). Additionally, the inventive devices can also be devices employed in a water system, such as water pipes and water reservoirs/containers, or machineries, whereas one or multiple surfaces of a particular device can be submerged in bacteria containing fluid (such as water). Regardless the applications of the devices, the contacting/submerging surfaces, suitable for modified plasma coating, can be metallic, such as, titanium (Ti), stainless steel (SS), or other metal alloys, and polymeric materials.
Specifically, trimethylsilane (TMS) is used as a monomer to coat the surface of 316L stainless steel (SS) and Grade 5 titanium alloy (Ti), which are widely used in implantable medical devices. As described further herein, the results of biofilm assays demonstrated that this TMS coating markedly decreased the formation of S. epidermidis as wells as other biofilms. In addition, bacterial cells on the TMS coated surfaces are more susceptible to antibiotic treatment than their counterparts in biofilms formed on uncoated surfaces. Therefore, TMS coating results in a surface that is resistant to biofilm development and also more sensitive to antibiotic therapy. Specifically, trimethylsilane (TMS) is used as a monomer to coat the surface of 316L stainless steel (SS) and Grade 5 titanium alloy (Ti), which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased the formation of Staphylococcus epidermidis (S. epidermidis) as wells as other biofilms. In addition, bacterial cells on the TMS coated surfaces are more susceptible to antibiotic treatment than their counterparts in biofilms formed on uncoated surfaces. Therefore, TMS coating results in a surface that is resistant to biofilm development and also more sensitive to antibiotic therapy.
According to one embodiment of the invention, the aforesaid modified plasma coating can comprise a continuous layer of organo-silicon monomers with thickness ranging between about 1 to 100 nm. Various organo-silicon monomers can be employed, including but not limited to the following: dimethylsilane, trimethylsilane (TMS), vinyltrichlorosilane, tetraethoxysilane, vinyltriethoxysilane, hexamethyldisilazane, tetramethylsilane, vinyldimethylethoxysilane, vinyltrimethoxysilane, tetravinylsilane, vinyltriacetoxysilane, and methyltrimethoxysilane. According to another embodiment of the invention, the modified plasma coating with properties of inhibiting bacterial attachment and preventing biofilm formation can also comprise at least one continuous layer or organo-silicon monomers and oxygen, O3 or CO2. According to an exemplary embodiment, the modified plasma coating comprises TMS and oxygen, whereas the ratio of TMS to oxygen is about 1 to 4.
The surface morphology and chemistry of the TMS-coated device of the present invention (1) preferably has a thickness of the coating layer ranging up to 100 nm and more preferably the coating adhesion is between 20 to 30 nm; (2) does not have a substantial change in surface roughness or morphology caused by the coating; and (3) has substantial surface chemistry change generated by the coating.
The bacterial adherence/attachment and colonization on the TMS-coated and TMS/O2-coated surfaces of the devices treated with the method of the present invention demonstrates that the organo-silicon plasma or organo-silicon/oxygen plasma coating markedly decreases biofilm formation by inhibiting the bacterial attachment to the coated contacting/submerging surface. Biofilm formation has been compared between the uncoated metallic wafer and the TMS-coated or TMS/O2-coated metallic wafer, in environments mimicking the devices' host environments (contacting with bodily fluids or tissues, or submerging in bacterial-containing fluids, such as tape water), via several methods, such as crystal violet staining, scanning electronic microscopy (SEM), and confocal laser scanning microscopy (CLSM).
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The foregoing is one example of a technique for depositing at least one layer of bacterial-inhibiting plasma coating on surfaces a device via low temperature plasma deposition technique. This layer provides for a reduction and prevention in biofilm formation on the contacting/submerging surfaces of the fluid-contacting device.
According to one embodiment of the invention, the inventive method comprises the step of depositing at least one layer of organo-silicon monomer coating on surfaces of a device via low temperature plasma deposition technique, where the thickness of the organo-silicon monomer coating ranges preferably from about 1 to 100 nm, and more preferably from about 20 to 30 nm. The organo-silicon monomer can be TMS and the coating can comprise TMS and oxygen in a ratio of about 1 to 4, respectively.
