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Novel coatings to prevent biofilm formation on urinary catheters based on nanoantibiotics and quorum quenching compounds

Final Report Summary - NANOQUENCH (Novel coatings to prevent biofilm formation on urinary catheters based on nanoantibiotics and quorum quenching compounds)

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide, inflicting a substantial financial burden on heath and social care system. In Europe, UTIs represent at least 40 % of all hospital acquired infections and are, in the majority of cases, catheter associated1. Bacteriuria develops in up to 25 % of patients who require a urinary catheter for one week or more2. The recent Global Prevalence Infection in Urology (GPIU) studies have shown that 10-12 % of patients hospitalized in urological areas has a healthcare-associated infection3. The primary cause of these infections is the biofilm that is formed on the surface of catheters once bacteria are attached. The inherent resistance of established biofilms to antibiotics and the host immune system poses serious problems when the treatment of these infections is required. There is a need to provide novel efficient strategies to prevent biofilm formation in successful prophylaxis of such infections.
NanoQuench addressed this concern by developing novel strategies to eradicate Gram-negative bacteria biofilms (from Escherichia coli and Pseudomonas aeruginosa) formed either on polystyrene surfaces (proof of concept) or on silicone-based strips and catheters. The approach relied on the creation of a platform able to: i) disrupt the bacterial quorum sensing (QS) - a mechanism that allows bacteria to communicate through specific signals called autoinducers (AIs) and set up pathogenesis and biofilm growth - using quorum quenching (QQ) compounds that degrade the AIs; and ii) kill susceptible bacteria using less concentration of a well-known, but physically modified antibiotics (nanoantibiotics). The main advantage of this approach is that QQ compounds attenuates virulenceand the nanoantibioticcs are effective at lower dosage and against bacteria with low susceptibility, therefore exerting less selective pressure on bacteria and reducing the risk of resistance development.
The main objectives proposed by Nanoquench and the resulting research achievements are outlined below:
- Identify the major bacteria found in UTI patients (WP1): P. aeruginosa was defined as the main target pathogen in NanoQuench because it could be isolated from a high number of catheterized patients with urinary tract infections (UTI) (collaboration Pirogov Hospital in Sofia, Bulgaria and a previous FP7 project NOVO – “Novel approaches for prevention of pathogenic bacteria biofilms formed on medical indwelling devices, e.g. catheters” FP7-278402.
- Recognise suitable enzymes for quenching bacterial QS and antibiotics for nanotransformation (WP1): Acylase was selected as the main QQ enzyme to be used in the project due to the fact that P. aeruginosa secretes QS signals that are easily degraded by the enzyme5. Regarding antibiotics, the susceptibility of P. aeruginosa to various antibiotics was performed and vancomycin and penicillin G were selected and further processed into nanospheres. P. aeruginosa possessed low susceptibility to these antibiotics and thus were set as the perfect candidates to study the expected boosting effect caused by the nanotransformation.
- Prove the concept of QQ enzyme and nanoantibiotics as antibacterial and antibiofilm agents (WP2): Acylase was proven to successfully degrade model QS signals and to inhibit the P. aeruginosa biofilm in more than 80 %5. Moreover, nanospheres of biopolymer (aminocellulose and thiolated chitosan) and antibiotics were successfully generated using a single step sonochemical method and were found to be more effective in killing gram-negative bacteria than their non-processed derivatives. The study of the interaction between the nanospheres and a bacterial membrane model, using a Langmuir monolayer technique, indicated that the high disturbance of the membrane caused by the spheres ruled the mechanism of action of the nanoantibiotics and nanobiopolymers as an antibacterial agent4,6,7.
- Develop multilayered coatings comprising QQ enzymes and/or nanoantibiotics (WP3/WP4): In order to develop antibacterial/antibiofilm surfaces in silicone-based catheters, silicone strips were first used to facilitate testing of the surfaces. The strips were provided by a company specialized in the manufacture of silicone catheters (Degania Medical, Israel). These were successfully pre-functionalized using (3-Aminopropyl)triethoxysilane to render the surface a positive charge and further coated with QQ acylase (polyanion) and polyethilenimine (polycation) using layer-by-layer technique. An efficient antibiofilm multilayer coating was built on these silicone strips first and then built on silicone catheters. The coatings were found to efficiently inhibit the adhesion of bacteria in the first hours and counteract the biofilm formation in more than 60 % after 24 h. Moreover, they were biologically and physically stable after contact with urine and PBS for 7 days as well as biocompatible after 7 days in direct contact with skin fibroblast5.
The combination of both QQ acylase and nanoantibiotics (nanovancomycin) was finally applied on silicone to attain an efficient antibacterial/antibiofilm surface that also avoids the development of bacteria resistance. The rationale for this relies on the knowledge that QQ acylase makes the biofilm more susceptible to antimicrobial compounds, but do not kill bacteria, which minimizes the emergence of resistant strains. On the other hand, nanoantibiotics are not recognized as a threat by the bacteria, thus diminishing the bacterial evolutionary stress and consequently the resistance to the coating.
First, vancomycin nanospheres (nanovancomycin) were proven to possess better antibacterial activity against P. aeruginosa when compared to vancomycin (as described above). The addition of acylase increased the susceptibility of P. aeruginosa proving that lower dosages are needed to eradicate bacteria. In fact, this combined strategy resulted in a diminution in culture medium of a virulence factor associated with bacterial QS. The virulence factor was the electrochemically active pyocianin, which was tracked by electrochemistry in P. aeruginosa culture medium using cyclic voltammetry and linear sweep voltammetry. The acylase and nanovancomycin were then applied on silicone using a LbL technique. The activity of acylase within the coatings containing the nanovancomycin was maintained, proved by the ability of the coating to inhibit in 50 % the QS violacein production in the biosensor strain Chromobacterium violaceum CECT 5999. Additionally, the P. aeruginosa biofilm formation on these coatings was inhibited in more than 70 % when compared to the control coatings7.

