scholarly journals Effect of rpoB Mutations Conferring Rifampin Resistance on Fitness of Mycobacterium tuberculosis

2004 ◽  
Vol 48 (4) ◽  
pp. 1289-1294 ◽  
Author(s):  
Deneke H. Mariam ◽  
Yohannes Mengistu ◽  
Sven E. Hoffner ◽  
Dan I. Andersson
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Problem ◽  
Relative Fitness ◽  
Macrophage Cell ◽  
Gene Encoding ◽  
Laboratory Medium ◽  
Rifampin Resistance ◽  
Resistant Mutants ◽  
Major Drug

ABSTRACT Rifampin is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the rpoB gene, encoding the β-subunit of RNA polymerase. We examined the effect of three different rpoB mutations on the fitness of Mycobacterium tuberculosis. Rifampin-resistant mutants were isolated from a virulent clinical isolate of M. tuberculosis (strain Harlingen) in vitro at a mutation frequency of 2.3 × 10−8. Mutations in the rpoB gene were identified, and the growth rates of three defined mutants were measured by competition with the susceptible parent strain in laboratory medium and by single cultures in a macrophage cell line and in laboratory medium. All of the mutants showed a decreased growth rate in the three assays. The relative fitness of the mutants varied between 0.29 and 0.96 (that of the susceptible strain was set to 1.0) depending on the specific mutant and assay system. Unexpectedly, the relative fitness ranking of the mutants differed between the different assays. In conclusion, rifampin resistance is associated with a cost that is conditional.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

scholarly journals Rifampin Resistance in Mycobacterium kansasii Is Associated with rpoB Mutations

2001 ◽  
Vol 45 (11) ◽  
pp. 3056-3058 ◽  
Author(s):  
John L. Klein ◽  
Timothy J. Brown ◽  
Gary L. French
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Basis ◽  
Strong Association ◽  
Missense Mutations ◽  
Β Subunit ◽  
Mycobacterium Kansasii ◽  
Bacterial Rna ◽  
Rifampin Resistance ◽  
Potent Drug

ABSTRACT Rifampin is the most potent drug used in the treatment of disease due to Mycobacterium kansasii. A 69-bp fragment ofrpoB, the gene that encodes the β subunit of the bacterial RNA polymerase, was sequenced and found to be identical in five rifampin-susceptible clinical isolates of M. kansasii. This sequence showed 87% homology with the Mycobacterium tuberculosis gene, with an identical deduced amino acid sequence. In contrast, missense mutations were detected in the same fragment amplified from five rifampin-resistant isolates. A rifampin-resistant strain generated in vitro also harbored an rpoB gene missense mutation that was not present in the parent isolate. All mutations detected (in codons 513, 526, and 531) have previously been described in rifampin-resistant M. tuberculosis isolates. Rifampin MICs determined by E-test were <1 mg/liter for all rifampin-susceptible isolates and >256 mg/liter for all rifampin-resistant ones. In addition, four of the five rifampin-resistant isolates were also resistant to rifabutin. We have thus shown a strong association between rpoB gene missense mutations and rifampin resistance in M. kansasii. Although our results are derived from a small number of isolates and confirmation with larger numbers would be useful, they strongly suggest that mutations within rpoB form the molecular basis of rifampin resistance in this species.

scholarly journals Characteristic Resistance Mechanism ofMycobacterium tuberculosisto DC-159a, a New Respiratory Quinolone

2011 ◽  
Vol 55 (8) ◽  
pp. 3958-3960 ◽  
Author(s):  
Jun-ichiro Sekiguchi ◽  
Areeya Disratthakit ◽  
Shinji Maeda ◽  
Norio Doi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Double Mutation ◽  
Resistant Mutants ◽  
Characteristic Resistance

ABSTRACTA G88C mutation in GyrA is one of the key alterations by whichMycobacterium tuberculosismutants acquire DC-159a resistancein vitro. A novel double mutation in GyrA, G88C D94H, conferred high DC-159a resistance. Different mutation patterns in GyrA were demonstrated for DC-159a-resistant mutants and quinolone-resistant multidrug-resistant (QR-MDR)M. tuberculosisisolates, with a mutation either at position 90 or 94 and double mutations at 90 and 91 or at 90 and 94. DC-159a might be promising for QRM. tuberculosistreatment.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals Strains of the East Asian (W/Beijing) Lineage of Mycobacterium tuberculosis Are DosS/DosT-DosR Two-Component Regulatory System Natural Mutants

