Tuberculosis chemotherapy is dependent on the use of the antibiotic pyrazinamide, which is being threatened by emerging drug resistance. Resistance is mediated through mutations in the bacterial gene ...pncA. Methods for testing pyrazinamide susceptibility are difficult and rarely performed, and this means that the full spectrum of pncA alleles that confer clinical resistance to pyrazinamide is unknown. Here, we performed in vitro saturating mutagenesis of pncA to generate a comprehensive library of PncA polymorphisms resultant from a single-nucleotide polymorphism. We then screened it for pyrazinamide resistance both in vitro and in an infected animal model. We identify over 300 resistance-conferring substitutions. Strikingly, these mutations map throughout the PncA structure and result in either loss of enzymatic activity and/or decrease in protein abundance. Our comprehensive mutational and screening approach should stand as a paradigm for determining resistance mutations and their mechanisms of action.The antibiotic pyrazinamide is central to tuberculosis treatment regimens, globally. Despite its efficacy, resistance to the drug is increasing. Here, Eric Rubin and colleagues characterise the genetic basis of pyrazinamide resistance.
Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what ...extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture.
DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance.
Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.
Bedaquiline, a diarylquinoline, improved cure rates when added to a multidrug-resistant tuberculosis (MDR-TB) treatment regimen in a previous placebo-controlled, phase 2 trial (TMC207-C208; ...NCT00449644). The current phase 2, multicenter, open-label, single-arm trial (TMC207-C209; NCT00910871) reported here was conducted to confirm the safety and efficacy of bedaquiline.Newly diagnosed or previously treated patients with MDR-TB (including pre-extensively drug-resistant (pre-XDR)-TB or extensively drug-resistant (XDR)-TB) received bedaquiline for 24 weeks with a background regimen of anti-TB drugs continued according to National TB Programme treatment guidelines. Patients were assessed during and up to 120 weeks after starting bedaquiline.Of 233 enrolled patients, 63.5% had MDR-TB, 18.9% had pre-XDR-TB and 16.3% had XDR-TB, with 87.1% having taken second-line drugs prior to enrolment. 16 patients (6.9%) died. 20 patients (8.6%) discontinued before week 24, most commonly due to adverse events or MDR-TB-related events. Adverse events were generally those commonly associated with MDR-TB treatment. In the efficacy population (n=205), culture conversion (missing outcome classified as failure) was 72.2% at 120 weeks, and 73.1%, 70.5% and 62.2% in MDR-TB, pre-XDR-TB and XDR-TB patients, respectively.Addition of bedaquiline to a background regimen was well tolerated and led to good outcomes in this clinically relevant patient cohort with MDR-TB.
Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome ...sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.
Summary
Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular ...basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock‐in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1‐encoded proteins were localized in the cell wall, and two of them, the immunodominant T‐cell antigens ESAT‐6 and CFP‐10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock‐ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock‐in, whereas BCG controls were cleared. Knocking‐in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.
...we would like to emphasize that the introduction of a uniform gene nomenclature for other secretion systems in Gram-negative bacteria (type II, type III) has facilitated comparative analysis of ...these systems. Amongst the different topology prediction programs that were used (TMHMM Server v. 2.0, MEMSAT3, Philius, SCAMPI, HMMTOP and Phobius) MEMSAT3 gave the correct prediction for the highest number of Ecc membrane proteins. ...only the topology prediction results of TMHMM (used on the TubercuList server) and MEMSAT3 are shown.
The distribution of
-acetyltransferase 2 gene (
) polymorphisms varies considerably among different ethnic groups. Information on
single-nucleotide polymorphisms in the South African population is ...limited. We investigated
polymorphisms and their effect on isoniazid pharmacokinetics (PK) in Zulu black HIV-infected South Africans in Durban, South Africa. HIV-infected participants with culture-confirmed pulmonary tuberculosis (TB) were enrolled from two unrelated studies. Participants with culture-confirmed pulmonary TB were genotyped for the
polymorphisms 282C>T, 341T>C, 481C>T, 857G>A, 590G>A, and 803A>G using Life Technologies prevalidated TaqMan assays (Life Technologies, Paisley, UK). Participants underwent sampling for determination of plasma isoniazid and
-acetyl-isoniazid concentrations. Among the 120 patients, 63/120 (52.5%) were slow metabolizers (
), 43/120 (35.8%) had an intermediate metabolism genotype (
), and 12/120 (11.7%) had a rapid metabolism genotype (
,
, and
). The
alleles evaluated in this study were
,
,
,
,
,
,
,
,
,
, and
was the most frequent allele (70.4%), followed by
(27.9%). Fifty-eight of 60 participants in study 1 had PK results. The median area under the concentration-time curve from 0 to infinity (AUC
) was 5.53 (interquartile range IQR, 3.63 to 9.12 μg h/ml), and the maximum concentration (
) was 1.47 μg/ml (IQR, 1.14 to 1.89 μg/ml). Thirty-four of 40 participants in study 2 had both PK results and
genotyping results. The median AUC
was 10.76 μg·h/ml (IQR, 8.24 to 28.96 μg·h/ml), and the
was 3.14 μg/ml (IQR, 2.39 to 4.34 μg/ml). Individual polymorphisms were not equally distributed, with some being represented in small numbers. The genotype did not correlate with the phenotype, with those with a rapid acetylator genotype showing higher AUC
values than those with a slow acetylator genotype, but the difference was not significant (
= 0.43). There was a high prevalence of slow acetylator genotypes, followed by intermediate and then rapid acetylator genotypes. The poor concordance between genotype and phenotype suggests that other factors or genetic loci influence isoniazid metabolism, and these warrant further investigation in this population.
A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict
(Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To ...determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.
In 2019, the WHO tuberculosis (TB) treatment guidelines were updated to recommend only limited use of streptomycin, in favor of newer agents or amikacin as the preferred aminoglycoside for ...drug-resistant
However, the emergence of resistance to newer drugs, such as bedaquiline, has prompted a reanalysis of antitubercular drugs in search of untapped potential. Using 211 clinical isolates of
from South Africa, we performed phenotypic drug susceptibility testing (DST) to aminoglycosides by both critical concentration and MIC determination in parallel with whole-genome sequencing to identify known genotypic resistance elements. Isolates with low-level streptomycin resistance mediated by
were frequently misclassified with respect to streptomycin resistance when using the WHO-recommended critical concentration of 2 μg/ml. We identified 29
isolates from South Africa with low-level streptomycin resistance concomitant with high-level amikacin resistance, conferred by
and
1400, respectively. Using a large global data set of
genomes, we observed 95 examples of this corresponding resistance genotype (
1400), including identification in 81/257 (31.5%) of extensively drug resistant (XDR) isolates. In a phylogenetic analysis, we observed repeated evolution of low-level streptomycin and high-level amikacin resistance in multiple countries. Our findings suggest that current critical concentration methods and the design of molecular diagnostics need to be revisited to provide more accurate assessments of streptomycin resistance for
-containing isolates. For patients harboring isolates of
with high-level amikacin resistance conferred by
1400, and for whom newer agents are not available, treatment with streptomycin may still prove useful, even in the face of low-level resistance conferred by
.
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