Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that ...the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the world's highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.
Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or ...repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria.
In light of the marked global health impact of tuberculosis (TB), strong focus has been on identifying biosignatures. Gene expression profiles in blood cells identified so far are indicative of a ...persistent activation of the immune system and chronic inflammatory pathology in active TB. Definition of a biosignature with unique specificity for TB demands that identified profiles can differentiate diseases with similar pathology, like sarcoidosis (SARC). Here, we present a detailed comparison between pulmonary TB and SARC including whole-blood gene expression profiling, microRNA expression, and multiplex serum analytes. Our analysis reveals that previously disclosed gene expression signatures in TB show highly similar patterns in SARC, with a common up-regulation of proinflammatory pathways and IFN signaling and close similarity to TB-related signatures. microRNA expression also presented a highly similar pattern in both diseases, whereas cytokines in the serum of TB patients revealed a slightly elevated proinflammatory pattern compared with SARC and controls. Our results indicate several differences in expression between the two diseases, with increased metabolic activity and significantly higher antimicrobial defense responses in TB. However, matrix metallopeptidase 14 was identified as the most distinctive marker of SARC. Described communalities as well as unique signatures in blood profiles of two distinct inflammatory pulmonary diseases not only have considerable implications for the design of TB biosignatures and future diagnosis, but they also provide insights into biological processes underlying chronic inflammatory disease entities of different etiology.
Antibiotic-resistant tuberculosis poses a global threat, causing the deaths of hundreds of thousands of people annually. While whole-genome sequencing (WGS), with its unprecedented level of detail, ...promises to play an increasingly important role in diagnosis, data analysis is a daunting challenge. Here, we present a simple-to-use web service (free for academic use at http://phyresse.org). Delineating both lineage and resistance, it provides state-of-the-art methodology to life scientists and physicians untrained in bioinformatics. It combines elaborate data processing and quality control, as befits human diagnostics, with a treasure trove of validated resistance data collected from well-characterized samples in-house and worldwide.
Whole-genome sequencing (WGS) has the potential to accelerate drug-susceptibility testing (DST) to design appropriate regimens for drug-resistant tuberculosis (TB). Several recently developed ...automated software tools promise to standardize the analysis and interpretation of WGS data. We assessed five tools (CASTB, KvarQ, Mykrobe Predictor TB, PhyResSE, and TBProfiler) with regards to DST and phylogenetic lineage classification, which we compared with phenotypic DST, Sanger sequencing, and traditional typing results for a collection of 91 strains. The lineage classifications by the tools generally only differed in the resolution of the results. However, some strains could not be classified at all and one strain was misclassified. The sensitivities and specificities for isoniazid and rifampicin resistance of the tools were high, whereas the results for ethambutol, pyrazinamide, and streptomycin resistance were more variable. False-susceptible DST results were mainly due to missing mutations in the resistance catalogues that the respective tools employed for data interpretation. Notably, we also found cases of false-resistance because of the misclassification of polymorphisms as resistance mutations. In conclusion, the performance of current WGS analysis tools for DST is highly variable. Sustainable business models and a shared, high-quality catalogue of resistance mutations are needed to ensure the clinical utility of these tools.
Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a ...major method for the epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible at http://www.MIRU-VNTRplus.org. The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.
Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of ...Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.
Contact tracing complemented with genotyping is considered an important means of understanding person-to-person transmission of tuberculosis (TB). It still remains unclear whether Whole Genome ...Sequencing (WGS) of Mycobacterium tuberculosis can rule in transmission and how it performs in different human populations, risk groups and across TB lineages. This systematic review aimed to determine the sensitivity and specificity of WGS for detection of recent transmission using conventional epidemiology as the gold standard and investigate if WGS identifies previously undetected transmission events.
Systematic review was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses group. A compound search strategy was developed to identify all relevant studies published between 01/01/2005 and 30/11/2014 using three online databases. Publications satisfying specific criteria have been identified and data extracted.
A total of 12 publications were included. We established that WGS has a higher discriminatory power compared to conventional genotyping and detects transmission events missed by epidemiological investigations. A cut-off value of <6 SNPs between isolates may predict recent transmission. None of the studies performed a head-to-head comparison between WGS and conventional genotyping using unselected prospectively collected isolates. Minimum reporting criteria for WGS studies have been proposed and quality control parameters considered.
Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve ...large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.
During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.
Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.
Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we ...reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.
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