Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model.

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.

Heterogeneous fitness landscape cues, pknG low expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth.

Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 µg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

Population structure of multidrug-resistant Mycobacterium tuberculosis clinical isolates in Colombia.

Emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) isolates is a major public health problem that threatens progress made in tuberculosis (TB) care and control worldwide. In Colombia, the prevalence of MDR tuberculosis (MDR-TB) has increased slowly but steadily since 2001. However, the population structure of the MDR-TB strains circulating in Colombia is sparsely known. In this work, 203 MDR isolates isolated in 2012-2013 were collected, and characterized by spoligotyping, followed by 24-loci MIRU-VNTR (data available for 190 isolates). The most prevalent genotypes corresponded to SIT42/LAM9 (12.81%), SIT62/H1 (10.34%), and SIT190/Beijing (10.34%). A fine analysis showed that although the MDR strains came from 29 of the 33 departments of Colombia, the distribution of these main lineages was not at random and depended on the city of isolation (p-value <0.000001). Both LAM and Beijing lineage strains were significantly associated with MDR-TB (p-value <0.0001): LAM lineage was associated with 2 patterns of MDR, namely combined resistance to INH + Rifampin (HR), and to SHRE (Streptomycin + INH + Rifampin + Ethambutol), while the Beijing lineage strains were essentially associated with MDR (SHRE). Interestingly, distribution of genotypic lineages in function of drug resistance information (e.g. pansusceptible vs. MDR) was different in our setting as compared to other countries in Latin America. However, MIRU-VNTR patterns were unique for all strains, an observation that did not support active transmission of circulating MDR clones.

Genetic diversity of Mycobacterium tuberculosis clinical isolates from HIV-TB patients from two public hospitals at Bogotá, Colombia.

The co-infection of TB/HIV is an increasing problem for public health worldwide. In Colombia, of 13.871 confirmed cases of TB in 2016 (prevalence of 0,028%) 14% correspond to HIV co-infection. However, we have scarce information regarding genetic diversity of strains infecting HIV patients. In this study, we carried-out an active search of cases of TB in 356 HIV-infected individuals, who were enrolled in two Public Hospitals at Bogotá-Colombia, between 2014 and 2015. We found 49 patients with HIV-TB co-infection. Genetic characterization of Mycobacterium tuberculosis (Mtb) isolates from these patients showed a predominance of three major sub-lineages: Haarlem (n = 26), LAM (n = 12) and T (n = 11). Remarkably, the most predominant pattern in the present study (SIT62/H1, n = 11) is very specific to this country. Indeed, taking in account distribution in countries with at least 3% of SIT62/H1, 36% of all such patterns collected worldwide were from Colombia. Furthermore, Colombia alone is responsible for almost all the SIT62/H1 strains in South America, suggesting a successful transmission of this genotype inside TB/HIV population from Colombia.

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance.

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia.