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1.
Antimicrob Agents Chemother ; 65(11): e0026221, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34424047

RESUMO

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump, EfpA, from the major facilitator superfamily, that is highly conserved among both slow-growing and fast-growing Mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis. The gene encoding EfpA from Mycobacterium smegmatis was overexpressed under the control of both a constitutive and an inducible promoter. The expression of the efpA gene under the control of both promoters resulted in >32-fold-increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid, and streptomycin) and second-line (amikacin) antituberculosis drugs. Notably, the drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was overexpressed. The increase in MICs correlated with the decreased uptake of drugs, including norfloxacin, moxifloxacin, and ethidium bromide, and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MICs of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose. The efpA gene is conserved across both pathogenic and nonpathogenic mycobacteria. The efpA gene from Mycobacterium bovis BCG/M. tuberculosis, which is 80% identical to efpA from M. smegmatis, also led to decreased antimicrobial efficacy of many drugs, although the fold change was lower. When overexpressed in M. bovis BCG, 8-fold-higher drug tolerance to moxifloxacin was observed. This is the first report of an efflux pump from Mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.


Assuntos
Mycobacterium smegmatis , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tolerância a Medicamentos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo
2.
FEMS Microbiol Lett ; 368(14)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34240144

RESUMO

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.


Assuntos
Técnicas Bacteriológicas/métodos , Moxifloxacina/farmacologia , Mycobacterium smegmatis/isolamento & purificação , Mycobacterium smegmatis/fisiologia , Rifampina/farmacologia , Antituberculosos/farmacologia , Tolerância a Medicamentos/genética , Perfilação da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Reprodutibilidade dos Testes
3.
Int J Biol Macromol ; 182: 2087-2096, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087298

RESUMO

The aim of our study was to prepare nanoparticles of disulfide bridged thiolated chitosan and eudragit RS100 using the air oxidation method for controlled drug delivery. The developed nanoparticles were characterized by FTIR, DSC, TGA, zeta sizer, zeta potential, SEM and 1H NMR. The loading, entrapment efficiency and in-vitro release of moxifloxacin from nanoparticles was determined. Toxicity was studied using Caco-2 cell line and pharmacokinetics of moxifloxacin from the developed nanoparticles was studied in albino rats. The FTIR analysis showed no chemical interaction of the drug with the thiolated polymers. The DSC and TGA showed the thermal stability of nanoparticles. The average particle size of nanoparticles was 87 nm, zeta potential of NTC3 was ± 19 and SEM showed the spherical shape of nanoparticles. The 1H NMR spectra confirmed the structure of thiolated chitosan and eudragit RS100. The loading, encapsulation efficiency and release of moxifloxacin from NTC3 were 100.3%, 89.67% and 88.49% respectively. The nanoparticles in culture medium did not affect the viability of Caco-2 cells. The NTC3 formulation showed a greater bioavailability of moxifloxacin compared to the reference formulation. The study reports a convenient and effective way to prepare a chitosan and eudragit RS100 based drug delivery system with a controlled release pattern.


Assuntos
Resinas Acrílicas/química , Quitosana/química , Dissulfetos/química , Sistemas de Liberação de Medicamentos , Moxifloxacina/farmacologia , Nanopartículas/química , Compostos de Sulfidrila/química , Animais , Células CACO-2 , Varredura Diferencial de Calorimetria , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/farmacologia , Liberação Controlada de Fármacos , Humanos , Hidrodinâmica , Cinética , Moxifloxacina/farmacocinética , Tamanho da Partícula , Espectroscopia de Prótons por Ressonância Magnética , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Termogravimetria
4.
Sci Rep ; 11(1): 9582, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953262

