RESUMO
Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.
Assuntos
Mycobacterium tuberculosis , Humanos , Filogenia , Colômbia/epidemiologia , Genoma Bacteriano , Surtos de Doenças , Povos IndígenasRESUMO
BACKGROUND: Mycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. RESULTS: We found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth. CONCLUSIONS: This study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server ( http://microbiomics.ibt.unam.mx/tools/aar/index.php ).