Final Report Summary - NOVO (Novel approaches for prevention and degeneration of pathogenic bacteria biofilms formed on medical devices e.g. catheters)
Executive Summary:
Executive summary
The resistance of microbial biofilms to the immune system and to antibiotics has is a common cause of medical infections, and of difficult-to-treat infections caused by colonized foreign bodies. More than 60% of bacterial infections currently treated in hospitals are caused by bacterial biofilms while catheter-associated biofilm urinary tract infections (UTI) alone account for around 40% of all hospital-acquired infections. Within the NOVO project three strategies for biofilm prevention on urinary catheters were developed, namely, inorganic nanoparticles (NPs) coatings, coatings with phenolic molecules and grafting with antimicrobial enzymes.
Within the first strategy an ultrasound based process for grafting antibacterial ZnO and MgF2 nanoparticles onto catheters was successfully developed. The coating was characterised with various techniques including ICP, EDS and electron microscopy indicating an extend of ~ 0.05% of the catheter weight. The coating survived the standard sterilization process (by γ-irradiation and by ethylene oxide) while in turn the mechanical properties of the catheters remained unchanged during coating. The ZnO coated catheters showed a reduction of more than 50% in the biofilm mass using a lab model of the bladder and in artificial urine solution while the remaining 50% biofilm was composed of mostly dead cells. No leaching of NPs from the surface was observed over seven days while tests based on several cell lines (epithelial, fibroblast) indicated no cytotoxicity.
The second strategy involved coating of catheters with poly(catechin) and poly(catechin)-antibiotic conjugates using laccase for polymerization and grafting. The most significant reduction in bacterial adhesion was observed with poly(catechin)-trimethoprim (gram-negative: - 85% and gram-positive: - 87%) and with poly(catechin)-trimethoprim-sulfamethoxazole (gram-negative: - 85% and gram-positive: - 91%). Likewise for this coating the cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90% of viable cells.
The third strategy involved grafting of antimicrobial enzymes. Cellobiose dehydrogenase releasing hydrogen peroxide was assessed against clinical isolates commonly found in catheter associated infections and proved to be very effective both in the prevention of biofilms and in the killing of bacteria embedded already formed biofilms. Grafting of the enzyme onto catheter surfaces was achieved both chemically and by using the developed ultrasound based technique. The bound enzyme was able to produce hydrogen peroxide in concentrations sufficient to inhibit biofilm formation on the coated surface by up to 70% when tested against Staphylococcus aureus. Again, this coating did not show any signs of cytotoxicity.
Complementary functionalizations with tailored protective inert polymers of two different architectures and two different grafting/coating methods (adsorption/entrapment and layer-by-layer) had been established and also successfully been used in combination with NP coating and enzyme immobilization.
For ethical reasons, only one of the developed strategies, namely nanoparticles, proven to prevent biofilm formation without being cytotoxic was evaluated in an in vivo model. The model developed was based on the New Zealand white rabbit well-tolerated to catheterize the animals without need for restraint allowing them to move freely. Catheters were recovered after 7 days and demonstrated significant reductions in both colonisation and urinary tract infection.
In summary, the Novo project thus demonstrated the great potential of the developed novel approaches for biofilm prevention on urinary catheters. The next steps towards a product would be larger scale production of functionalised catheters allowing clinical trials with concomitant detailed economic evaluation. In parallel, alternative applications as outlined below for the developed antimicrobial functionalization strategies will be evaluated.
Project Context and Objectives:
Requirements and Specifications
Biofilms are bacterial communities encased in a self-produced hydrated polymeric matrix. More than 50% of bacterial infections are caused by bacterial biofilms. Catheter-associated urinary tract infections (UTI) alone account for approximately 40% of all hospital acquired infections. The aim of NOVO had been to provide a coating for catheters which can prolong the in vivo use by effectively reducing the biofilm formation. The strategy was to use different approaches alone and in combination and consequently in the first phase the requirements and specifications were defined for:
- sonochemical process for biocidal metal oxide and metal fluoride nanoparticle coating
- antibiofouling protective coatings based on bio-inert hydrogels
- enzymatic process based on enzymatic oxidation of phenolics leading to biocidal coatings
- process using enzymes i.e. cellobiose dehydrogenase (CDH) in suited coating for biocidal activity
- methodology and tests for coating efficiency and the enzymatic processes
- functional effect of a successful coating in terms of bio-compatibility, durability and bio-activity
These requirements and specifications were then used as guidelines in the following work packages.
Sonochemical coating of inorganic antibacterial agents
The main task of the project was to coat latex and silicon catheters with the antibacterial ZnO and MgF2 nanoparticles. The coating was achieved using the sonochemical technique.
By using the sonochemical technique a chemical reaction occur under ultrasound irradiation. The reaction is dependent on the development of an acoustic bubble in the solution. In the process that follows the creation of the bubble (acoustic cavitation), the bubble expands and compresses until it eventually collapses. The extreme conditions (temperature and pressure) that develop when the bubbles collapse, cause the chemical reactions to occur. In addition when the bubble collapses near a solid surface microjets moving at a very high speed (500 m/sec.) are formed. They move towards the solid surface. The microjets throw the newly formed particles onto the surface forming a very stable coating. The formation of coating was analyzed by electron microscopy techniques. The analysis of the elements present on the catheter was done by ICP as well as EDS. Using the sonochemical technique a very homogenous coating was obtained on the full catheter, the amount of coating was ~ 0.05% of the catheter weight. The ZnO coated catheters were tested for antibiofilm activity using a laboratory model of the bladder and in artificial urine solution, a reduction of more than 50% in the biofilm mass was observed. The remaining 50% biofilm was composed of mostly dead cells. The same system was also used for detection of leaching (removal of NPs) from the surface. Following incubation for seven days, no leaching of NPs was observed. Complementary functionalization with tailored protective inert polymers with a diblock structure comprising PDMS anchor block and PEG or polyzwitterionic functional blocks and grafting methods (adsorption/entrapment) had been established and also successfully used in combination with NP coating.
Several additional antibiofilm approaches were tested. They include:ultrasound assisted immobilization of antibacterial enzymes (Amyalse and Cellobiose dehydrogenase). Both enzymes showed excellent antibiofilm activity, the combined coating of the two enzymes, showed a synergistic effect (higher biofilm reduction was observed).
The mechanical properties of the catheter were tested to probe if the sonochemical coating process affect the catheter. No mechanical change was observed. The coating was tested for durability under sterilization process (by γ-irradiation and by ethylene oxide) no effect on the coating was observed. Toxicity tests of the coated catheters were performed using several cell lines (epithelial, fibroblast) - cell morphology and viability remained unchanged. Finally the sonochemical coating process was up-scaled to coat simultaneously several catheters.
Enzymatic based polymerization of oxidized phenolics as antibacterial coating
Urinary polyurethane and silicone catheters were coated with poly(catechin) and poly(catechin)-antibiotic conjugates to induce reduction of bacteria adhesion onto the devices surface. Laccase was used as biocatalyst to oxidize the catechin and catechin-antibiotic conjugates monomers and produce the corresponding polymers. Four antimicrobial coatings were produced, namely with poly(catechin), poly(catechin)-trimethoprim, poly(catechin)-sulfamethoxazole and poly(catechin)-trimethoprim-sulfamethoxazole. The bacterial adhesion reduction was tested on the functionalized devices using gram-negative and gram-positive strains. The most significant reduction in adhesion was observed with poly(catechin)-trimethoprim (gram-negative: - 85% and gram-positive: - 87%) and with poly(catechin)-trimethoprim-sulfamethoxazole (gram-negative: - 85% and gram-positive: - 91%). The cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90% of viable cells. A promising approach for the increase of the indwelling catheters life-span was developed aiming to reduce catheter-associated chronic infections.
Enzymatic biofilm prevention by CDH
The main goal of this work package was to explore the possibility of producing urinary catheter with antimicrobial and antibiofilm properties using an the enzyme Cellobiose Dehydrogenase (CDH). CDH is an enzyme that is able to produce hydrogen peroxide (a strong antimicrobial agent) using various cello- and oligosaccharides as electron donors. The antimicrobial and antibiofilm effect of the free enzyme was assessed against clinical isolates commonly found colonizing catheters and causing infections. The CDH proved to be very effective both in the prevention of biofilms and in the killing of bacteria already embedded in biofilms. The enzyme was successfully immobilized on the surface of urinary catheters using different techniques (ultrasound, layer by layer and covalent binding) of which the covalent immobilization lead to the highest amount of enzyme deposited on the surface. The bound enzyme was able to produce hydrogen peroxide in concentrations sufficient to inhibit biofilm formation on the coated surface by up to 70% when tested against Staphylococcus aureus. Moreover, two other enzymes, namely amylase and acylase were investigated concerning their antimicrobial and antibiofilm properties. Amylase is an enzyme capable of hydrolyzing sugars present in the biofilm matrix and acylase interferes with the cell to cell communication of bacteria. They showed strong antibiofilm activities in free form and were successfully immobilized on urinary catheters using layer by layer technique retaining their antibiofilm activities. Complementary functionalizations with tailored protective inert polymers comprising cationic anchor groups and zwitterionic functional groups and a coating method (polyelectrolyte layer-by-layer) had been established and also successfully used for enzyme immobilization. All three investigated approaches show a great potential for developing urinary catheters with antimicrobial and antibiofilm properties since they are stable in synthetic urine over long periods of time and don´t show any signs of detaching from the surface. Further, the developed enzyme based coating didn´t show any signs of cytotoxicity and therefore could be a valuable alternative to solving the increasing catheter associated infections problems.
Validation: up scaling and in vivo tests of the novel coatings
Upscaling was performed primarily on silicone catheters given that these have the highest existing tolerance to biofilm formation. The coating was performed in bulk in a 10L Sonication vessel with stirring capabilities. 20 silicone catheters were produced in the F8 size for in vivo studies. The metal oxide used was Zinc oxide, as this coating demonstrated to be highly active on common genitourinary bacteria. The next step was to demonstrate that the coatings were not toxic to cells. This was done using cultured cells from the epithelium and the immune system. Having established that the catheters were both effective on bacteria, and harmless to human cells, we set out to test them in an in vivo model. The standard model is the New Zealand white rabbit. We developed a well-tolerated method to catheterize the animals without need for restraint allowing them to move freely. Catheters were recovered after 7 days and demonstrated significant reductions in both colonisation and urinary tract infection, reaching the goal specified.
Project Results:
Requirements and Specifications
Biofilms are bacterial communities encased in a self-produced hydrated polymeric matrix. More than 50% of bacterial infections are caused by bacterial biofilms. Catheter-associated urinary tract infections (UTI) alone account for approximately 40% of all hospital acquired infections. The aim of NOVO had been to provide a coating for catheter which can prolong the in vivo use by effectively reducing the biofilm formation. The strategy was to use different approaches alone and in combination and consequently in the first phase the requirements and specifications were defined for:
- sonochemical process for biocidal metal oxide and metal fluoride nanoparticle coating
- antibiofouling protective coatings based on bio-inert polymeric hydrogels
- enzymatic process based on enzymatic oxidation of phenolics leading to biocidal coatings
- process using enzymes cellobiose dehydrogenase (CDH) in suited coating for biocidal activity
- methodology and tests for coating efficiency and the enzymatic processes
- functional effect of a successful coating in terms of bio-compatibility, durability and bio-activity
Sonochemical process. ZnO and MgF2 nanoparticles are known to have intrinsic antibiofilm properties and were selected for this project. Sonochemistry was used for preparing coatings using two different approaches, i) the in situ method, and ii) the “throwing stones” method. The most important parameter is nanoparticle size; the optimal size had been identified in the range of 50–100 nm. The morphology and particle size will be probed by high resolution scanning electron microscopy, and complementary methods for quantification (e.g. atom absorption spectroscopy) were also identified. The coating amount and the particle size can be controlled during the sonochemical reactions by varying different reaction parameters such as: i) reagents and their concentration, ii) time of sonication, iii) intensity of sonication, or iv) temperature.
Bio-inert polymeric hydrogels. In order to minimize biofouling in long term applications, ultrathin surface-attached hydrophilic polymer (hydrogel) layers will be created which will largely reduce protein adsorption and bacteria adhesion. A “grafting-to” approach called adsorption/entrapment was focused. This method is a two-step process where the polymeric base material (silicone) is swelled with a compatible solvent containing the polymeric modifier (“protective inert polymer”; PIP), followed by a deswelling in water where the PIP is entrapped on the surface of the silicone. The method is well compatible with surface modification by ultrasound; the preferred version would be an integration of both modifications in one step. The modifiers will be designed as block co-polymers, containing two (or more) different blocks: the anchor block (to be entrapped into the silicone) and the functional block (facing outwards). Polyethylene oxide (PEO) and zwitterionic polymers containing sulfobetaine were chosen as functional blocks. Polydimethylsiloxane (PDMS), the catheter material, and polypropylene oxide (PPO) were used as anchors blocks. Various strategies for polymer synthesis were developed and the requirements to surface modification methods and evaluation of modified surfaces were defined (main chemical methods: contact angle and zeta potential analyses).
Enzymatic oxidation of phenolics. Possible substrates for the formation of biocidal coatings by enzymatic oxidation of phenolics have been identified: i) catechin, a flavan-3-ol, a type of natural phenol and antioxidant; ii) ellagic acid, a natural phenol antioxidant found in numerous fruits and vegetables; iii) tannic acid, a complex substance, usually extracted from plants; iv) galangin, a flavonol which has been shown to slow the increase and growth of tumor cells in vitro; v) apigenin, a natural product belonging to the flavone class that is the aglycone of several naturally occurring glycosides.
Cellobiose dehydrogenase (CDH). A mutant CDH with increased activity for hydrogen peroxide (H2O2) production has been established based on a wild-type enzyme from Myriococcum thermophilum and can be produced in larger amounts. The increased oxygen reactivity had been verified by different methods. The fermentation process was adapted from the Pichia Fermentation Process Guidelines (Invitrogen) and optimized during a lot of fermentation trials. Two assays for CDH enzymatic activity were proposed because that is crucial for the evaluation of CDH coated catheters: i) the 2,6-dichlorophenolindophenol assay, and ii) the analysis of H2O2 production with the AMPLEX Red Assay.
Amylase and acylase with antibiofilm activity were also identified. The action of acylase over model quorum sensing molecules and the α-amylase activity upon model exopolysaccharides were assayed. Both enzymes were able to inhibit the static biofilm formation of medically relevant bacteria on polymer surfaces.
For both enzyme groups, CDH and amylase/acylase, methods for immobilization on catheter surfaces shall be developed. The focus is using polyelectrolyte “layer-by-layer” technique where the enzymes are co-assembled with polyelectrolytes. PIPs can also be used as designed building blocks for such coating techniques. Another strategy is the combination of the sonochemical modification with immobilization of CDH and/or PIPs.
Anti-biofilm coating activity shall be demonstrated by microscopic images using Live/Dead kit cell viability staining and quantitative total biomass determination using crystal violet. Coating efficiency is also evaluated by using the methods developed for characterization of the individual modifications.
The functional effect of a successful coating must not be in conflict with the already established property profile:
• A catheter must be physically stable enough to allow insertion.
• A catheter should allow a reasonable flow of urine
• The balloon should inflate reliably,
• The balloon should deflate reliably, and return to a minimal size, even after a long time
• No parts of the catheter should break off, even after a long period of in-dwelling
• It should be easy to withdraw the catheter
• A catheter should not induce a local inflammatory reaction
• A catheter should not induce allergies, such that there is a massive reaction the second time they are used.
• A catheter should not be contaminated with foreign materials (particles, oil, etc.) or packaging remains
• A catheter should not leach plasticizers into the body
Established standard tests will be used to assess those properties, and list of the respective guidelines (ASTMA, ISO, EN etc.) had been compiled. Functional effects will be determined at four general levels:
- at a physical level (i.e. composition, surface charge, hydrophilicity)
- in vitro biological (impact on mammalian cells or on bacteria)
- in vivo in animals (resistance to infection, including artificial infection)
- in clinical practice (clinical outcomes, patient welfare, reduction in antibiotic use)
If the stated problem is an excess in biofilm formation, one may measure biofilm. However, such physical measures may be functional, but not be directly related to an actual economically quantifiable problem. Thus, they are easy to measure but difficult to convert into a probable economic gain. Similarly, stating the full economic problem as the functional goal, i.e. a reduction in the cost of urinary tract infections due to catheters by half, while precise, is extremely difficult to test without large scale blind trials. In this context, one has to accept that a model will have finite degree of translation to the clinical setting. The question then becomes which surrogate of the clinical problem is an acceptable indicator.
All these requirements and specifications were then used as guidelines in the following work packages.
(WP2) Sonochemical coating of inorganic antibacterial agents
In the current part the different parameters such as time of the sonication process, output power, and initial reagent concentration were evaluated for their effect on coating morphology and quality. The optimal initial reagent concentration of Zn(CH3COO)2 for the formation of uniform coating with ZnO nanoparticles was found to be 0.1M (figure 2.1). Coating of silicone catheters with magnesium fluoride NPs was also achieved. ZnO and MgF2 coated catheters were tested for their antibiofilm activity; the reduction in the biofilm mass was calculated by crystal violet (CV) staining. The purple dye is attached to the polysaccharide components of the biofilm (Figure 2.2) enabling visualization of the biofilm. For quantification, the dye is removed from the catheter and its amount is measured via spectrophotometer. The results showed that both MgF2 and ZnO coatings, showed excellent antibiofilm activity by reducing 70% and 60%of the biofilm mass, respectively. Moreover, it was observed that the cells that were able to attach to the surface were killed by the active coating.