The strategies used in Nanoquench for the prevention of biofilm formation on urinary catheters involved modalities that are completely un-related to the modes of action employed by current drugs, thus it significantly improved the state-of-art in this research area. Further in vivo studies should be performed to allow reaching the commercialization of catheters with prolonged life-span while significantly reducing the risk for the development of urinary tract infections (UTI). If successful, the outcomes from Nanoquench will surely contribute to decrease both the patient’s trauma and the treatment cost.

References:
(1) Rüden, H.; Gastmeier, P.; Daschner, F. D.; Schumacher, M. Nosocomial and Community-Acquired Infections in Germany. Summary of the Results of the First National Prevalence Study (NIDEP). Infection 1997, 25 (4), 199–205.
(2) Maki, D. G.; Tambyah, P. A. Engineering out the Risk for Infection with Urinary Catheters. In Emerging Infectious Diseases; 2001; Vol. 7, pp 342–347.
(3) Johansen, T. E. B.; Çek, M.; Naber, K. G.; Stratchounski, L.; Svendsen, M. V.; Tenke, P. Hospital Acquired Urinary Tract Infections in Urology Departments: Pathogens, Susceptibility and Use of Antibiotics. Data from the PEP and PEAP-Studies. Int. J. Antimicrob. Agents 2006, 28 (SUPPL. 1), 91–107.
(4) Fernandes, MM; Francesko, A.; Torrent-Burgués, J.; Carrión-Fité, F. J.; Heinze, T.; Tzanov, T. Sonochemically Processed Cationic Nanocapsules: Efficient Antimicrobials with Membrane Disturbing Capacity. Biomacromolecules 2014, 15 (4), 1365–1374.
(5) Ivanova, K.; Fernandes, MM; Mendoza, E.; Tzanov, T. Enzyme Multilayer Coatings Inhibit Pseudomonas Aeruginosa Biofilm Formation on Urinary Catheters. Appl. Microbiol. Biotechnol. 2015, 99 (10), 4373–4385.
(6) Fernandes MM, Ivanova K, A, Rivera D, Torrent-Burgués J, Gedanken A, Tzanov T, Escherichia coli and Pseudomonas aeruginosa Eradication by Nano-sized Penicillin G, submitted to Advanced Healthcare Materials.
(7) Fernandes MM, Ivanova K, Tzanov T, Synergy between quorum quenching acylase and nanoantibiotics (nanovancomycin) for the prevention of Pseudomonas aeruginosa biofilms on urinary catheters, in preparation.