scholarly journals Spontaneous mutations conferring antibiotic resistance to antitubercular drugs at a range of concentrations in Mycobacterium smegmatis

2018 ◽  
Author(s):  
Iveren W Nyinoh ◽  
Johnjoe McFadden
Keyword(s):  
Antibiotic Resistance ◽  
Mutation Rate ◽  
Mycobacterium Smegmatis ◽  
Future Generations ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Spontaneous Mutations ◽  
Likelihood Estimator ◽  
Antitubercular Drugs

AbstractMycobacteria population can undergo mutations in their DNA sequence during replication, which if not repaired, would be transferred to future generations. In this study, in vitro spontaneous mutations in Mycobacterium smegmatis mc2155 (Msm) conferring resistance to isoniazid (INHr), rifampicin (RIFr), kanamycin (KANr) and streptomycin (STRr) were determined at several concentrations in a fluctuation assay. Mutation rate was estimated using the P₀ method, and estimates were then compared with the Lea-Coulson method of the median and Ma-Sandri-Sarkar Maximum Likelihood Estimator (MSS-MLE) method available on the Fluctuation analysis calculator (FALCOR). The mutation rates of RIFr ranged from 9.24 × 10-8 - 2.18 × 10-10, INHr 1.2 × 10-7 - 1.20×10-9, STRr 2.77 × 10-8 - 5.31 × 10-8 and KANr 1.7 × 10-8 mutations per cell division. This study provides mutation rate estimates to key antitubercular drugs at a range of concentrations.

Seventy Years on from the Luria and Delbrûck Fluctuation Analysis: A Comparison of three Methods for Estimating Mutation Rate

2015 ◽  
Vol 6 ◽  
pp. 50-58
Author(s):  
I W Nyinoh
Keyword(s):  
Mutation Rate ◽  
Resistance Mutation ◽  
Model Organism ◽  
Relative Fitness ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Cellular Processes ◽  
Scientific Disciplines ◽  
Rate Fluctuation ◽  
Contemporary Interpretation

Seventy years ago, Luria and Delbrûck discovered fluctuation assay for estimating mutation rates. While this method is slightly dated, it is one of the few methods for estimating mutation rates in batch culture. Mutation rates when determined expose information on cellular processes and fundamental mutagenic mechanisms. Formerly, inferences drawn from fluctuation assay were sufficient to answer a specific question inbacterial genetics. However, contemporary interpretation of results goes far beyond the motive originally intended. As the fluctuation assay has gained popularity in various scientific disciplines, analyses of results obtained are not same. This study aims to compare the estimation of mutation rates using the Poison distribution (Po) method with, the Ma-Sarka Sandri maximum likelihood estimator and the Lea-Coulson median estimator. Mycobacterium smegmatismc 2 155was used as a model organism for Mycobacterium tuberculosis, and spontaneous mutations that arose in stationary phase cells exposed to antibiotic stress were investigated. Ten to twenty-four parallel cultures were tested with various anti-tuberculosis drugs; isoniazid, kanamycin, rifampicin and streptomycin. Minimum Inhibitory Concentration (MIC) of the drugs were also determinedto be; 8 ìg/mL, 0.24 ìg/mL, 16 ìg/mL and 0.5 ìg/mL for isoniazid, kanamycin, rifampicin and streptomycin respectively. The mutation rates obtained with the methods were very similar. To improve the power of deductions drawn from fluctuation assay, efforts should be made to experimentally determine the relative fitness of wild-type to mutant bacteria.This comparison is only a guide providing evidence regarding the authenticity of some of the methods currently available to researchers interested in estimating bacterial mutation rates.Keywords: antibiotic resistance, mutation rate, fluctuation assay, fluctuation analysis calculator.

scholarly journals Epigenetic modifications affect the rate of spontaneous mutations in a pathogenic fungus

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Habig ◽  
Cecile Lorrain ◽  
Alice Feurtey ◽  
Jovan Komluski ◽  
Eva H. Stukenbrock
Keyword(s):  
Temperature Stress ◽  
Mutation Rate ◽  
Cytosine Methylation ◽  
Pathogenic Fungus ◽  
Epigenetic Modifications ◽  
Mutation Rates ◽  
Adaptive Mutation ◽  
Spontaneous Mutations ◽  
Evolutionary Trajectory ◽  
Genome Wide