RESUMO

Stenotrophomonas maltophilia exhibits wide spectrum of fluoroquinolone resistance using different mechanisms as multidrug efflux pumps and Smqnr alleles. Here, the role of smeDEF, smeVWX efflux genes and contribution of Smqnr alleles in the development of fluoroquinolone resistance was assessed. Ciprofloxacin, levofloxacin and moxifloxacin resistance were found in 10.9%, 3.5%, and 1.6% of isolates, respectively. More than four-fold differences in ciprofloxacin MICs were detected in the presence of reserpine and smeD, F, V expression was significantly associated with ciprofloxacin resistance (p = 0.017 for smeD, 0.003 for smeF, and 0.001 for smeV). Smqnr gene was found in 52% of the ciprofloxacin-resistant isolates and Smqnr8 was the most common allele detected. Fluoroquinolone resistance in S. maltophilia clinical isolates was significantly associated with active efflux pumps. There was no correlation between the Smqnr alleles and ciprofloxacin resistance; however, contribution of the Smqnr genes in low-level levofloxacin resistance was revealed.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Stenotrophomonas maltophilia/genética , Alelos , Ciprofloxacina/farmacologia , Irã (Geográfico) , Moxifloxacina/farmacologia , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/isolamento & purificação
5.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805837

RESUMO

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.


Assuntos
Antituberculosos/farmacologia , Desferroxamina/farmacologia , Diarilquinolinas/farmacologia , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Macrófagos/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Amicacina/farmacologia , Carga Bacteriana/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clofazimina/farmacologia , Ciclosserina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Linezolida/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/metabolismo , Cultura Primária de Células , Pirazinamida/farmacologia
6.
Antimicrob Agents Chemother ; 65(7): e0007921, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33846136

RESUMO

Nontuberculous mycobacterial (NTM) infections are increasing globally. Mycobacterium avium complex (MAC) and M. abscessus complex are the most commonly reported NTM. Oral treatment options are limited, especially for the M. abscessus complex. We tested delafloxacin, a new oral fluoroquinolone, against 131 isolates of NTM. Delafloxacin microdilution MICs were performed as recommended by the Clinical and Laboratory Standards Institute using cation adjusted Mueller-Hinton broth. The rapidly growing mycobacteria tested included M. abscessus subsp. abscessus (n = 16) and subsp. massiliense (n = 5), M. chelonae (n = 11), M. immunogenum (n = 5), M. fortuitum group (n = 13), M. porcinum (n = 7), M. senegalense (n = 7), M. mucogenicum group (n = 5), and M. goodii (n = 1). For the slowly growing NTM (SGM), M. avium (n = 16), M. intracellulare (n = 13), M. chimaera (n = 9), M. arupense (n = 5), M. simiae (n = 5), M. lentiflavum (n = 4), M. kansasii (n = 6), and M. marinum (n = 3) were tested. Delafloxacin was most active in vitro against the M. fortuitum and M. mucogenicum groups and M. kansasii, with MIC50 values of 0.12 to 0.5 µg/ml (MIC range, 0.001 to 4 µg/ml) compared to ≤0.06 to >4 µg/ml for ciprofloxacin and ≤0.06 to >8 µg/ml for moxifloxacin. For other SGM (including MAC), and the M. abscessus/M. chelonae, the delafloxacin MIC range was 8 to >16 µg/ml compared to ciprofloxacin and moxifloxacin of 0.5 to >4 µg/ml and ≤0.06 to 8 µg/ml, respectively. To our knowledge, this is the first MIC study with delafloxacin to use Clinical and Laboratory Standards Institute (CLSI) recommended methods. This study illustrates the potential utility of delafloxacin in treatment of infections due to some NTM.


Assuntos
Anti-Infecciosos , Infecções por Mycobacterium não Tuberculosas , Ciprofloxacina/farmacologia , Fluoroquinolonas , Humanos , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Micobactérias não Tuberculosas
7.
Int J Infect Dis ; 106: 295-299, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33864922

RESUMO

OBJECTIVE: WFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. METHODS: We selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. RESULTS: MFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. CONCLUSION: WFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.


Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Levofloxacino/farmacologia , Moxifloxacina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação
8.
PLoS One ; 16(3): e0248857, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33739996

RESUMO

In situ gels have been extensively explored as ocular drug delivery system to enhance bioavailability and efficacy. The objective of present study was to design, formulate and evaluate ion-activated in situ gel to enhance the ocular penetration and therapeutic performance of moxifloxacin in ophthalmic delivery. A simplex lattice design was utilized to examine the effect of various factors on experimental outcomes of the in situ gel system. The influence of polymers (independent variables) such as gellan gum (X1), sodium alginate (X2), and HPMC (X3) on gel strength, adhesive force, viscosity and drug release after 10 h (Q10) were assessed. Selected formulation (MH7) was studied for ex vivo permeation, in vivo irritation and pharmacokinetics in rabbits. Data revealed that increase in concentration of polymers led to higher gel strength, adhesive force and viscosity, however, decreases the drug release. MH7 exhibited all physicochemical properties within acceptable limits and was stable for 6 months. Release profile of moxifloxacin from MH7 was comparable to the check point batches and followed Korsmeyer-Peppas matrix diffusion-controlled mechanism. Ocular irritation study signifies that selected formulation is safe and non-irritant for ophthalmic administration. In vivo pharmacokinetics data indicates significant improvement of moxifloxacin bioavailability (p < 0.0001) from MH7, as evidenced by higher Cmax (727 ± 56 ng/ml) and greater AUC (2881 ± 108 ng h/ml), when compared with commercial eye drops (Cmax; 503 ± 85 ng/ml and AUC; 978 ± 86 ng h/ml). In conclusion, developed in situ gel system (MH7) could offers a more effective and extended ophthalmic therapy of moxifloxacin in ocular infections when compared to conventional eye drops.


Assuntos
Composição de Medicamentos , Infecções Oculares/tratamento farmacológico , Géis/administração & dosagem , Géis/uso terapêutico , Projetos de Pesquisa , Adesividade , Administração Oftálmica , Administração Tópica , Animais , Varredura Diferencial de Calorimetria , Córnea/efeitos dos fármacos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Cabras , Moxifloxacina/administração & dosagem , Moxifloxacina/farmacologia , Permeabilidade , Coelhos , Reologia , Viscosidade
9.
EBioMedicine ; 65: 103281, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33721817

RESUMO

BACKGROUND: Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. METHODS: We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT). FINDINGS: Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.05-7.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post-treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. INTERPRETATION: Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials. FUNDING: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 850450).


Assuntos
Linezolida/farmacologia , Treponema pallidum/efeitos dos fármacos , Animais , Área Sob a Curva , Clofazimina/farmacologia , Clofazimina/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Linezolida/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Penicilina G Benzatina/farmacologia , Penicilina G Benzatina/uso terapêutico , Curva ROC , Coelhos , Sífilis/tratamento farmacológico , Sífilis/patologia
10.
Eur J Clin Microbiol Infect Dis ; 40(8): 1767-1771, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33604720

RESUMO

In this study, we demonstrate that fluroquinolone (FQ) is at risk of acquired drug resistance after continuous exposure. The reduced susceptibility is observed in subsequent Mycobacterium tuberculosis isolates from patients without FQ exposure. The stepwise selection of mutation of increasing FQ resistance highlights the urgent need for monitoring FQ resistance in multidrug-resistant tuberculosis patients throughout the entire treatment course.


Assuntos
Antituberculosos/farmacologia , Moxifloxacina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Estudos de Coortes , DNA Girase/genética , DNA Girase/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Estudos Retrospectivos , Seleção Genética
11.
Sci Rep ; 11(1): 181, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420301

RESUMO

Cataract surgery is the most common intraocular procedure. To decrease postsurgical inflammation, prevent infection and reduce the incidence of secondary cataract, we built a temperature-sensitive drug delivery system carrying dexamethasone, moxifloxacin and genistein nanostructured lipid carrier (GenNLC) modified by mPEG-PLA based on F127/F68 as hydrogel. Characterizations and release profiles of the drug delivery system were studied. In vitro functions were detected by CCK-8 test, immunofluorescence, wound-healing assay, real time-PCR and western blotting. The size of GenNLCs was 39.47 ± 0.69 nm in average with surface charges of - 4.32 ± 0.84 mV. The hydrogel gelation temperature and time were 32 °C, 20 s with a viscosity, hardness, adhesiveness and stringiness of 6.135 Pa.s, 54.0 g, 22.0 g, and 3.24 mm, respectively. Transmittance of the gel-release medium was above 90% (93.44 ± 0.33% to 100%) at range of 430 nm to 800 nm. Moxifloxacin released completely within 10 days. Fifty percent of dexamethasone released at a constant rate in the first week, and then released sustainably with a tapering down rate until day 30. Genistein released slowly but persistently with a cumulative release of 63% at day 40. The thermoresponsive hydrogel inhibited the proliferation, migration and epithelial-mesenchymal transition of SRA 01/04 cells, which were confirmed by testing CCK-8, wound-healing assay, western blot, real time-PCR (RT-PCR) and immunofluorescence. These results support this intracameral thermoresponsive in situ multi-drug delivery system with programmed release amounts and release profiles to cut down the need of eye drops for preventing inflammation or infection and to reduce posterior capsular opacification following cataract surgery.


