scholarly journals Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

1998 ◽  
Vol 42 (7) ◽  
pp. 1853-1857 ◽  
Author(s):  
D. L. Williams ◽  
L. Spring ◽  
L. Collins ◽  
L. P. Miller ◽  
L. B. Heifets ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cross Resistance ◽  
In Vitro Mutagenesis ◽  
Missense Mutations ◽  
Resistance Mutations ◽  
Development Of Resistance ◽  
High Level ◽  
The Impact ◽  
Level Resistance

ABSTRACT The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutantrpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoBmutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specificrpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

scholarly journals Correlation of molecular resistance mechanisms and phenotypic resistance levels in streptomycin-resistant Mycobacterium tuberculosis.

1996 ◽  
Vol 40 (11) ◽  
pp. 2452-2454 ◽  
Author(s):  
A Meier ◽  
P Sander ◽  
K J Schaper ◽  
M Scholz ◽  
E C Böttger
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Active Agent ◽  
Cross Resistance ◽  
Permeability Barrier ◽  
Wild Type ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Level Resistance

Quantitative susceptibility testing of clinical isolates of streptomycin-resistant Mycobacterium tuberculosis demonstrated that there is a close correlation between the molecular resistance mechanism and the in vitro activity of streptomycin: mutations in rpsL were mainly associated with high-level resistance, mutations in rrs were associated with an intermediate level of resistance, and streptomycin-resistant isolates with wild-type rpsL and rrs exhibited a low-level resistance phenotype. Investigations of streptomycin-resistant isolates with wild-type rpsL and rrs revealed that (i) there is no cross-resistance to other drugs and (ii) a permeability barrier may contribute to resistance, because resistance was significantly lowered in the presence of a membrane-active agent.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals In Vitro Study of Stepwise Acquisition of rv0678 and atpE Mutations Conferring Bedaquiline Resistance

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Nabila Ismail ◽  
Nazir A. Ismail ◽  
Shaheed V. Omar ◽  
Remco P. H. Peters
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Solid Medium ◽  
In Vitro Study ◽  
Whole Genome ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Atcc Strain ◽  
High Level ◽  
Level Resistance

ABSTRACT Bedaquiline resistance within Mycobacterium tuberculosis may arise through efflux-based (rv0678) or target-based (atpE) pathway mutations. M. tuberculosis mutant populations from each of five sequential steps in a passaging approach, using a pyrazinamide-resistant ATCC strain, were subjected to MIC determinations and whole-genome sequencing. Exposure to increasing bedaquiline concentrations resulted in increasing phenotypic resistance (up to >2 μg/ml) through MIC determination on solid medium (Middlebrook 7H10). rv0678 mutations were dynamic, while atpE mutations were fixed, once occurring. We present the following hypothesis for in vitro emergence of bedaquiline resistance: rv0678 mutations may be the first transient step in low-level resistance acquisition, followed by high-level resistance due to fixed atpE mutations.

scholarly journals Selection of High-Level Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors

2003 ◽  
Vol 47 (2) ◽  
pp. 759-769 ◽  
Author(s):  
Terri Watkins ◽  
Wolfgang Resch ◽  
David Irlbeck ◽  
Ronald Swanstrom
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Selective Pressure ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
High Level ◽  
Level Resistance

ABSTRACT Protease inhibitors represent some of the most potent agents available for therapeutic strategies designed to inhibit human immunodeficiency virus type 1 (HIV-1) replication. Under certain circumstances the virus develops resistance to the inhibitor, thereby negating the benefits of this therapy. We have carried out selections for high-level resistance to each of three protease inhibitors (indinavir, ritonavir, and saquinavir) in cell culture. Mutations accumulated over most of the course of the increasing selective pressure. There was significant overlap in the identity of the mutations selected with the different inhibitors, and this gave rise to high levels of cross-resistance. Virus particles from the resistant variants all showed defects in processing at the NC/p1 protease cleavage site in Gag. Selections with pairs of inhibitors yielded similar patterns of resistance mutations. A virus that could replicate at near-toxic levels of the three protease inhibitors combined was selected. The pro sequence of this virus was similar to that of the viruses that had been selected for high-level resistance to each of the drugs singly. Finally, a molecular clone carrying the eight most common resistance mutations seen in these selections was characterized. The sequence of this virus was relatively stable during selection for revertants in spite of displaying poor processing at the NC/p1 site and having significantly reduced fitness. These results reveal patterns of drug resistance that extend to near the limits of attainable selective pressure with these inhibitors and confirm the patterns of cross-resistance for these three inhibitors and the attenuation of virion protein processing and fitness that accompanies high-level resistance.

scholarly journals TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

2005 ◽  
Vol 79 (20) ◽  
pp. 12773-12782 ◽  
Author(s):  
Johan Vingerhoets ◽  
Hilde Azijn ◽  
Els Fransen ◽  
Inky De Baere ◽  
Liesbet Smeulders ◽  
...  
Keyword(s):  
Wild Type Virus ◽  
Cross Resistance ◽  
Resistant Virus ◽  
Wild Type ◽  
Genetic Barrier ◽  
Development Of Resistance ◽  
Selection Experiments ◽  
The Impact ◽  
Hiv 1

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

scholarly journals Low-Level Resistance to Rifampin in Streptococcus pneumoniae

2003 ◽  
Vol 47 (3) ◽  
pp. 863-868 ◽  
Author(s):  
Patricia Stutzmann Meier ◽  
Silvia Utz ◽  
Suzanne Aebi ◽  
Kathrin Mühlemann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Point Mutations ◽  
Low Level ◽  
Development Of Resistance ◽  
Rifampin Resistance ◽  
High Level ◽  
Tissue Compartments ◽  
Level Resistance

ABSTRACT Rifampin is recommended for combination therapy of meningitis due to β-lactam-resistant Streptococcus pneumoniae. High-level rifampin resistance (MIC, ≥4 mg/liter) has been mapped to point mutations in clusters I and III of rpoB of the pneumococcus. The molecular basis of low-level resistance (MICs, ≥0.5 and <4 mg/liter) was analyzed. Spontaneous mutants of clinical pneumococcal isolates were selected on Columbia sheep blood agar plates containing rifampin at 0.5, 4, 10, or 50 mg/liter. Low-level resistance could be assigned to mutations in cluster II (I545N, I545L). Sensitive (MIC, <0.048 mg/liter) wild-type strains acquired low-level resistance at a rate approximately 10 times higher than that at which they acquired high-level resistance (average mutation frequencies, 2.4 × 10−7 for low-level resistance versus 2.9 × 10−8 for high-level resistance [P < 0.0001]). In second-step experiments, the frequencies of mutations from low- to high-level resistance were over 10 times higher than the frequencies of mutations from susceptibility to high-level resistance (average mutation frequencies, 7.2 × 10−7 versus 5.0 × 10−8 [P < 0.001]). Mutants with low-level resistance were stable upon passage. Sequencing of a clinical isolate with low-level resistance (MIC, 0.5 mg/liter) revealed a Q150R mutation upstream of cluster I. The frequencies of mutations to high-level resistance for this strain were even higher than the rates observed for the in vitro mutants. Therefore, a resistance-mediating mutation located outside clusters I, II, and III has been described for the first time in the pneumococcus. In vitro low-level rifampin resistance in S. pneumoniae could be mapped to cluster II of rpoB. Mutants of pneumococcus with low-level resistance may be selected in vivo during therapy in tissue compartments with low antibiotic concentrations and play a role in the development of resistance.

scholarly journals Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

2006 ◽  
Vol 50 (8) ◽  
pp. 2860-2862 ◽  
Author(s):  
Emma Huitric ◽  
Jim Werngren ◽  
Pontus Juréen ◽  
Sven Hoffner
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Point Mutations ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Clinical Isolates ◽  
Large Deletions ◽  
High Level ◽  
Level Resistance

ABSTRACT The distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.

scholarly journals Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae

2001 ◽  
Vol 45 (12) ◽  
pp. 3517-3523 ◽  
Author(s):  
L. M. Weigel ◽  
G. J. Anderson ◽  
R. R. Facklam ◽  
F. C. Tenover
Keyword(s):  
Amino Acid ◽  
Antimicrobial Agents ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Genetic Analyses ◽  
High Level ◽  
Susceptibility Profiles ◽  
Level Resistance

ABSTRACT Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA,gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrAwas associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

scholarly journals In vitro cross-resistance to doravirine in a panel of HIV-1 clones harbouring multiple NNRTI resistance mutations

2020 ◽  
Vol 76 (1) ◽  
pp. 130-134
Author(s):  
Francesco Saladini ◽  
Federica Giammarino ◽  
Behnaz A Hosseini ◽  
Alessia Giannini ◽  
Adele Boccuto ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Cross Resistance ◽  
Reference List ◽  
Resistance Mutations ◽  
Nnrti Resistance ◽  
Infectious Clones ◽  
High Level ◽  
Reduced Activity ◽  
Hiv 1

Abstract Objectives Doravirine is a recently licensed HIV-1 NNRTI with improved efficacy, pharmacokinetics and safety profile compared with efavirenz and limited cross-resistance with rilpivirine and etravirine. In this in vitro study, cross-resistance to doravirine was analysed in a representative panel of NNRTI-resistant clones. Methods In vitro phenotypic susceptibility to doravirine was assessed in 10 clinically derived infectious clones with intermediate- to high-level resistance to rilpivirine, etravirine, efavirenz and nevirapine, and in NL4-3 site-directed mutants harbouring K103N, Y181C, M230L or K103N/Y181C NNRTI mutations. Results Although none of the infectious clones harboured any of the major doravirine resistance-associated mutations (RAMs) included in the IAS-USA reference list, doravirine fold change (FC) values were comparable to or higher than those calculated for other NNRTIs, particularly etravirine and rilpivirine. As expected, single NNRTI mutations K103N and Y181C did not impair doravirine susceptibility (FC 1.4 and 1.8, respectively), while reduced activity was observed with the single M230L or double K103N/Y181C mutations (FC 7.6 and 4.9, respectively). Median FC values increased significantly with increasing numbers of NNRTI RAMs (P = 0.005) and were &gt;10 in 4/4 and 1/4 clones harbouring four and three NNRTI RAMs, respectively. FC values correlated well with predicted susceptibility as inferred by Stanford HIV Drug Resistance Database (HIVdb) and ANRS algorithms (both P &lt; 0.001). Conclusions Substantial cross-resistance to doravirine was detected in NNRTI-resistant viruses harbouring complex mutational patterns, even in the absence of major IAS-USA doravirine RAMs. Therefore, based on the simple IAS-USA reference list, doravirine resistance may be underestimated in viruses harbouring multiple NNRTI mutations.

scholarly journals Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor

2009 ◽  
Vol 53 (6) ◽  
pp. 2424-2431 ◽  
Author(s):  
Ming-Tain Lai ◽  
Vandna Munshi ◽  
Sinoeun Touch ◽  
Robert M. Tynebor ◽  
Thomas J. Tucker ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Nnrti Resistance ◽  
Development Of Resistance ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection.

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