In addition to the special chemistry of the coated catheter, causing their strong antibiofilm activity, the formation of nano-rough surface (Figure 2.3) also contributed to the inhibition in biofilm formation. The rigid bacteria cell is not able to attach to the nano-rough surface and to form biofilm.
The catheters that underwent the sonochemical process were tested for changes in their mechanical properties- and were found unchanged. Sterilization with Gama –irradiation or by ethylene oxide was found not to harm the coating. The leaching of the coated NPs from the catheters was tested in a lab scale experiment and on an artificial bladder model. Leaching of NPs was not observed, some ions (up to 15% of their content in the coating) were detected in the leaching solution (Figure 2.4).
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Figure 2.4 Leaching of Zn+2 from ZnO coated catheters
The sonochemical coating was up-scaled – to be able to coat several catheters simultaneously. In an experiment in which 5 catheters were coated simultaneously, all the catheters were coated with a uniform coating (see Figure 2.5 for example of coating on 2 catheters). The weight of the coating was ~ 0.05% of the catheter weight. The catheters continued to show excellent antibiofilm activity.
Figure 2.5 ZnO coating on catheters from up scaled reaction (left mage catheter number 1, right image catheter number 5).
Finally, the antibiofilm activity of the coated catheters were tested in-vivo by using a rabbit model, the coated catheters showed good antibiofilm activity. The significant result of the rabbit experiment is that the rabbits that were treated with the coated catheters showed less urine contamination.
Enhanced antifouling activity by grafted protective inert polymers (PIP)
An extension of antibiofilm activity was achieved by the functionalization with grafted protective inert polymers (PIP) with hydrophilic moieties to protect the antibiofouling activity. Commercially available amphiphilic triblock copolymers Pluronics and a newly designed and synthesized PIP comprising a hydrophilic functional block and a hydrophobic anchor block which is compatible with the catheter material (PDMS) were used.
Figure 2.6 Synthesis of the novel amphiphilic diblock copolymer with PDMS as anchor block and poly-zwitterions as functional block
Three novel PIPs with different degree of polymerization of the zwitterionic block, i.e. different chain length (block size), were synthesized and tested. The structure was determined via NMR, GPC, elemental analysis and IR. With these PIPs the silicone catheter material was successfully modified via the non-covalent adsorption/entrapment mechanisms. The contact angle measurements showed that the surface became significantly more hydrophilic and remained unchanged for longer than 10 days. For all tested PIPs, the same contact angle was achieved and no trend for a better prevention against biofilm was found with the longer chains of zwitterion.
Figure 2.7 Results of contact angle measurements for silicon (PDMS) samples after adsorption/entrapment modification and storing over time in synthetic urine
The observation was also confirmed by zeta potential measurements which proved the coverage of the PDMS surface with a hydrophilic, overall neutral polymer.
Figure 2.8 Results of zeta-potential measurements for samples after adsorption/entrapment modification, exemplary for the smallest PIP (PDMS-b-PSPE20)
The biofilm formation was tested using the Alamar blue assay.
Figure 2.9 Result of the biofilm formation assay using Alamar blue
For the tube material a small antifouling effect in comparison to the control sample namely a reduction between 23 and 31% was found. But this result could not be confirmed with the balloon material. However, this antifouling strategy was used in combination with the “throwing stones” method with ZnO nanoparticles. After the coating with NPs the samples were modified via adsorption/entrapment with the novel PIPs. After this combined modification the samples were much more hydrophilic compared to each individual modification alone. On the other hand, biofilm assays showed that introduction of the PIP reduced the antibiofilm activity of the ZnO NPs. Protective inert polymers were applied to the ZnO coated catheters to protect the coating and thus enhance its activity. However, our results showed that introduction of the PIP reduced the antibiofilm activity of the ZnO NPs.
Synergistic activity of several active (enzymes) coatings
Another part of our work was to test the synergistic activity of several active coatings. Two enzymes (CDH and amylase) were successfully applied to the catheter surface using sonochemistry. The coated CDH retained its specific activity and produced the antibacterial H2O2. The immobilized enzyme showed excellent antibiofilm activity. OXY enzyme coating reduced the biofilm amount by more than 50%, moreover, the surviving biofilm mass showed the presence of mostly dead cells (Figure 2.10). The combination of the CDH with amylase further increased the biofilm inhibition to 65%.
Figure 2.10 Antibiofilm activity of CDH enzyme
Enzymatic based polymerization of oxidized phenolics as antibacterial coating
The remarkable antimicrobial properties of phenolic compounds as flavonoids make them an interesting alternative to the conventional antibiotic treatments. Flavonoids, a class of natural products that possess a diverse range of pharmacological properties, have been studied as active agents to prevent biofilm formation with promising results. The position and number of hydroxyl groups on these phenolic compounds were found to be related to their antimicrobial activity. The phenolic hydroxyl groups are thought to react with enzymes and inhibit various metabolic processes, while the hydrophilicity conferred by the hydroxyl groups is also critical for their ability to pass through bacterial cell walls. Prevention of biofilm formation can be achieved either by killing or repelling bacteria, with steric and electrostatic repulsion being involved in the latter process. Moreover, the polyphenols become negatively charged and reactive at pH greater than 7.5 increasing electrostatic repulsion of bacteria. Besides, the use of these polyphenols in combination with other antimicrobial agents has also been cited as a successful practice to fight the drug resistance problem. The synergistic effect provided by both may potentiate the antibiotic efficacy at lower concentrations decreasing adverse reactions.
Catechins are polyphenols susceptible to enzymatic and non-enzymatic oxidations, giving rise to a variety of dimeric, oligomeric and polymeric products. As o-dihydroxylated compounds, catechins are potential substrates for laccase, an oxidative enzyme able to oxidize a wide range of substrates as phenolic compound, their derivates and aromatic amines. Aiming to generate new antibiofilm molecules that can prolong the life-span of urinary catheters, the laccase-catalyzed oxidation of catechin was studied during this project for the production of polymeric antimicrobials (Fig.3.1) that can be useful to coat these medical devices and prevent the bacterial colonization onto the corresponding surfaces.
Fig. 3.1: Schematic representation of poly(catechin) production through laccase oxidation.
In addition, the in situ enzymatically oxidation of catechin-antibiotic conjugates by laccase (Fig.3.2) for catheters coating was also assessed in this project aiming potentiate the bacterial adhesion reduction onto the functionalized catheters.
Fig. 3.2: In situ laccase functionalization of polyurethane (PU) and silicone (SI) catheters using catechin-TMP and/or catechin-SMZ conjugates (TMP: trimethoprim; SMZ: sulfamethoxazole).
Overall, from this research resulted four novel antimicrobial coatings for medical devices. Taking the advantage of poly(catechin) and antibiotics properties, antimicrobial conjugates were designed, and the obtained synergism allowed the inhibition of planktonic bacteria growth and the bacterial adhesion reduction onto the coated surfaces (Fig.3.3).
Fig. 3.3: Inhibition of planktonic bacteria growth (left) and bacterial adhesion reduction (right) resulted from the poly(catechin) (PCAT) and poly(catechin)-antibiotic conjugates action.
This synergism was more evident on poly(catechin)-TMP and poly(catechin)-TMP-SMZ where a higher bacteria reduction was observed. Moreover, the cellular metabolism assays demonstrate that the antimicrobial coatings developed are not harmful for the cellular metabolism, maintaining more than 90 % of cell viability even after the highest incubation time tested (Fig.3.4).
Fig. 3.4: Relative L929 cell viability after 24, 48 and 72 hours of culture with DMEM solutions pre-inoculated with functionalized polyurethane (A-C) and silicone (D-F) catheters for 1, 3 and 5 days. Non-coated sample was used as CONTROL. Poly(catechin) (PCAT), poly(catechin)-trimethoprim (PCAT-TMP), poly(catechin)-sulfamethoxazole (PCAT-SMZ) and poly(catechin)-trimethoprim-sulfamethoxazole (PCAT-TMP-SMZ) were the coated samples. Data were achieved in relation to the control for 1 incubation day and show the mean values of three replicates from four experiments carried out independently. Statistical significant differences are indicated. *** = significantly different from the control (P < 0.001); ** = significantly different from the control (P < 0.01); * = significantly different from control (P < 0.05)
Therefore, a promising approach for extending the life-span of indwelling catheters was developed during this project and, concomitantly, the often catheter-associated infections could be reduced and the life quality of catheterized patients improved.
Enzymatic biofilm prevention by CDH
Another approach pursued in this project was the biofilm prevention/inhibition with the help of enzymes. Three different classes of enzymes were used: Cellobiose Dehydrogenase, producing the antimicrobial agent hydrogen peroxide, Amylase, an enzyme capable of hydrolysing sugars present in the biofilm matrix and Acylase, an enzyme that interferes with the cell to cell communication of bacteria. All three enzymes were first tested concerning their antimicrobial and antibiofilm properties in free form against various gram positive and gram negative bacteria. Once their antimicrobial effectiveness was proven several strategies were implied in order to deposit them on the surface of silicone urinary catheters. The main focus was set on the enzyme Cellobiose Dehydrogenase (CDH). The first step was to engineer and produce a genetic version that is able to produce high amounts of hydrogen peroxide in the presence of poly- and oligosaccharides and oxygen. This enzyme was then tested against gram positive and gram negative clinical isolates commonly causing catheter associated urinary tract infection in order to exploit its potential use as an antimicrobial enzyme. Results showed that CDH has a broad spectrum antimicrobial activity and that only very low amounts of substrate (which are consequently converted to hydrogen peroxide) are needed to be antimicrobially active against planktonic bacteria (Figure 4.1) and bacteria already embedded in biofilms (Figure 4.2) and is therefore a promising candidate for the use in antimicrobial coatings.
Figure 4 1: Inhibition of growth of S. aureus growing in liquid cultures and on agar plates in the presence of varying substrate concentrations (Cellobiose)
One possible application that was exploited is the incorporation of CDH into lubricant, commonly used for the gentle introduction of urinary catheters. Figure 4.2 shows that CDH is still active when incorporated into lubricants and even acts on bacteria already embedded in biofilms. It is therefore a promising candidate for the use in antimicrobial coatings.
A B C
Figure 4. 2: LIVE/DEAD staining images of S. aureus biofilms formed on silicone sheets: (A) positive control, incubated at 65°C for 1 h, (B) negative control, untreated biofilm, (C) biofilm treated with CDH and Cellobiose
The next step was the covalently bind the enzyme to the silicone urinary catheters surface. A five step protocol was developed (Figure 4.3) yielding a surface capable of producing high concentrations of the antimicrobial agent hydrogen peroxide.
Figure 4.3: Stepwise immobilization of CDH onto PDMS surfaces; (1) plasma treatment, (2) grafting APTES,(3) grafting glutaraldehyde and (4) grafting the enzyme
The newly created surface was tested concerning its antimicrobial activity, long term stability and biocompatibility. The enzyme deposited on the surface was able to reduce S. aureus biofilm formation by up to 70% when incubated for short periods (3 hours) as can be seen on the microscopic images in Figure 4.3 and longer periods (7 days).
Figure 4.4: Atomic force microscopy images of S. aureus biofilms deposited on uncoated (left) and CDH coated (right) silicone surfaces within 3 hours
The CDH coated silicone catheters were also incubated in artificial urine for 16 days at 37 °C in order to test their long term stability in a realistic environment. The enzyme was able to retain 20% of its activity over the entire period which shows again that this system is robust enough to apply it in the coating of the urinary catheters. In a last step the biocompatibility of the antimicrobial surface was tested with the help of two mammalian cell lines. The surface showed no signs of cytotoxicity at substrate concentrations needed by the enzyme to show antimicrobial activity. In summary it can be stated that all the results presented here make this approach a very interesting and valuable alternative to already existing coatings to fight catheter associated urinary tract infections.
Another immobilization of the enzymes was tested via polyelectrolyte “Layer-by-Layer”-deposition. Here novel PIPs were developed which have antifouling properties and can be used as polymeric building block for layer-by-Layer modification. The idea for the novel PIP is to combine segments with quaternized amino groups for electrostatic assembly and those with zwitterionic groups to achieved antifouling properties. PIPs with different fraction of zwitterionic segments (0%, 25% and 50% of zwitterions) were synthesized and completely characterized with NMR, IR, GPC and elementary analysis. For the layer-by-layer-deposition the novel PIPs were used as polycation and poly(4 styrenesulfonat) (PSS) as polyanion.
Figure 4.5: Schematic representation of silicone surface functionalization with cationic and zwitterionic PIPs via layer-by-layer deposition
Due to the low isoelectric point, the enzyme CDH was assembled as polyanion in one of the layers. After three more layers the enzyme was firmly embedded and the outer layer was a polycation with the zwitterionic segements for antifouling. This modification was successfully analyzed on a model system (silicon wafer) and on both catheter materials.
Figure 4.6: Contact angle measurement after modification with 6 layers of PIP, PSS and CDH over 10 days, here exemplary for tube material
After the deposition the surface become more hydrophilic than the unmodified base material. Because the contact angle remained constant over the time it seemed that the modification is stable in synthetic urine for about 10 days. The zetapotential of the last layer of a six-layer system with immobilized enzymes showed clearly the difference between the three PIPs with different zwitterionic fraction.
Figure 4.7: Zeta-potential of LbL-modified silicone with PIP with different zwitterion fraction on the outer surface, here exemplary for tube material
Figure 4.7 clearly showed a shift of the isoelectric point, which means that the surface becomes more neutral with more zwitterionic groups in the cationic PIP layer. The biological studies showed a very high enzyme activity with the enzyme on top of the layer. For the embedded enzyme the results were not very clear. Perhaps during the drying process the surface changed the structure, so that the diffusion of the substrate to the immobilized enzyme was hindered and reduced the enzyme activity. This conclusion can be supported by results obtained with atomic force microscopy. Further microscopic analyses with live/dead kit indicated improvements of the antifouling properties of the modified surfaces.
Figure 4.8: Microscopic analyzes with the live/dead staining, exemplary for tube material
Figure 4.8 shows a very low antibiofouling activity of the enzymes. For the control sample and the sample without zwitterions there was a very uniform distribution of individual colonies which were fairly close together. For the samples with zwitterions there were more irregular cluster formations, but generally less bacteria growth. These measurements show clearly that the zwitterion had a positive effect for antifouling.
The third approach included the immobilization of two other antibiofilm enzymes, namly amylase and acylase. Acylase inactivates acyl homoserine lactone (AHL) signals of Gram-negative bacteria, suggesting the enzyme application as antibiofilm coating on indwelling medical devices. Coatings comprising the Quorum sensing (QS) disrupting enzyme acylase were built on silicone urinary catheters using a Layer-by-Layer (LbL) deposition technique. The LbL assembly was achieved by alternate deposition of negatively charged enzyme and positively charged polyethyleneimine. After immobilization on silicone surface, the enzyme biological activity and stability were evaluated. The acylase-coated catheters efficiently quenched the QS process in C. violaceum through the degradation of AHLs in the extracellular environment, demonstrated by 50 % decrease of QS-regulated violacein production. Moreover, the QQ activity of the surface attached acylase was similar to what was observed for the free enzyme, suggesting that the approach used to construct the coatings could preserve the protein folding and activity (Figure 4.9).
Figure 4.9: Violacein production by C. violaceum in presence of free acylase solution and acylase multilayer coatings on silicone catheters. The control - untreated silicone was set as 100 % violacein production.
Furthermore, the potential of acylase-coated urinary catheters to inhibit QS-regulated biofilm formation was assessed in dynamic conditions using a catheterized bladder model, where the shear stress and flow rate vary similarly to the situation in the human bladder during catheterization. P. aeruginosa biofilm was allowed to grow for 7 days and then the biofilm formation on acylase-based coating was analyzed using Live/Dead cells viability kit. The fluorescence images of live (stained in green) and dead (stained in red) bacteria attached to the balloon and catheter shaft (called urethra) demonstrated significant reduction of P. aeruginosa biofilm, when compared to pristine Foley catheters (Figure 4.10A). Quantitatively, the total biofilm mass was decreased by 80 % and 45 % on the acylase-coated balloon and urethra, respectively (Figure 4.10B). Therefore, such enzyme-based approach relying on disruption of QS process via an enzymatic inactivation of AHLs in the extracellular environment could be a viable alternative to the antibiotics for controlling the biofilm occurrence on indwelling medical devices. While antibiotics kill or inhibit bacteria by targeting their structure or survival processes, interfering with QS pathways is thought to exert less selective pressure on bacterial population reducing the threat of resistance emergence.
Figure 4.10: A) Microscopic images of untreated and acylase coated Foley catheters after 7 days incubation in dynamic bladder model system (x40 magnification). B) Crystal violet assessment of total biomass formed on urinary catheters: untreated and acylase multilayer coatings on silicone Foley catheter
To increase the antibiofilm spectrum of the enzyme-coated catheters, an integrated biofilm inhibition strategy based on acylase and α-amylase able to interfere simultaneously with bacterial QS phenomenon and biofilm matrix, respectively, was investigated. Acylase would affect the AHLs based QS systems increasing the biofilm susceptibility to the amylase, which would exert its activity once the bacteria is attached and the polymeric matrix produced. The QQ acylase and matrix degrading α-amylase were individually and simultaneously deposited on urinary catheters in a LbL fashion. After ten sequential deposition steps on silicone, the hybrid coatings (amylase and acylase) showed higher antibiofilm activity against single- (P. aeruginosa) and duals-species (P. aeruginosa and E. coli) biofilm development, when compared to the enzymes applied individually on the surface (Figure 4.11). Interestingly, the biofilm was significantly reduced when the QQ enzyme was the outermost layer. Such outcomes could be related with the natural sequence of biofilm development – from bacterial communication to irreversible surface attachment
Figure 4.11: Inhibition of single (P. aeruginosa) and dual (P. aeruginosa and E. coli) species biofilm formation on enzyme coated silicone urinary catheters assessed with crystal violet.
