An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Long-Term Dominance of Carbapenem-Non-Susceptible Pseudomonas aeruginosa ST111 in Hematologic Malignancy Patients and Hematopoietic Cell Transplant Recipients.

An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD-deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains.

Recombinant Human Lactoferrin Reduces Inflammation and Increases Fluoroquinolone Penetration to Primary Granulomas During Mycobacterial Infection of C57Bl/6 Mice.

Infection with Mycobacterium tuberculosis (Mtb) results in the primary formation of a densely packed inflammatory foci that limits entry of therapeutic agents into pulmonary sites where organisms reside. No current therapeutic regimens exist that modulate host immune responses to permit increased drug penetration to regions of pathological damage during tuberculosis disease. Lactoferrin is a natural iron-binding protein previously demonstrated to modulate inflammation and granuloma cohesiveness, while maintaining control of pathogenic burden. Studies were designed to examine recombinant human lactoferrin (rHLF) to modulate histological progression of Mtb-induced pathology in a non-necrotic model using C57Bl/6 mice. The rHLF was oral administered at times corresponding to initiation of primary granulomatous response, or during granuloma maintenance. Treatment with rHLF demonstrated significant reduction in size of primary inflammatory foci following Mtb challenge, and permitted penetration of ofloxacin fluoroquinolone therapeutic to sites of pathological disruption where activated (foamy) macrophages reside. Increased drug penetration was accompanied by retention of endothelial cell integrity. Immunohistochemistry revealed altered patterns of M1-like and M2-like phenotypic cell localization post infectious challenge, with increased presence of M2-like markers found evenly distributed throughout regions of pulmonary inflammatory foci in rHLF-treated mice.

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

In Vitro Mechanistic Study of the Distribution of Lascufloxacin into Epithelial Lining Fluid.

The present study aimed to clarify the mechanism underlying the high distribution of lascufloxacin in epithelial lining fluid (ELF). Involvement of transporters was examined by transcellular transport across Calu-3 and transporter-overexpressing cells; the binding of lascufloxacin to ELF components was examined by an organic solvent-water partitioning system that employed pulmonary surfactant and phospholipids. Transcellular transport across the transporter-overexpressing cells indicated lascufloxacin to be a substrate of both P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP); therefore, its transport across Calu-3 cells was inhibited by P-gp and BCRP inhibitors. However, permeability and efflux ratios of lascufloxacin were similar to those of the other quinolones with relatively low ELF distribution, indicating the existence of another mechanism for lascufloxacin distribution in ELF. Amongst pulmonary surfactants, which are a primary component of ELF, lascufloxacin preferentially bound to phosphatidylserine (PhS) from several phospholipids, and the binding was significantly greater than that for other quinolones. This binding was saturable with two apparent classes of binding sites and inhibited by some weakly basic drugs, indicating the presence of an ionic bond. In conclusion, the results of this study suggest that the binding of lascufloxacin to PhS in the pulmonary surfactant is the major mechanism of the high distribution of lascufloxacin in the ELF.

Isothermal titration calorimetry and stopped flow circular dichroism investigations of the interaction between lomefloxacin and human serum albumin in the presence of amino acids.

The present study was designed to investigate the influence of two indispensable and two dispensable amino acids, including methionine, histidine, cysteine and proline, on the binding interaction between human serum albumin (HSA) and an antibiotic agent lomefloxacin (LMF). The fluorescence quenching experiments showed that the intrinsic emission of HSA was considerably quenched following binding to LMF in all the systems. Furthermore, in all the interactions the maximum wavelength of HSA was slightly decreased. The spectral changes observed in the binding systems we e all attributed to the alteration of the micro-environment around the tryptophan and tyrosine residues of HSA. The Kb values o HSA-LMF complex in the absence and presence of histidine, methionine, cysteine and proline have been obtained 6.02 × 105, 4.83 × 105, 5.05 × 105, 4.94 × 105 and 6.20 × 105 M-1 respectively. The various kind of Kb values showed the different interaction behavior between HSA and LMF in the absence and presence of amino acids mentioned. The data gathered by isothermal titration calorimetry (ITC) studies revealed that although all the binding interactions were exothermic, the amount of the heat exchanged during the HSA-LMF interaction increased in the presence of the amino acids especially cysteine. In the present study, the binding kinetics and affinity of LMF to HSA in the absence and presence of the amino acids were studies using stopped-flow circular dichroism and ITC techniques respectively. The results of these two techniques revealed that the bindig affinity and binding rate of the LMF-HSA interaction decreased in the presence of histidine, methionine and cysteine. In the presence of proline, the binding process of LMF-HSA was sped up and the affinity of LMF to HSA slightly increased. All the experimental results were then supported by the data collected from molecular modeling studies using density functional theory. Communicated by Ramaswamy H. Sarma.

Combatting implant-associated biofilms through localized drug synthesis.

Bacterial contamination of implantable biomaterials is a significant socioeconomic and healthcare burden. Indeed, bacterial colonization of implants after surgery has a high rate of incidence whereas concurrent prophylaxis using systemic antibiotics has limited clinical success. In this work, we develop enzyme-prodrug therapy (EPT) to prevent and to treat bacteria at interfaces. Towards the overall goal, novel prodrugs for fluoroquinolone antibiotics were developed on a privileged glucuronide scaffold. Whereas carbamoyl prodrugs were not stable and not suitable for EPT, glucuronides containing self-immolative linker between glucuronic acid masking group and the antibiotic were stable in solution and readily underwent bioconversion in the presence of ß-glucuronidase. Surface coatings for model biomaterials were engineered using sequential polymer deposition technique. Resulting coatings afforded fast prodrug conversion and mediated antibacterial measures against planktonic species as evidenced by pronounced zone of bacterial growth inhibition around the biomaterial surface. These biomaterials coupled with the glucuronide prodrugs also effectively combatted bacteria within established biofilms and also successfully prevented bacterial colonization of the surface. To our knowledge, this is the first report of EPT engineered to the surface of biomaterials to mediate antibacterial measures.

Identification and structural characterization of in vivo metabolites of balofloxacin in rat plasma, urine and feces samples using Q-TOF/LC/ESI/MS/MS : In silico toxicity studies.

Balofloxacin is a fluroquinolone antibiotic drug which has been used for the treatment of urinary tract infections (UTIs). Identification and structural characterization of metabolites is a critical component of both drug discovery and drug development research. In vivo metabolites of balofloxacin have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI-HR-MS/MS) experiments. To identify in vivo metabolites, blood, urine and feces samples were collected after oral administration of the drug to the female Sprague-Dawley rats (n = 3 per group). Protein precipitation, freeze liquid separation followed by solid-phase extraction methods were used for sample preparation. The extracted samples were subjected to LC-ESI/HRMS/MS analysis. The chromatographic separation of the drug and its metabolites were achieved on a XDB, C18 (50, 4.6 mm, 5 mm) column using gradient elution method in combination with 0.1% formic acid and acetonitrile at a flow rate of 0.4 mL/min. A total of 13 phase I and phase II metabolites of balofloxacin have been identified in plasma, urine and feces samples. Most of metabolites were observed in plasma and urine samples including dealkylated, desmethylated, decarbonylated, decarboxylated, hydroxylated, methylated, carboxylated, cysteine conjugated metabolites and high abundance glucuronidated metabolite. The structures of metabolites have been elucidated based on fragmentation patterns, accurate mass measurements and LC/MS/MS experiments. The main phase I metabolites of balofloxacin, decarbonylated, decarboxylated and desmethylated metabolites and phase II methylated metabolite undergo subsequent phase II glucuronidation pathways. In silico toxicity of the drug and its metabolites was determined using ProTox-II. Metabolites B-1, B-2, B-5, B-6, B-7, and B-8 to B-13 were predicted to possess immunotoxicity with high probability score. Additionally, Amine Oxidase A and Prostaglandin G/H Synthase 1 are predicted for metabolites B-1, B-3 to B-6 as toxicity targets with binding probability.

Two for one: Viral helicases as an ideal target for HIV and HCV co-infection.

Helicase enzyme is responsible for the unwinding of complementary nucleic acid strands, which is one of the preliminary steps in DNA replication. They are crucial for replication of an organism, including viruses. HCV and HIV are two clinically significant pathogens, responsible for millions of infections and deaths worldwide. Due to similar transmission routes, these viruses can establish co-infection in an individual. Individually, these infections are difficult to treat, however, in case of co-infection, the treatment becomes more difficult. Additionally, these viruses accumulate mutation in response to drug therapy that renders the treatment ineffective. HCV and HIV both encode enzyme containing helicase activity. The viral-encoded helicase plays a significant role in HIV and HCV life cycle. Here we propose viral helicases as an ideal single-hit target that can inhibit HIV and HCV co-infection. We also hypothesize that search for natural analogs sharing basic ring structure with a class of helicase inhibitors called fluoroquinolones can yield natural agents with superior antiviral (anti-helicase) activity with lower toxicity index. The fluoroquinolones and their analogs are currently not part of any antiviral regimens. Our proposal is to include fluoroquinolones-derived natural analogs as a conjugate therapy along with main regimens available against HCV and HIV co-infection.

Sustained molecular oxygen activation by solid iron doped silicon carbide under microwave irradiation: Mechanism and application to norfloxacin degradation.

Sustained molecular oxygen activation by iron doped silicon carbide (Fe/SiC) was investigated under microwave (MW) irradiation. The catalytic performance of Fe/SiC for norfloxacin (NOR) degradation was also studied. Rapid mineralization in neutral solution was observed with a pseudo-first-order rate constant of 0.2239 min-1 under 540 W of MW irradiation for 20 min. Increasing Fe/SiC rod and MW power significantly enhanced the degradation and mineralization rate with higher yield of reactive oxygen species (ROS). Fe shell corrosion and subsequent Fe0/II oxidation by molecular oxygen with MW activation was the key factor for NOR degradation through two-electron-transfer by Fe0 under acidic conditions and single-electron-transfer by FeII under neutral-alkaline solution. Removal rate of NOR was significantly affected by solution pH, showing higher degradation rates at both acidic and alkaline conditions. The highest removal efficiencies and rates at alkaline pH values were ascribed to the contribution of bound FeII species on the Fe shell surface due to the hydroxylation of Fe/SiC. ·OH was the main oxidizing specie for NOR degradation, confirmed by density functional theory (DFT) calculations and radical scavenger tests. DFT calculations were conducted on the reaction/activation energies of the transition/final states of NOR/degradation products, combined with intermediate identification with high performance liquid chromatography coupled with a triple-quadruple mass spectrometer (HPLC-MS/MS), the piperazinyl ring was the most reactive site for ·OH attack, followed by further ring-opening and stepwise oxidation. In this study, Fe/SiC were proved to be an excellent catalyst for the treatment of fluoroquinolone antibiotics with MW activation.

Orientation of the OmpF Porin in Planar Lipid Bilayers.

The outer-membrane protein OmpF is an abundant trimeric general diffusion porin that plays a central role in the transport of antibiotics and colicins across the outer membrane of E. coli. Individual OmpF trimers in planar lipid bilayers (PLBs) show one of two current-voltage asymmetries, thus implying that insertion occurs with either the periplasmic or the extracellular end first. A method for establishing the orientation of OmpF in PLB was developed, based on targeted covalent modification with membrane-impermeant reagents of peripheral cysteine residues introduced near the periplasmic or the extracellular entrance. By correlating the results of the modification experiments with measurements of current asymmetry or the sidedness of binding of the antibiotic enrofloxacin, OmpF orientation could be quickly determined in subsequent experiments under a variety of conditions. Our work will allow the precise interpretation of past and future studies of antibiotic permeation and protein translocation through OmpF and related porins.

Pharmacokinetics and distribution in interstitial and pulmonary epithelial lining fluid of danofloxacin in ruminant and preruminant calves.

The objective of this study was to compare active drug concentrations in the plasma vs. different effector compartments including interstitial fluid (ISF) and pulmonary epithelial lining fluid (PELF) of healthy preruminating (3-week-old) and ruminating (6-month-old) calves. Eight calves in each age group were given a single subcutaneous (s.c.) dose (8 mg/kg) of danofloxacin. Plasma, ISF, and bronchoalveolar lavage (BAL) fluid were collected over 96 h and analyzed by high-pressure liquid chromatography. PELF concentrations were calculated by a urea dilution assay of the BAL fluids. Plasma protein binding was measured using a microcentrifugation system. For most preruminant and ruminant calves, the concentration-time profile of the central compartment was best described by a two-compartment open body model. For some calves, a third compartment was also observed. The time to maximum concentration in the plasma was longer in preruminating calves (3.1 h) vs. ruminating calves (1.4 h). Clearance (CL/F) was 385.15 and 535.11 mL/h/kg in preruminant and ruminant calves, respectively. Ruminant calves maintained higher ISF/plasma concentration ratios throughout the study period compared to that observed in preruminant calves. Potential reasons for age-related differences in plasma concentration-time profiles and partitioning of the drug to lungs and ISF as a function of age are explored.