AbstractMutations are the source of genetic variation and the substrate for evolution. Genome-wide mutation rates appear to be affected by selection and are probably adaptive. Mutation rates are also known to vary along genomes, possibly in response to epigenetic modifications, but causality is only assumed. In this study we determine the direct impact of epigenetic modifications and temperature stress on mitotic mutation rates in a fungal pathogen using a mutation accumulation approach. Deletion mutants lacking epigenetic modifications confirm that histone mark H3K27me3 increases whereas H3K9me3 decreases the mutation rate. Furthermore, cytosine methylation in transposable elements (TE) increases the mutation rate 15-fold resulting in significantly less TE mobilization. Also accessory chromosomes have significantly higher mutation rates. Finally, we find that temperature stress substantially elevates the mutation rate. Taken together, we find that epigenetic modifications and environmental conditions modify the rate and the location of spontaneous mutations in the genome and alter its evolutionary trajectory.

scholarly journals Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance

2019 ◽  
Vol 117 (1) ◽  
pp. 629-634 ◽  
Author(s):  
Paulami Rudra ◽  
Kelley R. Hurst-Hess ◽  
Katherine L. Cotten ◽  
Andrea Partida-Miranda ◽  
Pallavi Ghosh
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycobacterium Abscessus ◽  
Macrolide Resistance ◽  
Gtp Hydrolysis ◽  
Ribosomal Subunits ◽  
Mutant Proteins ◽  
Splitting Factor

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of thehflXgene in the pathogenicMycobacterium abscessus, as well as the nonpathogenicMycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided byMab_hflXis equivalent to that conferred byerm41, implying thathflXconstitutes a significant resistance determinant inM. abscessus. We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in aΔMs_hflXstrain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence ofhflXgenes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.

scholarly journals In Vitro Analysis of Rates and Spectra of Mutations in a Polymorphic Region of the Rv0746 PE_PGRS Gene of Mycobacterium tuberculosis

2006 ◽  
Vol 189 (5) ◽  
pp. 2190-2195 ◽  
Author(s):  
Edith E. Machowski ◽  
Samantha Barichievy ◽  
Burkhard Springer ◽  
Steven I. Durbach ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Mutation Rates ◽  
Polymorphic Region ◽  
Replication Slippage ◽  
Vitro Analysis ◽  
In Vitro Analysis

ABSTRACT An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.

scholarly journals Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Toon Swings ◽  
Bram Van den Bergh ◽  
Sander Wuyts ◽  
Eline Oeyen ◽  
Karin Voordeckers ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Mutation Rate ◽  
Selective Pressure ◽  
Fast Response ◽  
Mutation Rates ◽  
Ethanol Stress ◽  
Adaptive Tuning ◽  
Stressful Environments ◽  
High Ethanol

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy.

scholarly journals Antibiotic sensitivity and mutation rates to antibiotic resistance inMycoplasma mycoidesssp.mycoides

1987 ◽  
Vol 98 (3) ◽  
pp. 361-368 ◽  
Author(s):  
D. H. Lee ◽  
R. J. Miles ◽  
J. R. M. Inal
Keyword(s):  
Antibiotic Resistance ◽  
Minimum Inhibitory Concentration ◽  
Mutation Rate ◽  
Parent Strain ◽  
Inhibitory Concentration ◽  
Antibiotic Sensitivity ◽  
Single Step ◽  
Mutation Rates ◽  
Mycoplasma Mycoides ◽  
Resistant Mutants

SUMMARYThe antibiotic resistance ofMycoplasma mycoidesssp.mycoidesstrain T1was investigated. This strain was resistant to high levels ( > 100 μg ml−1) of rifampicin and nalidixic acid. It was sensitive to streptomycin, spectinomycin and novobiocin; however, single step mutants with high levels of resistance ( > 100 μg ml−1) were readily isolated. With erythromycin and tylosin for which the minimum inhibitory concentration (MIC) for the parent strain was < 0·1 μg ml−1, mutants resistant to > 100 μg ml−1were obtained in two and three steps respectively. The MIC of tetracycline in single step resistant mutants (0·6 μg ml−1) was tenfold higher than the parent strain, but could not be increased further. There was only a twofold increase in resistance to chloramphenicol in single step mutants. The frequency of resistant mutants varied with the antibiotic and was between 4× 10minuss;6and 2× 10−8. The mutation rate to antibiotic resistance to streptomycin, spectinomycin, novobiocin, erythromycin and tylosin was between 3× 10−8and 5× 10−9per cell per generation. There was a fivefold decrease in mutation rate to resistance to 60 μg ml−1streptomycin compared to that to 20 μg ml−1.

scholarly journals Dynamic and Heterogeneous Mutations to Fluconazole Resistance in Cryptococcus neoformans