The hybrid coatings with better efficiency in inhibiting mono- and dual-species biofilm formation in static conditions (Figure 4.11) were further subjected to dynamic biofilm inhibition tests using an in vitro model of catheterized human bladder. Although, the different growth conditions in the model system the obtained results were comparable to that observed in static conditions. The total biofilm mass of single- and mixed- biofilms was significantly reduced on the LbL Hyb-Acy urethra parts of the catheters (Figure 4.12). Therefore, such integrated strategy might provide efficient control of single and multi-species biofilm formation on urinary catheters.
Figure 4.12: Fluorescence microscopy images of 7 days grown biofilms on enzyme coated silicone catheters analyzed after Live/Dead kit staining.
Validation: up scaling and in vivo tests of the novel coatings
The goal of the project is to deliver a viable product candidate. To this end, coating of catheters with ZnO was scaled up such that sufficient material was available for a panel of studies on safety and efficacy aspects. In vitro, studies were performed on antibacterial effect, and these data have been reported from earlier work packages. Similar studies were conducted with human epithelial and immune cells. For cell studies, the catheter surfaces were cut from scale up product and placed in a sterilizing ethanol bath, and then in culture with the cells. Cell density was assessed as the primary end point.
Figure 5.1. Hospital derived samples of the bacterium Acinetobacter baumanii were tested for sensitivity to various concentrations of ZnO nanoparticles, in this case, one can see that the bacteria grow well (dark blue line) in the absence of particles, but that at any concentration of particles, there is a reduction in growth.
Figure 5.2 Cultured U937 cells (immune derived) are able to grow to equal densities at equal rates irrespective of the coating density on silicone.
Figure 5.3 Cultured RAW 254 cells (immune derived, macrophage like) are not inhibited by the coating of ZnO on silicone.
Given that the two pre-conditions for further testing were met by the ZnO catheters, namely in vitro efficacy and biocompatibility, the catheters were further tested using an in vivo model. Catheters (ZnO and polyphenol) were placed in animals under anaesthesia and left in place for 7 days. Urine was sampled daily and at termination, catheters were recovered for biofilm evaluation. Treated catheters were generally less likely to have high levels of bacteria or biofilm.
Figure 5.4 Plates with different densities of bacteria from urine samples. Medium changes from green to yellow with changes in pH associated with bacterial metabolism. Low bacterial density left, vs. high right.
Figure 5.5 Micrographs of catheter balloons after removal showing signs of biofilm. Left, the uncoated control silicone catheter, vs. right the ZnO coated material where more base silicone is visible.
Figure 5.6 Micrographs of catheter balloons after removal showing signs of biofilm stained with crystal violet. Left, the uncoated control silicone catheter, vs. right the ZnO coated material.
Figure 5.7. The viable counts of bacteria in urine samples from catheterised rabbits. In this case, the mean is presented with N=8. Differences were significant to p>0.05 on all days after day 3 which is consistent with the normal course of infection in this model.
The ZnO coated catheters were active in both reduction of apparent biofilm on the balloon and other parts of the catheter, but also in reducing the rate with which bacteruria developed in the bulk urine. The rabbit model is a good correlate to the rates of infection development in people and these data are suggestive of a positive effect of the coating. In combination with the other observations, we conclude that the objective of reducing the rate of infection development by half has been achieved. Given that the coating process is scalable and technically cost effective, the next step in development is the estimation of clinical benefit and what, if any price difference could be justified for catheters incorporating this technology.
Potential Impact:
1) The potential impact
The NOVO project developed novel approaches (coatings with anti-fouling NPs, (poly) phenolics, specific CDH) to control biofilms on medical implants. In particular, the strategies applied within NOVO involve modalities that are completely un-related to the modes of action employed by current drugs. Thus, they prevent/degrade biofilm formation using effects that are not counteracted by the resistance mechanism that have arisen following selection by current drugs. The mechanisms employed in NOVO, therefore, provide for a means to attack infections that resist existing therapies.
The multidisciplinary consortium combined knowledge and resources of leading academics, hospitals, and manufactures of enzymes and plastic indwelling urinary systems -catheters (SMEs).
This project outputs have a potential to be successfully translated into clinical practice, introducing the novel breakthrough approach for anti-biofilm protection/destruction strategies in medical implants, resulting in better healthcare for the citizens along with reducing the cost of healthcare. NOVO’s technological platform can be implemented further for the biofilm prevention in wound dressings or as direct wound disinfectants, and for the non-medical products, facing the problem of biofilm formation, such as sewage pipes, pipelines of the offshore oil and gas industry, water treatment membranes et cetera.
1.1 Clinical and Scientific Impact
Main scientific and technological breakthroughs:
A user friendly and low cost one step ultrasound process for coating different antibiofouling agents (inorganic, organic and biologic):
• Inorganic: A single-step (US based) metal oxides and fluorides nanoparticles coating on latex/silicon surfaces.
• Organic: (poly)phenolics’.
• Enzymatic (CDH) coating with biofilm degradation activity (based on peroxide).
Simultaneous co-coating of a protective bio-inert polymer and anti-biofilm agents to protect their activity
General anti-biofilm strategies
While the technologies outlined within the project are for implants, there is good reason to expect that these technologies can be employed also in topical formulations for wounds. It is expected to be able to apply these procedures and anti-microbial agents, in slightly modified form for wound dressings and as direct topical disinfectants for colonized wound surfaces. In certain examples, there is also the potential for systemic application of biofilm specific polysaccharide binding enzymes to clear infections of heart valves and other difficult to clear sites.
The scientific impact is broader than its implementation in the project’s application:
The novel, single step, environmentally friendly US proposed nano-technological process can be implemented in different applications to avoid biofilm formation (e.g. water treatment membranes, antimicrobial textiles, etc).
The enzymatic processes could be also implemented in the above mentioned applications.
The protective coating is a novel and unique suggestion to protect the activity of the antibiofouling agents.
1.2 Socio-Economic impact
Medical implants and devices-associated infections
Infection associated with implantable devices such indwelling medical devices (e.g. contact lenses, central venous catheters and needleless connectors, urinary catheters, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes and voice prostheses) is serious problem as it means prolonged hospital admittance for the patient and possible multiple surgeries. The case-fatality rate for patients with ventilator-associated pneumonia is 42 percent, with an attributable mortality of 15 to 30 percent. For nosocomial bloodstream infection, the case fatality rate is 14 percent, with an estimated attributable mortality of 19 percent.
Avoiding infection is highly important for the patient's well-being as well as to minimize extended cost to the healthcare system. US Studies show that a post-surgical wound infection more than doubles the patient's hospital costs, with Staphylococcus aureus (Golden Staph) responsible for a trebling of patient's hospital costs.
The overall annual direct medical costs of healthcare-associated infections to U.S. hospitals ranges from $35.7 billion to $45 billion in 2007. With a 70 percent of effectiveness of possible infection control interventions, the benefits of prevention can be up to $31.5 billion for US only.
Catheter-associated urinary tract infection
Urinary tract infections are the most common type of healthcare-associated infection, accounting for more than 30% of infections reported by acute care hospitals. Virtually all healthcare-associated UTIs are caused by instrumentation of the urinary tract. Reported hospital wide prevalence rates for indwelling catheterization vary from 25% to 35%. Catheter-associated urinary tract infection (CAUTI) has been associated with increased morbidity, mortality, hospital cost, and length of stay.
CAUTI creates significant costs for the individual facility and the health care system at large. The cost of treating a single episode varies from $980 to $2900 depending on the presence of associated bacteremia. Cumulatively, CAUTI adds an additional 90 000 hospital days per year. With a collective annual cost of $424 million to $451 million in the United States alone.
In addition, bacteriuria commonly leads to unnecessary antimicrobial use, and urinary drainage systems are often reservoirs for multidrug-resistant bacteria and a source of transmission to other patients.
Moreover, from October, 2008, hospitals in USA no longer reimbursed for costs associated with nosocomial infections, giving them an incentive to reduce their frequency.
NOVO’s output will impact the entire implants related treatments. While implementing the developed technologies to different implants (one of the major causes of hospital-acquired infections), patients’ infections incidences will be significantly reduced and the usage time of the implants will be enlarged. NOVO will thus have a remarkable impact on the European citizens’ health care, economy and quality of life.
Employment
While the economy is adding jobs at lower levels than workers would like, during the last two years, analysts expect buds of growth in a wide range of service jobs this year-retail, information technology, professional, scientific and technical jobs, as well as continuing growth in the health-care industry.
With the aging population, health care remains the go-to field for job growth. "Health care is always adding jobs. That will clearly continue.
Among companies that expect to increase full-time, permanent workers, the top areas, by function, are: sales, information technology, customer service, engineering, technology, administrative, business development, marketing, research/development and accounting/finance.
NOVO is at the intersection of the above mentioned discipline thus having a positive impacting the employment in these fields.
Quality of life
At present, the vast majority of infections are associated with simple procedures like catheterization. In so far as a catheter is reliably and robustly more biocompatible, it will save significant numbers of procedures and nosicomial infections. In particular, it will be important within the growing aged care market where such procedures are increasingly common and where costs are correspondingly increasing.
The formation of biofilms affects sensitivity and resistance to antibacterial and antifungal drugs and therefore represents a clinical problem affecting the quality of life of the patients. By the newly developed effective methods to prevent the biofilm formation or destroy it, when adopted in practice, the quality of life will be enhanced since changing implants will be at a lower frequency, the antibiotics will be more effective thus lower doses will be applied for treatment. Furthermore the hospitalization periods will be shorter due to more effective treatments.
2) Main dissemination activities and exploitation of results
a. Dissemination
The NOVO consortium has allocated substantial efforts and resources for the purpose of dissemination of the project results within the scientific community and in the general public. The main efforts and dissemination activities are presented below.
Project Brochure:
OSM and BOKU have developed and produced the project brochure. NOVO Project Brochure was designed as a dissemination tool for the project. The brochure contains a general description of the project. The informative brochure promotes and describes the vision of the project, main objectives and outcomes as well as the project layout. The brochure also contains information about the project partners and their contact info. The brochure can be downloaded via the NOVO website domain and as prints during relevant conferences, meetings and workshops.
Website construction and maintenance
The NOVO website (http://www.fp7-novo.eu/) was launched by OSM on August 2012 and is updated regularly. The website is library of all the project documents including all deliverables and presentations from the meetings. The website contains two main sections (1) The public section which aims to introduce the NOVO consortium, project concept, objectives and vision; (2) The partners’ restricted section which offers an access to project records, e.g. submitted documents, deliverables, and meetings updated information including partners presentations and minutes. Every partner has received a private user and login password.
Scientific publications by NOVO consortium partners
BOKU:
1. Thallinger, B., Nugroho Prasetyo, Nyanhongo, G.S. Guebitz, G.M. Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms. Biotechnol J. 2013 Jan ;8(1):97-109. doi: 10.1002/biot.201200313.
2. Thallinger B, Argirova M, Lesseva M, Ludwig R, Sygmund C, Schlick A, Nyanhongo SG, Guebitz MG. 2014. Preventing microbial colonisation of catheters: Antimicrobial and antibiofilm activities of cellobiose dehydrogenase. 44:402–408 International Journal of Antimicrobial Agents, doi: 10.1016/j.ijantimicag.2014.06.016.
BOKU/UPC
1. Thallinger B, Brandauer B., Burger P., Sygmund C., Ludwig R., Ivanova K., Scaini D., Kun J., Burnet M., Tzanov T., Nyanhongo G., Guebitz G.M. Cellobiose dehydrogenase functionalized urinary catheter as novel antibofilm system” International Journal of Antimicrobial Agents (2014),
BIU
1. Upscaling sonochemical process for catheter coating with antibiofilm nanoparticles (in preparation)
BOKU/BIU
2. Lipovsky A., Thallinger B. Perelshtein I., Nyanhongo G., Guebitz GM., Gedanken A., Ultrasound assisted coating of PDMS with H2O2 producing enzymes, Langmuir, submitted (2015).
UDE:
1. Anne Vaterrodt, Mathias Ulbricht , Abstract (P3-121) in conference book of with the title “Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification” European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013;
TEC
1. I.Gonçalves, T. Matamá, A. Cavaco-Paulo, C. Silva (2013) Laccase coating of catheters with poly(catechin) for biofilm reduction, Biocatalysis and Biotransformation, Published Online (DOI: 10.3109/10242422.2013.828711). 2014, 32(1):2-12
2. I.Gonçalves, A. Abreu, T. Matamá, A. Ribeiro, A. Gomes, C. Silva, A. Cavaco-Paulo (2014) Enzymatic synthesis of poly(catechin)-antibiotic conjugates: an antimicrobial approach for indwelling catheters, Applied Microbiology and Biotechnology, Published Online (DOI:10.1007/s00253-014-6128-2)
3. I.Gonçalves, C. Silva, A. Cavaco-Paulo (2014) Ultrasound enhanced laccase applications, Green Chemistry, Published Online, DOI: 10.1039/C4GC02221A http://xlink.rsc.org/?doi=C4GC02221A
4. I. Gonçalves, C. Botelho, A. Teixeira, A. S. Abreu, L. Hilliou, C. Silva, A. Cavaco-Paulo, Antimicrobial lubricant formulations containing poly(hydroxybenzene)-trimethoprim conjugates synthesized by tyrosinase, Applied Microbiology and Biotechnology (2015) (submitted).
5. Polyphenol-antibiotic coating of urinary catheters. Idalina Gonçalves, Ana S. Abreu, Teresa Matamá, Carla Silva, Artur Cavaco-Paulo (in preparation).
UPC
1. Fernandes M M, Francesko A, Torrent-Burgués J, Tzanov T (2012), Effect of thiol-functionalisation on chitosan antibacterial activity: interaction with a bacterial membrane model. Reactive and Functional Polymers, Doi: 10.1016/j.reactfunctpolym.2013.01.004
2. Fernandes M. M, Francesko A, Torrent-Burgués J, Javier Carrión-Fité F, Heinze T, Tzanov T (2014), Sonochemically processed cationic nanocapsules: efficient antimicrobials with membrane disturbing capacity. Biomacromolecules, Doi: 10.1021/bm4018947 (Published)
3. Diaz Blanco C, Ortner A, Dimitrov R, Navarro A, Mendoza E, Tzanov T (2014), Building an antifouling zwitterionic coating on urinary catheters using an enzymatically triggered bottom-up approach, ACS Appl. Mater. Interfaces, DOI: 10.1021/am501961b. (Published)
4. Ivanova K, Fernandes M M, Mendonza E, Tzanov T (2015), Enzyme multilayer coatings inhibit Pseudomonas aeruginosa biofilm formation on urinary catheters. Appl Microbiology and Biotechnology, Doi: 10.1007/s00253-015-6378-7.
EMI
1. Leseva M., Arguirova M., Nashev D., Zamfirova E., Hadzhyiski O.: Annals of Burns and Fire Disasters - vol. XXVI - n. 1 - March 2013 Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control.
2. Leseva M., Arguirova M., Zamfirova E.: Study of the biofilm forming ability of microorganisms isolated from urine of catheterized patients. Proceedings of the XII National Congress in clinical microbiology and infections, p. 41, Sofia, Bulgaria, 25 Apr, 2014.
SYN:
Enzymatically modified hydrogels for prevention and treatment of biofilm on catheters. Guezguez, Pietrzik, Hahn, Burnet and potential partner inventors Patent application, in preparation
Presentations and posters at Conferences by NOVO Consortium partners:
BOKU:
1. Novel anti-biofilm system based on cellobiose dehydrogenase, Thallinger B., Schlick A., Nugroho Prasetyo E., Sygmund C., Ludwig R., Nyanhongo G.S. and Guebitz G.M. ÖGMBT Graz , (2012) (poster)
2. Multifunctional bioresponsive polymers for the management of chronic wounds, Gibson Nyanhongo, Endry Nugroho Prasetyo, Georg Guebitz, E-MRS 2012 Fall Meeting, , E-MRS 2012 Fall Meeting, September 17-21, 2012, Warsaw, Poland.(oral presentation)
3. Generating a new antimicrobial PDMS surface using cellobiose hydrogenase, Thallinger B., Schlick A., Brandauer M., Nyanhongo G., Sygmund C., Ludwig R. and Gübitz G.M , EPF2013 Pisa:
4. Immobilization of Cellobiose Dehydrogenase as an antibiofilm agent on silicone catheters, Thallinger, B., Sygmund, C, Brandauer, M.a Schlick, A., Ludwig, R, Nyanhongo, G. and Gübitz, G. Eurobiofilms 2013, Ghent, Belgium (poster)
5. Novel anti-biofilm system based on the degradation of Exopolysaccharides, Thallinger, B., Sygmund, C., Brandauer, M., Schlick, A., Nyanhongo, G. and Gübitz, G. , FEMS 2013, Leipzig Germany
6. Cellobiose Dehydrogenase- antimicrobial functionalization of polydimethylsiloxane, Thallinger, B., Brandauer, M., Schlick, A., Ludwig, R., Sygmund, C., Nyanhongo, G. and Gübitz, G , IPTB 2014, Braga Portugal (oral presentation)
7. Cellobiose Dehydrogenase- antimicrobial functionalization of polydimethylsiloxane, Thallinger Barbara, Brandauer Martin, Schlick Angelika, Ludwig Roland, Sygmund, Christoph, Nyanhongo Gibson1 and Gübitz Georg, 2nd DocDay Tulln, Tulln Austria (oral presentation).
