Kristina Ivanova

Quorum-Quenching and Matrix-Degrading Enzymes in Multilayer Coatings Synergistically Prevent Bacterial Biofilm Formation on Urinary Catheters

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

Bacteria-responsive multilayer coatings comprising polycationic nanospheres for bacteria biofilm prevention on urinary catheters

UNLABELLED This work reports on the development of infection-preventive coatings on silicone urinary catheters that contain in their structure and release on demand antibacterial polycationic nanospheres. Polycationic aminocellulose conjugate was first sonochemically processed into nanospheres to improve its antibacterial potential compared to the bulk conjugate in solution (ACSol). Afterward the processed aminocellulose nanospheres (ACNSs) were combined with the hyaluronic acid (HA) polyanion to build a layer-by-layer construct on silicone surfaces. Although the coating deposition was more effective when HA was coupled with ACSol than with ACNSs, the ACNSs-based coatings were thicker and displayed smoother surfaces due to the embedment of intact nanospheres. The antibacterial effect of ACNSs multilayers was 40% higher compared to ACSol coatings. This fact was further translated into more effective prevention of Pseudomonas aeruginosa biofilm formation. The coatings were stable in the absence of bacteria, whereas their disassembling occurred gradually during incubation with P. aeruginosa, and thus eradicate the biofilm upon release of antibacterial agents. Only 5 bilayers of HA/ACNSs were sufficient to prevent the biofilm formation, in contrast to the 10 bilayers of ACSol required to achieve the same effect. The antibiofilm efficiency of (HA/ACNSs)10 multilayer construct built on a Foley catheter was additionally validated under dynamic conditions using a model of the catheterized bladder in which the biofilm was grown during seven days. STATEMENT OF SIGNIFICANCE Antibacterial layer-by-layer coatings were fabricated on silicone that efficiently prevents Pseudomonas aeruginosa biofilm formation during time beyond the useful lifetime of the currently employed urinary catheters in medical practice. The coatings are composed of intact, highly antibacterial polycationic nanospheres processed from aminated cellulose and bacteria-degrading glycosaminoglycan hyaluronic acid. The importance of incorporating nanoscale structures within bacteria-responsive surface coatings to impart durable antibacterial and self-defensive properties to the medical indwelling devices is highlighted.

Cellobiose dehydrogenase functionalized urinary catheter as novel antibiofilm system

Urinary catheters expose patients to a high risk of acquiring nosocomial infections. To prevent this risk of infection, cellobiose dehydrogenase (CDH), an antimicrobial enzyme able to use various oligosaccharides as electron donors to produce hydrogen peroxide using oxygen as an electron acceptor, was covalently grafted onto plasma-activated urinary polydimethylsiloxane (PDMS) catheter surfaces. Successful immobilization of CDH on PDMS was confirmed by Fourier transformed-infrared spectrometry and production of H2 O2 . The CDH functionalized PDMS surfaces reduced the amount of viable Staphylococcus aureus by 60%, total biomass deposited on the surface by 30% and 70% of biofilm formation. The immobilized CDH was relatively stable in artificial urine over 16 days, retaining 20% of its initial activity. The CDH coated PDMS surface did not affect the growth and physiology of HEK 239 and RAW 264,7 mammalian cells. Therefore this new CDH functionalized catheter system shows great potential for solving the current problems associated with urinary catheters.

Escherichia coli and Pseudomonas aeruginosa eradication by nano-penicillin G

The transformation of penicillin G into nano/micro-sized spheres (nanopenicillin) using sonochemical technology was explored as a novel tool for the eradication of Gram-negative bacteria and their biofilms. Known by its effectiveness only against Gram-positive microorganisms, the penicillin G spherization boosted the inhibition of the Gram-negative Pseudomonas aeruginosa 10-fold (from 0.3 to 3.0 log-reduction) and additionally induced 1.2 log-reduction of Escherichia coli growth. The efficient penetration of the spheres within a Langmuir monolayer sustained the theory that nanopenicillin is able to cross the membrane and reach the periplasmic space in Gram-negative bacteria where they inhibit the β-lactam targets: the transferases that build the bacteria cell wall. Moreover, it considerably suppressed the growth of both bacterial biofilms on a medically relevant polystyrene surface, leaving majority of the adhered cells dead compared to the treatment with the non-processed penicillin G. Importantly, nanopenicillin was found innocuous towards human fibroblasts at the antibacterial-effective concentrations.

Strategies to prevent the occurrence of resistance against antibiotics by using advanced materials

Drug resistance occurrence is a global healthcare concern responsible for the increased morbidity and mortality in hospitals, time of hospitalisation and huge financial loss. The failure of the most antibiotics to kill “superbugs” poses the urgent need to develop innovative strategies aimed at not only controlling bacterial infection but also the spread of resistance. The prevention of pathogen host invasion by inhibiting bacterial virulence and biofilm formation, and the utilisation of bactericidal agents with different mode of action than classic antibiotics are the two most promising new alternative strategies to overcome antibiotic resistance. Based on these novel approaches, researchers are developing different advanced materials (nanoparticles, hydrogels and surface coatings) with novel antimicrobial properties. In this review, we summarise the recent advances in terms of engineered materials to prevent bacteria-resistant infections according to the antimicrobial strategies underlying their design.

Nanotransformation of Vancomycin Overcomes the Intrinsic Resistance of Gram-Negative Bacteria

The increased emergence of antibiotic-resistant bacteria is a growing public health concern, and although new drugs are constantly being sought, the pace of development is slow compared with the evolution and spread of multidrug-resistant species. In this study, we developed a novel broad-spectrum antimicrobial agent by simply transforming vancomycin into nanoform using sonochemistry. Vancomycin is a glycopeptide antibiotic largely used for the treatment of infections caused by Gram-positive bacteria but inefficient against Gram-negative species. The nanospherization extended its effect toward Gram-negative Escherichia coli and Pseudomonas aeruginosa, making these bacteria up to 10 and 100 times more sensitive to the antibiotic, respectively. The spheres were able to disrupt the outer membranes of these bacteria, overcoming their intrinsic resistance toward glycopeptides. The penetration of nanospheres into a Langmuir monolayer of bacterial membrane phospholipids confirmed the interaction of the nanoantibiotic with the membrane of E. coli cells, affecting their physical integrity, as further visualized by scanning electron microscopy. Such mechanism of antibacterial action is unlikely to induce mutations in the evolutionary conserved bacterial membrane, therefore reducing the possibility of acquiring resistance. Our results indicated that the nanotransformation of vancomycin could overcome the inherent resistance of Gram-negative bacteria toward this antibiotic and disrupt mature biofilms at antibacterial-effective concentrations.

Innovative Approaches for Controlling Clinically Relevant Biofilms: Current Trends and Future Prospects

Bacteria that colonize and form biofilms on living tissues and medical devices are a global healthcare concern. They cause life threatening infections and are associated with increased mortality and morbidity in the hospitals. Although antibiotics have been successfully applied for treatment of bacterial diseases, the adaptive and genetic changes of the microorganisms within the biofilms make them inherently resistant to all known antibacterial agents. Therefore, novel antimicrobial strategies that do not exert selective pressure on bacterial population and minimize the risk of resistance occurrence have been sought to prevent and treat biofilm related infections. A critical overview of the numerous groups and the rationale of advanced materials and surfaces with antibacterial and antibiofilm properties is the aim of this review. The development of antibiofilm coatings based on molecules interfering with bacterial cell-to-cell communication and biofilm integrity are discussed. Nano-scale transformation of obsolete antibiotics and surface functionalization with bacteriophages and natural antibacterials including enzymes, antimicrobial peptides, and polyphenols are also considered. Finally, recent efforts to design new generation of integrated antibacterial materials are reported.

Strategies for Silencing Bacterial Communication

Recent advances in microbiology have revealed that bacteria are able to communicate and cooperate in a wide range of multicellular behaviors such as dispersal, foraging and biofilm formation, in a process called quorum sensing (QS). In the QS-regulated communication, bacteria produce and secrete small signaling molecules – autoinducers that are recognised by specific receptors. Gram-negative bacteria secrete acyl-homoserine lactones (AHLs) that in threshold concentrations penetrate into the cells and activate cognate intracellular AHL receptors, while gram-positive bacteria produce autoinducing peptides that are detected by the cell membrane histidine kinase receptor. However, not all bacterial communication is species specific. Bacteria also possess a receptor for the signals sent out by other bacteria species. The knowledge about how this intra- and inter-species communication occurs has been increasingly used to develop new strategies for fighting infectious diseases. This chapter summarises recent advances for silencing cell-to-cell communication as a new approach for the prevention of bacterial diseases.

Inhibition of Quorum-Sensing: A New Paradigm in Controlling Bacterial Virulence and Biofilm Formation

Bacterial pathogens coordinate the expression of multiple virulence factors and formation of biofilms in cell density dependent manner, through a phenomenon named quorum sensing (QS). Protected in the biofilm community, bacterial cells resist the antibiotic treatment and host immune responses, ultimately resulting in difficult to treat infections. The high incidence of biofilm-related infections is a global concern related with increased morbidity and mortality in healthcare facilities, prolonged time of hospitalization and additional financial cost. This has led to the urgent need for innovative strategies to control bacterial diseases and drug resistance. In this review, we outline the disruption of QS pathways as a novel strategy for attenuation of bacterial virulence and prevention of resistant biofilms formation on medical devices and host tissues. Unlike the traditional antibiotics, inhibiting the QS signaling in bacteria will not kill the pathogen or affect its growth, but will block the targeted genes expression, making the cells less virulent and more vulnerable to host immune response and lower dosage of antimicrobials. We summarize the recent successes and failures in the development of novel anti-QS drugs as well as their application in controlling bacterial infections in healthcare facilities. The inhibitory targeting of the production of QS signals, their transduction and recognition by the other cells in the surrounding are discussed. Special focus is also given to the anti-QS nanomaterials with improved effectiveness and specificity towards the pathogens.

Antibacterial Coatings on Medical Devices

In the actual health-care threat of superbugs surge, the indwelling device-associated infections are a global concern for increased morbidity and mortality, prolonged hospitalization, and ultimately additional financial cost. The current treatment strategies of such recalcitrant infections are being impaired by not only the antibiotic resistant pathogens but also the risk to generate new resistant strains. To face this panorama, researchers are focusing on alternative strategies that while preventing the device-associated infections do not apply evolutionary pressure to evolve resistance mechanisms. Such strategies include disrupting bacterial cell-to-cell communication and biofilm integrity and developing antifouling surfaces or novel bactericidals. This chapter summarizes how researchers make use of different available technologies to translate these new antimicrobial concepts to real applications for fighting indwelling device-related infections.