RESUMO
Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.
Assuntos
Antituberculosos , Biologia Computacional , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biologia Computacional/métodos , Genoma Bacteriano , Farmacorresistência Bacteriana/genética , Mutação , Tuberculose/microbiologia , Tuberculose/tratamento farmacológicoRESUMO
Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.
Assuntos
Genoma Bacteriano , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biblioteca Gênica , DNA Bacteriano/genética , Tuberculose/microbiologia , Tuberculose/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
BACKGROUND: The Mycobacterium avium complex (MAC) comprises the most frequent non-tuberculous mycobacteria (NTM) in Central Europe and currently includes twelve species. M. avium (MAV), M. intracellulare subsp. intracellulare (MINT), and M. intracellulare subsp. chimaera (MCH) are clinically most relevant. However, the population structure and genomic landscape of MAC linked with potential pathobiological differences remain little investigated. METHODS: Whole genome sequencing (WGS) was performed on a multi-national set of MAC isolates from Germany, France, and Switzerland. Phylogenetic analysis was conducted, as well as plasmids, resistance, and virulence genes predicted from WGS data. Data was set into a global context with publicly available sequences. Finally, detailed clinical characteristics were associated with genomic data in a subset of the cohort. RESULTS: Overall, 610 isolates from 465 patients were included. The majority could be assigned to MAV (n = 386), MCH (n = 111), and MINT (n = 77). We demonstrate clustering with less than 12 SNPs distance of isolates obtained from different patients in all major MAC species and the identification of trans-European or even trans-continental clusters when set into relation with 1307 public sequences. However, none of our MCH isolates clustered closely with the heater-cooler unit outbreak strain Zuerich-1. Known plasmids were detected in MAV (325/1076, 30.2%), MINT (62/327, 19.0%), and almost all MCH-isolates (457/463, 98.7%). Predicted resistance to aminoglycosides or macrolides was rare. Overall, there was no direct link between phylogenomic grouping and clinical manifestations, but MCH and MINT were rarely found in patients with extra-pulmonary disease (OR 0.12 95% CI 0.04-0.28, p < 0.001 and OR 0.11 95% CI 0.02-0.4, p = 0.004, respectively) and MCH was negatively associated with fulfillment of the ATS criteria when isolated from respiratory samples (OR 0.28 95% CI 0.09-0.7, p = 0.011). With 14 out of 43 patients with available serial isolates, co-infections or co-colonizations with different strains or even species of the MAC were frequent (32.6%). CONCLUSIONS: This study demonstrates clustering and the presence of plasmids in a large proportion of MAC isolates in Europe and in a global context. Future studies need to urgently define potential ways of transmission of MAC isolates and the potential involvement of plasmids in virulence.
Assuntos
Genoma Bacteriano , Genômica , Complexo Mycobacterium avium , Infecção por Mycobacterium avium-intracellulare , Filogenia , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/isolamento & purificação , Humanos , Infecção por Mycobacterium avium-intracellulare/microbiologia , Infecção por Mycobacterium avium-intracellulare/epidemiologia , Europa (Continente) , Masculino , Feminino , Genômica/métodos , Sequenciamento Completo do Genoma , Idoso , Pessoa de Meia-Idade , Plasmídeos/genética , Polimorfismo de Nucleotídeo Único , Farmacorresistência Bacteriana/genética , Adulto , Virulência/genéticaRESUMO
Mycobacterium tuberculosis complex (MTBC) Lineage 3 (L3) strains are abundant in world regions with the highest tuberculosis burden. To investigate the population structure and the global diversity of this major lineage, we analyzed a dataset comprising 2682 L3 strains from 38 countries over 5 continents, by employing 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping (MIRU-VNTR) and drug susceptibility testing. We further combined whole-genome sequencing (WGS) and phylogeographic analysis for 373 strains representing the global L3 genetic diversity. Ancestral state reconstruction confirmed that the origin of L3 strains is located in Southern Asia and further revealed multiple independent introduction events into North-East and East Africa. This study provides a systematic understanding of the global diversity of L3 strains and reports phylogenetic variations that could inform clinical trials which evaluate the effectivity of new drugs/regimens or vaccine candidates.