8. Modification of PDMS with Cellobiose Dehydrogenase yielding an antimicrobial surface. Thallinger, B.a Brandauer, M.a Schlick, A.a Burger P.a Ludwig, R.b Sygmund, C.b Nyanhongo, G.a and Guebitz, G.M ÖGMBT 2014, Vienna Austria (oral presentation)
9. Novel enzymatic antimicrobial and anti-biofilm systemThallinger, A. Schlick, C. Sygmund, R. Ludwig, G.S. Nyanhongo and G.M. Guebitz, ICAAR 2014, Madrid Spain (poster).
UPC
1. Novel strategies for prevention of pathogenic bacterial biofilm formation on indwelling medical devices. T. Tzanov, International Conference on Antimicrobial Research- ICAR2012, Lisbon, Portugal November 21-23, 2012 (oral presentation).
2. A dual polyphenolic/zwitterionic coating prevents microbial biofilm formation on urinary catheters. T. Tzanov 11th E-MRS Fall meeting September 17th-21st, 2012 Warsaw, Poland (oral presentation).
3. Thiolation effect on the antibacterial activity of a low molecular weight chitosan: interactions with a bacterial membrane model. T Tzanov E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
4. Bioactive multilayer coatings for prevention of bacterial biofilm formation on indwelling medical devices. T. Tzanov E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
5. Enzymatic-induced degradation of bacterial biofilms. T. Tzanov T E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
6. Nanostructured multilayer coatings with antibiofilm activity for biomedical applications Tzanov T, Fernandes M M, Gamerith C, Ivanova K 245th ACS National Meeting & Exposition April 7-11, 2013 New Orleans, Louisiana, USA
7. Nanobiopolymers: efficient antimicrobials with membrane disturbant capacity.Fernandes M M, Francesko A, Torrent J, Tzanov T. 245th ACS National Meeting & Exposition April 7-11, 2013 New Orleans, Louisiana, USA
8. Enzymes to control bacterial biofilm formation, T Tzanov, 245th ACS National Meeting & Exposition, New Orleans, Louisiana, USA., April 7-11, 2013 (poster).
9. Combined antimicrobial/antifouling coatings using zwitterion-decorated phenolic nano-capsules Díaz Blanco C, Francesko A, Fernandes M M, Tzanov T. 245th ACS National Meeting & Exposition. April 7-11, 2013 New Orleans, Louisiana, USA (poster)
10. Industry-driven "bio-research". T. Tzanov, I Jornada de la recerca i la innovació tecnològica al Campus de la UPC a Terrassa, Terrassa,Spain, June, 2013 (oral communication).
11. Three examples of industry-university international collaborations, T. Tzanov, Jornada d’exposició de projectes per la visibilització de la innovació (Grups de recerca + empreses), Terrassa,Spain, June, 2013 (oral communication).
12. Enzyme-based nanoparticles to inhibit bacterial biofilm formation in urinary catheters Ivanova K, Fernandes M. M, Tzanov T 8th International Conference on Polymer and Fiber Biotechnology – IPFB 2014 May 25-27, 2014, Braga, Portugal (oral presentation).
13. Sonochemically processed cationic nanobiopolymers - efficient antimicrobials with membrane disturbant capacity, Fernandes M. M, Francesko A, Tzanov T, 8th International Conference on Polymer and Fiber Biotechnology – IPFB 2014, May 25-27, 2014 Braga, Portugal (oral presentation.)
14. Building a dual antimicrobial/antifouling coating on urinary catheters using a laccase-triggered bottom-up approach, Diaz Blanco C, Tzanov T, 7th International Congress on Biocatalysis - Biocat2014, August 31st - September 4th, 2014, Hamburg, Germany (oral presentation)
15. Enzyme multilayer coatings inhibit quorum sensing-regulated Pseudomonas aeruginosa biofilm formation on silicone urinary catheters, Ivanova K, Fernandes M M, Tzanov T, III International Conference on Antimicrobial Research - ICAR2014, October 1-3, 2014, Madrid, Spain. (oral presentation).
16. Enzyme multilayer coatings inhibit quorum sensing-regulated Pseudomonas aeruginosa biofilm formation on silicone urinary catheters, T. Tzankov, III International Conference on Antimicrobial Research - ICAR2014, Madrid, Spain. October 1-3, 2014 (oral presentation).
17. Convectional antibiotics in form of nanospheres prevent biofilm formation and provide infection control, Fernandes M. M, Ivanova K, Francesko A, Tzanov T, III International Conference on Antimicrobial Research - ICAR2014,October 1-3, 2014, Madrid, Spain. (oral presentation).
UDE
18. Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification, Anne Vaterrodt, Mathias Ulbricht, 29th European Membrane Society (EMS) Summer School, Essen, 22-26 July, 2013 Poster number P-57.
19. Abstract (P3-121) in conference book of the EPF Conference, “Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification” Anne Vaterrodt, Mathias Ulbricht, European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013
20. Synthesis of amphiphilic diblock-copolymer via ATRP polymerization for antibiofouling functionalizable surface modification” - Anne Vaterrodt, Mathias Ulbricht European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013
21. Novel antifouling surface functionalizations using polymeric zwitterions via adsorption/entrapment and layer-by-layer deposition, Anne Vaterrodt, Mathias Ulbricht , IPTB 2014 Braga.
BIU
1. "Novel methods for the fabrication of Nanomateirals and their Application" Lecture at NCKU, Tainan, Taiwan, February 2012
2. "Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Coatings on Textiles and Glasses" Lecture at the University of Milano Bicocca, April 4 2012.
3. "Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Coatings on Textiles and Glasses" Lecture at Palacky University, RCPTM, Olomouc, Czech Republic, May 12, 2012.
4. "Novel methods for the fabrication of Nanomateirals and their Application" 2 lectures in EuCheMS-SCI School on “ Synthesis and Characterization of Novel Nano-Sized InorganicMaterials” 17-22 June, 2012, Villa Larocca and Department of Chemistry, Campus Universitario, Bari
5. "Novel methods for the fabrication of Nanomateirals and their Application" 2 Lectures in ASON-2 Dubrovnik, Croatia, September 3-7, 2012.
6. "Anything you can do I can do better" What can be done better with Sonochemistry? Plenary Lecture at NANOCON conference in Brno, The Czech Republic, October 23, 2012.
7. "Novel methods for the fabrication of Nanomateirals and their Application" lecture before the Graduate students of the Department of Mater. Sci. &Eng. of NCKU, Tainan, Taiwan Nov. 23, 2012
8. "Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Nanoparticles and their Sonochemical Coating on Surfaces (Textiles and Glasses)" Lecture at Comenius University, Bratislava, Slovakia. January 30, 2013.
9. "Anything you can do I can do better" What can be done better using Sonochemistry? A lecture before the 78th meeting of the Israeli Chemical Society, Tel-Aviv, February 12, 2013.
10. "Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Nanoparticles and their Sonochemical Coating on Surfaces (Textiles and Glasses)" Lecture at Comenius University, Bratislava, Slovakia. January 30, 2013.
11. "Anything you can do I can do better" What can be done better using Sonochemistry? A lecture before the 78th meeting of the Israeli Chemical Society, Tel-Aviv, February 12, 2013.
12. "The sonochemical coating of textiles with antibacterial nanoparticles" Lecture at the 23 IFATCC Conference in Budapest, Hungary, May 09, 2013
13. "Coating Antibacterial Nanoparticles on Flat and Curved Surfaces and fighting Resistant Bacteria Empolying the Sonochemical Method" Lecture at the AOSS-2013 conference in Melbourne, Australia July 12, 2013.
14. "What is Sonochemistry? Emphasis on Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti Biofilms Coatings on Textiles and Glasses" Lecture at the University of Cagliari Sardinia, Italy, July 24, 2013.
15. "Coating antibacterial NPs on flat and curved Surfaces" ANBRE13, Seoul, Korea, August 26, 2013.
16. "Coating Antibacterial Nanoparticles on Flat and Curved Surfaces and fighting Resistant Bacteria Empolying the Sonochemical Method" NAMF meeting a section of the EMRS fall meeting at WARSAW, Poland September 17, 2013.
17. "Antibacterial, Antiviral, Anti Biofilm, and antifungi NPs and their Sonochemical Coatings on Surfaces" NANOVED conference , Svit, Slovakia, September 23, 2013.
18. "Coating Anti Bacterial, Anti Viral, Antibiofilm and Antifungi Nanoparticles on Flat and Curved Surfaces Employing the Sonochemical Method" Polaris workshop, Porto, Portugal, October 9, 2013.
19. "Coating antibacterial NPs on flat and curved Surfaces" Plenary lecture at the ICAFM conference at Trivandrum , India, Feb. 20, 2014.
20. "Coating antibacterial NPs on flat and curved Surfaces" Goa University, India, Feb. 24, 2014.
21. "Sonochemical coating" Lecture at MIRDC. Kaohsiung Taiwan March 5, 2014.
22. "Coating Anti Bacterial, Anti Viral, Antibiofilm and Antifungi Nanoparticles on Flat and Curved Surfaces Employing the Sonochemical Method" Lecture at Tzhjiang University Madical Hospital Number 2, April 28, Hangzhou, China.
23. “Making the Hospital a Safer Place by the Sonochemical Coating of the Textiles and Other Medical Devices with Antibacterial, Antibiofilm, Antiviral and Antigungal nanoparticles” Lecture in the ESS (European Society on Sonochemistry)14 in Avignon, France June June 6, 2014.
24. “Making the Hospital a Safer Place by the Sonochemical Coating of the Textiles and Other Medical Devices with Antibacterial, Antibiofilm, Antiviral and Antigungal nanoparticles” Lecture at the 6th PCGMR conference in Tainan, Taiwan on September 3, 2014.
25. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” a Plenary lecture at Ultrasonics 2014, in Caparica, Portugal on Sept. 15, 2014.
26. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the Capital Med Univ, Dept Pharmacol, Beijing 100069, Peoples R China, Nov. 10, 2014.
27. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at DGIST, Dept Energy Syst Engn, Taegu 711873, South Korea. On November 11, 2014.
28. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at Samsung Division of thin films, Tsuwon, Korea on November 12, 2014.
29. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the MOLMED 2014 conference at Haikou, China, Nov. 14, 2014.
30. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the NANOTEK -2014 San Francisco, December 3, 2014
TEC
1. Laccase coating of catheters with poly(catechin) for biofilm reduction, Oxizymes 2014, July 1-4, 2014, Vienna, Austria.
2. Enzymatic oxidation of phenolic compounds: a green strategy to enhance antimicrobial properties in urinary catheters, 248th ACS National Meeting & Exposition, August 10-14, 2014, San Francisco, USA.
EMI
1. Vacheva-Dobrevska R., I.Ivanov M.Leseva et al.: Emergence and persistance of Proteus mirabilis carrying VIM-1 metallo-beta-lactamases and carbapenem-resistant Klebsiella pneumoniae in Bulgarian Hospitals; XII National Congress in clinical microbiology and infections of BAM, 24-26 April 2014,
2. Leseva M., M. Arguirova et al.: A prospective study of the biofilm forming ability of microorganisms isolated from urine of catheterized patients with severe burns; XII National Congress in clinical microbiology and infections, 25 Apr, 2014, Symposium of scientific articles (oral presentation),
3. Chalashkanov Tz., M. Leseva, E. Zamfirova, A. Ivanov: Emergence and control on spread of multiple resistant strains of Klebsiella pneumoniae in three clinics of UMHATEM “Pirogov”, XII National Congress in clinical microbiology and infections, 24-26 April, 2014
4. Leseva M., A. Ivanov, E. Zamfirova, Tz. Chalashkanov: Current problems in nosocomial infections and antimicrobial resistance in UMHATEM “N.I. Pirogov”. Symposium dedicated to the European Antibiotic Awareness Day. Bulgaria, Kostenets, 19-20 November, 2014.
5. Chalashkanov Tz., M. Leseva et all.: Dynamics of nosocomial infections in the Trauma Clinics of UMHATEM “N.I. Pirogov”, Ist Trauma Symposium, 20-22 Nov., 2014, Sofia.
Media broadcast
UPC:
1. Oral communication addressed to academic audience. Title: Antimicrobial biotechnology Speaker: Tzanko Tzanov http://www.terrassa.upc.edu/noticies/biotecnologia-antimicrobiana-al-campus-de-la-upc-terrassa Live-broadcasted at UPCtv: http://tv.upc.edu/ Date: 06/03/2013
2. Oral communication addressed to academic and industrial audience 13/06/2013; Title: Industry-driven "bio-research“ Speaker: Tzanko Tzanov
3. Oral communication addressed to academic and industrial audience 09/10/2013; Title: Three examples of industry-university international collaborations, Speaker: Tzanko Tzanov
b. Exploitation strategy and activities
i) The markets of indwelling medical devices
Catheterization procedures have steadily increased as interventional and less-invasive procedures have become more popular. Catheters are vital to the completion of many procedures, and some procedures or interventions, particularly in cardiovascular surgery and neurosurgery, could not be done without the use of catheters.
The global catheter market amounted is expected to reach $32.1 billion in 2014, for a 5-year compound annual growth rate (CAGR) of 12.3% .
• The US and EU market’s for cardiovascular segment (includes: pacemakers, implantable cardioverter-defibrillators (ICDs), cardiac resynchronization therapy devices (CRTs), cardiac leads, external defibrillators, ablation catheters, intracardiac echocardiography (ICE) catheters and diagnostic electrophysiology (EP) catheters) was valuated at $10.6 billion in 2010 and it is projected to rise at a CAGR of 10.2%.
• The second-largest segment is urology, with expected to reach $13.2 billion in 2014, for the highest CAGR among all segments at 17.1%.89
• The US and EU market’s for peripheral vascular devices (includes: PTA balloon catheters, embolic protection devices, stent-grafts, surgical grafts, inferior vena cava filters, diagnostic catheters, interventional catheters, diagnostic guidewires, hydrophilic guidewires, Peripheral vascular closure devices) is valuated at $3 billion at 2010 .
Large diversity of indwelling implants is widely used by modern medicine.
• The global orthopedic implants market is forecast to grow to $41.8 billion by 2016 at the Compounded Annual Growth Rate (CAGR) rate of 7.8% during 2009-2016 .
• The worldwide market for cochlear implants valued at $1.59 billion in 2012, a 22 percent compound annual growth rate .
• The global dental implants market is expected to grow from $3.2 billion in 2010 to $4.2 billion in 2015 at a CAGR of 6% from 2010 to 2015. Europe currently forms the world’s largest market for dental implants with a 42% market share, and is also expected to have the highest CAGR 7.0% from 2010 to 2015 .
This potential huge market is the one targeted by the NOVO project’s outputs – thus it will positively influence the medical large and SMEs industries, in Europe.
The hospitalization periods will decrease due to the improved treatment, thus lowering health care cost and increasing working days.
ii) Exploitation by the R&D centers
The research organizations and universities involved in the project produced scientific and technological results of commercial value and will be involved in their further exploitation both directly and indirectly.
The direct output of this project (a) protected by patents and by granting licenses to the relevant industries for its exploitation (or establish spin-off)
(b) Increasing their leadership in their respective areas of research on a global scale.
iii) Exploitation by the SMEs
Main advantages of new products:
1. High market demand
2. Use of FDA approved components (MgF2, ZnO)
3. Unique US based one-stage method for anti-biofilm layer anchoring to the polymer
4. Combinatorial approach –preventive and bacteriostatic/ bactericidal anti-biofilm coating in one product
5. Relatively competitive price -it is estimated that the cost of the new catheters will be only 10% higher than the existing products. This estimation is based on Life Cycle Cost assessments done in SONO FP7 project for coating textiles with antibacterial agents
6. Potential use for non-medical markets.(e.g. water purification membranes)
Main envisaged obstacles:
1. Time to market
2. Competition.
3. Price
The SMEs in the consortium will develop strategies based on the outputs of the project with the aim to introduce the new proven antifouling technology to the market.
List of Websites:
Project website address: http://www.fp7-novo.eu/
List of partners
Par.
No. Name of principal Investigator Organization legal name Acronym Country email
1. Prof. Georg Guebitz University of Natural Resources and Life Scienes BOKU AT guebitz@boku.ac.at
2. Prof. Aharon Gedanken Bar-Ilan University BIU IL gedanken@mail.biu.ac.il
3. Prof. Tzanov Tzanko Universitat Politècnica de Catalunya UPC ES tzanko.tzanov@upc.edu
4. Prof. Mathias Ulbricht University of Duisburg-Essen UDE DE mathias.ulbricht@uni-due.de
5. Prof. Dr Maya Argirova Emergency Medicine Institute "Pirogov". EMI BG maya_arguirova@yahoo.com
6. Michael Burnet Synovo GmbH SYN DE Michael.Burnet@synovo.com
7. Prof. Artur Cavaco TECMINHO TEC PT artur@deb.uminho.pt
8. Dr. Andreas Paar Qualizyme Biotechnology QUA AT office@qualizyme.com
9. Assunção Mascarenhas PRONEFRO,S.A. PRO PT assuncao@pronefro.pt
10. Oded Stein Degania Silicon Ltd. DEG IL oded-s@ds-il.com
11. Dr. Pnina Dan OSM-DAN Ltd. OSM IL pninadan@osmdan.com
Executive summary
The resistance of microbial biofilms to the immune system and to antibiotics has is a common cause of medical infections, and of difficult-to-treat infections caused by colonized foreign bodies. More than 60% of bacterial infections currently treated in hospitals are caused by bacterial biofilms while catheter-associated biofilm urinary tract infections (UTI) alone account for around 40% of all hospital-acquired infections. Within the NOVO project three strategies for biofilm prevention on urinary catheters were developed, namely, inorganic nanoparticles (NPs) coatings, coatings with phenolic molecules and grafting with antimicrobial enzymes.
