STRATEGIE PER IL MIGLIORAMENTO DELLA PRODUZIONE E LO SVILUPPO DI MOLECOLE AD ATTIVITÀ ANTIBIOTICA DI ORIGINE NATURALE O DI SINTESI CHIMICA

The emergence of multi-drug resistant (MDR) bacterial strains is an urgent problem derived from the widespread and uncontrolled use of antibiotics. Therefore, new arrays of lead compounds exerting antimicrobial activity are necessary to contrast the spreading of MDR pathogens. Between 1980 and 2003, the interest in scientific research programs aimed to the new drug discovery by large pharmaceutical companies progressively decreased due to increasing costs in the respect of i) the low discovery rate of new leads, ii) the small amounts of product recovery needing process optimization and, finally, iii) regulatory obstacles associated with long-lasting pre-clinical and clinical trials for therapeutic use. Anyhow, during this time, natural products and synthetic compounds have contributed almost equally to the development of all drugs approved for therapeutic use including antibiotics. This PhD thesis describes results obtained in the within of two different topics related to the need of new antibiotics, in particular the development of: 1. strategies aimed at increment of antibiotic yield obtained from producing bacterial strains by fermentation; 2. novel synthetic compounds with antimicrobial activity. Concerning the topic (1), actinomycetes, Gram-positive filamentous mycelial bacteria, are very prolific sources of naturally bioactive molecules, including most of clinically relevant antibiotics as well as a wide range of enzymes of industrial interest, with Streptomycetes strains the major producers. The industrial production of bioactive molecules by actinomycetes, is performed in bioreactors using liquid growth media (i.e. submerged fermentations). However, the submerged fermentation does not reproduce the usual lifestyle of terrestrial actinomycetes which usually grow on the surface of organic or inorganic matters. In fact, one of the most frequent factors that negatively affects bio-production yield is the formation of mycelial clumps or pellets. As a consequence, immobilization of filamentous microorganisms in submerged fermentations for the production of biologically active compounds has become an attractive strategy. In this PhD thesis, three different supports were tested to perform immobilized-cell cultivations using Streptomyces coelicolor as a model actinomycete for the study of antibiotic production. Indeed, S. coelicolor produces different bioactive compounds, including the water-soluble blue-pigmented actinorhodin (ACT) and mycelium-associated red-pigmented undecylprodigiosin (RED). In particular, polycaprolactone (PCL), polyethylene glycol (PEG), and sodium chloride (NaCl) with different grain sizes were used to prepare PCL/PEG/NaCl blends in the melt. These blends were then leached to obtain PCL-based porous membranes with different porous size that were used as solid support for the growth of S. coelicolor in submerged cultivations. The results showed that ACT production is strongly dependent on the pore size. In particular, pore diameters ranging from 90 to 110 μm were associated with an two-fold improvement in volumetric production of ACT if compared to conventional (i.e. planktonic cell) submerged liquid cultures. The second kind of devices for cell-immobilization was obtained by electrospinning method. So, the use of electrospun polycaprolactone (PCL) and polylactic acid (PLA) membranes, subjected or not to O2-plasma treatment (PCL- or PLA-plasma), implied that S. coelicolor immobilized-cells produced more than 4-fold ACT yield in comparison with free-cell cultivations, with PLA-plasma membranes as the most effective ones. Indeed, immobilized-cells created a dense “biofilm-like” mycelial network on all kinds of membranes as observed by scanning electron microscope (SEM). In addition, ACT, produced by immobilized cells, was adsorbed on the PLA fibers of membranes by Raman spectroscopy. In addition, a differential proteome analysis, based on two dimensional Difference In Gel Electrophoresis (2D-DIGE) and mass spectrometry (MS) analyses, was carried out to highlight metabolic and molecular processes differentially regulated in immobilized- and planktonic-cell cultivations. The third kind of supports used for cell-immobilization was represented by two types of expanded perlite. In particular, Randalite W9 ™ (RW9) and Randalite W7 ™ (RW7), which differ in the degree of permeability and in the porosity, were chosen. Likewise other support, mycelial cells form a biofilm-like network on RW9, as showed by SEM observation. The quantitative analysis of antibiotic production revealed that S. coelicolor immobilized-cells on RW9 stimulated the production of ACT and RED, about 1.6 times more than control. It was also analyzed the production level of calcium-dependent antibiotic (CDA) by evaluating Randalite W7 and W9 antimicrobial activity by means of microbiological assay. Like before, RW9 shows more than 3.5 fold inhibition in comparison with planktonic cells. Moreover, PLA-based membranes and expanded perlite were used to perform immobilized-cell fermentation using selected industrial bacterial in collaboration with a Zoetis Manufacturing Italia S.R.L. from Catania. In addition to topic (1), the Antarctic bacterial strain Pseudoalteromonas haloplanktis TAC125 was also investigated. Indeed, this microorganism is a model for cold-adapted bacteria and is currently exploited for numerous biotechnological applications. Interestingly, this bacterium has been reported to be able to inhibit the growth of Burkholderia cepacia complex (Bcc) strains, opportunistic pathogens responsible for the infection of immune-compromised patients. Most likely, this occurs through the synthesis of several different compounds, including Volatile Organic Compounds (VOCs), whose nature and characteristics are currently mostly unknown. Bcc growth inhibition capability is deeply linked to the medium used to cultivate P. haloplanktis TAC125. Therefore, proteomic data were used in order to highlight metabolic and molecular processes differentially regulated in P. haloplanktis TAC125 associated to capability of inhibiting Bcc growth. Regarding topic (2), in the last few years, the chemical synthesis of novel chemotherapeutical leads is evolving thanks to possibility to design molecules with desired physical-chemical and, thus, biological properties. The imidazolium salts, recently proven effective to inhibit bacterial and/or cancer cell growth, possess an amphiphilic nature that is conferred by the imidazolium cation having a polar head generally coupled with aliphatic side chains. So, an array of 23 diimidazolium organic salts (DOS) has been synthesised and used to investigate their antimicrobial activity. In particular, salts based on the 3,3’-di-n-alkyl-1,1’-(1,n-phenylenedimethylene)-diimidazolium cation and differing in the alkyl chain length on the imidazolium ion, the isomeric substitution on the aromatic spacer and in the anion nature were used. The antimicrobial activity was investigated using both Gram-negative (Escherichia coli) and Gram-positive (Kokuria rhizophila, Staphilococcus aureus and Bacillus subtilis) strains. Data obtained demonstrate that biological activity is the result of the combined action of both cation and anion structure. In general, the cation hydrophobicity plays the most significant role with structural features of the anion becoming more relevant in the presence of shorter alkyl chain on the cationic head. Moreover, diimidazolium-based organic salts, bearing peptides or amino acids as anions have been synthesised and tested for their gelling ability in biocompatible solvents. These low molecular weight salts were successfully used as gelators in phosphate buffered saline (PBS) solution and ionic liquids. Furthermore, bioassays revealed that the obtained diimidazolium organic salts possessed antimicrobial activity, against Gram-negative and Gram-positive tester strains. In particular and noteworthy, the diimidazolium organic salts exerted a bactericidal capability, which was retained even if they are included in the gel phase. Thus, a novel kind of bioactive soft material was obtained that could be fruitfully employed as a non-covalent coating exerting antibacterial capability.