Assuntos
Extração de Catarata/efeitos adversos , Dexametasona/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Genisteína/farmacologia , Cápsula do Cristalino/efeitos dos fármacos , Moxifloxacina/farmacologia , Complicações Pós-Operatórias/prevenção & controle , Linhagem Celular , Dexametasona/administração & dosagem , Genisteína/administração & dosagem , Moxifloxacina/administração & dosagem , Temperatura
12.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430065

RESUMO

Mesoporous silica-based nanoparticles (MSNs) are considered promising drug carriers because of their ordered pore structure, which permits high drug loading and release capacity. The dissolution of Si and Ca from MSNs can trigger osteogenic differentiation of stem cells towards extracellular matrix calcification, while Mg and Sr constitute key elements of bone biology and metabolism. The aim of this study was the synthesis and characterization of sol-gel-derived MSNs co-doped with Ca, Mg and Sr. Their physico-chemical properties were investigated by X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), Brunauer Emmett Teller and Brunauer Joyner Halenda (BET/BJH), dynamic light scattering (DLS) and ζ-potential measurements. Moxifloxacin loading and release profiles were assessed with high performance liquid chromatography (HPLC) cell viability on human periodontal ligament fibroblasts and their hemolytic activity in contact with human red blood cells (RBCs) at various concentrations were also investigated. Doped MSNs generally retained their textural characteristics, while different compositions affected particle size, hemolytic activity and moxifloxacin loading/release profiles. All co-doped MSNs revealed the formation of hydroxycarbonate apatite on their surface after immersion in simulated body fluid (SBF) and promoted mitochondrial activity and cell proliferation.


Assuntos
Sistemas de Liberação de Medicamentos , Moxifloxacina/farmacologia , Nanopartículas/química , Engenharia Tecidual , Proliferação de Células/efeitos dos fármacos , Difusão Dinâmica da Luz , Humanos , Magnésio/química , Microscopia Eletrônica de Varredura , Moxifloxacina/química , Osteogênese/efeitos dos fármacos , Porosidade , Dióxido de Silício/química , Difração de Raios X
13.
Biochem J ; 478(3): 647-668, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33459338

RESUMO

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.


Assuntos
Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/isolamento & purificação , Células Cultivadas , Sinergismo Farmacológico , Células Epiteliais , Epitélio Corneano/citologia , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Moxifloxacina/farmacologia , Conformação Proteica , Proteínas Recombinantes/efeitos dos fármacos , Transfecção
14.
J Hazard Mater ; 409: 124518, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33191018

RESUMO

Moxifloxacin (MOX) and gatifloxacin (GAT) are fourth-generation fluoroquinolone antibiotics that are frequently detected in surface water environments and pose a threat to aquatic organisms. However, research into their toxicity to Microcystis aeruginosa, a cyanobacterium, has thus far been limited. In the present study, we investigated the effects of these antibiotics on M. aeruginosa growth, photosynthesis, oxidative stress, and microcystin (MC) release. The results of the 96 h EC50 values of MOX and GAT were 60.34 and 25.30 µg/L, respectively, and the risk quotients calculated indicated that these antibiotics could pose considerable ecological risks at actual environmental concentrations. Photosynthetic fluorescence intensity was shown to decline markedly, and Fv/Fm significantly decreased without any evidence of recovery, suggesting that the organism's photosystems were irreversibly damaged. Chlorophyll a and carotenoid content decreased, whereas the ratio of carotenoids to chlorophyll a increased, indicating that carotenoids were less susceptible to damage than chlorophyll a. The reactive oxygen species and malondialdehyde content significantly increased, as well as the superoxide dismutase and catalase activities, indicating that exposure caused serious oxidative stress. Additionally, MC release increased. These results demonstrate that the environmental risks posed by MOX and GAT should be given serious consideration, particularly as their use is increasing.