Within the first strategy an ultrasound based process for grafting antibacterial ZnO and MgF2 nanoparticles onto catheters was successfully developed. The coating was characterised with various techniques including ICP, EDS and electron microscopy indicating an extend of ~ 0.05% of the catheter weight. The coating survived the standard sterilization process (by γ-irradiation and by ethylene oxide) while in turn the mechanical properties of the catheters remained unchanged during coating. The ZnO coated catheters showed a reduction of more than 50% in the biofilm mass using a lab model of the bladder and in artificial urine solution while the remaining 50% biofilm was composed of mostly dead cells. No leaching of NPs from the surface was observed over seven days while tests based on several cell lines (epithelial, fibroblast) indicated no cytotoxicity.
The second strategy involved coating of catheters with poly(catechin) and poly(catechin)-antibiotic conjugates using laccase for polymerization and grafting. The most significant reduction in bacterial adhesion was observed with poly(catechin)-trimethoprim (gram-negative: - 85% and gram-positive: - 87%) and with poly(catechin)-trimethoprim-sulfamethoxazole (gram-negative: - 85% and gram-positive: - 91%). Likewise for this coating the cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90% of viable cells.
The third strategy involved grafting of antimicrobial enzymes. Cellobiose dehydrogenase releasing hydrogen peroxide was assessed against clinical isolates commonly found in catheter associated infections and proved to be very effective both in the prevention of biofilms and in the killing of bacteria embedded already formed biofilms. Grafting of the enzyme onto catheter surfaces was achieved both chemically and by using the developed ultrasound based technique. The bound enzyme was able to produce hydrogen peroxide in concentrations sufficient to inhibit biofilm formation on the coated surface by up to 70% when tested against Staphylococcus aureus. Again, this coating did not show any signs of cytotoxicity.
Complementary functionalizations with tailored protective inert polymers of two different architectures and two different grafting/coating methods (adsorption/entrapment and layer-by-layer) had been established and also successfully been used in combination with NP coating and enzyme immobilization.
For ethical reasons, only one of the developed strategies, namely nanoparticles, proven to prevent biofilm formation without being cytotoxic was evaluated in an in vivo model. The model developed was based on the New Zealand white rabbit well-tolerated to catheterize the animals without need for restraint allowing them to move freely. Catheters were recovered after 7 days and demonstrated significant reductions in both colonisation and urinary tract infection.
In summary, the Novo project thus demonstrated the great potential of the developed novel approaches for biofilm prevention on urinary catheters. The next steps towards a product would be larger scale production of functionalised catheters allowing clinical trials with concomitant detailed economic evaluation. In parallel, alternative applications as outlined below for the developed antimicrobial functionalization strategies will be evaluated.
Project Context and Objectives:
Requirements and Specifications
Biofilms are bacterial communities encased in a self-produced hydrated polymeric matrix. More than 50% of bacterial infections are caused by bacterial biofilms. Catheter-associated urinary tract infections (UTI) alone account for approximately 40% of all hospital acquired infections. The aim of NOVO had been to provide a coating for catheters which can prolong the in vivo use by effectively reducing the biofilm formation. The strategy was to use different approaches alone and in combination and consequently in the first phase the requirements and specifications were defined for:
- sonochemical process for biocidal metal oxide and metal fluoride nanoparticle coating
- antibiofouling protective coatings based on bio-inert hydrogels
- enzymatic process based on enzymatic oxidation of phenolics leading to biocidal coatings
- process using enzymes i.e. cellobiose dehydrogenase (CDH) in suited coating for biocidal activity
- methodology and tests for coating efficiency and the enzymatic processes
- functional effect of a successful coating in terms of bio-compatibility, durability and bio-activity
These requirements and specifications were then used as guidelines in the following work packages.
Sonochemical coating of inorganic antibacterial agents
The main task of the project was to coat latex and silicon catheters with the antibacterial ZnO and MgF2 nanoparticles. The coating was achieved using the sonochemical technique.
By using the sonochemical technique a chemical reaction occur under ultrasound irradiation. The reaction is dependent on the development of an acoustic bubble in the solution. In the process that follows the creation of the bubble (acoustic cavitation), the bubble expands and compresses until it eventually collapses. The extreme conditions (temperature and pressure) that develop when the bubbles collapse, cause the chemical reactions to occur. In addition when the bubble collapses near a solid surface microjets moving at a very high speed (500 m/sec.) are formed. They move towards the solid surface. The microjets throw the newly formed particles onto the surface forming a very stable coating. The formation of coating was analyzed by electron microscopy techniques. The analysis of the elements present on the catheter was done by ICP as well as EDS. Using the sonochemical technique a very homogenous coating was obtained on the full catheter, the amount of coating was ~ 0.05% of the catheter weight. The ZnO coated catheters were tested for antibiofilm activity using a laboratory model of the bladder and in artificial urine solution, a reduction of more than 50% in the biofilm mass was observed. The remaining 50% biofilm was composed of mostly dead cells. The same system was also used for detection of leaching (removal of NPs) from the surface. Following incubation for seven days, no leaching of NPs was observed. Complementary functionalization with tailored protective inert polymers with a diblock structure comprising PDMS anchor block and PEG or polyzwitterionic functional blocks and grafting methods (adsorption/entrapment) had been established and also successfully used in combination with NP coating.
Several additional antibiofilm approaches were tested. They include:ultrasound assisted immobilization of antibacterial enzymes (Amyalse and Cellobiose dehydrogenase). Both enzymes showed excellent antibiofilm activity, the combined coating of the two enzymes, showed a synergistic effect (higher biofilm reduction was observed).
The mechanical properties of the catheter were tested to probe if the sonochemical coating process affect the catheter. No mechanical change was observed. The coating was tested for durability under sterilization process (by γ-irradiation and by ethylene oxide) no effect on the coating was observed. Toxicity tests of the coated catheters were performed using several cell lines (epithelial, fibroblast) - cell morphology and viability remained unchanged. Finally the sonochemical coating process was up-scaled to coat simultaneously several catheters.
Enzymatic based polymerization of oxidized phenolics as antibacterial coating
Urinary polyurethane and silicone catheters were coated with poly(catechin) and poly(catechin)-antibiotic conjugates to induce reduction of bacteria adhesion onto the devices surface. Laccase was used as biocatalyst to oxidize the catechin and catechin-antibiotic conjugates monomers and produce the corresponding polymers. Four antimicrobial coatings were produced, namely with poly(catechin), poly(catechin)-trimethoprim, poly(catechin)-sulfamethoxazole and poly(catechin)-trimethoprim-sulfamethoxazole. The bacterial adhesion reduction was tested on the functionalized devices using gram-negative and gram-positive strains. The most significant reduction in adhesion was observed with poly(catechin)-trimethoprim (gram-negative: - 85% and gram-positive: - 87%) and with poly(catechin)-trimethoprim-sulfamethoxazole (gram-negative: - 85% and gram-positive: - 91%). The cytotoxicity to mammalian cells was tested by indirect contact for 5 days and revealed that all the tested coatings supported more than 90% of viable cells. A promising approach for the increase of the indwelling catheters life-span was developed aiming to reduce catheter-associated chronic infections.
Enzymatic biofilm prevention by CDH
The main goal of this work package was to explore the possibility of producing urinary catheter with antimicrobial and antibiofilm properties using an the enzyme Cellobiose Dehydrogenase (CDH). CDH is an enzyme that is able to produce hydrogen peroxide (a strong antimicrobial agent) using various cello- and oligosaccharides as electron donors. The antimicrobial and antibiofilm effect of the free enzyme was assessed against clinical isolates commonly found colonizing catheters and causing infections. The CDH proved to be very effective both in the prevention of biofilms and in the killing of bacteria already embedded in biofilms. The enzyme was successfully immobilized on the surface of urinary catheters using different techniques (ultrasound, layer by layer and covalent binding) of which the covalent immobilization lead to the highest amount of enzyme deposited on the surface. The bound enzyme was able to produce hydrogen peroxide in concentrations sufficient to inhibit biofilm formation on the coated surface by up to 70% when tested against Staphylococcus aureus. Moreover, two other enzymes, namely amylase and acylase were investigated concerning their antimicrobial and antibiofilm properties. Amylase is an enzyme capable of hydrolyzing sugars present in the biofilm matrix and acylase interferes with the cell to cell communication of bacteria. They showed strong antibiofilm activities in free form and were successfully immobilized on urinary catheters using layer by layer technique retaining their antibiofilm activities. Complementary functionalizations with tailored protective inert polymers comprising cationic anchor groups and zwitterionic functional groups and a coating method (polyelectrolyte layer-by-layer) had been established and also successfully used for enzyme immobilization. All three investigated approaches show a great potential for developing urinary catheters with antimicrobial and antibiofilm properties since they are stable in synthetic urine over long periods of time and don´t show any signs of detaching from the surface. Further, the developed enzyme based coating didn´t show any signs of cytotoxicity and therefore could be a valuable alternative to solving the increasing catheter associated infections problems.
Validation: up scaling and in vivo tests of the novel coatings
Upscaling was performed primarily on silicone catheters given that these have the highest existing tolerance to biofilm formation. The coating was performed in bulk in a 10L Sonication vessel with stirring capabilities. 20 silicone catheters were produced in the F8 size for in vivo studies. The metal oxide used was Zinc oxide, as this coating demonstrated to be highly active on common genitourinary bacteria. The next step was to demonstrate that the coatings were not toxic to cells. This was done using cultured cells from the epithelium and the immune system. Having established that the catheters were both effective on bacteria, and harmless to human cells, we set out to test them in an in vivo model. The standard model is the New Zealand white rabbit. We developed a well-tolerated method to catheterize the animals without need for restraint allowing them to move freely. Catheters were recovered after 7 days and demonstrated significant reductions in both colonisation and urinary tract infection, reaching the goal specified.
Project Results:
Requirements and Specifications
Biofilms are bacterial communities encased in a self-produced hydrated polymeric matrix. More than 50% of bacterial infections are caused by bacterial biofilms. Catheter-associated urinary tract infections (UTI) alone account for approximately 40% of all hospital acquired infections. The aim of NOVO had been to provide a coating for catheter which can prolong the in vivo use by effectively reducing the biofilm formation. The strategy was to use different approaches alone and in combination and consequently in the first phase the requirements and specifications were defined for:
- sonochemical process for biocidal metal oxide and metal fluoride nanoparticle coating
- antibiofouling protective coatings based on bio-inert polymeric hydrogels
- enzymatic process based on enzymatic oxidation of phenolics leading to biocidal coatings
- process using enzymes cellobiose dehydrogenase (CDH) in suited coating for biocidal activity
- methodology and tests for coating efficiency and the enzymatic processes
- functional effect of a successful coating in terms of bio-compatibility, durability and bio-activity
Sonochemical process. ZnO and MgF2 nanoparticles are known to have intrinsic antibiofilm properties and were selected for this project. Sonochemistry was used for preparing coatings using two different approaches, i) the in situ method, and ii) the “throwing stones” method. The most important parameter is nanoparticle size; the optimal size had been identified in the range of 50–100 nm. The morphology and particle size will be probed by high resolution scanning electron microscopy, and complementary methods for quantification (e.g. atom absorption spectroscopy) were also identified. The coating amount and the particle size can be controlled during the sonochemical reactions by varying different reaction parameters such as: i) reagents and their concentration, ii) time of sonication, iii) intensity of sonication, or iv) temperature.
Bio-inert polymeric hydrogels. In order to minimize biofouling in long term applications, ultrathin surface-attached hydrophilic polymer (hydrogel) layers will be created which will largely reduce protein adsorption and bacteria adhesion. A “grafting-to” approach called adsorption/entrapment was focused. This method is a two-step process where the polymeric base material (silicone) is swelled with a compatible solvent containing the polymeric modifier (“protective inert polymer”; PIP), followed by a deswelling in water where the PIP is entrapped on the surface of the silicone. The method is well compatible with surface modification by ultrasound; the preferred version would be an integration of both modifications in one step. The modifiers will be designed as block co-polymers, containing two (or more) different blocks: the anchor block (to be entrapped into the silicone) and the functional block (facing outwards). Polyethylene oxide (PEO) and zwitterionic polymers containing sulfobetaine were chosen as functional blocks. Polydimethylsiloxane (PDMS), the catheter material, and polypropylene oxide (PPO) were used as anchors blocks. Various strategies for polymer synthesis were developed and the requirements to surface modification methods and evaluation of modified surfaces were defined (main chemical methods: contact angle and zeta potential analyses).
Enzymatic oxidation of phenolics. Possible substrates for the formation of biocidal coatings by enzymatic oxidation of phenolics have been identified: i) catechin, a flavan-3-ol, a type of natural phenol and antioxidant; ii) ellagic acid, a natural phenol antioxidant found in numerous fruits and vegetables; iii) tannic acid, a complex substance, usually extracted from plants; iv) galangin, a flavonol which has been shown to slow the increase and growth of tumor cells in vitro; v) apigenin, a natural product belonging to the flavone class that is the aglycone of several naturally occurring glycosides.
Cellobiose dehydrogenase (CDH). A mutant CDH with increased activity for hydrogen peroxide (H2O2) production has been established based on a wild-type enzyme from Myriococcum thermophilum and can be produced in larger amounts. The increased oxygen reactivity had been verified by different methods. The fermentation process was adapted from the Pichia Fermentation Process Guidelines (Invitrogen) and optimized during a lot of fermentation trials. Two assays for CDH enzymatic activity were proposed because that is crucial for the evaluation of CDH coated catheters: i) the 2,6-dichlorophenolindophenol assay, and ii) the analysis of H2O2 production with the AMPLEX Red Assay.
Amylase and acylase with antibiofilm activity were also identified. The action of acylase over model quorum sensing molecules and the α-amylase activity upon model exopolysaccharides were assayed. Both enzymes were able to inhibit the static biofilm formation of medically relevant bacteria on polymer surfaces.
For both enzyme groups, CDH and amylase/acylase, methods for immobilization on catheter surfaces shall be developed. The focus is using polyelectrolyte “layer-by-layer” technique where the enzymes are co-assembled with polyelectrolytes. PIPs can also be used as designed building blocks for such coating techniques. Another strategy is the combination of the sonochemical modification with immobilization of CDH and/or PIPs.
Anti-biofilm coating activity shall be demonstrated by microscopic images using Live/Dead kit cell viability staining and quantitative total biomass determination using crystal violet. Coating efficiency is also evaluated by using the methods developed for characterization of the individual modifications.
The functional effect of a successful coating must not be in conflict with the already established property profile:
• A catheter must be physically stable enough to allow insertion.
• A catheter should allow a reasonable flow of urine
• The balloon should inflate reliably,
• The balloon should deflate reliably, and return to a minimal size, even after a long time
• No parts of the catheter should break off, even after a long period of in-dwelling
• It should be easy to withdraw the catheter
• A catheter should not induce a local inflammatory reaction
• A catheter should not induce allergies, such that there is a massive reaction the second time they are used.
• A catheter should not be contaminated with foreign materials (particles, oil, etc.) or packaging remains
• A catheter should not leach plasticizers into the body
Established standard tests will be used to assess those properties, and list of the respective guidelines (ASTMA, ISO, EN etc.) had been compiled. Functional effects will be determined at four general levels:
- at a physical level (i.e. composition, surface charge, hydrophilicity)
- in vitro biological (impact on mammalian cells or on bacteria)
- in vivo in animals (resistance to infection, including artificial infection)
- in clinical practice (clinical outcomes, patient welfare, reduction in antibiotic use)
If the stated problem is an excess in biofilm formation, one may measure biofilm. However, such physical measures may be functional, but not be directly related to an actual economically quantifiable problem. Thus, they are easy to measure but difficult to convert into a probable economic gain. Similarly, stating the full economic problem as the functional goal, i.e. a reduction in the cost of urinary tract infections due to catheters by half, while precise, is extremely difficult to test without large scale blind trials. In this context, one has to accept that a model will have finite degree of translation to the clinical setting. The question then becomes which surrogate of the clinical problem is an acceptable indicator.
All these requirements and specifications were then used as guidelines in the following work packages.
(WP2) Sonochemical coating of inorganic antibacterial agents
In the current part the different parameters such as time of the sonication process, output power, and initial reagent concentration were evaluated for their effect on coating morphology and quality. The optimal initial reagent concentration of Zn(CH3COO)2 for the formation of uniform coating with ZnO nanoparticles was found to be 0.1M (figure 2.1). Coating of silicone catheters with magnesium fluoride NPs was also achieved. ZnO and MgF2 coated catheters were tested for their antibiofilm activity; the reduction in the biofilm mass was calculated by crystal violet (CV) staining. The purple dye is attached to the polysaccharide components of the biofilm (Figure 2.2) enabling visualization of the biofilm. For quantification, the dye is removed from the catheter and its amount is measured via spectrophotometer. The results showed that both MgF2 and ZnO coatings, showed excellent antibiofilm activity by reducing 70% and 60%of the biofilm mass, respectively. Moreover, it was observed that the cells that were able to attach to the surface were killed by the active coating.
In addition to the special chemistry of the coated catheter, causing their strong antibiofilm activity, the formation of nano-rough surface (Figure 2.3) also contributed to the inhibition in biofilm formation. The rigid bacteria cell is not able to attach to the nano-rough surface and to form biofilm.
The catheters that underwent the sonochemical process were tested for changes in their mechanical properties- and were found unchanged. Sterilization with Gama –irradiation or by ethylene oxide was found not to harm the coating. The leaching of the coated NPs from the catheters was tested in a lab scale experiment and on an artificial bladder model. Leaching of NPs was not observed, some ions (up to 15% of their content in the coating) were detected in the leaching solution (Figure 2.4).
.
Figure 2.4 Leaching of Zn+2 from ZnO coated catheters
The sonochemical coating was up-scaled – to be able to coat several catheters simultaneously. In an experiment in which 5 catheters were coated simultaneously, all the catheters were coated with a uniform coating (see Figure 2.5 for example of coating on 2 catheters). The weight of the coating was ~ 0.05% of the catheter weight. The catheters continued to show excellent antibiofilm activity.
Figure 2.5 ZnO coating on catheters from up scaled reaction (left mage catheter number 1, right image catheter number 5).
Finally, the antibiofilm activity of the coated catheters were tested in-vivo by using a rabbit model, the coated catheters showed good antibiofilm activity. The significant result of the rabbit experiment is that the rabbits that were treated with the coated catheters showed less urine contamination.
Enhanced antifouling activity by grafted protective inert polymers (PIP)
An extension of antibiofilm activity was achieved by the functionalization with grafted protective inert polymers (PIP) with hydrophilic moieties to protect the antibiofouling activity. Commercially available amphiphilic triblock copolymers Pluronics and a newly designed and synthesized PIP comprising a hydrophilic functional block and a hydrophobic anchor block which is compatible with the catheter material (PDMS) were used.
Figure 2.6 Synthesis of the novel amphiphilic diblock copolymer with PDMS as anchor block and poly-zwitterions as functional block
Three novel PIPs with different degree of polymerization of the zwitterionic block, i.e. different chain length (block size), were synthesized and tested. The structure was determined via NMR, GPC, elemental analysis and IR. With these PIPs the silicone catheter material was successfully modified via the non-covalent adsorption/entrapment mechanisms. The contact angle measurements showed that the surface became significantly more hydrophilic and remained unchanged for longer than 10 days. For all tested PIPs, the same contact angle was achieved and no trend for a better prevention against biofilm was found with the longer chains of zwitterion.
Figure 2.7 Results of contact angle measurements for silicon (PDMS) samples after adsorption/entrapment modification and storing over time in synthetic urine
The observation was also confirmed by zeta potential measurements which proved the coverage of the PDMS surface with a hydrophilic, overall neutral polymer.
Figure 2.8 Results of zeta-potential measurements for samples after adsorption/entrapment modification, exemplary for the smallest PIP (PDMS-b-PSPE20)
The biofilm formation was tested using the Alamar blue assay.
Figure 2.9 Result of the biofilm formation assay using Alamar blue
For the tube material a small antifouling effect in comparison to the control sample namely a reduction between 23 and 31% was found. But this result could not be confirmed with the balloon material. However, this antifouling strategy was used in combination with the “throwing stones” method with ZnO nanoparticles. After the coating with NPs the samples were modified via adsorption/entrapment with the novel PIPs. After this combined modification the samples were much more hydrophilic compared to each individual modification alone. On the other hand, biofilm assays showed that introduction of the PIP reduced the antibiofilm activity of the ZnO NPs. Protective inert polymers were applied to the ZnO coated catheters to protect the coating and thus enhance its activity. However, our results showed that introduction of the PIP reduced the antibiofilm activity of the ZnO NPs.
Synergistic activity of several active (enzymes) coatings
Another part of our work was to test the synergistic activity of several active coatings. Two enzymes (CDH and amylase) were successfully applied to the catheter surface using sonochemistry. The coated CDH retained its specific activity and produced the antibacterial H2O2. The immobilized enzyme showed excellent antibiofilm activity. OXY enzyme coating reduced the biofilm amount by more than 50%, moreover, the surviving biofilm mass showed the presence of mostly dead cells (Figure 2.10). The combination of the CDH with amylase further increased the biofilm inhibition to 65%.
Figure 2.10 Antibiofilm activity of CDH enzyme
Enzymatic based polymerization of oxidized phenolics as antibacterial coating
The remarkable antimicrobial properties of phenolic compounds as flavonoids make them an interesting alternative to the conventional antibiotic treatments. Flavonoids, a class of natural products that possess a diverse range of pharmacological properties, have been studied as active agents to prevent biofilm formation with promising results. The position and number of hydroxyl groups on these phenolic compounds were found to be related to their antimicrobial activity. The phenolic hydroxyl groups are thought to react with enzymes and inhibit various metabolic processes, while the hydrophilicity conferred by the hydroxyl groups is also critical for their ability to pass through bacterial cell walls. Prevention of biofilm formation can be achieved either by killing or repelling bacteria, with steric and electrostatic repulsion being involved in the latter process. Moreover, the polyphenols become negatively charged and reactive at pH greater than 7.5 increasing electrostatic repulsion of bacteria. Besides, the use of these polyphenols in combination with other antimicrobial agents has also been cited as a successful practice to fight the drug resistance problem. The synergistic effect provided by both may potentiate the antibiotic efficacy at lower concentrations decreasing adverse reactions.
Catechins are polyphenols susceptible to enzymatic and non-enzymatic oxidations, giving rise to a variety of dimeric, oligomeric and polymeric products. As o-dihydroxylated compounds, catechins are potential substrates for laccase, an oxidative enzyme able to oxidize a wide range of substrates as phenolic compound, their derivates and aromatic amines. Aiming to generate new antibiofilm molecules that can prolong the life-span of urinary catheters, the laccase-catalyzed oxidation of catechin was studied during this project for the production of polymeric antimicrobials (Fig.3.1) that can be useful to coat these medical devices and prevent the bacterial colonization onto the corresponding surfaces.
Fig. 3.1: Schematic representation of poly(catechin) production through laccase oxidation.
In addition, the in situ enzymatically oxidation of catechin-antibiotic conjugates by laccase (Fig.3.2) for catheters coating was also assessed in this project aiming potentiate the bacterial adhesion reduction onto the functionalized catheters.
Fig. 3.2: In situ laccase functionalization of polyurethane (PU) and silicone (SI) catheters using catechin-TMP and/or catechin-SMZ conjugates (TMP: trimethoprim; SMZ: sulfamethoxazole).
Overall, from this research resulted four novel antimicrobial coatings for medical devices. Taking the advantage of poly(catechin) and antibiotics properties, antimicrobial conjugates were designed, and the obtained synergism allowed the inhibition of planktonic bacteria growth and the bacterial adhesion reduction onto the coated surfaces (Fig.3.3).
Fig. 3.3: Inhibition of planktonic bacteria growth (left) and bacterial adhesion reduction (right) resulted from the poly(catechin) (PCAT) and poly(catechin)-antibiotic conjugates action.
This synergism was more evident on poly(catechin)-TMP and poly(catechin)-TMP-SMZ where a higher bacteria reduction was observed. Moreover, the cellular metabolism assays demonstrate that the antimicrobial coatings developed are not harmful for the cellular metabolism, maintaining more than 90 % of cell viability even after the highest incubation time tested (Fig.3.4).
Fig. 3.4: Relative L929 cell viability after 24, 48 and 72 hours of culture with DMEM solutions pre-inoculated with functionalized polyurethane (A-C) and silicone (D-F) catheters for 1, 3 and 5 days. Non-coated sample was used as CONTROL. Poly(catechin) (PCAT), poly(catechin)-trimethoprim (PCAT-TMP), poly(catechin)-sulfamethoxazole (PCAT-SMZ) and poly(catechin)-trimethoprim-sulfamethoxazole (PCAT-TMP-SMZ) were the coated samples. Data were achieved in relation to the control for 1 incubation day and show the mean values of three replicates from four experiments carried out independently. Statistical significant differences are indicated. *** = significantly different from the control (P < 0.001); ** = significantly different from the control (P < 0.01); * = significantly different from control (P < 0.05)
Therefore, a promising approach for extending the life-span of indwelling catheters was developed during this project and, concomitantly, the often catheter-associated infections could be reduced and the life quality of catheterized patients improved.
Enzymatic biofilm prevention by CDH
Another approach pursued in this project was the biofilm prevention/inhibition with the help of enzymes. Three different classes of enzymes were used: Cellobiose Dehydrogenase, producing the antimicrobial agent hydrogen peroxide, Amylase, an enzyme capable of hydrolysing sugars present in the biofilm matrix and Acylase, an enzyme that interferes with the cell to cell communication of bacteria. All three enzymes were first tested concerning their antimicrobial and antibiofilm properties in free form against various gram positive and gram negative bacteria. Once their antimicrobial effectiveness was proven several strategies were implied in order to deposit them on the surface of silicone urinary catheters. The main focus was set on the enzyme Cellobiose Dehydrogenase (CDH). The first step was to engineer and produce a genetic version that is able to produce high amounts of hydrogen peroxide in the presence of poly- and oligosaccharides and oxygen. This enzyme was then tested against gram positive and gram negative clinical isolates commonly causing catheter associated urinary tract infection in order to exploit its potential use as an antimicrobial enzyme. Results showed that CDH has a broad spectrum antimicrobial activity and that only very low amounts of substrate (which are consequently converted to hydrogen peroxide) are needed to be antimicrobially active against planktonic bacteria (Figure 4.1) and bacteria already embedded in biofilms (Figure 4.2) and is therefore a promising candidate for the use in antimicrobial coatings.
Figure 4 1: Inhibition of growth of S. aureus growing in liquid cultures and on agar plates in the presence of varying substrate concentrations (Cellobiose)
One possible application that was exploited is the incorporation of CDH into lubricant, commonly used for the gentle introduction of urinary catheters. Figure 4.2 shows that CDH is still active when incorporated into lubricants and even acts on bacteria already embedded in biofilms. It is therefore a promising candidate for the use in antimicrobial coatings.
A B C
Figure 4. 2: LIVE/DEAD staining images of S. aureus biofilms formed on silicone sheets: (A) positive control, incubated at 65°C for 1 h, (B) negative control, untreated biofilm, (C) biofilm treated with CDH and Cellobiose
The next step was the covalently bind the enzyme to the silicone urinary catheters surface. A five step protocol was developed (Figure 4.3) yielding a surface capable of producing high concentrations of the antimicrobial agent hydrogen peroxide.
Figure 4.3: Stepwise immobilization of CDH onto PDMS surfaces; (1) plasma treatment, (2) grafting APTES,(3) grafting glutaraldehyde and (4) grafting the enzyme
The newly created surface was tested concerning its antimicrobial activity, long term stability and biocompatibility. The enzyme deposited on the surface was able to reduce S. aureus biofilm formation by up to 70% when incubated for short periods (3 hours) as can be seen on the microscopic images in Figure 4.3 and longer periods (7 days).
Figure 4.4: Atomic force microscopy images of S. aureus biofilms deposited on uncoated (left) and CDH coated (right) silicone surfaces within 3 hours
The CDH coated silicone catheters were also incubated in artificial urine for 16 days at 37 °C in order to test their long term stability in a realistic environment. The enzyme was able to retain 20% of its activity over the entire period which shows again that this system is robust enough to apply it in the coating of the urinary catheters. In a last step the biocompatibility of the antimicrobial surface was tested with the help of two mammalian cell lines. The surface showed no signs of cytotoxicity at substrate concentrations needed by the enzyme to show antimicrobial activity. In summary it can be stated that all the results presented here make this approach a very interesting and valuable alternative to already existing coatings to fight catheter associated urinary tract infections.
Another immobilization of the enzymes was tested via polyelectrolyte “Layer-by-Layer”-deposition. Here novel PIPs were developed which have antifouling properties and can be used as polymeric building block for layer-by-Layer modification. The idea for the novel PIP is to combine segments with quaternized amino groups for electrostatic assembly and those with zwitterionic groups to achieved antifouling properties. PIPs with different fraction of zwitterionic segments (0%, 25% and 50% of zwitterions) were synthesized and completely characterized with NMR, IR, GPC and elementary analysis. For the layer-by-layer-deposition the novel PIPs were used as polycation and poly(4 styrenesulfonat) (PSS) as polyanion.
Figure 4.5: Schematic representation of silicone surface functionalization with cationic and zwitterionic PIPs via layer-by-layer deposition
Due to the low isoelectric point, the enzyme CDH was assembled as polyanion in one of the layers. After three more layers the enzyme was firmly embedded and the outer layer was a polycation with the zwitterionic segements for antifouling. This modification was successfully analyzed on a model system (silicon wafer) and on both catheter materials.
Figure 4.6: Contact angle measurement after modification with 6 layers of PIP, PSS and CDH over 10 days, here exemplary for tube material
After the deposition the surface become more hydrophilic than the unmodified base material. Because the contact angle remained constant over the time it seemed that the modification is stable in synthetic urine for about 10 days. The zetapotential of the last layer of a six-layer system with immobilized enzymes showed clearly the difference between the three PIPs with different zwitterionic fraction.
Figure 4.7: Zeta-potential of LbL-modified silicone with PIP with different zwitterion fraction on the outer surface, here exemplary for tube material
Figure 4.7 clearly showed a shift of the isoelectric point, which means that the surface becomes more neutral with more zwitterionic groups in the cationic PIP layer. The biological studies showed a very high enzyme activity with the enzyme on top of the layer. For the embedded enzyme the results were not very clear. Perhaps during the drying process the surface changed the structure, so that the diffusion of the substrate to the immobilized enzyme was hindered and reduced the enzyme activity. This conclusion can be supported by results obtained with atomic force microscopy. Further microscopic analyses with live/dead kit indicated improvements of the antifouling properties of the modified surfaces.
Figure 4.8: Microscopic analyzes with the live/dead staining, exemplary for tube material
Figure 4.8 shows a very low antibiofouling activity of the enzymes. For the control sample and the sample without zwitterions there was a very uniform distribution of individual colonies which were fairly close together. For the samples with zwitterions there were more irregular cluster formations, but generally less bacteria growth. These measurements show clearly that the zwitterion had a positive effect for antifouling.
The third approach included the immobilization of two other antibiofilm enzymes, namly amylase and acylase. Acylase inactivates acyl homoserine lactone (AHL) signals of Gram-negative bacteria, suggesting the enzyme application as antibiofilm coating on indwelling medical devices. Coatings comprising the Quorum sensing (QS) disrupting enzyme acylase were built on silicone urinary catheters using a Layer-by-Layer (LbL) deposition technique. The LbL assembly was achieved by alternate deposition of negatively charged enzyme and positively charged polyethyleneimine. After immobilization on silicone surface, the enzyme biological activity and stability were evaluated. The acylase-coated catheters efficiently quenched the QS process in C. violaceum through the degradation of AHLs in the extracellular environment, demonstrated by 50 % decrease of QS-regulated violacein production. Moreover, the QQ activity of the surface attached acylase was similar to what was observed for the free enzyme, suggesting that the approach used to construct the coatings could preserve the protein folding and activity (Figure 4.9).
Figure 4.9: Violacein production by C. violaceum in presence of free acylase solution and acylase multilayer coatings on silicone catheters. The control - untreated silicone was set as 100 % violacein production.
Furthermore, the potential of acylase-coated urinary catheters to inhibit QS-regulated biofilm formation was assessed in dynamic conditions using a catheterized bladder model, where the shear stress and flow rate vary similarly to the situation in the human bladder during catheterization. P. aeruginosa biofilm was allowed to grow for 7 days and then the biofilm formation on acylase-based coating was analyzed using Live/Dead cells viability kit. The fluorescence images of live (stained in green) and dead (stained in red) bacteria attached to the balloon and catheter shaft (called urethra) demonstrated significant reduction of P. aeruginosa biofilm, when compared to pristine Foley catheters (Figure 4.10A). Quantitatively, the total biofilm mass was decreased by 80 % and 45 % on the acylase-coated balloon and urethra, respectively (Figure 4.10B). Therefore, such enzyme-based approach relying on disruption of QS process via an enzymatic inactivation of AHLs in the extracellular environment could be a viable alternative to the antibiotics for controlling the biofilm occurrence on indwelling medical devices. While antibiotics kill or inhibit bacteria by targeting their structure or survival processes, interfering with QS pathways is thought to exert less selective pressure on bacterial population reducing the threat of resistance emergence.
Figure 4.10: A) Microscopic images of untreated and acylase coated Foley catheters after 7 days incubation in dynamic bladder model system (x40 magnification). B) Crystal violet assessment of total biomass formed on urinary catheters: untreated and acylase multilayer coatings on silicone Foley catheter
To increase the antibiofilm spectrum of the enzyme-coated catheters, an integrated biofilm inhibition strategy based on acylase and α-amylase able to interfere simultaneously with bacterial QS phenomenon and biofilm matrix, respectively, was investigated. Acylase would affect the AHLs based QS systems increasing the biofilm susceptibility to the amylase, which would exert its activity once the bacteria is attached and the polymeric matrix produced. The QQ acylase and matrix degrading α-amylase were individually and simultaneously deposited on urinary catheters in a LbL fashion. After ten sequential deposition steps on silicone, the hybrid coatings (amylase and acylase) showed higher antibiofilm activity against single- (P. aeruginosa) and duals-species (P. aeruginosa and E. coli) biofilm development, when compared to the enzymes applied individually on the surface (Figure 4.11). Interestingly, the biofilm was significantly reduced when the QQ enzyme was the outermost layer. Such outcomes could be related with the natural sequence of biofilm development – from bacterial communication to irreversible surface attachment
Figure 4.11: Inhibition of single (P. aeruginosa) and dual (P. aeruginosa and E. coli) species biofilm formation on enzyme coated silicone urinary catheters assessed with crystal violet.
