Genomic signatures of pre-resistance in Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Torres Ortiz A
PROVIDER: S-EPMC8674244 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance in Mycobacterium tuberculosis. Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistant M. tuberculosis arises solely by de novo chromosomal mutation. Using whole-genome sequencing data from two natural populations of M. tuberculosis, we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant subpopulations to be less diverse than susceptible subpopulations, consistent with ongoing transmission of resistant M. tuberculosis. A subset of resistance genes ("sloppy targets") were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For "tight targets" of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times in the phylogeny and segregated at markedly different frequencies in resistant and susceptible subpopulations. These results suggest that diverse genetic architectures underlie drug resistance in M. tuberculosis and that combined approaches are needed to identify causal mutations. Extrapolating from patterns observed for well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria. IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tuberculosis populations. Identifying the mutations that cause drug resistance in M. tuberculosis is important for guiding TB treatment and halting the spread of drug resistance. Whole-genome sequencing (WGS) of M. tuberculosis isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug-resistant M. tuberculosis to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tuberculosis and other clonal pathogens.
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Project description:Fluoroquinolones (FQs) are effective second-line drugs for treating antibiotic-resistant tuberculosis (TB) and are being considered for use as first-line agents. Because FQs are used to treat a range of infections, in a setting of undiagnosed TB, there is potential to select for drug-resistant Mycobacterium tuberculosis mutants during FQ-based treatment of other infections, including pneumonia. Here we present a detailed characterization of ofloxacin-resistant M. tuberculosis samples isolated directly from patients in Taiwan, which demonstrates that selection for FQ resistance can occur within patients who have not received FQs for the treatment of TB. Several of these samples showed no mutations in gyrA or gyrB based on PCR-based molecular assays, but genome-wide next-generation sequencing (NGS) revealed minority populations of gyrA and/or gyrB mutants. In other samples with PCR-detectable gyrA mutations, NGS revealed subpopulations containing alternative resistance-associated genotypes. Isolation of individual clones from these apparently heterogeneous samples confirmed the presence of the minority drug-resistant variants suggested by the NGS data. Further NGS of these purified clones established evolutionary links between FQ-sensitive and -resistant clones derived from the same patient, suggesting de novo emergence of FQ-resistant TB. Importantly, most of these samples were isolated from patients without a history of FQ treatment for TB. Thus, selective pressure applied by FQ monotherapy in the setting of undiagnosed TB infection appears to be able to drive the full or partial emergence of FQ-resistant M. tuberculosis, which has the potential to confound diagnostic tests for antibiotic susceptibility and limit the effectiveness of FQs in TB treatment.
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Project description:Mycobacterium tuberculosis has acquired drug resistance to all drugs that have been used against it, including those only recently introduced into clinical practice. Compared to other bacteria, it has a well conserved genome due to its role as an obligate human pathogen that has adapted to a niche over five to ten thousand years. These features facilitate reconstruction and dating of M. tuberculosis phylogenies, giving key insights into how resistance has been acquired and spread globally. Resistance to each new drug has occurred within five to ten years of clinical use and has occurred even more rapidly with recently introduced drugs. In most cases, resistance-conferring mutations come with a fitness cost, but this can be overcome by compensatory mutations which restore fitness to that of wild-type bacteria. It is likely that M. tuberculosis acquires drug resistance while maintaining limited genomic variability due the generation of low frequency within-host variation, combined with ongoing purifying selection causing loss of variants without a clear fitness advantage. However, variants that do confer an advantage, such as drug resistance, can increase in prevalence amongst all bacteria within a host and become the dominant clone. These resistant strains can then be transmitted leading to primary drug resistant infection in a new host. As many countries move towards genomic methods for diagnosis of M. tuberculosis infection and drug resistance, it is important to be aware of the implications for the evolution of resistance. Currently, understanding of resistance-conferring mutations is incomplete, and some targeted genetic diagnostics create their own selective pressures. We discuss an example where a rifampicin resistance-conferring mutation which was not routinely covered by standard testing became dominant. Finally, resistance to new drugs such as bedaquiline and delamanid is caused by individually rare mutations occurring across a large mutational genomic target that have been detected over a short time, and do not provide statistical power for genotype-phenotype correlation - in contrast to longer-established drugs that form the backbone of drug-sensitive antituberculosis therapy. Therefore, we need a different approach to identify resistance-conferring mutations of new drugs before their resistance becomes widespread, abrogating their usefulness.
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Project description:Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment. Mice were challenged intravenously with Mycobacterium tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction. Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis ribonucleic acid in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB. Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlight the possible role of these genes for survival of M. tuberculosis in the ocular environment, thus contributing to pathogenesis of IOTB.
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Project description:Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG?TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10?? to 2.49×10??) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10??, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10??). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.
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Project description:Mycobacterium tuberculosis is a serious human pathogen threat exhibiting complex evolution of antimicrobial resistance (AMR). Accordingly, the many publicly available datasets describing its AMR characteristics demand disparate data-type analyses. Here, we develop a reference strain-agnostic computational platform that uses machine learning approaches, complemented by both genetic interaction analysis and 3D structural mutation-mapping, to identify signatures of AMR evolution to 13 antibiotics. This platform is applied to 1595 sequenced strains to yield four key results. First, a pan-genome analysis shows that M. tuberculosis is highly conserved with sequenced variation concentrated in PE/PPE/PGRS genes. Second, the platform corroborates 33 genes known to confer resistance and identifies 24 new genetic signatures of AMR. Third, 97 epistatic interactions across 10 resistance classes are revealed. Fourth, detailed structural analysis of these genes yields mechanistic bases for their selection. The platform can be used to study other human pathogens.
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Project description:A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.
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Project description:Multidrug resistance is a major threat to global elimination of tuberculosis (TB). We performed phenotypic drug-susceptibility testing and whole-genome sequencing for 309 isolates from 342 consecutive patients who were given a diagnosis of TB in Yangon, Myanmar, during July 2016‒June 2018. We identified isolates by using the GeneXpert platform to evaluate drug-resistance profiles. A total of 191 (62%) of 309 isolates had rifampin resistance; 168 (88%) of these rifampin-resistant isolates were not genomically related, indicating the repeated emergence of resistance in the population, rather than extensive local transmission. We did not detect resistance mutations to new oral drugs, including bedaquiline and pretomanid. The current GeneXpert MTB/RIF system needs to be modified by using the newly launched Xpert MTB/XDR cartridge or line-probe assay. Introducing new oral drugs to replace those currently used in treatment regimens for multidrug-resistant TB will also be useful for treating TB in Myanmar.
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Project description:Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.
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