Domenico Setacci

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.

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.

Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43

OBJECTIVES The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240-->Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. METHODS Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. RESULTS CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. CONCLUSIONS Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240-->Gly allows a better accommodation of the bulky C7beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.

Bactericidal activity of levofloxacin and ciprofloxacin on clinical isolates of different phenotypes of Pseudomonas aeruginosa

Levofloxacin has been reported to have in vitro activity against both gram-positive and gram-negative bacteria. A recent survey carried out at our Institution showed clinical isolates of Pseudomonas aeruginosa to be more susceptible to levofloxacin than to ciprofloxacin. The in vitro activity of the two fluoroquinolones was evaluated further by looking at their bactericidal activity against two strains of each of the following antibio-phenotypes of P. aeruginosa: levofloxacin- and ciprofloxacin-susceptible, levofloxacin-susceptible/ciprofloxacin-resistant, levofloxacin-susceptible/ciprofloxacin-susceptible and ceftazidime-resistant, (National Committee for Clinical Laboratory Standards susceptibility breakpoints were used). MIC and MBC values were measured and time-kill experiments were carried out. Drugs were used at susceptibility or resistance breakpoint concentrations in the time-kill experiments and results were recorded over 12 h in an attempt to link in vitro results with the clinical situation The polypeptide profiles of outer membrane preparations of the six strains were examined by gel electrophoresis. Levofloxacin was shown to be more bactericidal than ciprofloxacin in the time-kill experiments. No differences were observed between the outer membrane proteins of the six strains. Levofloxacin showed greater bactericidal activity against P. aeruginosa clinical isolates than ciprofloxacin.

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 β-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.

Antibacterial activity of selected metabolites from Chilean lichen species against methicillin-resistant staphylococci.

The in vitro antibacterial activities of eight compounds isolated from lichens, collected in several Southern regions of Chile (including Antarctica), were evaluated against methicillin-resistant clinical isolates strains of Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus warneri. The minimum inhibitory concentrations, calculated in microdilution, were ranging from 8 µg mL−1 for sphaerophorin to 1024 µg mL−1 for fumarprotocetraric acid. 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.

In Vitro Selection and Characterization of Mutants in TEM-1-Producing Escherichia coli by Ceftazidime and Ceftibuten

Abstract The present work was undertaken to study the ability of ceftazidime and ceftibuten to select In Vitro resistant mutants of Escherichia coli HB101 harboring blaTEM-1 β-lactamase gene. Minimum inhibitory concentrations (MICs) of ceftazidime and ceftibuten were increased by a factor of 32, overcoming in the case of ceftazidime the breakpoint for clinical resistance. Outer membrane protein analysis and PCR for bla TEM alleles revealed that ceftazidime and ceftibuten select for different resistance mechanisms. Ceftazidime created mutants that encode an extended-spectrum β-lactamase (TEM-12) and exhibit decreased expression of OmpF. Ceftibuten was unable to select for extended-spectrum β-lactamase expressing mutants but reduced the expression of two porins, OmpC and OmpF. The stability of ceftibuten to hydrolysis and the difference in the structure of these β-lactam antibiotics could be responsible for the selection of different mechanisms of resistance.

In vitro activity of cefodizime (HR-221) in combination with beta-lactamase inhibitors.

Cefodizime (formerly HR221) was tested either for in vitro microbiological activity or for its stability to beta-lactamases in the presence of two beta-lactamase inhibitors (clavulanic acid, tazobactam). Cefodizime was a poor substrate of class C enzymes but hyperproducer strains were generally resistant with or without a beta-lactamase inhibitor used in combination. On the contrary, class A enzymes were able to hydrolyze cefodizime. However, strains expressing class A beta-lactamase were susceptible to cefodizime in combination with clavulanic acid.

Ceftibuten stability to active-site serine and metallo-ß-lactamases

Abstract Ceftibuten is an oral third-generation cephalosporin active against a wide range of bacteria and shows an improved stability to hydrolysis by several s-lactamases because of the carboxyethilidine moiety at position 7 of the s-acyl side chain. The kinetic interactions between ceftibuten and active-site serine and metallo-s-lactamases were investigated. The activity of several TEM-derived extended spectrum s-lactamases (ESsLs) against ceftibuten, cefotaxime and ceftazidime was compared using K m , K cat and K cat / K m . Ceftibuten behaved as a poor substrate for class A and B s-lactamases compared with cefotaxime. The chromosomal class C s-lactamase from Enterobacter cloacae 908R gave a high K cat value (21 s −1 ), whereas there was poor activity with enzymes from Acinetobacter baumannii and Morganella morganii and ceftibuten. Ceftibuten resists hydrolysis in the presence of typical respiratory or urogenital-tract pathogens producing s-lactamases.

Ceftaroline in vitro activity against methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococci: a short report from Italy

Introduction Ceftaroline is a new parenteral cephalosporin recently approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for the treatment of complicated skin and soft tissue infections (cSSTIs). This is the first cephalosporin approved for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Ceftaroline, developed by modifying the structure of the fourth-generation cephalosporin, cefozopran, is the bioactive metabolite of ceftaroline fosamil, an N-phosphonoamino water-soluble cephalosporin prodrug, which is rapidly converted by plasma phosphatases to active ceftaroline. The compound has high affinity not only for PBP2a but also for PBP 1, 2 and 3 also for methicillin-susceptible S. aureus. Moreover, ceftaroline inhibits the activity of PBP2a more efficiently than other beta-lactams including imipenem. In the present study, we investigated the in vitro activity of ceftaroline compared with gentamicin, levofloxacin and ceftriaxone against various selected strains of MRSA and coagulase-negative Staphylococci (CoNS) clinical isolates.