Structurally Related Liposomes Containing N-Oxide Surfactants: Physicochemical Properties and Evaluation of Antimicrobial Activity in Combination with Therapeutically Available Antibiotics.

Although liposomes are largely investigated as drug delivery systems, they can also exert a pharmacological activity if devoid of an active principle as a function of their composition. Specifically, charged liposomes can electrostatically interact with bacterial cells and, in some cases, induce bacterial cell death. Moreover, they also show a high affinity toward bacterial biofilms. We investigated the physicochemical and antimicrobial properties of liposomes formulated with a natural phospholipid and four synthetic l-prolinol-derived surfactants at 9/1 and 8/2 molar ratios. The synthetic components differ in the nature of the polar headgroup (quaternary ammonium salt or N-oxide) and/or the length of the alkyl chain (14 or 16 methylenes). These differences allowed us to investigate the effect of the molecular structure of liposome components on the properties of the aggregates and their ability to interact with bacterial cells. The antimicrobial properties of the different formulations were assessed against Gram-negative and Gram-positive bacteria and fungi. Drug-drug interactions with four classes of available clinical antibiotics were evaluated against Staphylococcus spp. The target of each class of antibiotics plays a pivotal role in exerting a synergistic effect. Our results highlight that the liposomal formulations with an N-oxide moiety are required for the antibacterial activity against Gram-positive bacteria. In particular, we observed a synergism between oxacillin and liposomes containing 20 molar percentage of N-oxide surfactants onStaphylococcus haemolyticus, Staphylococcus epidermidis, andStaphylococcus aureus. In the case of liposomes containing 20 molar percentage of the N-oxide surfactant with 14 carbon atoms in the alkyl chain for S. epidermidis, the minimum inhibitory concentration was 0.125 µg/mL, well below the breakpoint value of the antibiotic.

Targeting the Class A Carbapenemase GES-5 via Virtual Screening.

The worldwide spread of ß-lactamases able to hydrolyze last resort carbapenems contributes to the antibiotic resistance problem and menaces the successful antimicrobial treatment of clinically relevant pathogens. Class A carbapenemases include members of the KPC and GES families. While drugs against KPC-type carbapenemases have recently been approved, for GES-type enzymes, no inhibitors have yet been introduced in therapy. Thus, GES carbapenemases represent important drug targets. Here, we present an in silico screening against the most prevalent GES carbapenemase, GES-5, using a lead-like compound library of commercially available compounds. The most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5 leading to four derivatives active as high micromolar competitive inhibitors. For the best inhibitors, the ability to inhibit KPC-2 was also evaluated. The discovered inhibitors constitute promising starting points for hit to lead optimization.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

BACKGROUND: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PURPOSE: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. METHODS: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. RESULTS: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. CONCLUSION: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Interaction between lichen secondary metabolites and antibiotics against clinical isolates methicillin-resistant Staphylococcus aureus strains.

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Curcumin inhibits the SOS response induced by levofloxacin in Escherichia coli.

The role of RecA protein in bacterial resistance to antibiotics makes this protein attractive from a pharmacological point of view. In this study we demonstrate that curcumin is able to inhibit the SOS response in Escherichia coli induced by levofloxacin. The blaTEM-1 gene has been placed under the control of the LexA-binding box and used as reporter gene. The expression of TEM-1 ß-lactamase enzyme was increased in the presence of ssDNA induced by levofloxacin, while, the presence of curcumin at 8µg/ml, reduced dramatically the expression of the reporter gene. Moreover a simple microplate assay suitable for high-throughput screening has been developed.

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates.

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC(90), MIC(50), as well as MBC(90) and MBC(50), were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔE model methods.

The in vitro antimicrobial activities of usnic acid were evaluated in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, were evaluated. A synergistic action was observed in combination with gentamicin, while antagonism was observed with levofloxacin. The combination with erythromycin showed indifference, while variability was observed for clindamycin and oxacillin. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index (FICI) and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings, beside confirm the well known antimicrobial activity of usnic acid, suggest, however, that this substance might be a good candidate for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Occurrence of class 1 and 2 integrons in resistant Enterobacteriaceae collected from a urban wastewater treatment plant: first report from central Italy.

The purpose of this study was to investigate the presence and distribution of integron-carrying isolates among Enterobacteriaceae resistant to ß-lactam antibiotics collected from a wastewater effluent of the city of L'Aquila (Italy). A total of 471 Enterobacteriaceae were collected during a period of 2 years (2005-2006). The presence and distribution of class 1 and 2 integrons was investigated by colony blot hybridization using specific probes labelled with dUTP-fluoresceine kit. The variable region of class 1 and 2 integrons was analyzed by polymerase chain reaction and DNA sequencing in 24 isolates with different random amplified polymorphic DNA profile. The characterization of class 1 and 2 integrons gene cassettes of 24 nonrelated strains showed the presence of four different arrays: dfr17-aadA5; aadA10; dfr1-sat1; dfr1-sat1-aadA1. This is the first report from Italy in which the authors confirm the presence of Enterobacteriaceae carrying class 1 and 2 integrons in a wastewater treatment plant that collects the urban and hospital discharges.