RESUMO
In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.
Assuntos
Antituberculosos/farmacologia , DNA Girase/genética , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Mutação , Mycobacterium tuberculosis/genética , Bioensaio , DNA Girase/metabolismo , Reações Falso-Positivas , Expressão Gênica , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/metabolismo , Filogenia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.
Assuntos
Mycobacterium abscessus , Mycobacterium tuberculosis , Mycobacterium abscessus/genética , Sistemas CRISPR-Cas , Streptococcus thermophilus/genética , Mycobacterium tuberculosis/genéticaRESUMO
BACKGROUND: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. METHODS: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005-2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. FINDINGS: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200â¯years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10â¯years for a 5 SNP/allele cut-off. INTERPRETATION: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting.