NEW STRATEGIES BASED ON NATURAL COMPOUNDS FOR CONTROLLING BIOFILM FORMATION

Direct observations of a wide variety of natural habitats have established that 99% of microorganisms grow in form of biofilms, complex structured communities of microorganisms associated with surfaces and embedded in a self-produced extracellular polymeric substances. Biofilms are able to colonize every surface that offers minimal conditions for the life included all human artefact surfaces. Consequently, industrial installations, work benches, water distribution systems and also medical devices can be covered by biofilm with devastating consequences in terms of social and economic impact. The most detrimental property of bio\ufb01lms is that conventional biocidal practices often prove inadequate. Indeed, adopting the sessile mode of life, microorganisms improve their resistance to antimicrobial agents up to several orders of magnitude. In addition, increasingly restrictive regulations limiting the use of substances hazardous to human health and the environment, have resulted in several biocides being banned. In this contest the development of new improved effective solutions able to replace the presently dominating drug/device products is becoming imperative. An innovative approach could be the use of biocide-free anti-biofilm compounds with novel targets, unique modes of action and properties that are different from those of the currently used antimicrobials. Recently, the use of sub-lethal doses of bio-inspired molecules able to interfere with specific key steps that are needed to establish bio\ufb01lms have been proposed as an alternative strategy to prevent biofilm formation. Since this approach deprives microorganisms of their virulence properties without affecting their existence, the selection pressure for antibiotics and biocides resistance mutations decreases. In this contest, nature represents an immense source of new bioactive substances with unrivalled structural diversity and complexity. Recently, the natural compounds zosteric acid (ZA) and salicylic acid (SA) have been proposed as alternative biocide-free agents able to significantly reduce, at sub-lethal concentrations, both bacterial and fungal adhesion and to play a role in shaping biofilm architecture, in reducing biofilm biomass and thickness and in extending the antimicrobial agents performance. In this project, ZA and SA were selected as suitable anti-biofilm compounds to provide new effective preventive or integrative solutions against bacterial and fungal biofilm formation available for a wide range of applications. Specifically, the aim of the project was to develop new anti-biofilm materials by covalently anchoring ZA and SA to commercial available polymeric materials, allowing the anti-biofilm compounds to preserve the biological function upon the conjugation process and over a working timescale. The conjugation reaction was performed in such a way to orient the molecules with their active scaffold externally exposed on the surface so that to ease the interaction with the target microorganism. In this study, a detailed chemical and functional information necessary to ZA and SA to exert their anti-biofilm activity was obtained without affecting the active structure of the investigated molecules. In Chapter 3 a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to identify important structural chemical determinants for ZA anti-biofilm activity in order to select potential functional groups that could be exploited for the covalent linkage to an abiotic surface. The compounds were characterized by: i) the introduction of substituents at different positions on phenyl ring; ii) the removal of the insaturation; iii) the substitution of the carboxylic acid with an alcohol, an aldehyde and ester functionalities. Both E/Z isomers were also prepared in order to explore the role of the double bond. All the molecules were submitted to biological assays in order to evaluate their ability to be a carbon and energy source and to affect both planktonic growth and microbial adhesion at several concentrations. Structure/anti-biofilm activity relationship considerations revealed that the carboxylic acid moiety conjugated to the double bond in trans configuration was an essential requirement in ZA chemical structure to guarantee a good anti-biofilm performance. In contrast, the deletion of the sulphate ester group seemed not to compromise the ZA anti-biofilm activity. Moreover, the biological results revealed that the para position on the phenyl ring could be considered a good point for the coupling to a polymer. Indeed, p-aminocinnamic (p-ACA) acid bearing the same ZA active scaffold and an amino group in the para position of the phenyl ring was selected as suitable compound to form a covalent linkage with appropriate functional groups of the polymeric material. Besides cinnamic acid analogs, another class of derivatives related to the scaffold of salicylic acid has been investigated. Also in this case, the addition of an amino group in the para position