Novel Approach for Comparing the Abilities of Quinolones To Restrict the Emergence of Resistant Mutants during Quinolone Exposure

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The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays. O ne approach to help stem the emergence of new antimicrobial resistance is to kill bacterial pathogens rapidly, thereby quickly reducing bacterial burden and restricting effects of stressinduced mutagenesis (1, 2). Reactive oxygen species (ROS) have been proposed to be key factors in antimicrobial lethality (3-5), and substantial evidence supports this proposition (3-19). However, their role in lethality has been challenged (20, 21). If ROS are indeed integral to antimicrobial-mediated killing, compounds that act as antioxidants and radical scavengers should reduce antimicrobial lethality. We chose to examine this hypothesis using the radical scavenger dimethyl sulfoxide (DMSO) (22, 23), because it is also a popular solvent that is widely used in the pharmaceutical industry and in antimicrobial research due to its (i) low toxicity, (ii) ability to dissolve both organic and inorganic compounds, (iii) ability to remain in a liquid state over a broad temperature range (e.g., from 19°C to 189°C), (iv) ability to enhance cell membrane permeability, and (v) miscibility in water and a wide range of organic solvents. We report here that DMSO interferes with rapid killing of Escherichia coli and Acinetobacter baumannii by members of three antimicrobial classes.E. coli K-12 strains BW25113 and ATCC 25922 and A. baumannii strain ATCC 17978 were grown in Luria-Bertani (LB) broth or on LB agar at 37°C. LB medium, ampicillin, and kanamycin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Oxolinic acid, ciprofloxacin, and DMSO were acquired from Sigma-Aldrich Co. (St. Louis, MO). Meropenem (Sumitomo Dainippon Pharma Co. Ltd.) was obtained from Zhongshan Hospital Pharmacy. The fluorescent probe carboxy-H 2 DCFDA [5(6)-carboxy-2=,7=-dichlorodihydrofluorescein diacetate] was purchased from Invitrogen (Grand Island, NY). All chemical stock solutions were dissolved in sterile water (except carboxy-H 2 DCFDA, which was dissolved in DMSO) and stored at Ϫ80°C until use. MICs were assayed by broth dilution according to CLSI protocols (24); exponentially growing cultures were diluted to 10 5 CFU/ml for MIC determinations. To measure rapid bacterial killing, exponentially growing cultures at about 5 ϫ 10 8 CFU/ml were treated with antimicrobials, after which they were serially diluted and plated on drug-free agar. Viable colony counts were determined after an overnight incubation at 37°C; percentage survival rates were calculat...

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confidence: 80%

Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing

Wang

Xue

et al. 2016

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“…The concept of dual targeting as an advantage for topoisomerase inhibitors has been recognized from the fluoroquinolone precedent (33)(34)(35). In Gram-positive pathogens, fluoroquinolones in general display greater potency toward TopoIV than gyrase, and as a result of this asymmetric target profile, earlier generations of quinolones readily selected for first-step ParC mutants.…”

Section: Discussionmentioning

confidence: 99%

Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

Nayar

Dougherty

Reck

et al. 2015

), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4-to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16-and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

“…These MIC-based measurements reflect only bacteriostatic activity. Methods are also available to compare agents by two types of rapid killing, one that requires ongoing protein synthesis and one that does not (10); these lethal properties are likely to be important for limiting the induction of resistant mutants (27). Thus, MIC measured with wild-type cells, which is universally used for antimicrobial discovery, is but one of several ways to evaluate compounds early in drug development.…”

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confidence: 99%

Fluoroquinolone and Quinazolinedione Activities against Wild-Type and Gyrase Mutant Strains of Mycobacterium smegmatis

Malik¹,

Marks

Mustaev

et al. 2011

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Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the MIC for wild-type cells, were lower. Addition of a 3-amino group to the 2,4-dione core increased relative activity against mutants, while alteration of the 8-methoxy group to a methyl or of the 2,4-dione core to a 1,3-dione core lowered activity against mutants. A GyrA G89C bacterial variant was strikingly susceptible to most of the diones tested; in contrast, low susceptibility to fluoroquinolones was observed. Many of the bacteriostatic differences between diones and fluoroquinolones were explained by interactions at the N terminus of GyrA helix IV revealed by recently published X-ray structures of drug-topoisomerase-DNA complexes. When lethal activity was normalized to the MIC in order to minimize the effects of drug uptake, efflux, and ternary complex formation, a 3-amino-2,4-dione exhibited killing activity comparable to that of a cognate fluoroquinolone. Surprisingly, the lethal activity of the dione was inhibited less by chloramphenicol than that of the cognate fluoroquinolone. This observation adds the 2,4-dione structural motif to the list of structural features known to impart lethality to fluoroquinolone-like compounds in the absence of protein synthesis, a phenomenon that is not explained by X-ray structures of drug-enzyme-DNA complexes.Fluoroquinolones are lethal antibacterial agents that are widely used to control many bacterial infections (23); for some diseases, such as multidrug-resistant tuberculosis, fluoroquinolones are key to successful treatment (4). As with other antimicrobials, fluoroquinolone use is threatened by an increasing prevalence of resistance (1). One way to address the resistance problem is to identify new derivatives that are particularly active with resistant mutants. For example, with some bacterial species, addition of an 8-methoxy substituent increases fluoroquinolone activity against mutants (8,45,46). Other examples include quinazolinediones, which have shown good activity with gyrase resistance mutants selected by fluoroquinolone treatment of Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae (11,16,34). We found with E. coli that a quinazoline-2,4-dione was almost as active with gyrase mutants as with wild-type cells (12). How quinazolinediones behave with mycobacteria is unknown.To compare quinolone-like compounds with regard to the likely effects of fluoroquinolone resistance mutations, sets of gyrase mutants are prepared in which the strains are otherwise isogenic. The MIC for the mutant is measured and related to the MIC for the wild type to correct for differences in uptake and efflux. The ratio of the MIC for the mutant to the MIC for the wild type de...