Assuntos
Gatifloxacina/farmacologia , Microcistinas/metabolismo , Microcystis , Moxifloxacina/farmacologia , Antioxidantes , Clorofila , Clorofila A , Microcystis/efeitos dos fármacos , Microcystis/metabolismo , Fotossíntese
15.
Comb Chem High Throughput Screen ; 24(3): 328-341, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32342810

RESUMO

BACKGROUND: Considering the low ocular bioavailability of conventional formulations used for ocular bacterial infection treatment, there is a need to design efficient novel drug delivery systems that may enhance precorneal retention time and corneal permeability. AIM AND OBJECTIVE: The current research focuses on developing nanosized and non-toxic Eudragit® RL 100 and Kollidon® SR nanoparticles loaded with moxifloxacin hydrochloride (MOX) for its prolonged release to be promising for effective ocular delivery. METHODS: In this study, MOX incorporation was carried out by spray drying method aiming ocular delivery. In vitro characteristics were evaluated in detail with different methods. RESULTS: MOX was successfully incorporated into Eudragit® RL 100 and Kollidon® SR polymeric nanoparticles by a spray-drying process. Particle size, zeta potential, entrapment efficiency, particle morphology, thermal, FTIR, NMR analyses and MOX quantification using HPLC method were carried out to evaluate the nanoparticles prepared. MOX loaded nanoparticles demonstrated nanosized and spherical shape while in vitro release studies demonstrated modified-release pattern, which followed the Korsmeyer-Peppas kinetic model. Following the successful incorporation of MOX into the nanoparticles, the formulation (MOX: Eudragit® RL 100, 1:5) (ERL-MOX 2) was selected for further studies because of its better characteristics like cationic zeta potential, smaller particle size, narrow size distribution and more uniform prolonged release pattern. Moreover, ERLMOX 2 formulation remained stable for 3 months and demonstrated higher cell viability values for MOX. CONCLUSION: In vitro characterization analyses showed that non-toxic, nano-sized and cationic ERL-MOX 2 formulation has the potential of enhancing ocular bioavailability.


Assuntos
Moxifloxacina/farmacologia , Nanopartículas/química , Ácidos Polimetacrílicos/química , Povidona/química , Células 3T3 , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Cinética , Camundongos , Moxifloxacina/química , Tamanho da Partícula
16.
Anaerobe ; 67: 102314, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33359396

RESUMO

We have a vast knowledge on human intestinal microbiota but it can still be regarded incomplete. One of the objectives of scientists using so-called "omics" techniques is to be interested in the consequences that drugs can have on the composition of the intestinal microbiota and inversely. To date, few publications have reported the effects of drugs on the growth of bacteria composing this microbiota using a "culturomics" approach. We focused on antibiotics commonly prescribed for which the only published are the susceptibility of the pathogenic strains and not that of the commensal strains. The aim of our study was to determine the sensitivity of 30 strains considered to represent the intestinal core microbiota to 8 antibiotics and to study the possible modification of these molecules by bacteria. The 30 bacterial strains were cultured under anaerobic conditions in order to determine their sensitivity to the antibiotics. After 48 h of culture, the supernatants were also analyzed via UHPLC-MS/MS in order to determine if the antibiotics have been chemically modified. Under the current experimental conditions, cefpodoxime, metronidazole, erythromycin, sulfamethozaxole, trimethoprim and the trimethoprim/sulfamethozaxole combination have little impact on the core microbiota strain growth. On the contrary, moxifloxacin and amoxicillin inhibit the growth of numerous strains of our panel. Using UHPLC-MS/MS analyses, we have shown that some antibiotics can be modifed by the bacteria composing the intestinal core microbiome. The bacteria that make up the intestinal microbiota core are impacted by the antibiotics most commonly prescribed in clinics today and inversely.


