Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Microbiol Spectr ; 10(3): e0082822, 2022 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-35647694

RESUMEN

Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health requiring the development of novel therapies. TP0586532 is a novel non-hydroxamate LpxC inhibitor that inhibits the synthesis of lipopolysaccharides, which are components of the outer membranes of Gram-negative bacteria. Based on the mechanism of action of TP0586532, we hypothesized that it might enhance the antibacterial activity of other antibiotics by increasing the permeability of the outer bacterial membrane. The combination of TP0586532 with meropenem, amikacin, cefepime, piperacillin, and tigecycline showed synergistic and additive effects against carbapenem-susceptible Klebsiella pneumoniae and Escherichia coli. Checkerboard experiments against 21 carbapenem-resistant K. pneumoniae and E. coli strains (13 blaKPC+, 5 blaNDM-1+, 2 blaVIM+, and 1 blaIMP+) showed that the combination of TP0586532 with meropenem yielded synergistic and additive effects against 9 and 12 strains, respectively. In a time-kill assay examining 12 CRE strains, synergistic effects were observed when TP0586532 was combined with meropenem against many of the strains. A membrane permeability assay using ethidium bromide (EtBr) was performed to investigate the mechanism of the potentiating effect. TP0586532 increased the influx of EtBr into a CRE strain, suggesting that TP0586532 increased membrane permeability and facilitated intracellular access for the antibiotics. Our study demonstrates that TP0586532 potentiates the in vitro antibacterial activity of meropenem against CRE. Combination therapy consisting of TP0586532 and meropenem has potential as a treatment for CRE infections. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health threat, as therapeutic options are limited. TP0586532 is a novel LpxC inhibitor that inhibits the synthesis of lipopolysaccharides in the outer membranes of Gram-negative bacteria. Here, we demonstrated the potentiating effects of TP0586532 on the antibacterial activity of meropenem against CRE harboring various types of carbapenemase genes (blaKPC+, blaNDM-1+ blaVIM+, and blaIMP+). TP0586532 also augmented the bactericidal effects of meropenem against CRE strains, even against those with a high level of resistance to meropenem. The potentiating effects were suggested to be mediated by an increase in bacterial membrane permeability. Our study revealed that a combination therapy consisting of TP0586532 and meropenem has the potential to be a novel therapeutic option for CRE infections.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Enterobacteriaceae , Humanos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , beta-Lactamasas/genética , Butanoles/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Carbapenémicos/farmacología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/genética , Bacterias Gramnegativas , Imidazoles/farmacología , Klebsiella pneumoniae/genética , Meropenem/farmacología , Pruebas de Sensibilidad Microbiana
2.
J Infect Chemother ; 28(5): 635-642, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35131156

RESUMEN

INTRODUCTION: TP0586532 is a novel non-hydroxamate UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) inhibitor. Pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitude of index that correlated with the efficacy of TP0586532 were determined and used to estimate the clinically effective doses of TP0586532. METHODS: Dose-fractionation studies were conducted using a murine neutropenic lung infection model caused by carbapenem-resistant Enterobacteriaceae. The relationships between the efficacy and the PK/PD index (the maximum unbound plasma concentration divided by the MIC [fCmax/MIC], the area under the unbound plasma concentration-time curve from 0 to 24 h divided by the MIC, and the cumulative percentage of a 24-h period that the unbound plasma concentration exceeds the MIC) were determined using an inhibitory sigmoid maximum-effect model. In addition, the magnitudes of fCmax/MIC were evaluated using the dose-response relationships for each of the seven carbapenem-resistant strains of Enterobacteriaceae. Furthermore, the clinically effective doses of TP0586532 were estimated using the predicted human PK parameters, the geometric mean of fCmax/MIC, and the MIC90 for carbapenem-resistant Klebsiella pneumoniae. RESULTS: The PK/PD index that best correlated with the efficacy was the fCmax/MIC. The geometric means of the fCmax/MIC associated with the net stasis and 1-log reduction endpoints were 2.30 and 3.28, respectively. The clinically effective doses of TP0586532 were estimated to be 1.24-2.74 g/day. CONCLUSION: These results indicate the potential for TP0586532 to have clinical efficacy at reasonable doses against infections caused by carbapenem-resistant Enterobacteriaceae. This study provided helpful information for a clinically effective dosing regimen of TP0586532.