2010 ◽  
Vol 192 (8) ◽  
pp. 2228-2238 ◽  
Author(s):  
Ashley Fallow ◽  
Pilar Domenech ◽  
Michael B. Reed
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Frameshift Mutation ◽  
East Asian ◽  
Regulatory System ◽  
Bacterial Respiration ◽  
Low Oxygen ◽  
Gene Encoding ◽  
Two Component ◽  
Dosr Regulon

ABSTRACT As part of our ongoing efforts to uncover the phenotypic consequences of genetic variability among clinical Mycobacterium tuberculosis isolates, we previously reported that isolates of the “East Asian” or “W/Beijing” lineage constitutively overexpress the coordinately regulated transcriptional program known as the DosR regulon under standard in vitro conditions. This phenotype distinguishes the W/Beijing lineage from all other M. tuberculosis lineages, which normally induce expression of this regulon only once exposed to low oxygen or nitric oxide, both of which result in inhibition of bacterial respiration and replication. Transcription of the DosR regulon is controlled through a two-component regulatory system comprising the transcription factor DosR and two possible cognate histidine sensor kinases, DosS and DosT. Through sequence analysis of a carefully selected set of isolates representing each of the major M. tuberculosis lineages, we describe herein a naturally occurring frameshift mutation in the gene encoding the DosT sensor kinase for isolates of the most recently evolved W/Beijing sublineages. Intriguingly, the occurrence of the frameshift mutation correlates precisely with the appearance of the constitutive DosR regulon phenotype displayed by the same “modern” W/Beijing strains. However, complementation studies have revealed that the mutation in dosT alone is not directly responsible for the constitutive DosR regulon phenotype. Our data serve to highlight the evolutionary pressure that exists among distinct M. tuberculosis lineages to maintain tight control over DosR regulon expression.

scholarly journals The relative fitness of drug-resistant Mycobacterium tuberculosis : a modelling study of household transmission in Peru

2018 ◽  
Vol 15 (143) ◽  
pp. 20180025 ◽  
Author(s):  
Gwenan M. Knight ◽  
Mirko Zimic ◽  
Sebastian Funk ◽  
Robert H. Gilman ◽  
Jon S. Friedland ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Credible Interval ◽  
Relative Fitness ◽  
Drug Resistant ◽  
Household Transmission ◽  
Prevalence Estimates ◽  
Control Programmes ◽  
Mdr Tb ◽  
Large Prospective Cohort Study

The relative fitness of drug-resistant versus susceptible bacteria in an environment dictates resistance prevalence. Estimates for the relative fitness of resistant Mycobacterium tuberculosis ( Mtb ) strains are highly heterogeneous and mostly derived from in vitro experiments. Measuring fitness in the field allows us to determine how the environment influences the spread of resistance. We designed a household structured, stochastic mathematical model to estimate the fitness costs associated with multidrug resistance (MDR) carriage in Mtb in Lima, Peru during 2010–2013. By fitting the model to data from a large prospective cohort study of TB disease in household contacts, we estimated the fitness, relative to susceptible strains with a fitness of 1, of MDR- Mtb to be 0.32 (95% credible interval: 0.15–0.62) or 0.38 (0.24–0.61), if only transmission or progression to disease, respectively, was affected. The relative fitness of MDR- Mtb increased to 0.56 (0.42–0.72) when the fitness cost influenced both transmission and progression to disease equally. We found the average relative fitness of MDR- Mtb circulating within households in Lima, Peru during 2010–2013 to be significantly lower than concurrent susceptible Mtb . If these fitness levels do not change, then existing TB control programmes are likely to keep MDR-TB prevalence at current levels in Lima, Peru.

scholarly journals Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance

2009 ◽  
Vol 64 (3) ◽  
pp. 515-523 ◽  
Author(s):  
Indra L. Bergval ◽  
Anja R. J. Schuitema ◽  
Paul R. Klatser ◽  
Richard M. Anthony
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Isoniazid Resistance ◽  
Resistant Mutants

scholarly journals Novel Conjugate of Moxifloxacin and Carboxymethylated Glucan with Enhanced Activity against Mycobacterium tuberculosis

2006 ◽  
Vol 50 (6) ◽  
pp. 1982-1988 ◽  
Author(s):  
Y. S. Schwartz ◽  
M. I. Dushkin ◽  
V. A. Vavilin ◽  
E. V. Melnikova ◽  
O. M. Khoschenko ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Targeted Delivery ◽  
Scavenger Receptors ◽  
Intracellular Pathogen ◽  
Macrophage Cell ◽  
Macrophage Receptors ◽  
Mycobacterial Growth ◽  
Dose Dependent