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The invention further demonstrates that the organo-silicon plasma coating markedly improves the sensitivities of the coated contacting/submerging surface to antibiotic therapies. The susceptibility of bacterial cells on both TMS coated and uncoated wafers to antibiotic treatment has tested with both vacomycin and ciprofloxacin, and it is found that the bacterial cells on the TMS coated surfaces are significantly more sensitive to antibiotic treatments than those on the uncoated surfaces.
The result suggests that bacteria on the TMS-coated surface have yet to adopt the distinct biofilm phenotype that made them highly resistant to antibiotic treatment. While bacteria in biofilms exhibited great tolerance to ciprofloxacin with MBCs of more than 128 μg/ml, bacteria on the surfaces of TMS coated materials demonstrated significantly increased susceptibility.
In addition, another explanation for the increased susceptibility of bacteria on TMS-coated surfaces is attributable to its structure. While S. epidermidis develops a multilayer biofilm structure, which prevents penetration of antibiotics, or render antibiotics inactivate on uncoated surfaces, S. epidermidis biofilm on TMS-coated surfaces comprises mostly scattered cells with occasional cell clusters with both dead and living cells, which are easily accessible to antibiotics. This result is in agreement with previous study that S. epidermidis cells in monolayer biofilm had a comparatively low tolerance to antibiotics than cells in multilayer biofilm. Reduced slime production also contributed to increased susceptibility of biofilm to antibiotics since substance of exopolysaccharide biofilm matrix has been known to hamper penetrance of antibiotics into biofilm.
In summary, the organo-silicon or organo-silicon/O2 coated devices, specifically TMS coated devices, are able to inhibit or reduce greatly the bacterial attachment to the surface, and the same time, make the residual bacterial on the surface susceptible to antibiotic treatments. The beneficial properties of organo-silicon coated devices can be employed in various clinical applications.
SS and Ti substrates. 316L SS and high-strength Ti (Grade 5, also known as Ti-6Al-4V because of the addition of aluminum and vanadium alloying elements) coupons with approximate dimensions 10 mm×10 mm×1 mm were cleaned with a 3% (v/v) Detergent 8 (Alconox, Inc., White Plains, N.Y., USA) solution for 3 hours at 50° C. in an ultrasonic bath. During the cleaning time, samples were removed from the solution every 30 min, rinsed with distilled water, and placed into fresh detergent solution. After cleaning, the metallic coupons were rinsed with acetone and blotted dry with Kimwipes paper.
TMS plasma coating on SS and Ti wafers. The coupons were then attached to an aluminum panel of surface area 15.3 cm×7.6 cm using silver epoxy. Additionally, a silicon wafer was attached to the aluminum panel for coating thickness assessment. The panel was then placed inside an 80 l bell jar-type reactor. This panel was situated between two SS or Ti plates. In this configuration, the central aluminum panel served as the cathode, whereas the two outer SS or Ti panels served as the electrically grounded anodes. This arrangement is typical of a substrate-as-electrode type arrangement in which the sample to be modified serves as the working electrode (cathode). In this scheme, the electrodes were connected to the output of a MDX-1K magnetron drive (Advanced Energy Industries, Inc., Fort Collins, Colo.), which served as a DC power source. The entire reactor setup is indicated in
Oxygen plasma was used to remove organic contaminants on the SS or Ti surface. The reactor was sealed and evacuated to base pressure (<2 mTorr) using a series mechanical pump and booster pump. Pure oxygen (Praxair Inc., Danbury, Conn.) was then introduced to the reactor at a flow rate of 1 sccm (standard cubic centimeters per minute) using an MKS mass flow controller (MKS Instruments Inc., Andover, Maas.) and an MKS 247C readout to set the flow rate. Pressure was allowed to stabilize at 50 mTorr using an MKS pressure controller. The oxygen was then excited with the DC power supply at 20 W in order to form the plasma. The treatment time was 2 min. Following surface cleaning, the reactor was evacuated to base pressure and TMS (Gelest Inc., Morrisville, Pa.) was introduced to the reactor at 1 sccm. The reactor pressure was allowed to reach 50 mTorr, and the TMS was excited by the DC power supply at 5 W for 15 s.
Surface characterization. Surface characterization was performed using a non-contact optical profilometer, X-ray Photoelectron Spectroscopy (XPS), and contact angle to evaluate the surface topography, chemical composition, and surface wettability respectively.
