In Vitro Antibacterial Resistance Selection and Quantitation

Young, Katherine

doi:10.1002/0471141755.ph13a06s34

Cited by 14 publications

(9 citation statements)

References 16 publications

(28 reference statements)

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“…Determination of resistance rates. The spontaneous rates of resistance to the test compounds were determined according to published methods (35,38). Agar selection plates were made by adding compound from DMSO stocks into molten Mueller-Hinton agar in a 55°C water bath.…”

Section: Methodsmentioning

confidence: 99%

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Faghih

Zhang

Ranade

et al. 2017

Antimicrob Agents Chemother

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Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against ,, and strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

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Section: Methodsmentioning

confidence: 99%

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Faghih

Zhang

Ranade

et al. 2017

Antimicrob Agents Chemother

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“…The mutation rates of Hfr2xSFXϪ and 2xOriT were measured using a standard fluctuation test (23) under 0.55 M (32 g/liter) NaCl stress to determine if the strains have unequal mutation rates under osmotic stress, which would influence their relative adaptation rates. For 2xOriT, the mutation rate is 1.43 mutants per 10 9 cells (95% confidence interval of 0.72 to 2.31); for Hfr-2xSFXϪ, the mutation rate is 1.52 mutants per 10 9 cells (95% confidence interval of 0.71 to 2.54).…”

Section: Methodsmentioning

confidence: 99%

Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

Winkler

Garcı́a

Olson

et al. 2014

Appl Environ Microbiol

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c Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. Escherichia coli, an important industrial microorganism for the production of a wide variety of fine chemicals, fuels, and proteins, has been extensively targeted to improve its suitability as a biofactory. Strain development efforts have focused on improving tolerance of feedstocks containing toxic compounds (1, 2) or products (3, 4). Many environmental variables, including osmotic pressure, can negatively impact biocatalyst performance (5). Use of nonconventional waste streams, such as waste glycerol or brackish water sources, to support microbial growth can also reduce process costs (6, 7) while reducing pressure on fresh water resources; however, these carbon and water sources generally contain high concentrations of salt that may be inhibitory to microbial growth. In addition to osmotic stresses, excess Na ϩ can disrupt the ion homeostasis in E. coli as well (8). Previous studies have attempted to engineer improved osmotic tolerance in E. coli (9, 10), but overall, knowledge of the genetic mechanisms that confer tolerance of osmotic stress in general or to specific osmolytes remains limited. A detailed analysis of E. coli osmotolerance to osmolytes would therefore provide new insight into the molecular mechanisms underlying this complex phenotype.Adaptive laboratory evolution (11) is a promising approach to identify potentially novel osmotic tolerance mechanisms, as this technique requires no assumptions about the underlying genotype-phenotype relationship. Complex phenotypes, such as enhanced resistance to biofuels (3,4,12), lignocellulosic hydrolysates (2, 13), antibiot...

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“…To assess the spontaneous resistance rate for Myx, we performed fluctuation assays (14,24,26,57), plating defined numbers of cells (ϳ10 9 CFU/plate) of S. aureus ATCC 12600 on agar containing 1ϫ, 2ϫ, 4ϫ, 8ϫ, or 16ϫ MIC of Myx, counting numbers of resistant colonies after 24 h of incubation at 37°C, and calculating resistance rates using the MSS-MLE method (16,22,27,44,50,51). For comparison, we assayed the resistance rate for Rif under the identical experimental conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Resistance rates were determined using fluctuation assays (14,24,26,57). Defined numbers of cells of S. aureus ATCC 12600 (1 ϫ 10 9 CFU/plate) were plated on Mueller-Hinton II cation-adjusted agar containing amounts of test compound equivalent to 1ϫ MIC, 2ϫ MIC, 4ϫ MIC, 8ϫ MIC, or 16ϫ MIC, and numbers of colonies were counted after 24 h at 37°C (at least five independent deter-minations for each concentration of each test compound).…”

Section: Methodsmentioning

confidence: 99%

Frequency, Spectrum, and Nonzero Fitness Costs of Resistance to Myxopyronin in Staphylococcus aureus

Srivastava

Degen

Ebright

2012

Antimicrob Agents Chemother

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The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 ؋ 10 ؊8 to 7 ؋ 10 ؊8 per generation, which is equal within error to the resistance rate for Rif (3 ؋ 10 ؊8 to 10 ؋ 10 ؊8 per generation). Substitutions conferring Myx resistance were obtained in the RNAP ␤ subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127 (1322) Mf50 (18,20,23,49). Myx exhibits broad-spectrum antibacterial activity, with potent antibacterial activity against most Gram-positive species and some Gram-negative species. Myx is under investigation as a potential lead compound for broad-spectrum antibacterial therapy. M yxopyronin (Myx) is an ␣-pyrone antibiotic produced by Myxococcus fulvusMyx functions by inhibiting bacterial RNA polymerase (RNAP) (3,18,20,32,49). The binding site on RNAP for Myx is located in the RNAP "switch region" and comprises the RNAP switch region structural elements termed "switch 1" and "switch 2," (switch region SW1/SW2 subregion) (3,18,32,49). The binding site on RNAP for Myx is different from the binding site on RNAP for the RNAP inhibitor in current use in broad-spectrum antibacterial therapy, rifampin (Rif) (3,18,32,49). Accordingly, Myx exhibits no cross-resistance with Rif (18,19,32,33,49).Previous studies have provided information about spontaneous resistance frequencies and resistance spectra for Myx (31, 32). However, the fitness costs of resistance have not previously been assessed.In this work, we comprehensively evaluate the resistance properties of Myx in Staphylococcus aureus. We report (i) the spontaneous resistance rate for Myx in S. aureus, (ii) the spontaneous resistance spectrum of Myx in S. aureus, and (iii) the fitness costs of substitutions that confer Myx resistance. Hu et al. (19). Rif was purchased from Sigma, Inc. Bacterial strains were obtained from the American Type Culture Collection. MATERIALS AND METHODS Materials. (Ϯ)-Myx B was synthesized as described inMICs. Minimal inhibitory concentrations (MICs) in column 2 of Table 1 were quantified using broth microdilution assays (8). MICs in column 3 of Table 1, and in Tables 3 and 4, were quantified using spiral gradient endpoint assays, essentially as described previously (38,45,54). Spiral gradient endpoint assays employed 150-mm by 4-mm exponentialgradient plates containing Mueller-Hinton II cation-adjusted agar and 0.5 to 100 g/ml of Myx, 0.0008 to 0.2 g/ml of Rif, or 0.2 to 40 g/ml of Rif. Plates were prepared using an Autoplate 4000 spiral plater (Spiral Biotech, Inc.). Cells were grown to early log phase, adjusted to 1 ϫ 10 8 CFU/ml, and swabbed radially onto plates. Plates were incubated for 16 h at 3...

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In Vitro Antibacterial Resistance Selection and Quantitation

Cited by 14 publications

References 16 publications

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections

Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

Frequency, Spectrum, and Nonzero Fitness Costs of Resistance to Myxopyronin in Staphylococcus aureus

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