Strong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium’s metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3 , dosR , and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs’ activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules. IMPORTANCE Mycobacterium tuberculosis establishes long-lasting highly prevalent infection inside the human body, called latent tuberculosis. The known involvement of fatty acids is changing our understanding of that silent infection; however, question of how tubercle bacilli globally adapt to a lipid-enriched environment is still an unanswered. With the single change of providing fatty acids as carbon sources, the bacilli switch on their program related to dormant stage: slowed growth, accumulation of lipid bodies, and development of drug tolerance. In this stage, unexpected and previously unknown participants were found to play putatively important roles during the process. For the first time, this work compares the global transcriptomics of bacteria by using strand-specific RNA sequencing under two different growth conditions. This study suggests novel targets for the control of tuberculosis and provides a new straightforward in vitro model that could help to test the activity of drugs against dormant bacilli from a novel perspective.

Changes in respiratory tract microbiota have been associated with diseases such as tuberculosis, a global public health problem that affects millions of people each year. This pilot study was carried out using sputum, oropharynx, and nasal respiratory tract samples collected from patients with pulmonary tuberculosis and healthy control individuals, in order to compare sample types and their usefulness in assessing changes in bacterial and fungal communities.

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.

Background: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. Results: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. Conclusions: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

Tuberculosis is the world's leading cause of death due to a single infectious agent, and efforts aimed at its control require a better understanding of host, environmental, and bacterial factors that govern disease outcome. Growing evidence indicates that certain Mycobacterium tuberculosis strains of distinct phylogeographic lineages elicit unique immunopathological events. However, identifying the genetic basis of these phenotypic peculiarities has proven difficult. Here we report the presence of six large sequence polymorphisms which, together with two single-nucleotide changes previously described by our group, consistently differentiate Haarlem strains from the remaining M. tuberculosis lineages. The six newly found Haarlem-specific genetic events are four deletions, which altogether involve more than 13 kb, and two intragenic insertions of the element IS 6110 . The absence of the genes involved in these polymorphisms could have an important physiological impact on Haarlem strains, i.e., by affecting key genes, such as Rv1354c and cyp121 , which have been recently proposed as plausible drug targets. These lineage-specific polymorphisms can serve as genetic markers for the rapid PCR identification of Haarlem strains, providing a useful tool for strain surveillance and molecular epidemiology studies. Strain variability such as that described here underscores the need for the definition of a core set of essential genes in M. tuberculosis that are ubiquitously present in all circulating lineages, as a requirement in the development of effective antituberculosis drugs and vaccines.

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from t