Surface chemistry analysis. In order to better understand how plasma coating affect the surface chemistry of SS and Ti, all the plasma coated wafers and uncoated controls were analyzed using XPS (X-ray Photoelectron Spectroscopy) at the Material Research Center, Missouri University of Science & Technology, Rolla, Mo. The XPS analysis of a surface provides qualitative and quantitative information on all the elements present (except hydrogen and helium) from the binding energies of the main lines and the peak area, respectively. A Kratos AXIS 165 X-ray Photoelectron Spectrometer (Kratos Analytical Inc., Chestnut Ridge, N.Y.) equipped with a monochromatic A1 Kα X-ray (1486.6 eV) source operating at 150 W was used to characterize the elemental composition and chemical bonding states of the elements present at the substrate surfaces. The take-off angle of the X-ray source was fixed at 90° to the substrate surface for an area of 200 μm×200 μm to be analyzed. Survey spectra, from a 0 to 1200 eV binding energy, were recorded at 160 eV pass energy, a dwell time of 500 ms, and one scan. Whereas the high resolution spectra were taken at 20 eV pass energy, 0.1 eV/step, a dwell time of 500 ms, and a total of twelve scans averaged. The relative atomic concentration of elements detected by XPS was quantified on the basis of the peak area in the survey spectra with sensitivity factors for the Kratos instrument used. High-resolution spectra were charge-compensated by setting the binding energy of the Cls peak to 284.5 eV. Peaks (Cis) were fitted (Gaussian/Lorentzian curves) after background subtraction (Shirley type with CASA XPS (Casa Software Ltd) Version 2.3.15, taking in consideration Scofield sensitivity factors, so as to determine the peak components or chemical states and their elemental concentrations.
The elemental composition of wafers with and without TMS plasma coating is listed in Tables 1 and 2. Those data were calculated from survey scans of substrate surfaces. The major elements on the SS surface were C, O, Fe, Cr, and Si, a trace amount of N, Mo, Ni, and F, which were expected considering the chemical composition of SS (Table 1). The presence of C was mainly due to the contaminant organic species adsorbed onto the metal surface. Oxygen can be attributed to the protective oxide layer that always forms on SS surface. With TMS plasma coating deposited on SS surface, more C and Si were detected.
The data in Table 2 indicate that on the surface of Ti control without plasma coating, besides the Ti and Al from the bulk material, a large percent of carbon was present primarily due to organic contaminants on the surface. Oxygen was believed to come from the protective oxide layer that forms on the metal surface quite often. The presence of Zn in a trace amount is due to the contamination happened during the sample handling process. With TMS coating, it is expected to see more carbon and silicon at the surface because of monomer TMS contains three carbon atoms and one Si atom. The oxygen on the TMS coating could be attributed to the oxidation of the coating material after its exposure to the atmosphere, which has been reported to be observed on the surface of many other plasma deposited coatings.
Surface contact angle. The static water contact angle was determined at room temperature using deionized water. The contact angle formed between a sessile drop and its supporting surface is directly related to the forces at the liquid/solid interface, indicating the hydrophilic or hydrophobic characteristics of the surface. The water droplet size used in the contact measurements was 1 μl, and the measurements were performed and recorded using a computer-aided VCA-2500XE Video Contact Angle System (AST Products Inc., Billerica, Mass.).
Contact angle measurements of uncoated controls and wafers coated with TMS plasma coatings indicated that the TMS coating turned the surface of both 316L SS and Ti (Grade 5) into hydrophobic, reflected by the contact angle increase from about 70° to around 100° (Table 3).
Surface profilometry. Surface morphology was measured using a Wyko NT9100 Optical Profilometer (Veeco Instruments, Inc., New York) and Vision (version 4.10) software. This optical profiler performs non-contact, 3-D surface measurements using a vertical scanning interferometry (VSI) mode for a wide variety of topologies. The samples were mounted in horizontal position for measurements. The measurements were made over an approximately 125 μm×94 μm area. Each sample was scanned in 5 locations. The scan resolution is 500 nm laterally and 0.5 nm vertically. Before calculating the topography parameters, raw data were processed with a tilt correction. From the corrected and smoothed data the surface roughness parameters Ra (arithmetic average of the absolute values of vertical deviations from a mean plane), Rq (the root mean square roughness) were derived. The Ra and Rq will be used to characterize the surface roughness before and after TMS plasma coating. They are expressed in units of height. It is understood that surface roughness of biomaterials will affect cell attachment and proliferation.
