RESUMO
Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.
Assuntos
Aptâmeros de Nucleotídeos/genética , Hepacivirus/genética , RNA Polimerase Dependente de RNA/genética , Aptâmeros de Nucleotídeos/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Genótipo , Hepacivirus/enzimologia , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Técnica de Seleção de Aptâmeros , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Integrons were detected in 37 (72.5%) of 51 Klebsiella spp. producing extended-spectrum beta-lactamases by PCR with primers that targeted integrase genes and cassette regions. PCR and amplicon sequencing of the cassette regions revealed aadB and aadA2 gene cassettes that confer resistance to a range of aminoglycosides. aadB was associated with a class 1 integron on a 28-kb plasmid, pES1, that also contained bla(SHV-12) and IS26.
Assuntos
Genes Bacterianos/genética , Klebsiella/genética , beta-Lactamases/genética , Aminoglicosídeos/farmacologia , Transferência Genética Horizontal/genética , Integrases/genética , Integrons/genética , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Integrons have been recognised as important contributors to the acquisition and dissemination of antibiotic resistance in Gram-negative bacteria. In a collection of 19 multi-antibiotic resistant Gram-negative clinical isolates, 47 per cent (9/19) of strains were found to contain one or more integron, using a polymerase chain reaction (PCR) based screening method. Resistance gene cassettes within the integrons were amplified, sequenced and characterised. Antibiotic susceptibility testing demonstrated that resistance phenotypes correlated with the resistance conferred by gene cassettes identified. PCR-screening for integrons and gene cassettes provides a rapid technique for the identification of genetic determinants of resistance in Gram-negative bacteria. Such screening could assist in guiding treatment regimens and complement existing antibiotic resistance surveillance programs by providing information on molecular mechanisms of both resistance and resistance dissemination.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Integrons/genética , Reação em Cadeia da Polimerase , Testes de Sensibilidade MicrobianaRESUMO
Class 2 integrons (Tn7) were found in all randomly selected epidemic (n = 27) and preepidemic (n = 13) strains of multiresistant Shigella sonnei biotype g. A class 1 integron was also found in two epidemic strains. Gene cassettes within these integrons account for resistance to commonly used therapeutic agents.