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Point mutations in the rrs gene and eis promoter are known to confer resistance to second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer a majority of SLID-resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1184 clinical M. tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, 270 AMK-R, CAP-R, and KAN-R isolates, 264 (81.2%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 111 of the phenotypically resistant isolates. A gene-wise method identified three genes and promoters with mutations that, on aggregate, associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 promoter mutations with KAN resistance, supporting clinical relevance for the previously demonstrated role of whiB7 overexpression in KAN resistance. We also provide evidence for the novel association of ppe51 (a gene previously associated with various antimicrobial compounds) with AMK resistance, and for the novel association of thrB with AMK and CAP resistance. The use of gene-wise association can provide additional insight, and therefore is recommended for identification of rare mechanisms of resistance when individual mutations carry insufficient statistical power.

This study assembles DNA adenine methylomes for 93 Mycobacterium tuberculosis complex (MTBC) isolates from seven lineages paired with fully-annotated, finished, de novo assembled genomes. Integrative analysis yielded four key results. First, methyltransferase allele-methylome mapping corrected methyltransferase variant effects previously obscured by reference-based variant calling. Second, heterogeneity analysis of partially active methyltransferase alleles revealed that intracellular stochastic methylation generates a mosaic of methylomes within isogenic cultures, which we formalize as ‘intercellular mosaic methylation’ (IMM). Mutation-driven IMM was nearly ubiquitous in the globally prominent Beijing sublineage. Third, promoter methylation is widespread and associated with differential expression in the ΔhsdM transcriptome, suggesting promoter HsdM-methylation directly influences transcription. Finally, comparative and functional analyses identified 351 sites hypervariable across isolates and numerous putative regulatory interactions. This multi-omic integration revealed features of methylomic variability in clinical isolates and provides a rational basis for hypothesizing the functions of DNA adenine methylation in MTBC physiology and adaptive evolution.

Tuberculosis (TB) represents a significant challenge to public health authorities, especially with the emergence of drug-resistant (DR) and multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis. We sought to examine the genomic variations among recently isolated strains of M. tuberculosis in two closely related countries with different population demography in the Middle East. Clinical isolates of M. tuberculosis from both Egypt and Saudi Arabia were subjected to phenotypic and genotypic analysis on gene and genome-wide levels. Isolates with MDR phenotypes were highly prevalent in Egypt (up to 35%) despite its relatively stable population structure (sympatric pattern). MDR-TB isolates were not identified in the isolates from Saudi Arabia despite its active guest worker program (allopatric pattern). However, tuberculosis isolates from Saudi Arabia, where lineage 4 was more prevalent (>65%), showed more diversity than isolates from Egypt, where lineage 3 was the most prevalent (>75%). Phylogenetic and molecular dating analyses indicated that lineages from Egypt were recently diverged (~78 years), whereas those from Saudi Arabia were diverged by over 200 years. Interestingly, DR isolates did not appear to cluster together or spread more widely than drug-sensitive isolates, suggesting poor treatment as the main cause for emergence of drug resistance rather than more virulence or more capacity to persist.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne’s assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8–10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

Pyrazinamide (PZA) is an important first-line drug in the treatment of tuberculosis (TB) and of significant interest to the HIV-infected community due to the prevalence of TB-HIV coinfection in some regions of the world. The mechanism of resistance to PZA is unlike that of any other anti-TB drug. The gene pncA , encoding pyrazinamidase (PZase), is associated with resistance to PZA. However, because single mutations in PZase have a low prevalence, the individual sensitivities are low. Hundreds of distinct mutations in the enzyme have been associated with resistance, while some only appear in susceptible isolates. This makes interpretation of molecular testing difficult and often leads to the simplification that any PZase mutation causes resistance. This systematic review reports a comprehensive global list of mutations observed in PZase and its promoter region in clinical strains, their phenotypic association, their global frequencies and diversity, the method of phenotypic determination, their MIC values when given, and the method of MIC determination and assesses the strength of the association between mutations and phenotypic resistance to PZA. In this systematic review, we report global statistics for 641 mutations in 171 (of 187) codons from 2,760 resistant strains and 96 mutations from 3,329 susceptible strains reported in 61 studies. For diagnostics, individual mutations (or any subset) were not sufficiently sensitive. Assuming similar error profiles of the 5 phenotyping platforms included in this study, the entire enzyme and its promoter provide a combined estimated sensitivity of 83%. This review highlights the need for identification of an alternative mechanism(s) of resistance, at least for the unexplained 17% of cases.

Background By using whole genome sequencing (WGS), researchers are beginning to understand the genetic diversity of Mycobacterium tuberculosis (MTB) and its consequences for the diagnosis of multidrug-resistant tuberculosis (MDR–TB) on a genomic scale. The Global Consortium for Drug-resistant TB Diagnostics (GCDD) conducted a genome scale variant analyses of 366 clinical MTB genomes (mostly MDR/XDR [extensively drug resistant]) from four countries in order to inform the development of rapid molecular diagnostics. This project has been extended by performing an evolutionary analysis of isoniazid (INH)-resistant isolates for prognostic purposes. Methods 151 (130 INH R , 21 INH S ) clinical MTB isolates from India (19: 17 INH R , 2 INH S ), Moldova (48: 42 INH R , 6 INH S ), the Philippines (26: 20 INH R , 6 INH S ), and South Africa (58: 51 INH R , 7 INH S ) were included in this study. INH drug susceptibility was determined by using MGIT 960 and WHO (World Health Organization)-recommended critical concentration of 0.1 mg/L. Isolates were sequenced using PacBio RS WGS platform. A genome-wide variant analysis was conducted using a proprietary pipeline (PacDAP) developed at San Diego State University. To infer the amino acid changes in katG that confer resistance, PAML was utilized to detect sites in silico that are under positive selection. The dN/dS method was used in combination with Bayes empirical Bayes to determine sites under positive selection and Chi-Squared analysis to determine the significance of the selected sites. Results PacDAP variant analysis revealed 22 novel catalase-peroxidase ( katG product) mutations. Of these, 14 were single nucleotide polymorphisms, while 8 novel mutations appeared in combination with katG S315T and/or with inhA promoter C-15T. These SNPs have not been previously reported. Additionally, 11 previously observed, but uncommon, katG mutations were also observed in these clinical isolates. These results suggest that 17 amino acids in the enzyme are under positive selective pressure; most significantly in South Africa and the Philippines. No selective pressure on codons other than 315 was observed in isolates from Moldova. Due to the low number of isolates from India, the significance of the sites under positive selection was low and no prediction for India could be made based on this study. Conclusions Eleven of the 14 SNPs are resistance conferring, and it is believed that the remaining 8 combinatorial mutations are either compensatory in nature or, in combination with known SNPs, could increase resistance levels. Positive selection results indicate a diversifying evolutionary path to resistance more in line with long tail statistics and therefore indicate a departure from the traditional point mutation (or “hotspot”) model that current molecular diagnostics are based on. Positive selection pressures indicate a future with elevated diagnostic and prognostic significance of the “long tail” (i.e., alternative mechanisms of resistance) and potentially diminishing significance of the canonical mutations (especially in South Africa and the Philippines), which could have significant future implications on narrowly targeting molecular diagnostics.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.