The optical images of the surface topography of SS (316L) and Ti (Ti6Al4V) substrates before and after TMS plasma coating were presented in
SS and Ti wafers that had went through cleaning procedure were then mounted sample holder made from aluminum strip and stainless steel rod using small clips. Additionally, a silicon wafer was mounted to the sample holder for coating thickness assessment. The sample holder was then placed inside an 80 l bell jar-type reactor. This holder was situated between two titanium plates. In this configuration, the wafers and the sample holder served as the cathode, whereas the two outer titanium panels served as the electrically grounded anodes. This arrangement is typical of a substrate-as-electrode type arrangement in which the sample to be modified serves as the working electrode (cathode). In this scheme, the electrodes were connected to the output of a MDX-1K magnetron drive (Advanced Energy Industries, Inc., Fort Collins, Colo., USA), which served as a DC power source. The entire reactor setup is indicated in
Oxygen plasma was used to remove organic contaminants on the stainless steel surface. The reactor was sealed and evacuated to base pressure (<2 mTorr) using a series mechanical pump and booster pump. Pure oxygen (Praxair Inc., Danbury, Conn., USA) was then introduced to the reactor at a flow rate of 1 sccm (standard cubic centimeters per minute) using an MKS mass flow controller (MKS Instruments Inc., Andover, Mass., USA) and an MKS 247C readout to set the flow rate. Pressure was allowed to stabilize at 50 mTorr using an MKS pressure controller. The oxygen was then excited with the DC power supply at 20 W in order to form the plasma. The treatment time was 2 mM. Following surface cleaning, the reactor was evacuated to base pressure and TMS (Gelest Inc., Morrisville, Pa., USA) and oxygen were simultaneously introduced to the reactor at a mass flow ratio of 1:4 (TMS flow rate 1 sccm, and oxygen flow rate 4 sccm). The reactor pressure was allowed to reach 50 mTorr, and the TMS plus oxygen were excited by the DC power supply at 5 W to form coating on the substrates for 90 s.
The S. epidermidis strain ATCC35984/RP62A was isolated from a patient with device-associated sepsis. This strain was demonstrated to be a high biofilm producer. RP62A was kindly provided by Network on Antimicrobial Resistance in Staphylococcus aureus program (NARSA), which is supported under NIAID, NIH Contract No. HHSN272200700055C.
Biofilm Formation Assay.
Biofilm formation was first measured by crystal violet (CV) staining. Four groups of 1×1 cm wafers (SS uncoated, SS TMS coated, Ti uncoated and Ti TMS coated) were used in the experiments. Wafers were sterilized with ultraviolet lamps at a wavelength of 253.7 nm for 20 minutes on each side and then coated with 20% (v/v) human plasma in 50 mM carbonate buffer (pH 9.5) overnight. The content of one capsule for Carbonate-Bicarbonate Buffer (Sigma-Aldrich, St. Louis, Mo.) was dissolved in 80 ml distilled and deionized water and mixed with 20 ml human plasma to generate 20% human plasma in 50 mM carbonate buffer (pH9.5). The human plasma was purchased from Innovative Research (Innovative Research, Inc, Novi, MI), with sodium citrate as anticoagulant.
After human plasma adsorption, wafers were placed into wells of 24-well flat-bottomed sterile microtiter plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland). An overnight culture of S. epidermidis was diluted at 1:200 in Todd-Hewitt broth containing 0.2% yeast extract (THY) medium with 0.5% glucose. Aliquots (1 ml) of the diluted bacterial suspensions were inoculated into the wells containing wafers pre-coated with human plasma and incubated for 48 hours at 37° C., with medium changed every 12 hours. Triplicate wafers of each group (SS uncoated SS, SS TMS coated SS, Ti uncoated and Ti TMS coated) were used.
The wafers were washed four times with Phosphate-Buffered Saline (PBS) to remove non-adherent bacterial cells. Biofilms on wafers were dried at 37° C. for 1 hour, and stained at room temperature with 2.3% (w/v) crystal violet (CV) (Sigma-Aldrich, St Louis, Mo.) for 30 minutes. The wafers were rinsed four times with PBS to remove excess stain. Biofilm formation was quantified by solubilization of the CV stain in 100% ethanol The concentration of CV was determined by measuring OD595 nm with a microplate reader (Molecular Devices, Sunnyvale, Calif.). The experiment was performed three times to obtain the means and standard errors of means.
Biofilms were formed on the four groups of wafers in triplicate as described above with or without human plasma pretreatment. The wafers were washed four times with PBS to remove non-adherent bacterial cells. The wafers were put into tubes with PBS. The biofilms on wafers were detached and disaggregated with ultrasonic bath treatment. The wafers were sonicated 6 times in ultrasonic bath (120V, 50/60 Hz) (Fisher-Scientific, Pittsburgh, Pa.) for 30 seconds and vortexed on the highest setting for 30 seconds after each sonication. The number of bacterial cells in PBS was quantified using the spread plate technique. The experiment was performed three times to obtain the means and standard errors of means.
Biofilm was formed on SS and Ti wafers as described above and slime was measured as reported before. Biofilms were formed on four groups of wafers (SS uncoated, SS TMS coated, Ti uncoated and Ti TMS coated) as described above and slime was measured. To exclude the possibility that TMS coating could affect the binding of toluidine on metal wafers, wafers were also incubated with culture medium without S. epidermidis inoculum as blank control. Triplicate wafers of each group were used in each experiment. Biofilm samples on the wafers were fixed by Carnoy's solution (glacial acetic acid, chloroform and absolute alcohol (1:3:6, v/v)) for 30 minutes, and stained by 0.1% toluidine solution (Sigma-Aldrich, St Louis, Mo.) for 30 minutes. The wafers were subsequently incubated in 0.2 M NaOH solution, heated in a water bath at 85° C. for 1 hour and OD590 nm was measured. The mean of OD590 nm value of blank control wafers in each group was used as blank to calibrate the value of slime formation in each group. The experiment was performed three times to obtain the means and standard errors of means.
Biofilm attachment assay. The four groups of wafers in triplicate were sterilized and coated with human plasma as described above. Overnight cultures of S. epidermidis were diluted in fresh THY with 0.5% glucose to an OD600 nm value of 0.02 and grown at 37° C. to an OD600 nm value of 0.5. Aliquots (1 ml) were then pipetted into sterile wells containing wafers and incubated at 37° C. for 1 hour, 2 hours and 4 hours, respectively. Culture supernatants were gently removed with a pipette. The wafers were washed four times with PBS and then put into tubes with PBS. The wafers were sonicated twice in ultrasonic bath (120V, 50/60 Hz) (Fisher-Scientific, Pittsburgh, Pa.) for 30 seconds and vortexed twice on the highest setting for 30 seconds, respectively. The number of bacterial cells in PBS was quantified using the spread plate technique. The experiment was performed three times to obtain the means and standard errors of means.
Fluorescent staining of adherent bacteria. SS and Ti wafers (1×1 cm) were sterilized, coated with human plasma, and placed into the wells of 24-well microplates. Overnight culture of S. epidermidis was diluted 1:200 into fresh THY medium with 0.5% glucose. Aliquots (1 ml) of the cell suspensions were seeded into each well containing wafers and incubated at 37° C. to form biofilm as described above. The wafers were gently washed three times with PBS to remove non-adherent bacterial cells. Adherent bacterial cells were stained using the LIVE/DEAD BacLight viability kit (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions, followed by three PBS washes to remove nonspecific stain. Fluorescence-adherent bacteria were visualized by confocal laser scanning microscope Zeiss LSM 510 (Carl Zeiss Microlmaging GmbH, Jena, Germany). Images were acquired from random locations within the biofilm formed on SS and Ti wafers. 3D structural reconstruction of confocal laser scanning microscope (CLSM) image stacks was performed using Imaris 4.0 (Bitplane AG, Zurich,. Switzerland).