2001 ◽  
Vol 45 (2) ◽  
pp. 420-427 ◽  
Author(s):  
Jianping Xu ◽  
Chiatogu Onyewu ◽  
Heather J. Yoell ◽  
Rabia Y. Ali ◽  
Rytas J. Vilgalys ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mutation Rate ◽  
Antifungal Agent ◽  
Basidiomycetous Yeast ◽  
Mutation Rates ◽  
Significant Heterogeneity ◽  
Random Set ◽  
Fluconazole Resistance ◽  
Mutational Processes

ABSTRACT Infections with the human pathogenic basidiomycetous yeastCryptococcus neoformans are often treated with fluconazole. Resistance to this antifungal agent has been reported. This study investigated the patterns of mutation to fluconazole resistance inC. neoformans in vitro. The MIC of fluconazole was measured for 21 strains of C. neoformans. The MICs for these 21 strains differed (0.25 to 4.0 μg/ml), but the strains were selected for this study because they exhibited no growth on plates of yeast morphology agar (YMA) containing 8 μg of fluconazole per ml. To determine their mutation rates, six independent cultures from a single original colony were established for each of the 21 strains. Each culture was then spread densely on a YMA plate with 8 μg of fluconazole per ml. A random set of putative mutants was subcultured, and the MIC of fluconazole was determined for each mutant. The 21 strains evinced significant heterogeneity in their mutation rates. The MICs of the putative mutants ranged widely, from their original MIC to 64 μg of fluconazole per ml. However, for this set of 21 strains, there was no significant correlation between the original MIC for a strain and the mutation rate of that strain; the MIC for the mutant could not be predicted from the original MIC. These results suggest that dynamic and heterogeneous mutational processes are involved in generating fluconazole resistance in C. neoformans.

scholarly journals Production of MSTN mutated cattle using CRISPR--Cas9

2021 ◽  
Author(s):  
Gyeong-Mim Gim ◽  
Dong-Hyuk Kwon ◽  
Kyeong-Hyeon Eom ◽  
Joon-Ho Moon ◽  
Ji-Hyun Park ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mutation Rate ◽  
Genetically Modified ◽  
Muscle Growth ◽  
Transgenic Animals ◽  
Mutation Rates ◽  
Biallelic Mutation ◽  
Growth Phenotype ◽  
Synthetic Mrna

Many transgenic animals have been produced using CRISPR–Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique in cattle. The goal of this study was to produce trait-improved cattle using the genome editing technology CRISPR–Cas9. Myostatin (MSTN) was selected as a target locus and synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in vitro fertilized embryos. As a result, 17 healthy calves were born and 3 of these showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had biallelic mutation (-12bp) and a doubling muscle growth phenotype. In conclusion, we showed that the genome editing technology CRISPR–Cas9 can produce genetically modified calves with improved traits.

scholarly journals Identifying Mutator Phenotypes among Fluoroquinolone-Resistant Strains of Streptococcus pneumoniae Using Fluctuation Analysis

2007 ◽  
Vol 51 (9) ◽  
pp. 3225-3229 ◽  
Author(s):  
Carolyn V. Gould ◽  
Paul D. Sniegowski ◽  
Mikhail Shchepetov ◽  
Joshua P. Metlay ◽  
Jeffrey N. Weiser
Keyword(s):  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Mismatch Repair ◽  
Mutation Rate ◽  
In Vitro Selection ◽  
Clinical Isolates ◽  
Mutation Rates ◽  
Fluctuation Analysis ◽  
Missense Mutations ◽  
Resistant Strains

ABSTRACT The occurrence of mutator phenotypes among laboratory-generated and clinical levofloxacin-resistant strains of Streptococcus pneumoniae was determined using fluctuation analysis. The in vitro selection for levofloxacin-resistant mutants of strain D39, each with point mutations in both gyrA and parC or parE, was not associated with a significant change in the mutation rate. Two of eight clinical isolates resistant to levofloxacin (MIC, >8 μg/ml) had estimated mutation rates of 1.2 × 10−7 and 9.4 × 10−8 mutations per cell division, indicating potential mutator phenotypes, compared to strain D39, which had an estimated mutation rate of 1.4 × 10−8 mutations per cell division. The levofloxacin-resistant isolates with the highest mutation rates showed evidence of dysfunctional mismatch repair and contained missense mutations in mut genes at otherwise highly conserved sites. The association of hypermutability in levofloxacin-resistant S. pneumoniae clinical isolates with mutations in DNA mismatch repair genes provides further evidence that mismatch repair mutants may have a selective advantage in the setting of antibiotic pressure, facilitating the development of further antibiotic resistance.

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