HflX controls hypoxia-induced non-replicating persistence in slow growing mycobacteria

GTPase HflX is highly conserved in prokaryotes and is a ribosome splitting factor during heat shock in E. coli. Here we report that HflX produced by slow growing M. tuberculosis and M. bovis BCG is a GTPase that plays a critical role in the pathogen’s transition to a non-replicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model, that precipitated entry into dormancy. The KO displayed the hallmarks of dormant mycobacteria including phenotypic drug resistance, altered morphology, low intracellular ATP and up-regulated dormancy dos regulon. KO-infected mice displayed increased bacterial burden during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic phase. Unlike fast-growing mycobacteria, BCG HlfX was not involved in antibiotic resistance under normoxia. Proteomics, pull-down and ribo-sequencing supported that mycobacterial HflX is a ribosome binding protein that controls the translational activity of the cell. Collectively, our study provides further insights into the mechanisms deployed by mycobacteria to adapt to their hypoxic microenvironment.

Systematic Review of Whole-Genome Sequencing Data To Predict Phenotypic Drug Resistance and Susceptibility in Swedish Mycobacterium tuberculosis Isolates, 2016 to 2018

ABSTRACT In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was ∼99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future.

CHARACTERISTICS OF M.TUBERCULOSIS DRUG RESISTANCE DETERMINED BY MOLECULAR GENETIC AND PHENOTYPIC METHODS

Objective: to determine the genetic and phenotypic drug resistance of M . tuberculosis to first-line and second-line anti-TB drugs. Material and methods. Gene mutations in 247 strains of M. tuberculosis (MBT) associated with drug resistance to isoniazid, rifampicin, fluoroquinolones, and aminoglycosides were studied. Genetic resistance of a tuberculosis causative agent was determined by means of LPA (GenoType® MTBDRsl MTBDRplus and MTBDRsl, ver.2.0). The results of the study are confirmed by the determination of phenotypic drug resistance in the automated system BACTEC ™ MGIT ™ 960. Results. The drug resistant MBT strains circulating around Gomel region have been determined, and the high reliability of molecular and genetic determination of MBT drug resistance has been confirmed by microbiological methods (isoniazid and rifampicin - 97.2 %, fluoroquinolones - 85.1 %, aminoglycosides - 92.3 %). A considerable number of drug resistant MTB strains with gene mutations (45.1 %) which are not included in the GenoType® MTBDRsl system were detected. Conclusion. Тhe considerable genetic variability of drug-resistant MBT strains requires complex application of all the methods of drug resistance testing.

Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum

ABSTRACTTuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measurein situdrug susceptibility ofMycobacterium tuberculosisbacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe anex vivoassay developed to measure the cidal activity of anti-TB drugs againstM. tuberculosisbacilli present in cavity caseum obtained from rabbits with active TB. We show that caseumM. tuberculosisbacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseumM. tuberculosisexhibits higher drug tolerance than nonreplicatingM. tuberculosisin the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus,ex vivocaseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.

A standardised method for interpreting the association between mutations and phenotypic drug resistance inMycobacterium tuberculosis

A clear understanding of the genetic basis of antibiotic resistance inMycobacterium tuberculosisis required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype–phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6–90.9%), while for isoniazid it was 78.2% (77.4–79.0%) and their specificities were 96.3% (95.7–96.8%) and 94.4% (93.1–95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1–70.6%) for capreomycin to 88.2% (85.1–90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1–92.5%) for moxifloxacin to 99.5% (99.0–99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors ofM. tuberculosisdrug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.