RESUMO
OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.
Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Hanseníase/epidemiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Antibacterianos/efeitos adversos , Proteínas de Bactérias/genética , Biópsia por Agulha , Brasil/epidemiologia , Colômbia/epidemiologia , DNA Girase/genética , Dapsona/uso terapêutico , Doenças Endêmicas/estatística & dados numéricos , Monitoramento Epidemiológico , Saúde Global , Humanos , Índia/epidemiologia , Hanseníase/diagnóstico , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana , Mutação , Ofloxacino/uso terapêutico , Reação em Cadeia da Polimerase , Estudos Prospectivos , Recidiva , Rifampina/uso terapêutico , Vigilância de Evento Sentinela , Pele/microbiologia , Pele/patologia , Inquéritos e Questionários , Organização Mundial da SaúdeRESUMO
Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.
Assuntos
Farmacorresistência Bacteriana Múltipla , Mycobacterium leprae/genética , Análise de Sequência de DNA/métodos , Sudeste Asiático , Genoma Bacteriano , Genótipo , Humanos , Hanseníase/microbiologia , Dados de Sequência Molecular , Mycobacterium leprae/classificação , Filogenia , Polimorfismo de Nucleotídeo Único , VenezuelaRESUMO
Mycolic acids are major components of the cell envelope of mycobacteria, such as Mycobacterium tuberculosis, and play an important role in its architecture, impermeability and interaction with the environment. Synthesis of mycolic acids is carried out by two types of fatty acid synthases (FAS) working in concert: type I FAS, a multifunctional enzyme capable of de novo synthesis of medium-chain fatty acids, and type II FAS, responsible for their elongation. In this article we report the identification and characterization of a transcriptional regulator (MabR), whose binding to the FAS-II promoter region was demonstrated in vitro and in vivo. Overexpression and knock-down studies in Mycobacterium smegmatis revealed the repressor nature of MabR, with reduced amounts of FAS-II transcripts and fatty acids in the overproducing strain. Under these conditions, downregulation of fas transcription was also observed, thereby suggesting the existence of cross-talk between the two FAS, mediated by MabR. Finally, the finding that a mabR knock-out mutant could only be obtained in a merodiploid strain of M. smegmatis, confirmed the predicted essentiality, thus implying an essential role for MabR in mycobacterial fatty acid metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Metabolismo dos Lipídeos , Mycobacterium smegmatis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Ácido Graxo Sintase Tipo II/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Mycobacterium smegmatis/enzimologia , Óperon , Regiões Promotoras GenéticasRESUMO
Continuous subculture has been observed to produce changes in the virulence of micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for the last 100 years; however, in some instances its efficacy has been observed to be very low. In order to determine whether similar changes can be produced in Mycobacterium tuberculosis, we selected four isolates, M. tuberculosis H37Rv, a Beijing strain (DR-689), and two more isolates with deletion of the phospholipase C locus (plcA-plcB-plcC ), and subjected them to serial culturing on Middlebrook 7H9 medium, with or without ox bile. After 100 passages, we performed RFLP-IS6110 analysis to determine whether genomic changes were produced. We also checked their genomic composition by microarray analysis. Changes in virulence were studied by measuring the cytotoxic effect of parental and subcultured isolates on a THP-1 macrophage monolayer. The most visible change was the change of position of an IS6110 band of approximately 1400 bp to approximately 1600 bp in the Beijing isolate subcultured in the ox bile medium. Analysis by microarray and PCR confirmation did not reveal any genomic changes. Cytotoxic activity was decreased in the isolates at levels close to that of BCG, and more consistently in those subcultured in the presence of ox bile.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Bile/fisiologia , Células Cultivadas , Meios de Cultura , Humanos , Macrófagos/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Polimorfismo de Fragmento de Restrição , VirulênciaAssuntos
Análise de Sequência de DNA , DNA Bacteriano , Dados de Sequência Molecular , Evolução Molecular , Família Multigênica , Genoma Bacteriano , Hanseníase/microbiologia , Metabolismo Energético , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Tatus , Transferência Genética HorizontalRESUMO
The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.