Mycobacterium tuberculosis str. Haarlem/NITR202
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ABSTRACT:
PROVIDER: PRJNA192916 | ENA |
REPOSITORIES: ENA
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Project description:Streptomycin (STR) is a component of first-line drugs used to treat multidrug-resistant tuberculosis. The purpose of this study was to investigate the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to STR and their relationship with the STR-resistant phenotype and with the epidemiological molecular model of the isolates. A total of 302 clinical isolates, including 215 STR-resistant and 87 STR-susceptible isolates, were characterized using the proportion method with Lowenstein-Jensen medium. The genes rpsL, rrs and gidB were screened for mutations using DNA sequencing methodology. All strains were genotyped using the spoligotyping technique. Mutations in rpsL and in rrs were observed in 63.3% and 15.8% of the STR-resistance isolates, respectively. The most prevalent mutations were the Lys43Arg substitution in the rpsL gene and the A514C change in the rrs gene. Ten novel mutations were identified in gidB. These novel mutations might be new potential markers for predicting STR-resistance in clinical Mycobacterium tuberculosis isolates. Mutations in rpsL, rrs, and gidB had a sensitivity of 84.2% and a specificity of 77.0% for the detection of STR-resistance isolates. The Beijing lineage strains were associated with the rpsL mutation Lys43Arg (P = 0.051), as well as the dual gidB mutations Glu92Asp and Ala205Ala (P < 0.001). Our study suggested that rpsL and rrs can act as useful genetic markers for predicting STR-resistance, and gidB polymorphisms play an important role in STR-resistant clinical Mycobacterium tuberculosis isolates from Hebei, China.
2019-01-01 | S-EPMC6949554 | BioStudies
Project description:BACKGROUND:Mortality due to tuberculosis (TB) has increased due to the development of drug resistance, the mechanisms of which have not been fully elucidated. Our research group identified a low expression of lipF gene in Mycobacterium tuberculosis clinical isolates with drug resistance. The aim of this work was to evaluate the effect of lipase F (LipF) expression on mycobacterial drug resistance. RESULTS:The effects of expressing lipF from Mycobacterium tuberculosis in Mycobacterium smegmatis on resistance to antituberculosis drugs were determined with resazurin microtiter assay plate and growth kinetics. Functionality of ectopic LipF was confirmed. LipF expression reduced the rifampicin (RIF) and streptomycin (STR) minimum inhibitory concentration (MIC) from 3.12??g/mL to 1.6??g/mL and 0.25??g/mL to 0.06??g/mL respectively, moreover a reduced M. smegmatis growth in presence of RIF and STR compared with that of a control strain without LipF expression (p?<?0.05 and p?<?0.01) was shown. CONCLUSIONS:LipF expression was associated with increased RIF and STR sensitivity in mycobacteria. Reduced LipF expression may contribute to the development of RIF and STR resistance in Mycobacterium species. Our findings provide information pertinent to understanding mycobacterial drug resistance mechanisms.
2020-01-01 | S-EPMC7249682 | BioStudies
Project description:Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR) in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible) and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9%) of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5%) STR-resistant and 13 (40.6%) STR-susceptible isolates. Four (12.5%) STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T?G (L16R) mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A?C (E92D) and 615A?G (A205A) mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.
2014-01-01 | S-EPMC4061058 | BioStudies
Project description:The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses.The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.
2016-01-01 | S-EPMC4779234 | BioStudies
Project description:We analyzed 98 Mycobacterium tuberculosis complex isolates collected in 2 regions of Algeria in 2015-2018 from 93 cases of pulmonary tuberculosis. We identified 93/98 isolates as M. tuberculosis lineage 4 and 1 isolate as M. tuberculosis lineage 2 (Beijing). We confirmed 4 isolates as M. bovis by whole-genome sequencing.