Asunto(s)
Antibacterianos , Klebsiella pneumoniae , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Enterobacteriaceae , Humanos , Pulmón , Ratones , Pruebas de Sensibilidad Microbiana
3.
J Antibiot (Tokyo) ; 75(3): 136-145, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34987187

RESUMEN

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an essential enzyme in the biosynthesis of Lipid A, an active component of lipopolysaccharide (LPS), from UDP-3-O-acyl-N-acetylglicosamine. LPS is a major component of the cell surface of Gram-negative bacteria. LPS is known to be one of causative factors of sepsis and has been associated with high mortality in septic shock. TP0586532 is a novel non-hydroxamate LpxC enzyme inhibitor. In this study, we examined the inhibitory effect of TP0586532 on the LPS release from Klebsiella pneumoniae both in vitro and in vivo. Our results confirmed the inhibitory effect of TP0586532 on LPS release from the pathogenic bacterial species. On the other hand, meropenem and ciprofloxacin increase the level of LPS release. Furthermore, the effects of TP0586532 on LPS release and interleukin (IL)-6 production in the lung were determined using a murine model of pneumonia caused by K. pneumoniae. As observed in the in vitro study, TP0586532 showed the marked inhibitory effect on LPS release in the lungs, whereas meropenem- and ciprofloxacin-treated mice showed higher levels of LPS release and IL-6 production in the lungs as compared to those in the lungs of vehicle-treated mice. Moreover, TP0586532 used in combination with meropenem and ciprofloxacin attenuated the LPS release and IL-6 production induced by meropenem and ciprofloxacin in the lung. These results indicate that the inhibitory effect of TP0586532 on LPS release from pathogenic bacteria might be of benefit in patients with sepsis.


Asunto(s)
Amidohidrolasas/metabolismo , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Animales , Ciprofloxacina/farmacología , Femenino , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/metabolismo , Meropenem/metabolismo , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana/métodos
4.
J Antibiot (Tokyo) ; 75(2): 98-107, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34837061

RESUMEN

The emergence of multi-drug resistant pathogenic bacteria, especially Gram-negative bacteria, is a worldwide health problem. New antibiotics directed at previously unexplored targets are urgently needed to overcome resistance to existing antibiotic classes. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an attractive target for a new antibacterial agent. Although a number of LpxC inhibitors have been identified, none have been approved as antibacterial agents. These LpxC inhibitors contain a hydroxamate moiety, which is a robust zinc ion chelator. The nonspecific inhibition of metalloenzymes through zinc ion chelation is one of possibilities leading to unwanted side effects. Herein, we report that TP0586532, a non-hydroxamate LpxC inhibitor, has a broad spectrum of antibacterial activity against carbapenem-resistant Enterobacteriaceae. The MIC90 of TP0586532 against clinical isolates of carbapenem-resistant Klebsiella pneumoniae was 4 µg ml-1. TP0586532 also showed an in vivo efficacy against murine systemic, urinary tract and lung infection models caused by meropenem- or ciprofloxacin-resistant strains. The estimated maximum unbound plasma concentration value at the effective dose of TP0586532 in murine infection models was around 13 µg ml-1. TP0586532 is predicted to exhibit a in vivo efficacy without cardiovascular toxicity and showed the potential of non-hydroxamate LpxC inhibitors as antibacterial agents against carbapenem-resistant Enterobacteriaceae.


Asunto(s)
Amidohidrolasas , Antibacterianos , Enterobacteriaceae , Animales , Ratones , Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Antibacterianos/toxicidad , Quelantes/química , Quelantes/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Meropenem/farmacología , Pruebas de Sensibilidad Microbiana , Zinc/química
5.
Artículo en Inglés | MEDLINE | ID: mdl-33558293

