Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.

OBJECTIVES: To evaluate the contribution of cysK and cysM to the fluoroquinolone (ofloxacin) antibiotic resistance in Salmonella Typhimurium, and their impact on H2S and cysteine production through targeted mutagenesis. METHODS: Salmonella Typhimurium 14028s and its cysK and cysM mutants were tested for their susceptibility to ofloxacin, as determined by a broth microdilution test (to determine the MIC) and survival curves. H2S levels were measured by the Pb(AC)2 method and cysteine levels were determined using 5,5-dithio-bis-2-nitrobenzoic acid. DNA damage induced by antibiotic treatment was determined by PFGE. Finally, expression of cysK and cysM genes under antibiotic treatment was determined by real-time reverse transcription PCR. RESULTS: As determined by MIC, the ΔcysK strain was more resistant to ofloxacin, a reactive oxygen species (ROS)-producing fluoroquinolone, than the WT and ΔcysM strains, which correlates with survival curves. Moreover, the ΔcysK strain exhibited higher H2S levels and lower cysteine levels than the WT strain. Finally, the ΔcysK strain exhibited lower DNA damage upon challenge with ofloxacin than the WT and ΔcysM strains. These results are in accordance with lower expression of cysK under ofloxacin treatment in the WT strain. CONCLUSIONS: This work demonstrated that cysteine metabolism in Salmonella Typhimurium modulated H2S levels, conferring resistance to second-generation fluoroquinolones.

Depletion study and estimation of the withdrawal period for enrofloxacin in pacu (Piaractus mesopotamicus).

Defining the pharmacokinetic parameters and depletion intervals for antimicrobials used in fish will help in the development of important guidelines for future regulations by Brazilian agencies on the use of these substances in fish farming. This paper presents a depletion study for enrofloxacin (ENR) and its main metabolite, ciprofloxacin (CIP), in pacu (Piaractus mesopotamicus) fillets. The depletion study was carried out under monitored environmental conditions, with the temperature controlled at 27 °C to mimic the fish farming conditions in Brazil. ENR was administered orally via medicated feed for 10 consecutive days at daily dosages of 10 mg/kg body weight (b.w.). The fish were slaughtered at 6, 12, and 24 h and 2, 3, 5, 8, 12, 17, and 24 days after the medication period. Considering a maximum residue limit of 100 µg/kg for the sum of the ENR and CIP residues in the fillet, the results obtained in the depletion study allowed the estimation of a half-life for ENR of 2.75 days and a withdrawal period of 23 days. The results obtained in this study are important for the farming of pacu in tropical regions.

Copper(II)-fluoroquinolone complexes with anti-Trypanosoma cruzi activity and DNA binding ability.

Copper(II) complexes of fluoroquinolone antibacterial agents levofloxacin (LEV) and sparfloxacin (SPAR), containing or not a nitrogen donor heterocyclic ligand, 2,2'-bipyridine (bipy) or 1,10-phenathroline (phen), were prepared and characterized. The complexes are of the type [CuCl(2)(H(2)O)(L)], [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)], where L = LEV or SPAR. The data suggest that LEV and SPAR act as zwitterionic bidentade ligands coordinated to Cu(II) through the carboxylate and ketone oxygen atoms. The electron paramagnetic resonance spectra of the [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)] complexes (L = LEV and SPAR) in aqueous and DMSO solutions indicate mixture of mononuclear and binuclear forms. The Cu(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The assays performed against bloodstream trypomastigotes showed that all complexes were more active than their corresponding ligands. Complexes [CuCl(2)(phen)(LEV)] and [CuCl(2)(phen)(SPAR)] were revealed, among all studied compounds, to be the most active with IC(50) = 1.6 and 4.7 µM, respectively, both presenting a superior effect than benznidazole. The interactions of fluoroquinolones and their Cu(II) complexes with calf-thymus DNA were investigated. These compounds showed binding properties towards DNA, with moderated binding constants values, suggesting that this structure may represent a parasite target.

[Prevalence of plasmid-mediated quinolone resistance determinant aac(6')-Ib-cr among ESBL producing enterobacteria isolates from Chilean hospitals]. / Prevalencia del determinante de resistencia plasmídica a quinolonas aac(6')-Ib-cr en cepas de Escherichia coli y Klebsiella pneumoniae productoras de BLEE aisladas en diez hospitales de Chile.

INTRODUCTION: The frequency of aac(6')-Ib-cr gene in ESBL-producing strains of Klebsiella pneumoniae and Escherichia coli is unknown, in Chile. METHODOLOGY: The aac(6')-Ib and aac(6')-Ib-cr genes were investigated using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and sequencing, in strains isolated from 10 Chilean hospitals between 2008-2009. RESULTS: The aac(6')-Ib-cr gene was detected in 54% of K. pneumoniae and 74% of E. coli strains. The CIM(50) of CIP was higher among strains harboring aac(6')-Ib-cr, 8 times higher in K. pneumoniae and 4 times higher in E. coli. Moreover, both aac(6')-Ib and aac(6')-Ib-cr were simultaneously found in 13 K. pneumoniae and 3 E. coli isolates. CONCLUSION: This is the first report of aac(6')-Ib-cr in ESBL-producing strains of K. pneumoniae and E. coli isolated from in-patients in Chilean hospitals located along an area of more than 2,800 Km.

Identification and expression of nor efflux family genes in Staphylococcus epidermidis that act against gatifloxacin.

NorA, NorB, and NorC are efflux proteins in the Nor family that regulate the secretion of fluoroquinolones, and MgrA/NorR is a transcription factor of the Nor family. Overexpression of Nor family proteins provides fluoroquinolone resistance in Staphylococcus aureus. However, in coagulase-negative staphylococci (CNS), members of the Nor family had not been identified. In this work, the presence of Nor family proteins in Staphylococcus spp. and the expression of Nor family in gatifloxacin resistant S. epidermidis strains obtained from ocular infections (OI) were identified and analyzed. S. epidermidis strains from OIs (n = 44) and healthy skin (HS; n = 52) were isolated. The nor family genes were identified in CNS using PCR, sequencing and phylogenetic approaches. Nor family expression was determined by RT-PCR. NorA efflux activity was determined using the automated ethidium bromide method. In-silico analysis showed that norA, mgrA/norR, and "norB-like" and "norC-like" (norB/norC) genes are present in CNS. The nor family genes were distributed and constitutively expressed in all S. epidermidis strains studied. In one gatifloxacin resistant strain isolated from the endophthalmitis, treatment with gatifloxacin induced overexpression of the norA gene and resulted in high activity of NorA efflux. These results indicate that the Nor family of proteins is present in CNS, and the NorA efflux mechanism for gatifloxacin response occurs in at least one strain of S. epidermidis, contributing to gatifloxacin resistance.

Study of enrofloxacin and flumequine residues depletion in eggs of laying hens after oral administration.

Two groups of laying hens (each n=12) were administered 10 mg/kg enrofloxacin (ENRO) (group A) or 26.6 mg/kg flumequine (FLU) (group B) by gastric catheter daily for five consecutive days. A third group (n=6) was untreated controls. Eggs were collected from day one of treatment and up to 30 days after withdrawal of the drug. Egg white and yolk from each egg were separated, and ENRO, its metabolite ciprofloxacin (CIP) and FLU residues were analysed by a high-performance liquid chromatography method with fluorescence detection. The sum of ENRO and CIP was detectable in egg white on the first day of treatment in high-level concentrations (2007.7 µg/kg) and remained steady during administration. In egg yolk, residues were detectable at day one in lower concentrations (324.4 µg/kg), increasing to the end of treatment. After treatment, these residues decreased and were detectable up to day 8 in egg white, and day 10 in yolk. FLU residues during drug administration in white were detectable in high concentrations from day one to five (6788.4-6525.9 µg/kg), and in yolk, concentrations were lower during administration (629.6-853.9 µg/kg). After drug withdrawal, FLU residues remained longer in egg white (30 days) than in yolk (26 days). For both drugs, differences of concentrations between matrices were significant.

Enhanced bacterial uptake and bactericidal properties of ofloxacin loaded on bioadhesive hydrogels against Pseudomonas aeruginosa.

Carbomer hydrogels 971pNf, 934pNf and 940Nf loaded with ofloxacin were characterized and their antimicrobial properties evaluated. bactericidal profiles show improved efficacy and prolonged activity exhibited by ofloxacin-containing hydrogels against Pseudomonas aeruginosa. Analysis of bactericidal index (BI) values after a short time of drug exposure confirms the higher potency of hydrogels compared with that of ofloxacin. Increased BI values observed after 24 h indicate prolonged action against the microorganisms evaluated. The bacterial uptake of ofloxacin from hydrogels was higher than that obtained with a solution of free ofloxacin in both fluoroquinolone-sensitive and -resistant P. aeruginosa. The improved uptake in fluoroquinolone-resistant isolates was correlated with the viscosity of hydrogels. The performance of hydrogels seems to be related to their bioadhesive properties that allow prolonged contact time and the release of an effective amount of drug close to bacterial cells. Hence, hydrogels could be used in the development of more effective formulations for topical administration of antibiotics. Improved performance of an old antibiotic can preserve the use of new generation fluoroquinolones.

A topological substructural approach for the prediction of P-glycoprotein substrates.

A topological substructural molecular design approach (TOPS-MODE) has been used to predict whether a given compound is a P-glycoprotein (P-gp) substrate or not. A linear discriminant model was developed to classify a data set of 163 compounds as substrates or nonsubstrates (91 substrates and 72 nonsubstrates). The final model fit the data with sensitivity of 82.42% and specificity of 79.17%, for a final accuracy of 80.98%. The model was validated through the use of an external validation set (40 compounds, 22 substrates and 18 nonsubstrates) with a 77.50% of prediction accuracy; fivefold full cross-validation (removing 40 compounds in each cycle, 80.50% of good prediction) and the prediction of an external test set of marketed drugs (35 compounds, 71.43% of good prediction). This methodology evidenced that the standard bond distance, the polarizability and the Gasteiger-Marsilli atomic charge affect the interaction with the P-gp; suggesting the capacity of the TOPS-MODE descriptors to estimate the P-gp substrates for new drug candidates. The potentiality of the TOPS-MODE approach was assessed with a family of compounds not covered by the original training set (6-fluoroquinolones), and the final prediction had a 77.7% of accuracy. Finally, the positive and negative substructural contributions to the classification of 6-fluoroquinolones, as P-gp substrates, were identified; evidencing the possibilities of the present approach in the lead generation and optimization processes.