Mycobacterium abscessus complex (MABC) is a taxonomic group of rapidly growing, nontuberculous mycobacteria that are found as etiologic agents of various types of infections. They are considered as emerging human pathogens. MABC consists of 3 subspecies—M. abscessus subsp. bolletti, M. abscessus subsp. massiliense and M. abscessus subsp. abscessus. Here we present a novel method for subspecies differentiation of M. abscessus named Subspecies-Specific Sequence Detection (SSSD). This method is based on the presence of signature sequences present within the genomes of each subspecies of MABC. We tested this method against a virtual database of 1505 genome sequences of MABC. Further, we detected signature sequences of MABC in 45 microbiological samples through DNA hybridization. SSSD showed high levels of sensitivity and specificity for differentiation of subspecies of MABC, comparable to those obtained by rpoB sequence typing.

Mycobacterium tuberculosis is one of the deadliest and most challenging pathogens to study in current microbiological research. One of the issues that remains to be resolved is the importance of cobalamin in the metabolism of M. tuberculosis. The functionality of a vitamin B12 biosynthesis pathway in M. tuberculosis is under dispute, and the ability of this pathogen to scavenge vitamin B12 from the host is unknown. Here, we quantified the ratios of nonsynonymous and synonymous nucleotide substitution rates (dN/dS) in the genes involved in vitamin B12 biosynthesis and transport and in genes encoding cobalamin-dependent enzymes in nearly four thousand strains of M. tuberculosis. We showed that purifying selection is the dominant force acting on cobalamin-related genes at the levels of individual codons, genes and groups of genes. We conclude that cobalamin-related genes may not be essential but are adaptive for M. tuberculosis in clinical settings. Furthermore, the cobalamin biosynthesis pathway is likely to be functional in this species.

Cobalamin (vitamin B12) is a structurally complex molecule that acts as a cofactor for enzymes and regulates gene expression through so-called riboswitches. The existing literature on the vitamin B12 synthesis capacity in Mycobacterium tuberculosis is ambiguous, while in non-tuberculous mycobacteria (NTM) is rather marginal. Here we present the results of our investigation into the occurrence of vitamin B12 in mycobacteria. For detection purposes, immunoassay methods were applied to cell lysates of NTM and M. tuberculosis clinical and laboratory strains grown under different conditions. We show that whereas vitamin B12 is present in cells of various NTM species, it cannot be evidenced in strains of differently cultured M. tuberculosis, even though the genes responsible for vitamin B12 synthesis are actively expressed based on RNA-Seq data. In summary, we conclude that the production of vitamin B12 does occur in mycobacteria, with the likely exception of M. tuberculosis. Our results provide direct evidence of vitamin B12 synthesis in a clinically important group of bacteria.

Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBC^{CRISPRi/dCas9} strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBC^{CRISPRi/dCas9} strain at the transcript level.

Background: In many countries tuberculosis (TB) remains a highly prevalent disease and a major contributor to infectious disease mortality. The fight against TB requires surveillance of the population of strains circulating worldwide and the analysis of the prevalence of certain strains in populations. Nowadays, whole genome sequencing (WGS) allows for accurate tracking of TB transmission. Currently, there is a lack of a comprehensive summary of the characteristics of TB outbreaks. Methods: We systematically analyzed studies reporting TB outbreaks worldwide, monitored through WGS of Mycobacterium tuberculosis. We 1) mapped the reported outbreaks from 2011- 2020, 2) estimated the average size of the outbreaks, 3) indicated genetic lineages causing the outbreaks, and 4) determined drug-resistance patterns of M. tuberculosis strains involved in the outbreaks. Results: Most data originated from Europe, Asia, and North America. We found that TB outbreaks were reported throughout the globe, on all continents, and in countries with both high and low incidences. The detected outbreaks contained a median of five M. tuberculosis isolates. Most strains causing the outbreaks belonged to lineage four, more rarely to lineage two. Reported outbreak isolates were often drug resistant. Conclusions: We conclude that more WGS surveillance of M. tuberculosis outbreaks is needed. Globally standardized procedures might improve the control of M. tuberculosis infections.