RESUMEN

Gonorrhea is a common, sexually transmitted disease caused by Neisseria gonorrhoeae Multidrug-resistant N. gonorrhoeae is an urgent threat, and the development of a new antimicrobial agent that functions via a new mechanism is strongly desired. We evaluated the in vitro and in vivo activities of a DNA gyrase/topoisomerase IV inhibitor, TP0480066, which is a novel 8-(methylamino)-2-oxo-1,2-dihydroquinoline derivative. The MICs of TP0480066 were substantially lower than those of other currently or previously used antimicrobials against gonococcal strains demonstrating resistance to fluoroquinolones, macrolides, ß-lactams, and aminoglycosides (MICs, ≤0.0005 µg/ml). Additionally, no cross-resistance was observed between TP0480066 and ciprofloxacin. The frequencies of spontaneous resistance to TP0480066 for N. gonorrhoeae ATCC 49226 were below the detection limit (<2.4 × 10-10) at concentrations equivalent to 32× MIC. TP0480066 also showed potent in vitro bactericidal activity and in vivo efficacy in a mouse model of N. gonorrhoeae infection. These data suggest that TP0480066 is a candidate antimicrobial agent for gonococcal infections.


Asunto(s)
Gonorrea , Neisseria gonorrhoeae , Animales , Antibacterianos/farmacología , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Farmacorresistencia Bacteriana , Fluoroquinolonas , Gonorrea/tratamiento farmacológico , Ratones , Pruebas de Sensibilidad Microbiana
6.
Bioorg Med Chem ; 30: 115964, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33385955

RESUMEN

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.


Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Imidazoles/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Amidohidrolasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Imidazoles/síntesis química , Imidazoles/química , Klebsiella pneumoniae/enzimología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad
7.
J Med Chem ; 63(23): 14805-14820, 2020 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-33210531

RESUMEN

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 µg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.


Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Quelantes/farmacología , Inhibidores Enzimáticos/farmacología , Anilidas/farmacología , Antibacterianos/síntesis química , Quelantes/síntesis química , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Imidazoles/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperidinas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Relación Estructura-Actividad , Zinc/química
8.
Bioorg Med Chem ; 28(22): 115776, 2020 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-33032189

RESUMEN

The global increase in multidrug-resistant pathogens has caused severe problems in the treatment of infections. To overcome these difficulties, the advent of a new chemical class of antibacterial drug is eagerly desired. We aimed at creating novel antibacterial agents against bacterial type II topoisomerases, which are well-validated targets. TP0480066 (compound 32) has been identified by using structure-based optimization originated from lead compound 1, which was obtained as a result of our previous lead identification studies. The MIC90 values of TP0480066 against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and genotype penicillin-resistant Streptococcus pneumoniae (gPRSP) were 0.25, 0.015, and 0.06 µg/mL, respectively. Hence, TP0480066 can be regarded as a promising antibacterial drug candidate of this chemical class.


Asunto(s)
Antibacterianos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Quinolinas/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana/efectos de los fármacos , Células Hep G2 , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/enzimología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Regulador Transcripcional ERG/antagonistas & inhibidores , Regulador Transcripcional ERG/metabolismo , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Enterococos Resistentes a la Vancomicina/enzimología
9.
ACS Omega ; 5(17): 10145-10159, 2020 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-32391502

RESUMEN

DNA gyrase and topoisomerase IV are well-validated pharmacological targets, and quinolone antibacterial drugs are marketed as their representative inhibitors. However, in recent years, resistance to these existing drugs has become a problem, and new chemical classes of antibiotics that can combat resistant strains of bacteria are strongly needed. In this study, we applied our hit-to-lead (H2L) chemistry for the identification of a new chemical class of GyrB/ParE inhibitors by efficient use of thermodynamic parameters. Investigation of the core fragments obtained by fragmentation of high-throughput screening hit compounds and subsequent expansion of the hit fragment was performed using isothermal titration calorimetry (ITC). The 8-(methylamino)-2-oxo-1,2-dihydroquinoline derivative 13e showed potent activity against Escherichia coli DNA gyrase with an IC50 value of 0.0017 µM. In this study, we demonstrated the use of ITC for primary fragment screening, followed by structural optimization to obtain lead compounds, which advanced into further optimization for creating novel antibacterial agents.

10.
Anaerobe ; 57: 86-89, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30951830

RESUMEN

Aspiration pneumonia is an infectious disease of the lungs caused by inhalation of saliva or foods, associated with swallowing dysfunction. Therefore, the major causative organisms are oral or gastric bacteria. In this study, we evaluated the antimicrobial susceptibility patterns of the anaerobic bacteria which can cause aspiration pneumonia, Fusobacterium spp., Finegoldia magna, Bacteroides fragilis, Peptostreptococcus spp., Prevotella spp., and Streptococcus milleri group to ceftriaxone, cefmetazole, flomoxef, ampicillin/sulbactam, and ampicillin. We also tested the ß-lactamase activities of each of the bacterial strains. Fusobacterium spp. and Finegoldia magna were susceptible to all of the tested antimicrobial drugs, except ampicillin, and showed no ß-lactamase activity. The Streptococcus milleri group, Bacteroides fragilis, and Peptostreptococcus spp. showed decreased susceptibility to cefmetazole or flomoxef as compared to the susceptibility levels documented in a previous report. There was one strain of Peptostreptococcus anaerobius which was not susceptible to ampicillin/sulbactam, but also showed no ß-lactamase activity, suggesting that this strain harbored a mechanism of resistance other than the production of ß-lactamase. The susceptibility of Prevotella spp. to ceftriaxone was also decreased as compared to the susceptibility level documented in a previous report. Furthermore, ß-lactamase-positive strains were found even among ceftriaxone-susceptible strains. Elderly persons with swallowing dysfunction carry a risk of recurrent episodes of aspiration pneumonia and repeated use of antibiotics increases the risk of development of antibiotic resistance. In the present study, the antibiotic susceptibilities of some of organisms which can cause aspiration pneumonia were found to be decreased as compared to the susceptibility levels documented in a previous report. Therefore, surveillance of the antimicrobial susceptibility patterns of these bacteria is recommended to prevent the development of resistance.


Asunto(s)
Antibacterianos/farmacología , Bacterias Anaerobias/efectos de los fármacos , Bacterias Anaerobias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Pruebas de Sensibilidad Microbiana , Neumonía por Aspiración/microbiología , Humanos
11.
J Infect Chemother ; 24(1): 45-52, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29107652

RESUMEN

The increased prevalence of the virulence factor exoU + genotype among multidrug-resistant Pseudomonas aeruginosa has been previously reported. However, the genes that are related to the multidrug resistance of the exoU + genotype strain have not been analyzed and remain to be elucidated. The objective of this study was to analyze the correlations between virulence factors and resistance genes. The exoU + genotype was frequently found in carbapenem and fluoroquinolone non-susceptible strains. The imp carbapenemase genotype, the quinolone-resistance-determining region mutation in GyrA and ParC and the defective mutation in OprD were not frequently found in the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. On the other hand, mexY and ampC mRNA overexpressing strains were more frequently found in the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. Moreover, sequence type 235, a high risk clone of multidrug-resistant P. aeruginosa, was prevalent among the exoU + genotype and carbapenem and fluoroquinolone non-susceptible strains. ExoU is highly virulent protein, and the overexpression of efflux pumps and AmpC ß-lactamase induce a multidrug-resistant phenotype. Therefore, the increased prevalence of P. aeruginosa strains with an exoU + genotype and the overexpression of efflux pumps and AmpC ß-lactamase are likely to make P. aeruginosa infections difficult to treat. An understanding of the prevalence of both the exoU + genotype and the mRNA overexpression of resistance genes may help to select empirical therapy for the treatment of nosocomial infections caused by P. aeruginosa.


Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Genes MDR/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , ARN Mensajero/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Carbapenémicos/farmacología , Fluoroquinolonas/farmacología , Genotipo , Humanos , Mutación , Pseudomonas aeruginosa/aislamiento & purificación , ARN Mensajero/genética , Factores de Virulencia/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
12.
EMBO J ; 22(12): 2913-23, 2003 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-12805207

RESUMEN

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the alpha-ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimer's disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH-SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD-deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse-label experiment revealed that the loss of the subunits is a post-translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.


Asunto(s)
Aciltransferasas/genética , Aciltransferasas/metabolismo , Enfermedad de Alzheimer/genética , Transporte de Electrón/fisiología , ARN Mensajero/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Regulación Enzimológica de la Expresión Génica , Haplotipos , Humanos , Persona de Mediana Edad , Mitocondrias Hepáticas/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estrés Oxidativo , Células PC12 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/genética , Ratas , Células Tumorales Cultivadas
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...