Sustained Antibiotic-Eluting Intra-Ocular Lenses: A New Approach.

Currently, infections following cataract surgery are not as effectively managed with antibiotic eye drops, which suffer from poor bioavailability of drug and low patient compliance. The ideal solution, which can help to overcome the issue of drug wastage and poor bioavailabilty, as well as the need for frequent applications (patient inconvenience), is a drug-eluting intraocular lens (IOL). We describe a novel approach to such a drug-eluting lens by using a peripheral IOL attachment as a drug depot to deliver antibiotics, Levofloxacin (LFX) or Moxifloxacin (MFX). In this work, drug was entrapped within a fully-degradable polymer, poly(L-lactide-co-ɛ-caprolactone) (PLC). The effects of drug loading and solvent type on drug release and film morphology were investigated using cast films. The study clearly demonstrated that a slower-evaporating solvent tetrahydrofuran (THF) resulted in a better surface morphology, as well as lower initial burst compared to dichloromethane (DCM), and hence, was better suited to developing a drug-eluting attachment with sustained release of drug. When attachments were fabricated with drugs at high loading percentages (20% and 25% in polymer), significant burst was observed compared to films: this is attributed to the higher surface-to-volume ratio of the attachments. When the levofloxacin (LFX) loading percentage was decreased to 3% and 5%, the attachments presented lower burst and sustained release with therapeutic efficacy. This work has demonstrated the potential of using an IOL attachment as a more efficacious anti-infective option compared to daily eye drops.

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.

Radiation formation of functionalized polysaccharide-protein based skin mimicking semi- inter penetrating network for biomedical application.

Radiation treatment of chitosan, gelatin, polyvinyl alcohol (PVA) and polyacrylamide [poly(AAm)] will form the sterile hydrogel wound dressings which can mimic the artificial skin function in wound therapy. These polymers have been characterized by cryo-scanning electron micrographs (cryo-SEMs), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and 13C solid state nuclear magnetic resonance (NMR) spectroscopy and swelling studies. Some important properties of hydrogel wound dressings like drug delivery, blood compatibility, wound fluid absorption, antioxidant activity, oxygen permeability, water vapour permeability, microbial penetration, mucoadhesion and mechanical properties have also been determined. The release profile of moxifloxacin from the polyacrylamide functionalized chitosan-gelatin matrix followed Fickian diffusion mechanism and release profile best fitted in Korsmeyer-Peppas kinetic model. The hydrogel films are permeable to O2 and H2O vapour and impermeable to microbes in open environment and showed high wound absorption, good mucoadhesion and antioxidant activity. Beside release of antibiotic, the inherent wound healing potential of chitosan, adhesion capacity of gelatin, film forming ability of PVA and wound fluid absorption of poly(AAm), may enhance wound healing potential of these hydrogel wound dressings.

The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin.

P-glycoprotein ( P-GP: , encoding gene Abcb1) and Breast Cancer Resistance Protein ( BCRP: , encoding gene Abcg2) are transport proteins that play a major role in modulating the bioavailability of oral drugs in humans and rodents. It has been shown that rifampicin is the typical inducer of P-gp in rodents by activating the nuclear receptor. However, its effect on Abcb1, Abcg2, CYP3A, and chicken xenobiotic-sensing orphan nuclear receptor ( CXR: ) mRNA expression in broilers is poorly understood. This study explored the effect of rifampicin on mRNA expression of Abcb1, Abcg2, CYP3A37, CXR as well as its effect on the pharmacokinetics of enrofloxacin in broilers. The mRNA levels of Abcb1, Abcg2, CYP3A37, and CXR were significantly increased in the liver (except Abcg2), kidney, jejunum, and ileum (P < 0.05) but not significantly changed in the duodenum (P > 0.05) after treated with rifampicin. Further analysis revealed that the variation tendencies of Abcb1, Abcg2, and CYP3A37 expression levels were significantly correlated with CXR mRNA expression levels in liver, kidney, jejunum, and ileum. Coadministration of rifampicin significantly changed the pharmacokinetic behavior of enrofloxacin orally administered by showing clearly lower AUC0-∞, AUC0-t, and Cmax as well as longer Tmax. The bioavailability of orally administered enrofloxacin was decreased from 72.5% to 24.8% by rifampicin. However, rifampicin did not significantly change the pharmacokinetics of enrofloxacin following intravenous administration. Our study shows that rifampicin up-regulated the small intestinal level of P-gp and BCRP and suggests that P-gp and BCRP are key factors that affected pharmacokinetic behavior of orally administered enrofloxacin by limiting its absorption from the intestine in broilers.