ABSTRACT Mycobacterium tuberculosis is an intracellular pathogen that persists within macrophages of the human host. One approach to improving the treatment of tuberculosis (TB) is the targeted delivery of antibiotics to macrophages using ligands to macrophage receptors. The moxifloxacin-conjugated dansylated carboxymethylglucan (M-DCMG) conjugate was prepared by chemically linking dansylcadaverine (D) and moxifloxacin (M) to carboxymethylglucan (CMG), a known ligand of macrophage scavenger receptors. The targeted delivery to macrophages and the antituberculosis activity of the conjugate M-DCMG were studied in vitro and in vivo. Using fluorescence microscopy, fluorimetry, and the J774 macrophage cell line, M-DCMG was shown to accumulate in macrophages through scavenger receptors in a dose-dependent (1 to 50 μg/ml) manner. After intravenous administration of M-DCMG into C57BL/6 mice, the fluorescent conjugate was concentrated in the macrophages of the lungs and spleen. Analyses of the pharmacokinetics of the conjugate demonstrated that M-DCMG was more rapidly accumulated and more persistent in tissues than free moxifloxacin. Importantly, therapeutic studies of mycobacterial growth in C57BL/6 mice showed that the M-DCMG conjugate was significantly more potent than free moxifloxacin.

scholarly journals Consequences of Noncompliance for Therapy Efficacy and Emergence of Resistance in Murine Tuberculosis Caused by the Beijing Genotype of Mycobacterium tuberculosis

2012 ◽  
Vol 56 (9) ◽  
pp. 4937-4944 ◽  
Author(s):  
Jurriaan E. M. de Steenwinkel ◽  
Marian T. ten Kate ◽  
Gerjo J. de Knegt ◽  
Henri A. Verbrugh ◽  
Rob E. Aarnoutse ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutation Frequency ◽  
Evolutionary Development ◽  
Beijing Genotype ◽  
Great Effort ◽  
East African ◽  
Content Type ◽  
Resistant Mutants ◽  
Emergence Of Resistance

ABSTRACTDespite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development ofMycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotype strain to investigate with our mouse TB model whether the higher mutation frequency observed in a Beijing genotype strain is associated with treatment failure particularly during noncompliance therapy. Both genotype strains showed high virulence in comparison to that ofM. tuberculosisstrain H37Rv, resulting in a highly progressive infection with a rapid lethal outcome in untreated mice. Compliance treatment was effective without relapse of TB irrespective of the infecting strain, showing similar decreases in the mycobacterial load in infected organs and similar histopathological changes. Noncompliance treatment, simulated by a reduced duration and dosing frequency, resulted in a relapse of infection. Relapse rates were correlated with the level of noncompliance and were identical for Beijing infection and East African/Indian infection. However, only in Beijing-infected mice, isoniazid-resistant mutants were selected at the highest level of noncompliance. This is in line with the substantial selection of isoniazid-resistant mutantsin vitroin a wide isoniazid concentration window observed for the Beijing strain and not for the EAI strain. These results suggest that genotype diversity ofM. tuberculosismay be involved in emergence of resistance and indicates that genotype-tailor-made treatment should be investigated.

scholarly journals Mutation Analysis of MycobacterialrpoBGenes and Rifampin Resistance Using Recombinant Mycobacterium smegmatis

2012 ◽  
Vol 56 (4) ◽  
pp. 2008-2013 ◽  
Author(s):  
Noboru Nakata ◽  
Masanori Kai ◽  
Masahiko Makino
Keyword(s):  
Mycobacterium Smegmatis ◽  
Clinical Isolates ◽  
Recombinant Bacteria ◽  
Gene Encoding ◽  
Content Type ◽  
Rifampin Resistance ◽  
Recombinant Mycobacterium Smegmatis ◽  
The Relationship ◽  
Major Drug

ABSTRACTRifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance ofMycobacterium lepraeandMycobacterium tuberculosisresults from a mutation in therpoBgene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other thanrpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains ofMycobacterium smegmatiscarrying theM. lepraeorM. tuberculosis rpoBgene with or without mutation and disrupted their ownrpoBgenes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected inM. lepraeisolates or rifampin-resistantM. tuberculosisisolates are not involved in rifampin resistance.

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