The hybrid coatings with better efficiency in inhibiting mono- and dual-species biofilm formation in static conditions (Figure 4.11) were further subjected to dynamic biofilm inhibition tests using an in vitro model of catheterized human bladder. Although, the different growth conditions in the model system the obtained results were comparable to that observed in static conditions. The total biofilm mass of single- and mixed- biofilms was significantly reduced on the LbL Hyb-Acy urethra parts of the catheters (Figure 4.12). Therefore, such integrated strategy might provide efficient control of single and multi-species biofilm formation on urinary catheters.
Figure 4.12: Fluorescence microscopy images of 7 days grown biofilms on enzyme coated silicone catheters analyzed after Live/Dead kit staining.
Validation: up scaling and in vivo tests of the novel coatings
The goal of the project is to deliver a viable product candidate. To this end, coating of catheters with ZnO was scaled up such that sufficient material was available for a panel of studies on safety and efficacy aspects. In vitro, studies were performed on antibacterial effect, and these data have been reported from earlier work packages. Similar studies were conducted with human epithelial and immune cells. For cell studies, the catheter surfaces were cut from scale up product and placed in a sterilizing ethanol bath, and then in culture with the cells. Cell density was assessed as the primary end point.
Figure 5.1. Hospital derived samples of the bacterium Acinetobacter baumanii were tested for sensitivity to various concentrations of ZnO nanoparticles, in this case, one can see that the bacteria grow well (dark blue line) in the absence of particles, but that at any concentration of particles, there is a reduction in growth.
Figure 5.2 Cultured U937 cells (immune derived) are able to grow to equal densities at equal rates irrespective of the coating density on silicone.
Figure 5.3 Cultured RAW 254 cells (immune derived, macrophage like) are not inhibited by the coating of ZnO on silicone.
Given that the two pre-conditions for further testing were met by the ZnO catheters, namely in vitro efficacy and biocompatibility, the catheters were further tested using an in vivo model. Catheters (ZnO and polyphenol) were placed in animals under anaesthesia and left in place for 7 days. Urine was sampled daily and at termination, catheters were recovered for biofilm evaluation. Treated catheters were generally less likely to have high levels of bacteria or biofilm.
Figure 5.4 Plates with different densities of bacteria from urine samples. Medium changes from green to yellow with changes in pH associated with bacterial metabolism. Low bacterial density left, vs. high right.
Figure 5.5 Micrographs of catheter balloons after removal showing signs of biofilm. Left, the uncoated control silicone catheter, vs. right the ZnO coated material where more base silicone is visible.
Figure 5.6 Micrographs of catheter balloons after removal showing signs of biofilm stained with crystal violet. Left, the uncoated control silicone catheter, vs. right the ZnO coated material.
Figure 5.7. The viable counts of bacteria in urine samples from catheterised rabbits. In this case, the mean is presented with N=8. Differences were significant to p>0.05 on all days after day 3 which is consistent with the normal course of infection in this model.
The ZnO coated catheters were active in both reduction of apparent biofilm on the balloon and other parts of the catheter, but also in reducing the rate with which bacteruria developed in the bulk urine. The rabbit model is a good correlate to the rates of infection development in people and these data are suggestive of a positive effect of the coating. In combination with the other observations, we conclude that the objective of reducing the rate of infection development by half has been achieved. Given that the coating process is scalable and technically cost effective, the next step in development is the estimation of clinical benefit and what, if any price difference could be justified for catheters incorporating this technology.
Potential Impact:
1) The potential impact
The NOVO project developed novel approaches (coatings with anti-fouling NPs, (poly) phenolics, specific CDH) to control biofilms on medical implants. In particular, the strategies applied within NOVO involve modalities that are completely un-related to the modes of action employed by current drugs. Thus, they prevent/degrade biofilm formation using effects that are not counteracted by the resistance mechanism that have arisen following selection by current drugs. The mechanisms employed in NOVO, therefore, provide for a means to attack infections that resist existing therapies.
The multidisciplinary consortium combined knowledge and resources of leading academics, hospitals, and manufactures of enzymes and plastic indwelling urinary systems -catheters (SMEs).
This project outputs have a potential to be successfully translated into clinical practice, introducing the novel breakthrough approach for anti-biofilm protection/destruction strategies in medical implants, resulting in better healthcare for the citizens along with reducing the cost of healthcare. NOVO’s technological platform can be implemented further for the biofilm prevention in wound dressings or as direct wound disinfectants, and for the non-medical products, facing the problem of biofilm formation, such as sewage pipes, pipelines of the offshore oil and gas industry, water treatment membranes et cetera.
1.1 Clinical and Scientific Impact
Main scientific and technological breakthroughs:
A user friendly and low cost one step ultrasound process for coating different antibiofouling agents (inorganic, organic and biologic):
• Inorganic: A single-step (US based) metal oxides and fluorides nanoparticles coating on latex/silicon surfaces.
• Organic: (poly)phenolics’.
• Enzymatic (CDH) coating with biofilm degradation activity (based on peroxide).
Simultaneous co-coating of a protective bio-inert polymer and anti-biofilm agents to protect their activity
General anti-biofilm strategies
While the technologies outlined within the project are for implants, there is good reason to expect that these technologies can be employed also in topical formulations for wounds. It is expected to be able to apply these procedures and anti-microbial agents, in slightly modified form for wound dressings and as direct topical disinfectants for colonized wound surfaces. In certain examples, there is also the potential for systemic application of biofilm specific polysaccharide binding enzymes to clear infections of heart valves and other difficult to clear sites.
The scientific impact is broader than its implementation in the project’s application:
The novel, single step, environmentally friendly US proposed nano-technological process can be implemented in different applications to avoid biofilm formation (e.g. water treatment membranes, antimicrobial textiles, etc).
The enzymatic processes could be also implemented in the above mentioned applications.
The protective coating is a novel and unique suggestion to protect the activity of the antibiofouling agents.
1.2 Socio-Economic impact
Medical implants and devices-associated infections
Infection associated with implantable devices such indwelling medical devices (e.g. contact lenses, central venous catheters and needleless connectors, urinary catheters, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes and voice prostheses) is serious problem as it means prolonged hospital admittance for the patient and possible multiple surgeries. The case-fatality rate for patients with ventilator-associated pneumonia is 42 percent, with an attributable mortality of 15 to 30 percent. For nosocomial bloodstream infection, the case fatality rate is 14 percent, with an estimated attributable mortality of 19 percent.
Avoiding infection is highly important for the patient's well-being as well as to minimize extended cost to the healthcare system. US Studies show that a post-surgical wound infection more than doubles the patient's hospital costs, with Staphylococcus aureus (Golden Staph) responsible for a trebling of patient's hospital costs.
The overall annual direct medical costs of healthcare-associated infections to U.S. hospitals ranges from $35.7 billion to $45 billion in 2007. With a 70 percent of effectiveness of possible infection control interventions, the benefits of prevention can be up to $31.5 billion for US only.
Catheter-associated urinary tract infection
Urinary tract infections are the most common type of healthcare-associated infection, accounting for more than 30% of infections reported by acute care hospitals. Virtually all healthcare-associated UTIs are caused by instrumentation of the urinary tract. Reported hospital wide prevalence rates for indwelling catheterization vary from 25% to 35%. Catheter-associated urinary tract infection (CAUTI) has been associated with increased morbidity, mortality, hospital cost, and length of stay.
CAUTI creates significant costs for the individual facility and the health care system at large. The cost of treating a single episode varies from $980 to $2900 depending on the presence of associated bacteremia. Cumulatively, CAUTI adds an additional 90 000 hospital days per year. With a collective annual cost of $424 million to $451 million in the United States alone.
In addition, bacteriuria commonly leads to unnecessary antimicrobial use, and urinary drainage systems are often reservoirs for multidrug-resistant bacteria and a source of transmission to other patients.
Moreover, from October, 2008, hospitals in USA no longer reimbursed for costs associated with nosocomial infections, giving them an incentive to reduce their frequency.
NOVO’s output will impact the entire implants related treatments. While implementing the developed technologies to different implants (one of the major causes of hospital-acquired infections), patients’ infections incidences will be significantly reduced and the usage time of the implants will be enlarged. NOVO will thus have a remarkable impact on the European citizens’ health care, economy and quality of life.
Employment
While the economy is adding jobs at lower levels than workers would like, during the last two years, analysts expect buds of growth in a wide range of service jobs this year-retail, information technology, professional, scientific and technical jobs, as well as continuing growth in the health-care industry.
With the aging population, health care remains the go-to field for job growth. "Health care is always adding jobs. That will clearly continue.
Among companies that expect to increase full-time, permanent workers, the top areas, by function, are: sales, information technology, customer service, engineering, technology, administrative, business development, marketing, research/development and accounting/finance.
NOVO is at the intersection of the above mentioned discipline thus having a positive impacting the employment in these fields.
Quality of life
At present, the vast majority of infections are associated with simple procedures like catheterization. In so far as a catheter is reliably and robustly more biocompatible, it will save significant numbers of procedures and nosicomial infections. In particular, it will be important within the growing aged care market where such procedures are increasingly common and where costs are correspondingly increasing.
The formation of biofilms affects sensitivity and resistance to antibacterial and antifungal drugs and therefore represents a clinical problem affecting the quality of life of the patients. By the newly developed effective methods to prevent the biofilm formation or destroy it, when adopted in practice, the quality of life will be enhanced since changing implants will be at a lower frequency, the antibiotics will be more effective thus lower doses will be applied for treatment. Furthermore the hospitalization periods will be shorter due to more effective treatments.
2) Main dissemination activities and exploitation of results
a. Dissemination
The NOVO consortium has allocated substantial efforts and resources for the purpose of dissemination of the project results within the scientific community and in the general public. The main efforts and dissemination activities are presented below.
Project Brochure:
OSM and BOKU have developed and produced the project brochure. NOVO Project Brochure was designed as a dissemination tool for the project. The brochure contains a general description of the project. The informative brochure promotes and describes the vision of the project, main objectives and outcomes as well as the project layout. The brochure also contains information about the project partners and their contact info. The brochure can be downloaded via the NOVO website domain and as prints during relevant conferences, meetings and workshops.
Website construction and maintenance
The NOVO website (http://www.fp7-novo.eu/) was launched by OSM on August 2012 and is updated regularly. The website is library of all the project documents including all deliverables and presentations from the meetings. The website contains two main sections (1) The public section which aims to introduce the NOVO consortium, project concept, objectives and vision; (2) The partners’ restricted section which offers an access to project records, e.g. submitted documents, deliverables, and meetings updated information including partners presentations and minutes. Every partner has received a private user and login password.
Scientific publications by NOVO consortium partners
BOKU:
1. Thallinger, B., Nugroho Prasetyo, Nyanhongo, G.S. Guebitz, G.M. Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms. Biotechnol J. 2013 Jan ;8(1):97-109. doi: 10.1002/biot.201200313.
2. Thallinger B, Argirova M, Lesseva M, Ludwig R, Sygmund C, Schlick A, Nyanhongo SG, Guebitz MG. 2014. Preventing microbial colonisation of catheters: Antimicrobial and antibiofilm activities of cellobiose dehydrogenase. 44:402–408 International Journal of Antimicrobial Agents, doi: 10.1016/j.ijantimicag.2014.06.016.
BOKU/UPC
1. Thallinger B, Brandauer B., Burger P., Sygmund C., Ludwig R., Ivanova K., Scaini D., Kun J., Burnet M., Tzanov T., Nyanhongo G., Guebitz G.M. Cellobiose dehydrogenase functionalized urinary catheter as novel antibofilm system” International Journal of Antimicrobial Agents (2014),
BIU
1. Upscaling sonochemical process for catheter coating with antibiofilm nanoparticles (in preparation)
BOKU/BIU
2. Lipovsky A., Thallinger B. Perelshtein I., Nyanhongo G., Guebitz GM., Gedanken A., Ultrasound assisted coating of PDMS with H2O2 producing enzymes, Langmuir, submitted (2015).
UDE:
1. Anne Vaterrodt, Mathias Ulbricht , Abstract (P3-121) in conference book of with the title “Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification” European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013;
TEC
1. I.Gonçalves, T. Matamá, A. Cavaco-Paulo, C. Silva (2013) Laccase coating of catheters with poly(catechin) for biofilm reduction, Biocatalysis and Biotransformation, Published Online (DOI: 10.3109/10242422.2013.828711). 2014, 32(1):2-12
2. I.Gonçalves, A. Abreu, T. Matamá, A. Ribeiro, A. Gomes, C. Silva, A. Cavaco-Paulo (2014) Enzymatic synthesis of poly(catechin)-antibiotic conjugates: an antimicrobial approach for indwelling catheters, Applied Microbiology and Biotechnology, Published Online (DOI:10.1007/s00253-014-6128-2)
3. I.Gonçalves, C. Silva, A. Cavaco-Paulo (2014) Ultrasound enhanced laccase applications, Green Chemistry, Published Online, DOI: 10.1039/C4GC02221A http://xlink.rsc.org/?doi=C4GC02221A
4. I. Gonçalves, C. Botelho, A. Teixeira, A. S. Abreu, L. Hilliou, C. Silva, A. Cavaco-Paulo, Antimicrobial lubricant formulations containing poly(hydroxybenzene)-trimethoprim conjugates synthesized by tyrosinase, Applied Microbiology and Biotechnology (2015) (submitted).
5. Polyphenol-antibiotic coating of urinary catheters. Idalina Gonçalves, Ana S. Abreu, Teresa Matamá, Carla Silva, Artur Cavaco-Paulo (in preparation).
UPC
1. Fernandes M M, Francesko A, Torrent-Burgués J, Tzanov T (2012), Effect of thiol-functionalisation on chitosan antibacterial activity: interaction with a bacterial membrane model. Reactive and Functional Polymers, Doi: 10.1016/j.reactfunctpolym.2013.01.004
2. Fernandes M. M, Francesko A, Torrent-Burgués J, Javier Carrión-Fité F, Heinze T, Tzanov T (2014), Sonochemically processed cationic nanocapsules: efficient antimicrobials with membrane disturbing capacity. Biomacromolecules, Doi: 10.1021/bm4018947 (Published)
3. Diaz Blanco C, Ortner A, Dimitrov R, Navarro A, Mendoza E, Tzanov T (2014), Building an antifouling zwitterionic coating on urinary catheters using an enzymatically triggered bottom-up approach, ACS Appl. Mater. Interfaces, DOI: 10.1021/am501961b. (Published)
4. Ivanova K, Fernandes M M, Mendonza E, Tzanov T (2015), Enzyme multilayer coatings inhibit Pseudomonas aeruginosa biofilm formation on urinary catheters. Appl Microbiology and Biotechnology, Doi: 10.1007/s00253-015-6378-7.
EMI
1. Leseva M., Arguirova M., Nashev D., Zamfirova E., Hadzhyiski O.: Annals of Burns and Fire Disasters - vol. XXVI - n. 1 - March 2013 Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control.
2. Leseva M., Arguirova M., Zamfirova E.: Study of the biofilm forming ability of microorganisms isolated from urine of catheterized patients. Proceedings of the XII National Congress in clinical microbiology and infections, p. 41, Sofia, Bulgaria, 25 Apr, 2014.
SYN:
Enzymatically modified hydrogels for prevention and treatment of biofilm on catheters. Guezguez, Pietrzik, Hahn, Burnet and potential partner inventors Patent application, in preparation
Presentations and posters at Conferences by NOVO Consortium partners:
BOKU:
1. Novel anti-biofilm system based on cellobiose dehydrogenase, Thallinger B., Schlick A., Nugroho Prasetyo E., Sygmund C., Ludwig R., Nyanhongo G.S. and Guebitz G.M. ÖGMBT Graz , (2012) (poster)
2. Multifunctional bioresponsive polymers for the management of chronic wounds, Gibson Nyanhongo, Endry Nugroho Prasetyo, Georg Guebitz, E-MRS 2012 Fall Meeting, , E-MRS 2012 Fall Meeting, September 17-21, 2012, Warsaw, Poland.(oral presentation)
3. Generating a new antimicrobial PDMS surface using cellobiose hydrogenase, Thallinger B., Schlick A., Brandauer M., Nyanhongo G., Sygmund C., Ludwig R. and Gübitz G.M , EPF2013 Pisa:
4. Immobilization of Cellobiose Dehydrogenase as an antibiofilm agent on silicone catheters, Thallinger, B., Sygmund, C, Brandauer, M.a Schlick, A., Ludwig, R, Nyanhongo, G. and Gübitz, G. Eurobiofilms 2013, Ghent, Belgium (poster)
5. Novel anti-biofilm system based on the degradation of Exopolysaccharides, Thallinger, B., Sygmund, C., Brandauer, M., Schlick, A., Nyanhongo, G. and Gübitz, G. , FEMS 2013, Leipzig Germany
6. Cellobiose Dehydrogenase- antimicrobial functionalization of polydimethylsiloxane, Thallinger, B., Brandauer, M., Schlick, A., Ludwig, R., Sygmund, C., Nyanhongo, G. and Gübitz, G , IPTB 2014, Braga Portugal (oral presentation)
7. Cellobiose Dehydrogenase- antimicrobial functionalization of polydimethylsiloxane, Thallinger Barbara, Brandauer Martin, Schlick Angelika, Ludwig Roland, Sygmund, Christoph, Nyanhongo Gibson1 and Gübitz Georg, 2nd DocDay Tulln, Tulln Austria (oral presentation).
8. Modification of PDMS with Cellobiose Dehydrogenase yielding an antimicrobial surface. Thallinger, B.a Brandauer, M.a Schlick, A.a Burger P.a Ludwig, R.b Sygmund, C.b Nyanhongo, G.a and Guebitz, G.M ÖGMBT 2014, Vienna Austria (oral presentation)
9. Novel enzymatic antimicrobial and anti-biofilm systemThallinger, A. Schlick, C. Sygmund, R. Ludwig, G.S. Nyanhongo and G.M. Guebitz, ICAAR 2014, Madrid Spain (poster).
UPC
1. Novel strategies for prevention of pathogenic bacterial biofilm formation on indwelling medical devices. T. Tzanov, International Conference on Antimicrobial Research- ICAR2012, Lisbon, Portugal November 21-23, 2012 (oral presentation).
2. A dual polyphenolic/zwitterionic coating prevents microbial biofilm formation on urinary catheters. T. Tzanov 11th E-MRS Fall meeting September 17th-21st, 2012 Warsaw, Poland (oral presentation).
3. Thiolation effect on the antibacterial activity of a low molecular weight chitosan: interactions with a bacterial membrane model. T Tzanov E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
4. Bioactive multilayer coatings for prevention of bacterial biofilm formation on indwelling medical devices. T. Tzanov E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
5. Enzymatic-induced degradation of bacterial biofilms. T. Tzanov T E-MRS 2012 Fall Meeting September 17-21, 2012 Warsaw, Poland (oral presentation).
6. Nanostructured multilayer coatings with antibiofilm activity for biomedical applications Tzanov T, Fernandes M M, Gamerith C, Ivanova K 245th ACS National Meeting & Exposition April 7-11, 2013 New Orleans, Louisiana, USA
7. Nanobiopolymers: efficient antimicrobials with membrane disturbant capacity.Fernandes M M, Francesko A, Torrent J, Tzanov T. 245th ACS National Meeting & Exposition April 7-11, 2013 New Orleans, Louisiana, USA
8. Enzymes to control bacterial biofilm formation, T Tzanov, 245th ACS National Meeting & Exposition, New Orleans, Louisiana, USA., April 7-11, 2013 (poster).
9. Combined antimicrobial/antifouling coatings using zwitterion-decorated phenolic nano-capsules Díaz Blanco C, Francesko A, Fernandes M M, Tzanov T. 245th ACS National Meeting & Exposition. April 7-11, 2013 New Orleans, Louisiana, USA (poster)
10. Industry-driven "bio-research". T. Tzanov, I Jornada de la recerca i la innovació tecnològica al Campus de la UPC a Terrassa, Terrassa,Spain, June, 2013 (oral communication).
11. Three examples of industry-university international collaborations, T. Tzanov, Jornada d’exposició de projectes per la visibilització de la innovació (Grups de recerca + empreses), Terrassa,Spain, June, 2013 (oral communication).
12. Enzyme-based nanoparticles to inhibit bacterial biofilm formation in urinary catheters Ivanova K, Fernandes M. M, Tzanov T 8th International Conference on Polymer and Fiber Biotechnology – IPFB 2014 May 25-27, 2014, Braga, Portugal (oral presentation).
13. Sonochemically processed cationic nanobiopolymers - efficient antimicrobials with membrane disturbant capacity, Fernandes M. M, Francesko A, Tzanov T, 8th International Conference on Polymer and Fiber Biotechnology – IPFB 2014, May 25-27, 2014 Braga, Portugal (oral presentation.)
14. Building a dual antimicrobial/antifouling coating on urinary catheters using a laccase-triggered bottom-up approach, Diaz Blanco C, Tzanov T, 7th International Congress on Biocatalysis - Biocat2014, August 31st - September 4th, 2014, Hamburg, Germany (oral presentation)
15. Enzyme multilayer coatings inhibit quorum sensing-regulated Pseudomonas aeruginosa biofilm formation on silicone urinary catheters, Ivanova K, Fernandes M M, Tzanov T, III International Conference on Antimicrobial Research - ICAR2014, October 1-3, 2014, Madrid, Spain. (oral presentation).
16. Enzyme multilayer coatings inhibit quorum sensing-regulated Pseudomonas aeruginosa biofilm formation on silicone urinary catheters, T. Tzankov, III International Conference on Antimicrobial Research - ICAR2014, Madrid, Spain. October 1-3, 2014 (oral presentation).
17. Convectional antibiotics in form of nanospheres prevent biofilm formation and provide infection control, Fernandes M. M, Ivanova K, Francesko A, Tzanov T, III International Conference on Antimicrobial Research - ICAR2014,October 1-3, 2014, Madrid, Spain. (oral presentation).
UDE
18. Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification, Anne Vaterrodt, Mathias Ulbricht, 29th European Membrane Society (EMS) Summer School, Essen, 22-26 July, 2013 Poster number P-57.
19. Abstract (P3-121) in conference book of the EPF Conference, “Silicon-based amphiphilic diblock copolymers for antibiofouling surface modification” Anne Vaterrodt, Mathias Ulbricht, European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013
20. Synthesis of amphiphilic diblock-copolymer via ATRP polymerization for antibiofouling functionalizable surface modification” - Anne Vaterrodt, Mathias Ulbricht European Polymer Federation (EPF) Conference, Pisa, Italy; 16 21 June 2013
21. Novel antifouling surface functionalizations using polymeric zwitterions via adsorption/entrapment and layer-by-layer deposition, Anne Vaterrodt, Mathias Ulbricht , IPTB 2014 Braga.
BIU
1. "Novel methods for the fabrication of Nanomateirals and their Application" Lecture at NCKU, Tainan, Taiwan, February 2012
2. "Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Coatings on Textiles and Glasses" Lecture at the University of Milano Bicocca, April 4 2012.
3. "Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Coatings on Textiles and Glasses" Lecture at Palacky University, RCPTM, Olomouc, Czech Republic, May 12, 2012.
4. "Novel methods for the fabrication of Nanomateirals and their Application" 2 lectures in EuCheMS-SCI School on “ Synthesis and Characterization of Novel Nano-Sized InorganicMaterials” 17-22 June, 2012, Villa Larocca and Department of Chemistry, Campus Universitario, Bari
5. "Novel methods for the fabrication of Nanomateirals and their Application" 2 Lectures in ASON-2 Dubrovnik, Croatia, September 3-7, 2012.
6. "Anything you can do I can do better" What can be done better with Sonochemistry? Plenary Lecture at NANOCON conference in Brno, The Czech Republic, October 23, 2012.
7. "Novel methods for the fabrication of Nanomateirals and their Application" lecture before the Graduate students of the Department of Mater. Sci. &Eng. of NCKU, Tainan, Taiwan Nov. 23, 2012
8. "Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Nanoparticles and their Sonochemical Coating on Surfaces (Textiles and Glasses)" Lecture at Comenius University, Bratislava, Slovakia. January 30, 2013.
9. "Anything you can do I can do better" What can be done better using Sonochemistry? A lecture before the 78th meeting of the Israeli Chemical Society, Tel-Aviv, February 12, 2013.
10. "Anti Bacterial, Anti Viral, Anti biofilms and Antifungal Nanoparticles and their Sonochemical Coating on Surfaces (Textiles and Glasses)" Lecture at Comenius University, Bratislava, Slovakia. January 30, 2013.
11. "Anything you can do I can do better" What can be done better using Sonochemistry? A lecture before the 78th meeting of the Israeli Chemical Society, Tel-Aviv, February 12, 2013.
12. "The sonochemical coating of textiles with antibacterial nanoparticles" Lecture at the 23 IFATCC Conference in Budapest, Hungary, May 09, 2013
13. "Coating Antibacterial Nanoparticles on Flat and Curved Surfaces and fighting Resistant Bacteria Empolying the Sonochemical Method" Lecture at the AOSS-2013 conference in Melbourne, Australia July 12, 2013.
14. "What is Sonochemistry? Emphasis on Coating a Large Variety of Surfaces by the Sonochemical Methods: Anti Bacterial, Anti Viral, Anti Biofilms Coatings on Textiles and Glasses" Lecture at the University of Cagliari Sardinia, Italy, July 24, 2013.
15. "Coating antibacterial NPs on flat and curved Surfaces" ANBRE13, Seoul, Korea, August 26, 2013.
16. "Coating Antibacterial Nanoparticles on Flat and Curved Surfaces and fighting Resistant Bacteria Empolying the Sonochemical Method" NAMF meeting a section of the EMRS fall meeting at WARSAW, Poland September 17, 2013.
17. "Antibacterial, Antiviral, Anti Biofilm, and antifungi NPs and their Sonochemical Coatings on Surfaces" NANOVED conference , Svit, Slovakia, September 23, 2013.
18. "Coating Anti Bacterial, Anti Viral, Antibiofilm and Antifungi Nanoparticles on Flat and Curved Surfaces Employing the Sonochemical Method" Polaris workshop, Porto, Portugal, October 9, 2013.
19. "Coating antibacterial NPs on flat and curved Surfaces" Plenary lecture at the ICAFM conference at Trivandrum , India, Feb. 20, 2014.
20. "Coating antibacterial NPs on flat and curved Surfaces" Goa University, India, Feb. 24, 2014.
21. "Sonochemical coating" Lecture at MIRDC. Kaohsiung Taiwan March 5, 2014.
22. "Coating Anti Bacterial, Anti Viral, Antibiofilm and Antifungi Nanoparticles on Flat and Curved Surfaces Employing the Sonochemical Method" Lecture at Tzhjiang University Madical Hospital Number 2, April 28, Hangzhou, China.
23. “Making the Hospital a Safer Place by the Sonochemical Coating of the Textiles and Other Medical Devices with Antibacterial, Antibiofilm, Antiviral and Antigungal nanoparticles” Lecture in the ESS (European Society on Sonochemistry)14 in Avignon, France June June 6, 2014.
24. “Making the Hospital a Safer Place by the Sonochemical Coating of the Textiles and Other Medical Devices with Antibacterial, Antibiofilm, Antiviral and Antigungal nanoparticles” Lecture at the 6th PCGMR conference in Tainan, Taiwan on September 3, 2014.
25. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” a Plenary lecture at Ultrasonics 2014, in Caparica, Portugal on Sept. 15, 2014.
26. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the Capital Med Univ, Dept Pharmacol, Beijing 100069, Peoples R China, Nov. 10, 2014.
27. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at DGIST, Dept Energy Syst Engn, Taegu 711873, South Korea. On November 11, 2014.
28. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at Samsung Division of thin films, Tsuwon, Korea on November 12, 2014.
29. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the MOLMED 2014 conference at Haikou, China, Nov. 14, 2014.
30. “Making the Hospital a Safer Place by the Sonochemical Coating of all the Textiles and Other Medical Devices with Antibacterial, and Antibiofilm, nanoparticles” Lecture at the NANOTEK -2014 San Francisco, December 3, 2014
TEC
1. Laccase coating of catheters with poly(catechin) for biofilm reduction, Oxizymes 2014, July 1-4, 2014, Vienna, Austria.
2. Enzymatic oxidation of phenolic compounds: a green strategy to enhance antimicrobial properties in urinary catheters, 248th ACS National Meeting & Exposition, August 10-14, 2014, San Francisco, USA.
EMI
1. Vacheva-Dobrevska R., I.Ivanov M.Leseva et al.: Emergence and persistance of Proteus mirabilis carrying VIM-1 metallo-beta-lactamases and carbapenem-resistant Klebsiella pneumoniae in Bulgarian Hospitals; XII National Congress in clinical microbiology and infections of BAM, 24-26 April 2014,
2. Leseva M., M. Arguirova et al.: A prospective study of the biofilm forming ability of microorganisms isolated from urine of catheterized patients with severe burns; XII National Congress in clinical microbiology and infections, 25 Apr, 2014, Symposium of scientific articles (oral presentation),
3. Chalashkanov Tz., M. Leseva, E. Zamfirova, A. Ivanov: Emergence and control on spread of multiple resistant strains of Klebsiella pneumoniae in three clinics of UMHATEM “Pirogov”, XII National Congress in clinical microbiology and infections, 24-26 April, 2014
4. Leseva M., A. Ivanov, E. Zamfirova, Tz. Chalashkanov: Current problems in nosocomial infections and antimicrobial resistance in UMHATEM “N.I. Pirogov”. Symposium dedicated to the European Antibiotic Awareness Day. Bulgaria, Kostenets, 19-20 November, 2014.
5. Chalashkanov Tz., M. Leseva et all.: Dynamics of nosocomial infections in the Trauma Clinics of UMHATEM “N.I. Pirogov”, Ist Trauma Symposium, 20-22 Nov., 2014, Sofia.
Media broadcast
UPC:
1. Oral communication addressed to academic audience. Title: Antimicrobial biotechnology Speaker: Tzanko Tzanov http://www.terrassa.upc.edu/noticies/biotecnologia-antimicrobiana-al-campus-de-la-upc-terrassa Live-broadcasted at UPCtv: http://tv.upc.edu/ Date: 06/03/2013
2. Oral communication addressed to academic and industrial audience 13/06/2013; Title: Industry-driven "bio-research“ Speaker: Tzanko Tzanov
3. Oral communication addressed to academic and industrial audience 09/10/2013; Title: Three examples of industry-university international collaborations, Speaker: Tzanko Tzanov
b. Exploitation strategy and activities
i) The markets of indwelling medical devices
Catheterization procedures have steadily increased as interventional and less-invasive procedures have become more popular. Catheters are vital to the completion of many procedures, and some procedures or interventions, particularly in cardiovascular surgery and neurosurgery, could not be done without the use of catheters.
The global catheter market amounted is expected to reach $32.1 billion in 2014, for a 5-year compound annual growth rate (CAGR) of 12.3% .
• The US and EU market’s for cardiovascular segment (includes: pacemakers, implantable cardioverter-defibrillators (ICDs), cardiac resynchronization therapy devices (CRTs), cardiac leads, external defibrillators, ablation catheters, intracardiac echocardiography (ICE) catheters and diagnostic electrophysiology (EP) catheters) was valuated at $10.6 billion in 2010 and it is projected to rise at a CAGR of 10.2%.
• The second-largest segment is urology, with expected to reach $13.2 billion in 2014, for the highest CAGR among all segments at 17.1%.89
• The US and EU market’s for peripheral vascular devices (includes: PTA balloon catheters, embolic protection devices, stent-grafts, surgical grafts, inferior vena cava filters, diagnostic catheters, interventional catheters, diagnostic guidewires, hydrophilic guidewires, Peripheral vascular closure devices) is valuated at $3 billion at 2010 .
Large diversity of indwelling implants is widely used by modern medicine.
• The global orthopedic implants market is forecast to grow to $41.8 billion by 2016 at the Compounded Annual Growth Rate (CAGR) rate of 7.8% during 2009-2016 .
• The worldwide market for cochlear implants valued at $1.59 billion in 2012, a 22 percent compound annual growth rate .
• The global dental implants market is expected to grow from $3.2 billion in 2010 to $4.2 billion in 2015 at a CAGR of 6% from 2010 to 2015. Europe currently forms the world’s largest market for dental implants with a 42% market share, and is also expected to have the highest CAGR 7.0% from 2010 to 2015 .
This potential huge market is the one targeted by the NOVO project’s outputs – thus it will positively influence the medical large and SMEs industries, in Europe.
The hospitalization periods will decrease due to the improved treatment, thus lowering health care cost and increasing working days.
ii) Exploitation by the R&D centers
The research organizations and universities involved in the project produced scientific and technological results of commercial value and will be involved in their further exploitation both directly and indirectly.
The direct output of this project (a) protected by patents and by granting licenses to the relevant industries for its exploitation (or establish spin-off)
(b) Increasing their leadership in their respective areas of research on a global scale.
iii) Exploitation by the SMEs
Main advantages of new products:
1. High market demand
2. Use of FDA approved components (MgF2, ZnO)
3. Unique US based one-stage method for anti-biofilm layer anchoring to the polymer
4. Combinatorial approach –preventive and bacteriostatic/ bactericidal anti-biofilm coating in one product
5. Relatively competitive price -it is estimated that the cost of the new catheters will be only 10% higher than the existing products. This estimation is based on Life Cycle Cost assessments done in SONO FP7 project for coating textiles with antibacterial agents
6. Potential use for non-medical markets.(e.g. water purification membranes)
Main envisaged obstacles:
1. Time to market
2. Competition.
3. Price
The SMEs in the consortium will develop strategies based on the outputs of the project with the aim to introduce the new proven antifouling technology to the market.
List of Websites:
Project website address: http://www.fp7-novo.eu/
List of partners
Par.
No. Name of principal Investigator Organization legal name Acronym Country email
1. Prof. Georg Guebitz University of Natural Resources and Life Scienes BOKU AT guebitz@boku.ac.at
2. Prof. Aharon Gedanken Bar-Ilan University BIU IL gedanken@mail.biu.ac.il
3. Prof. Tzanov Tzanko Universitat Politècnica de Catalunya UPC ES tzanko.tzanov@upc.edu
4. Prof. Mathias Ulbricht University of Duisburg-Essen UDE DE mathias.ulbricht@uni-due.de
5. Prof. Dr Maya Argirova Emergency Medicine Institute "Pirogov". EMI BG maya_arguirova@yahoo.com
6. Michael Burnet Synovo GmbH SYN DE Michael.Burnet@synovo.com
7. Prof. Artur Cavaco TECMINHO TEC PT artur@deb.uminho.pt
8. Dr. Andreas Paar Qualizyme Biotechnology QUA AT office@qualizyme.com
9. Assunção Mascarenhas PRONEFRO,S.A. PRO PT assuncao@pronefro.pt
10. Oded Stein Degania Silicon Ltd. DEG IL oded-s@ds-il.com
11. Dr. Pnina Dan OSM-DAN Ltd. OSM IL pninadan@osmdan.com