Assuntos
Antibacterianos/farmacologia , Cromatografia Líquida/métodos , Microbioma Gastrointestinal/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Amoxicilina/farmacologia , Humanos , Programas de Rastreamento , Metronidazol/farmacologia , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Sulfametoxazol/farmacologia
17.
mSphere ; 5(6)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33361124

RESUMO

The cure rate of multidrug-resistant tuberculosis (MDR-TB) is relatively low in China. The reasons for the treatment failure and within-host evolution during treatment have not been sufficiently studied. All MDR-TB patients receiving standard treatment from January 2014 to September 2016 at a designated TB Hospital in Zhejiang Province were retrospectively included and grouped according to their known treatment outcome. Clinical information was collected. Baseline strains of all patients and serial strains of treatment-failure patients were revived. Drug susceptibility tests (DSTs) of 14 drugs and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were performed. The genetic distance and within-host evolution were investigated based on SNPs. In total, 20 treatment failure patients and 74 patients who succeeded in treatment were included. The number of effective drugs for patients who failed treatment was no more than three. Eighteen (90.0%) treatment-failure patients were characterized by a continuous infection of the primary strain, of which 14 patients (77.8%) developed phenotypic or genotypic acquired drug resistance under ineffective treatment. Acquired resistance to amikacin and moxifloxacin (2.0 mg/ml) was detected most frequently, in 5 and 4 patients, respectively. The insufficient number of effective drugs in the combined treatment regimen was the main reason for MDR-TB treatment failure. The study emphasizes the importance of DST for second-line drugs when implementing the second-line drug regimen in MDR-TB patients. For patients with risk factors for MDR-TB, DST of second-line antituberculosis drugs should be performed at initiation of treatment. Second-line drugs should be selected based on the results of DST to avoid acquired resistance. WGS detects low-frequency resistance mutations and heterogeneous resistance with high sensitivity, which is of great significance for guiding clinical treatment and preventing acquired resistance.IMPORTANCE Few studies have focused on the reasons for the low cure rate of multidrug-resistant tuberculosis in China and within-host evolution during treatment, which is of great significance for improving clinical treatment regimens. Acquired resistance events were common during the ineffective treatment, among which resistance to amikacin and high-level moxifloxacin were the most common. The main reason for the treatment failure of MDR-TB patients was insufficient effective drugs, which may lead to higher levels of drug resistance in MDR-TB strains. Therefore, the study emphasizes the importance of DST in the development of second-line treatment regimen when there is a risk of MDR. By performing whole-genome sequencing of serial strains from patients with treatment failure, we found that WGS can detect low-frequency resistance mutations and heterogeneous resistance with high sensitivity. It is thus recommended to conduct drug susceptibility tests at the beginning of treatment and repeat the DST when the sputum bacteria remain positive.


Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Sequenciamento Completo do Genoma/métodos , Amicacina/farmacologia , Antituberculosos/farmacologia , China , Farmacorresistência Bacteriana Múltipla , Humanos , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Estudos Retrospectivos , Falha de Tratamento , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
18.
Viruses ; 13(1)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374514

RESUMO

Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC50) of 126.4 µM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 µM enoxacin and 600 µM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 µM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.


Assuntos
Antibacterianos/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Infecções por Coronavirus/tratamento farmacológico , Fluoroquinolonas/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Células A549 , Enzima de Conversão de Angiotensina 2 , Animais , Linhagem Celular , Chlorocebus aethiops , Ciprofloxacina/farmacologia , Enoxacino/farmacologia , Humanos , Levofloxacino/farmacologia , Moxifloxacina/farmacologia , Células Vero
19.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352719

RESUMO

Phototoxicity of fluoroquinolones is connected with oxidative stress induction. Lomefloxacin (8-halogenated derivative) is considered the most phototoxic fluoroquinolone and moxifloxacin (8-methoxy derivative) the least. Melanin pigment may protect cells from oxidative damage. On the other hand, fluoroquinolone-melanin binding may lead to accumulation of drugs and increase their toxicity to skin. The study aimed to examine the antioxidant defense system status in normal melanocytes treated with lomefloxacin and moxifloxacin and exposed to UV-A radiation. The obtained results demonstrated that UV-A radiation enhanced only the lomefloxacin-induced cytotoxic effect in tested cells. It was found that fluoroquinolones alone and with UV-A radiation decreased superoxide dismutase (SOD) activity and SOD1 expression. UV-A radiation enhanced the impact of moxifloxacin on hydrogen peroxide-scavenging enzymes. In turn, lomefloxacin alone increased the activity and the expression of catalase (CAT) and glutathione peroxidase (GPx), whereas UV-A radiation significantly modified the effects of drugs on these enzymes. Taken together, both analyzed fluoroquinolones induced oxidative stress in melanocytes, however, the molecular and biochemical studies indicated the miscellaneous mechanisms for the tested drugs. The variability in phototoxic potential between lomefloxacin and moxifloxacin may result from different effects on the antioxidant enzymes.


Assuntos
Antioxidantes/metabolismo , Fluoroquinolonas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Melanócitos/patologia , Moxifloxacina/farmacologia , Pele/patologia , Raios Ultravioleta/efeitos adversos , Antibacterianos/farmacologia , Antioxidantes/efeitos da radiação , Catalase/metabolismo , Catalase/efeitos da radiação , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/efeitos da radiação , Humanos , Melaninas/metabolismo , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , Estresse Oxidativo , Pele/efeitos dos fármacos , Pele/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/efeitos da radiação
20.
PLoS One ; 15(11): e0241780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33141832

RESUMO

The DNA topoisomerase complement of Streptococcus pneumoniae is constituted by two type II enzymes (topoisomerase IV and gyrase), and a single type I enzyme (topoisomerase I). These enzymes maintain the DNA topology, which is essential for replication and transcription. While fluoroquinolones target the type II enzymes, seconeolitsine, a new antimicrobial agent, targets topoisomerase I. We compared for the first time the in vitro effect of inhibition of topoisomerase I by seconeolitsine and of the type II topoisomerases by the fluoroquinolones levofloxacin and moxifloxacin. We used three isogenic non-encapsulated strains and five non-vaccine serotypes isolates belonging to two circulating pneumococcal clones, ST638 (2 strains) and ST1569V (3 strains). Each group contained strains with diverse susceptibility to fluoroquinolones. Minimal inhibitory concentrations, killing curves and postantibiotic effects were determined. Seconeolitsine demonstrated the fastest and highest bactericidal activity against planktonic bacteria and biofilms. When fluoroquinolone-susceptible planktonic bacteria were considered, seconeolitsine induced postantibiotic effects (1.00-1.87 h) similar than levofloxacin (1.00-2.22 h), but longer than moxifloxacin (0.39-1.71 h). The same effect was observed in sessile bacteria forming biofilms. Seconeolitsine induced postantibiotic effects (0.84-2.31 h) that were similar to those of levofloxacin (0.99-3.32 h) but longer than those of moxifloxacin (0.89-1.91 h). The greatest effect was observed in the viability and adherence of bacteria in the postantibiotic phase. Seconeolitsine greatly reduced the thickness of the biofilms formed in comparison with fluoroquinolones: 2.91 ± 0.43 µm (seconeolitsine), 7.18 ± 0.58 µm (levofloxacin), 17.08 ± 1.02 µm (moxifloxacin). When fluoroquinolone-resistant bacteria were considered, postantibiotic effects induced by levofloxacin and moxifloxacin, but not by seconeolitsine, were shorter, decreasing up to 5-fold (levofloxacin) or 2-fold (moxifloxacin) in planktonic cells, and up to 1.7 (levofloxacin) or 1.4-fold (moxifloxacin) during biofilm formation. Therefore, topoisomerase I inhibitors could be an alternative for the treatment of pneumococcal diseases, including those caused by fluoroquinolone-resistant isolates.


Assuntos
Antibacterianos/farmacologia , DNA Topoisomerase IV/antagonistas & inibidores , Fluoroquinolonas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Benzodioxóis/farmacologia , DNA Girase/metabolismo , Levofloxacino/farmacologia , Moxifloxacina/farmacologia , Fenantrenos/farmacologia , Streptococcus pneumoniae/enzimologia
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