2021-01-01 | S-EPMC7920681 | BioStudies
Project description:Nonreplicating or dormant cells of Mycobacterium tuberculosis constitute a challenge to tuberculosis (TB) therapy because of their tolerance or phenotypic resistance to most drugs. Here, we propose a simple model for testing drugs against nongrowing cells that exploits the 18b strain of M. tuberculosis, a streptomycin (STR)-dependent mutant. Optimal conditions were established that allowed 18b cells to replicate in the presence of STR and to survive, but not multiply, following withdrawal of STR. In the presence of the antibiotic, M. tuberculosis 18b was susceptible to the currently approved TB drugs, isoniazid (INH) and rifampin (RIF), and to the experimental drugs TMC207, PA-824, meropenem (MER), benzothiazinone (BTZ), and moxifloxacin (MOXI). After STR depletion, the strain displayed greatly reduced susceptibility to the cell wall inhibitors INH and BTZ but showed increased susceptibility to RIF and PA-824, while MOXI and MER appeared equipotent under both conditions. The same potency ranking was found against nonreplicating M. tuberculosis 18b after in vivo treatment of chronically infected mice with five of these drugs. Despite the growth arrest, strain 18b retains significant metabolic activity in vitro, remaining positive in the resazurin reduction assay. Upon adaption to a 96-well format, this assay was shown to be suitable for high-throughput screening with strain 18b to find new inhibitors of dormant M. tuberculosis.
1000-01-01 | S-EPMC2944619 | BioStudies
Project description:Background:The interaction between different drug-resistant mutations is important to the development of drug resistance and its evolution. In this study, we aimed to reveal the potential relationships between mutations conferring resistance to two important antituberculosis drugs streptomycin (STR) and fluoroquinolones (FLQ). Materials and methods:We used an in vitro competitive fitness assay to reveal the interactions between different mutations of rpsL and gyrA in drug-resistant Mycobacterium smegmatis, followed by the analysis of the frequency of rpsL and gyrA mutation combinations in 213 STR-FLQ dual-resistant clinical Mycobacterium tuberculosis isolates from Sichuan region, which was also investigated by the whole genome data from 3,056 global clinical M. tuberculosis isolates. Results:The strains with K43R and K88R mutation in rpsL showed no difference in relative fitness compared with their susceptible ancestor, while K43N, K43M, K43T, and K88E exhibited a significantly lower relative fitness (P<0.05). For the FLQ-resistant mutants, all mutation types showed no difference in their relative fitness. Among STR-FLQ dual-resistant M. smegmatis strains, a lower fitness was detected in those with K43N/M/T and K88E instead of K43R and K88R mutations in rpsL. Among M. tuberculosis isolates harboring rpsL and gyrA dual mutations, the most two frequent combinatorial mutation types were K43R/D94G (n=37) and K43R/A90V (n=24), with the former being the most frequent one by both in vitro tests and clinical survey. Conclusion:Our results suggest that the interaction between rpsL and gyrA mutations affects the fitness cost in STR-FLQ dual-resistant M. smegmatis and also the predilection of mutation combinations in clinical M. tuberculosis isolates.
2018-01-01 | S-EPMC5877491 | BioStudies
Project description:Human tuberculosis is a life-threatening infection following the inhalation of Mycobacterium tuberculosis, while the closely related bacteria Mycobacterium bovis and Mycobacterium canettii are thought to be transmitted by ingestion. To explore whether M. tuberculosis could also infect individuals by ingestion, male BALBc mice were fed 2 x 106 CFUs of M. tuberculosis Beijing or phosphate-buffered saline as a negative control, over a 28-day experiment. While eight negative control mice remained disease-free, M. tuberculosis was identified in the lymph nodes and lungs of 8/14 mice and in the spleens of 4/14 mice by microscopy, PCR-based detection and culture. Whole-genome sequencing confirmed the identity of the inoculum and the tissue isolates. In these genetically identical mice, the dissemination of M. tuberculosis correlated with the results of the culture detection of four intestinal bacteria. These observations indicate that ingested M. tuberculosis mycobacteria can translocate, notably provoking lymphatic tuberculosis.
2019-01-01 | S-EPMC6936814 | BioStudies
Project description:Mycobacterium tuberculosis 18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model both in vitro and in vivo by comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18b in vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all active in vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis.
1000-01-01 | S-EPMC3486556 | BioStudies
Project description:Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.
1000-01-01 | S-EPMC4468706 | BioStudies