Scanning electron microscopy. SS and Ti wafers (1×1 cm) were sterilized by irradiating with UV for 15 minutes, coated with human plasma, and placed into the wells of 24-well microplates. Overnight culture of S. epidermidis was diluted 1:200 into fresh THY medium with 0.5% glucose. Aliquots (1 ml) of the cell suspensions were seeded into each well containing wafers and incubated at 37° C. to form biofilm as described above. The wafers were gently washed three times with PBS to remove non-adherent bacteria and fixed with 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) for 2 h at 4° C. The surfaces were washed twice with PBS for 1 hour and subsequently fixed with 0.1% osmium tetraoxide for 1 hour. The bacteria were then dehydrated by replacing the buffer with increasing concentrations of ethanol (20%, 50%, 70%, 90%, 95%, 100%, 100% and 100%) for 15 minutes each. After critical-point drying and coating by gold sputter, samples were examined using a scanning electron microscope.
Susceptibility of bacterial cells in biofilm to antibiotics was studied with a protocol established previously with modification. SS and Ti wafers (1×1 cm) were sterilized, coated with human plasma, and placed into the wells of 24-well microplates. Overnight culture of S. epidermidis was diluted 1:200 into fresh THY medium with 0.5% glucose. Aliquots (1 ml) of the cell suspensions were seeded into each well containing wafers and incubated at 37° C. for 16 hours. The wafers were gently washed three times with PBS to remove non-adherent bacterial cells. Then fresh Mueller Hinton broth (MHB) with and without 6 μg/ml vancomycin or ciprofloxacin was added into the wells containing wafers and cultured at 37° C. for 8 hours. The wafers were washed four times with PBS and then put into tubes with PBS. The biofilms on wafers were detached and disaggregated with ultrasonic bath treatment. The number of bacterial cells was quantified using the spread plate technique. Response to antibiotic treatment of a treated sample was defined as the percentage of untreated samples calculated by CFU numbers. Triplicate of wafers were used in each experiments and four independent experiments were performed for vacomycin and three independent experiments were performed for ciprofloxacin.
Antibiotic susceptibility assay of S. epidermidis cells attached on wafers with different dose of antibiotics. Biofilm formation on the four groups of wafers in triplicate was performed as described in biofilm formation assay section. The wafers with biofilms were gently washed four times with PBS to remove non-adherent bacterial cells. Then fresh Mueller Hinton broth (MHB) with 0, 2, 8, 32, and 128 μg/ml ciprofloxacin (Sigma-Aldrich, St Louis, Mo.) was added into the wells containing wafers and cultured at 37° C. for 48 hours, with MHB medium with corresponding concentration of ciprofloxacin changed every 12 hours. The wafers were washed four times with PBS and bacteria numbers on the wafers were counted.
Mouse infection model. Overnight cultures of S. epidermidis RP62A were diluted 1:100 into fresh THY medium and grown at 37° C. to OD600 nm 0.5. Fifty ml culture aliquots were sedimented by centrifugation, washed in PBS, and suspend in 2 ml Pluronic® F-127 solution. Balb/C female mice were anesthetized with ketamine/xylazine. Their flanks were shaved, and the skin was cleansed with ethanol. A 0.5 cm-1.0 cm incision was made. The uncoated or TMS-coated SS wafers were implanted into the subcutaneous tunnel and placed at a distance of about 2 cm from the incision. One hundred μl suspensions of S. epidermidis (about 2-3×108 CFU) were inoculated onto the wafers. The incision was closed with surgical sutures, with the skin disinfected. Animals were euthanized at day 7 after infections. Implanted SS wafers were harvested, rinsed with sterile PBS three times. The numbers of bacterial cells on the wafers were counted.
Biofilm formation assay on silicone wafer. 1×1 cm silicone wafers (uncoated, TMS coated) were used in the experiments. Biofilm formation assay were performed following the sample protocol for SS and Ti wafers measured by crystal violet staining.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent application, patents, and other references mentioned herein are incorporated by reference in their entirety. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While the invention has been described in connection with specific embodiments thereof, it will be understood that the inventive device is capable of further modifications. This patent application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth.
The application for patent claims priority to U.S. Provisional Application Ser. No. 61/631,892, filed Jan. 13, 2012 and is entitled “Low Temperature Plasma Coating for Anti-Biofilm Formation,” which is hereby incorporated by reference herein to the extent permitted by law.
This invention was made with Government support under Grant No. P01HL57346 and Grant No. 1R44HL097485-01A2 awarded by the National Institute of Health (NIH). The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61631892 | Jan 2012 | US |
