Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Evidence for the Effect of Vaccination on Host-Pathogen Interactions in a Murine Model of Pulmonary Tuberculosis by Mycobacterium tuberculosis.

The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guérin (BCG) -the most widely used vaccine in history-has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts.

Genetic profiling of Mycobacterium tuberculosis revealed "modern" Beijing strains linked to MDR-TB from Southwestern Colombia.

Beijing strains of Mycobacterium tuberculosis (lineage 2) have been associated with drug-resistance and transmission of tuberculosis worldwide. Most of the Beijing strains identified in the Colombian Pacific coast have exhibited a multidrug resistant (MDR) phenotype. We sought to evaluate the clonality and sublineage of Beijing strains circulating in Southwestern Colombia. Thirty-seven Beijing strains were identified through spoligotyping out of 311 clinical isolates collected in 9 years from 2002-2010. Further analysis by MIRU-VNTR 24 loci was conducted for the Beijing strains. For sublineage classification, deletions of RD105, RD207, and RD131 and point mutations at fbpB, mutT2, and acs were evaluated. Drug-resistance associated mutations to first- and second-line anti-TB drugs were also evaluated. Additionally, two Beijing strains were Illumina-whole genome sequenced (one MDR and one drug-susceptible). Among the 37 Beijing strains characterized, 36 belonged to the SIT190 type from which 28 were MDR, four pre-extensively drug resistant (XDR) TB, and four XDR-TB. The remaining strain was SIT1 and drug susceptible. MIRU-VNTR analysis allowed the identification of three Beijing clusters and two unique strains. Beijing strains were confirmed as "modern" sublineage. The mutations rpoB S531L and katG S315T were the most common among MDR strains. Moreover, the two strains evaluated by whole genome sequencing (WGS) shared most of the genetic features with the sublineage 2.2.1 "modern" Beijing previously characterized from Asian strains. WGS analysis of the MDR strain revealed the presence of eight SNPs previously reported in other MDR "Beijing-like" strains from Colombia. The presence of "modern" Beijing strains in Southwestern Colombia, most of them with MDR phenotype, suggests a different origin of this M. tuberculosis sublineage compared to other Beijing strains found in neighboring South American countries. This work may serve as a genetic baseline to study the evolution and spread of M. tuberculosis Beijing strains in Colombia, which play an important role in the propagation of MDR-TB.

Mycobacterium tuberculosis lineage 1 genetic diversity in Pará, Brazil, suggests common ancestry with east-African isolates potentially linked to historical slave trade.

Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sublineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48) and L1/EAI6-BGD1 (SIT591) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil.

Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey.

We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading.

Predictive value of molecular drug resistance testing of Mycobacterium tuberculosis isolates in Valle del Cauca, Colombia.

Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen's kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases.

Successful alternative treatment of extensively drug-resistant tuberculosis in Argentina with a combination of linezolid, moxifloxacin and thioridazine.

OBJECTIVES: Current drug choices to treat extensively drug-resistant (XDR) tuberculosis (TB) are scarce; therefore, information on the safety, tolerability and efficacy of alternative regimens is of utmost importance. The aim of this study was to describe the management, drug adverse effects and outcome of alternative combined treatment in a series of XDR-TB patients. PATIENTS AND METHODS: A retrospective study was performed on 17 non-AIDS, pulmonary adult patients with XDR-TB admitted to a referral treatment centre for infectious diseases in Buenos Aires from 2002 through 2008. Drug susceptibility testing was performed under regular proficiency testing and confirmed at the national TB reference laboratory. RESULTS: Linezolid was included in the drug regimens of all patients; moxifloxacin and/or thioridazine were included in the regimens of 14 patients. Clinically tractable drug adverse effects were observed in nine patients, the most frequent being haematological disorders and neurotoxicity. In two patients, thioridazine was discontinued. Negative culture conversion was achieved in 15 patients, 11 completed treatment meeting cure criteria, 4 are still on follow-up with good evolution, 1 defaulted treatment and 1 was lost to follow-up. CONCLUSIONS: The combination of linezolid, moxifloxacin and thioridazine is recommended for compassionate use in specialized centres with expertise in the management of XDR-TB.

Mycobacterium tuberculosis Beijing genotype induces differential cytokine production by peripheral blood mononuclear cells of healthy BCG vaccinated individuals.

Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could affect the cellular immune response. In order to address this issue, peripheral blood mononuclear cells (PBMC) from healthy BCG vaccinated individuals were stimulated with protein extracts from three Mycobacterium tuberculosis genotypes: Canetti, H37Rv and Beijing evaluating T cell proliferation and cytokine production. In this system both CD4+ and CD8+ proliferated in a similar manner independently of the Mtb genotype used for stimulation. Regarding cytokines, all strains induced similar levels of IFN-γ, but were unable to induce IL-4 and TGF-ß. Contrasting, Canetti strain induced lower production of IL-10, TNF-α and IL-12 compared to H37Rv and Beijing. Interestingly, PBMC stimulated with the Beijing strain produced the highest levels of IL-12 and IL-10 than those stimulated with other strains. This differential cytokine expression could affect the pathogenesis induced by Beijing strain through the modulation of inflammatory process in the host, but the precise mechanisms by which this cytokine environment affects the Beijing strain pathogenesis needs further characterization.

Multidrug-resistant Mycobacterium tuberculosis, Southwestern Colombia.

Using spoligotyping, we identified 13 genotypes and 17 orphan types among 160 Mycobacterium tuberculosis isolates from patients in Valle del Cauca, Colombia. The Beijing genotype represented 15.6% of the isolates and was correlated with multidrug-resistant tuberculosis, female sex of the patients, and residence in Buenaventura and may represent a new public health threat.

Thioridazine cures extensively drug-resistant tuberculosis (XDR-TB) and the need for global trials is now!

Thioridazine (TDZ) has been shown to have in vitro activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis, to promote the killing of intracellular MDR and XDR strains and to cure the mouse of antibiotic-susceptible and -resistant pulmonary tuberculosis (TB) infections. Recently, TDZ was used to cure 10 of 12 XDR-TB patients in Buenos Aires, Argentina. At the time of writing, it is being used for the therapy of non-antibiotic-responsive terminal XDR-TB patients in Mumbai, India, on the basis of compassionate therapy and although it is too early to determine a cure, the patients have improved appetite, weight gain, are afebrile and free of night sweats, and their radiological picture shows great improvement. Because XDR-TB is essentially a terminal disease in many areas of the world and no new effective agents have yet to yield successful clinical trials, global clinical trials for the therapy of XDR-TB are urgently required.

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America.

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6 percent) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9 percent), five of the 512 patients from Argentina (1.0 percent), two of the 252 Brazilian cases (0.8 percent), one of the 166 patients from Paraguay (0.6 percent) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

24-locus MIRU-VNTR genotyping is a useful tool to study the molecular epidemiology of tuberculosis among Warao Amerindians in Venezuela.

While the gold standard for molecular epidemiological studies on tuberculosis is changing towards MIRU-VNTR typing because this technique generates easily analyzed numerical results, it is less labor intensive and has a discriminative power comparable to that of IS6110-based RFLP, especially when 24 loci are analyzed; more extensive and representative validation studies are needed to confirm this. In this study we genotyped 41 Mycobacterium tuberculosis isolates, about 40% of the total case load of the study year, from the Warao people, an indigenous population who live in a geographically isolated area in Venezuela and have a high TB incidence of 450/100,000. IS6110-based RFLP analysis on these isolates indicates that 78% of the strains are in clusters, suggesting a very high transmission rate. We show that both the 15-locus MIRU-VNTR combined with spoligotyping, as well as the 24-locus MIRU-VNTR typing have sufficient discrimination power (an HGI of 0.93 and 0.95, respectively) to replace IS6110-based RFLP (HGI=0.93) and thus are useful tools to study the molecular epidemiology of tuberculosis in this high TB incidence population.

Mycobacterium tuberculosis lipids regulate cytokines, TLR-2/4 and MHC class II expression in human macrophages.

The interaction of macrophages with Mycobacterium tuberculosis through Toll-like receptors is critical in defining the cytokine profile that may or may not control disease progression. Cell-wall lipids are the main pathogen-associated molecular ligands of mycobacteria, in this paper, we analysed how lipid fractions of three different strains of the M. tuberculosis complex (genotypes Canetti, Beijing and H37Rv) affected the innate immunity by regulating TNF-alpha and IL-10 secretion, TLR2, TLR4, and MHC class II expression of human monocyte-derived macrophages. Of note, lipid fractions from the Beijing genotype (hypervirulent phenotype) preferentially induced macrophages to secrete high amounts of TNF-alpha and IL-10, but downregulated TLR2, TLR4 and MHC class II expression. In contrast, lipids from M. tuberculosis Canetti induced lower amounts of TNF-alpha and IL-10, upregulated TLR2 and TLR4 without modifying MHC class II expression. These results indicate that the virulent mycobacterial genotype Beijing expresses lipids that negatively modified cytokine, TLR and MHC class II expression. These findings may help to unravel the complex mechanisms used by virulent mycobacteria to evade and subvert the immune response.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.