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 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 Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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 Susceptibility trends of zoliflodacin against multidrug-resistant Neisseria gonorrhoeae clinical isolates in Nanjing, China (2014-2018)

2020 ◽  
Author(s):  
Wenjing Le ◽  
Xiaohong Su ◽  
Xiangdi Lou ◽  
Xuechun Li ◽  
Xiangdong Gong ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Dna Sequencing ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Extended Spectrum ◽  
High Level ◽  
Level Resistance

ABSTRACTPreviously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 gonococcal isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC and parE genes were determined by PCR and DNA sequencing. The MIC of zoliflodacin for N. gonorrhoeae ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 μg/ml); 21.2% (209/986) were resistant to azithromycin (≥1 μg/ml), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 143 isolates with higher zoliflodacin MICs (0.125-0.25 mg/L), all had quinolone resistance associated double or triple mutations in gyrA; 139/143 (97.2%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin has excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

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 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 Point Mutations within the Fatty Acid Synthase Type II Dehydratase Components HadA or HadC Contribute to Isoxyl Resistance in Mycobacterium tuberculosis

2012 ◽  
Vol 57 (1) ◽  
pp. 629-632 ◽  
Author(s):  
Laila Gannoun-Zaki ◽  
Laeticia Alibaud ◽  
Laurent Kremer
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Point Mutations ◽  
Mycolic Acid ◽  
Type Ii ◽  
Content Type ◽  
High Level ◽  
Fas Ii ◽  
Level Resistance

ABSTRACTThe mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO inMycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

scholarly journals Analysis of ahpC gene mutations in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

1997 ◽  
Vol 41 (9) ◽  
pp. 2057-2058 ◽  
Author(s):  
C L Kelley ◽  
D A Rouse ◽  
S L Morris
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleotide Sequence ◽  
Gene Mutations ◽  
Clinical Isolates ◽  
Sequence Analyses ◽  
Promoter Mutations

The ahpC genes of 57 clinical isolates and one in vitro mutant of Mycobacterium tuberculosis were evaluated by nucleotide sequence analyses. Although compensatory ahpC promoter mutations were identified in 8 catalase-negative, katG-defective strains, the ahpC genes of 25 catalase-positive, isoniazid-resistant isolates and 25 drug-sensitive strains were not altered.

scholarly journals Diverse Cytomegalovirus UL27 Mutations Adapt to Loss of Viral UL97 Kinase Activity under Maribavir

2008 ◽  
Vol 53 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Sunwen Chou
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Genetic Defect ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Drug Selection ◽  
Normal Expression ◽  
High Level ◽  
Level Resistance

ABSTRACT In vitro resistance to maribavir (MBV), a cytomegalovirus UL97 kinase inhibitor currently in clinical trials, is known to result from viral UL97 mutations that confer moderate to high-level resistance and UL27 mutations that confer low-level resistance. To add to the four reported UL27 mutations, cytomegalovirus isolates or strains were propagated under MBV. Four clinical isolates evolved UL27 mutations, which were first detected after 8 to 30 passages under drug selection. In three separate experiments, laboratory strain T2294, which contained an exonuclease mutation, developed UL27 mutations at 10 to 12 passages under MBV. Most of these isolates and strains also developed a UL97 mutation, commonly T409M, before or after the appearance of the UL27 mutation. The passage of two laboratory strains genetically defective in UL97, in the absence of MBV, likewise resulted in UL27 mutations. The nine UL27 mutations observed included multiple instances of point, stop, and frameshift mutations, which were individually transferred to a reference CMV strain and which were shown to confer two- to threefold increases in MBV inhibitory concentrations. In contrast, seven common UL27 amino acid changes found in baseline clinical isolates conferred no MBV resistance. The mutants with UL27 mutations had slightly attenuated growth. The frequent mutation of UL27 suggests that its normal expression is mildly disadvantageous to the virus in the absence of UL97 kinase activity, whether the latter results from MBV inhibition or a genetic defect. Although the function of UL27 is unknown, it does not appear to be a direct antiviral target for MBV.

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.

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