Immune response to antituberculosis drug-loaded gelatin and polyisobutyl-cyanoacrylate nanoparticles in macrophages.

AIM: Secondary toxicity of nanoparticles (NPs) in macrophages is a well-known phenomenon. The aim of the study was to investigate the immuneresponse of macrophages after NP treatment. METHODS & RESULTS: Antituberculosis drugs moxifloxacin and rifampicin were loaded into gelatin and polyisobutyl-cyanoacrylate NPs. The NPs were physicochemical characterized. Cellular immuneresponses and cellular viability were determined. The drug release kinetics vary depending on the type of NP, size and loading capacity. IC50 values of polyisobutyl-cyanoacrylate NPs were lower than for gelatin NPs. NPs treatment induced higher release of Th1 type cytokines compared with free drug. CONCLUSION: NPs together with chemotherapeutic drugs might be able to trigger an immune response in macrophages. The combined effect might be able to overcome mycobacteria infections.

Biotransformation of fluoroquinolone antibiotics by ligninolytic fungi--Metabolites, enzymes and residual antibacterial activity.

A group of white rot fungi (Irpex lacteus, Panus tigrinus, Dichomitus squalens, Trametes versicolor and Pleurotus ostreatus) was investigated for the biodegradation of norfloxacin (NOR), ofloxacin (OF) and ciprofloxacin (CIP). The selected fluoroquinolones were readily degraded almost completely by I. lacteus and T. versicolor within 10 and 14 d of incubation in liquid medium, respectively. The biodegradation products were identified by liquid chromatography-mass spectrometry. The analyses indicated that the fungi use similar mechanisms to degrade structurally related antibiotics. The piperazine ring of the molecules is preferably attacked via either substitution or/and decomposition. In addition to the degradation efficiency, attention was devoted to the residual antibiotic activities estimated using Gram-positive and Gram-negative bacteria. Only I. lacteus was able to remove the antibiotic activity during the course of the degradation of NOR and OF. The product-effect correlations evaluated by Principal Component Analysis (PCA) enabled elucidation of the participation of the individual metabolites in the residual antibacterial activity. Most of the metabolites correlated with the antibacterial activity, explaining the rather high residual activity remaining after the biodegradation. PCA of ligninolytic enzyme activities indicated that manganese peroxidase might participate in the degradation.

Molecular docking and molecular dynamics studies on the structure-activity relationship of fluoroquinolone for the HERG channel.

Fluoroquinolones play an important role in the treatment of serious bacterial infections, but at the same time they could lead to cardiac toxicity due to the blockage of the HERG potassium channel, which even leads to the withdrawal of some fluoroquinolones. Blockage of the HERG potassium channel by drugs or drug-like compounds has become a critical problem in drug discovery. Though there were large amounts of bioactivity data of fluoroquinolones on the blockage of HERG, little structural basis of binding of blockers to the HERG channel was known. Here, we combined molecular docking, molecular dynamics simulations, free energy calculations and binding energy decomposition analysis to explore the binding modes of fluoroquinolones in the HERG potassium channel. The calculated binding free energies were consistent with the experimental binding affinities. Our results showed that the CH3 group in MX was favorable for the binding to the HERG channel, while Tyr652 and Phe656 were critical for the hydrophobic interaction between fluoroquinolones and the HERG channel. We expected that our results of calculation could provide important insights for the rational design and discovery of drugs.