of the phenyl ring of SA structure did not affect the molecule anti-biofilm performance. Indeed, p-aminosalicylic acid (p-ASA) bearing the chemical structure of salicylic acid and an amino group in the para position of the phenyl ring was selected to be covalently coupled to a polymeric materials. In Chapter 4 the molecular mechanisms and pathways by which ZA and SA affect the biofilm formation in E. coli were investigated. In this study, a pull-down system combined with a mass spectrometry-based approach was successfully employed to screen the whole E. coli proteome in order to identify the molecular targets implicated in ZA and SA anti-biofilm responses. By successfully exploiting the information obtained from the previous work, p-ACA and p-ASA were chosen as suitable molecules to successfully immobilize ZA and SA active scaffold on a chromatography solid phase support via an amide bond. Mass spectrometry analysis of SDS-PAGE-resolved pull-down proteins revealed that both ZA and SA in E. coli directly interact with the same protein WrbA. On the basis of previous studies reported in the literature, we supposed a mechanism by which ZA and SA interact with WrbA negatively modulating its activity and leading to an enhanced production of both the cell signal indole and reactive oxygen species that cooperate in a complex network of events leading to the inhibition of biofilm formation. The blast search inside Bacteria and Fungi databases revealed that WrbA sequences exist in a diversity of microorganisms with a high percentage of identity, suggesting a possible important role of this protein in the microbial life, probably associated to the response against environmental stresses. Because of the presence inside other microorganisms of a protein with a similar sequence and activity, it can be supposed that ZA and SA interact with these proteins with a potential anti-biofilm effect also in these microorganisms. Only related to SA, FkpA and Tdh were also identified as possible molecular targets, suggesting the presence of a complex and overlapping regulatory network controlling quorum sensing, motility and biofilm formation. Based on the information provided by experiment reported in the previous chapters, in Chapter 5, ZA and SA were successfully covalently immobilized on low density polyethylene surface, providing new no-leaching bio-hybrid materials available for a wide range of applications. A specific protocol to expose ZA and SA active scaffold on the external surface of a polymeric material via an amide bond was set up, allowing the anti-biofilm compound active moieties to directly interact with bacterial cells and successfully exerting their function even immobilized. Firstly, oxygen plasma treatment was employed to activate the low density polyethylene surface. Subsequently, the graft-polymerization with 2-hydroxyethyl methacrylate and the immobilization of the selected bioactive molecules on the polymeric surface successfully provided the new materials. Attenuated total reflectance Fourier transform infrared spectroscopy and confocal laser scanner microscope (CSLM) analyses proved that the polyethylene surface was successfully functionalized with ZA and SA molecules. The anti-biofilm performance of the innovative materials was investigated against E. coli biofilm using the CDC reactor able to simulate flow conditions normally encountered in vivo. The number of adhered cells on both traditional and functionalized polyethylene surface was established by plate count assays. Biofilm grown on both treated and control material was also submitted to live/dead staining procedure and analyzed by epifluorescence microscope. Both plate count assays and microscope observations showed a 60% percentage reduction of biofilm growth on both ZA and SA functionalized materials, with a mechanism that did not affect bacterial viability. CLSM analysis also revealed a dramatic impact of immobilized zosteric acid and salicylic acid on biofilm morphology, showing a biofilm with a significantly reduced thickness and polysaccharide matrix amount. Moreover, biofilm grown on functionalized surfaces showed enhanced susceptibility to ampicillin and ethanol, that further reduced biofilm biomass up to 95 %. In conclusion, in this study a polyethylene surface was successfully functionalized with the antibiotic-free natural compounds ZA and SA, providing new, improved solutions able to reduce the incidence of biofilm formation. In this approach, no molecules are leached from the surface, sidestepping the problem of the compound kinetic release, providing long term protection against bacterial colonization, and reducing the risk of developing resistant microbial strains, as the concentration of the anti-biofilm agent is constantly below the lethal level. In addition, the knowledge of ZA and SA structure/anti-biofilm activity relationship and molecular targets involved in their anti-biofilm activity open the way to new additional studies to generate more potent derivatives based on the same target and activity structure