Gatifloxacin Activity against Quinolone-Resistant Gyrase: Allele-Specific Enhancement of Bacteriostatic and Bactericidal Activities by the C-8-Methoxy Group

ABSTRACT Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant, gyrA (gyrase) mutants ofEscherichia coli. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in α-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several gyrA alleles located outside α-helix 4. These data are consistent with α-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a parC(topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.

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Enhancement of Fluoroquinolone Activity by C-8 Halogen and Methoxy Moieties: Action against a Gyrase Resistance Mutant of Mycobacterium smegmatis and a Gyrase-Topoisomerase IV Double Mutant of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2703-2709.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2703-2709 ◽

Cited By ~ 34

Author(s):

Tao Lu ◽

Xilin Zhao ◽

Xinying Li ◽

Alex Drlica-Wagner ◽

Jian-Ying Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mycobacterium Smegmatis ◽

Double Mutant ◽

Structure Refinement ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Topoisomerase Iv ◽

Methoxy Groups ◽

Bactericidal Activities ◽

Mutant Cells

ABSTRACT The increasing prevalence of antibiotic resistance among bacterial pathogens prompted a microbiological study of fluoroquinolone structure-activity relationships with resistant mutants. Bacteriostatic and bactericidal activities for 12 fluoroquinolones were examined with a gyrase mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus. For both organisms C-8 halogen and C-8 methoxy groups enhanced activity. The MIC at which 99% of the isolates tested were inhibited (MIC99) was reduced three- to fivefold for the M. smegmatis mutant and seven- to eightfold for theS. aureus mutant by C-8 bromine, chlorine, and methoxy groups. With both organisms a smaller reduction in the MIC99 (two- to threefold) was associated with a C-8 fluorine moiety. In most comparisons with M. smegmatis the response to a C-8 substituent was similar (within twofold) for wild-type and mutant cells. In contrast, mutant S. aureuswas affected more than the wild type by the addition of a C-8 substituent. C-8 halogen and methoxy groups also improved the ability to kill the two mutants and the respective wild-type cells when measured with various fluoroquinolone concentrations during an incubation period equivalent to four to five doubling times. Collectively these data help define a group of fluoroquinolones that can serve (i) as a base for structure refinement and (ii) as test compounds for slowing the development of fluoroquinolone resistance during infection of vertebrate hosts.

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Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.6.1649-1653.2001 ◽

2001 ◽

Vol 45 (6) ◽

pp. 1649-1653 ◽

Cited By ~ 55

Author(s):

Hideyuki Fukuda ◽

Ryuta Kishii ◽

Masaya Takei ◽

Masaki Hosaka

Keyword(s):

Antibacterial Activity ◽

Streptococcus Pneumoniae ◽

Type Strain ◽

Methoxy Group ◽

Wild Type Strain ◽

Dna Gyrase ◽

Wild Type ◽

Topoisomerase Iv ◽

Mutant Strains

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

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Contribution of the 8-Methoxy Group to the Activity of Gatifloxacin against Type II Topoisomerases of Streptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.77-81.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 77-81 ◽

Cited By ~ 27

Author(s):

Ryuta Kishii ◽

Masaya Takei ◽

Hideyuki Fukuda ◽

Katsuhiko Hayashi ◽

Masaki Hosaka

Keyword(s):

Antibacterial Activity ◽

Streptococcus Pneumoniae ◽

Methoxy Group ◽

Dna Gyrase ◽

Type Ii ◽

Wild Type ◽

Topoisomerase Iv ◽

Methoxy Groups ◽

Dual Inhibition ◽

Inhibitory Activities

ABSTRACT The inhibitory activities (50% inhibitory concentrations [IC50s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC50s of 10 compounds ranged from 4.28 to 582 μg/ml against DNA gyrase and from 1.90 to 35.2 μg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC50s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC50s for topoisomerase IV were 1.5 times lower. Moreover, the IC50 ratios (IC50 for DNA gyrase/IC50 for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.

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Killing of Staphylococcus aureus by C-8-Methoxy Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.4.956 ◽

1998 ◽

Vol 42 (4) ◽

pp. 956-958 ◽

Cited By ~ 64

Author(s):

Xilin Zhao ◽

Jian-Ying Wang ◽

Chen Xu ◽

Yuzhi Dong ◽

Jianfeng Zhou ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Protein Synthesis ◽

Methoxy Group ◽

Wild Type ◽

Topoisomerase Iv

ABSTRACT C-8-methoxy fluoroquinolones were more lethal than C-8-bromine, C-8-ethoxy, and C-8-H derivatives for Staphylococcus aureus, especially when topoisomerase IV was resistant. The methoxy group also increased lethality against wild-type cells when protein synthesis was inhibited. These properties encourage refinement of C-8-methoxy fluoroquinolones to kill staphylococci.

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Target Preference of 15 Quinolones against Staphylococcus aureus, Based on Antibacterial Activities and Target Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3544-3547.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3544-3547 ◽

Cited By ~ 77

Author(s):

Masaya Takei ◽

Hideyuki Fukuda ◽

Ryuta Kishii ◽

Masaki Hosaka

Keyword(s):

Staphylococcus Aureus ◽

Type Strain ◽

Wild Type Strain ◽

Dna Gyrase ◽

Antibacterial Activities ◽

Type I ◽

Type Ii ◽

Wild Type ◽

Topoisomerase Iv ◽

Mutant Strains

ABSTRACT The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC50s) of quinolones against topoisomerase IV to those against DNA gyrase (IC50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC50ratios was observed (r = 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC50 ratios were approximately 2.

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In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1228-1237.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1228-1237 ◽

Cited By ~ 56

Author(s):

Nagraj Mani ◽

Christian H. Gross ◽

Jonathan D. Parsons ◽

Brian Hanzelka ◽

Ute Müh ◽

...

Keyword(s):

Bacterial Resistance ◽

Wide Spectrum ◽

Dna Gyrase ◽

Antibacterial Activities ◽

Mechanisms Of Action ◽

Fluoroquinolone Resistance ◽

Topoisomerase Iv ◽

Low Frequencies

ABSTRACT Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

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Green Synthesis of Multifunctional Carbon Dots with Antibacterial Activities

Nanomaterials ◽

10.3390/nano11020369 ◽

2021 ◽

Vol 11 (2) ◽

pp. 369 ◽

Cited By ~ 1

Author(s):

Arumugam Saravanan ◽

Moorthy Maruthapandi ◽

Poushali Das ◽

John H. T. Luong ◽

Aharon Gedanken

Keyword(s):

Carbon Dots ◽

Fluorescence Spectra ◽

Antibacterial Effect ◽

Curcuma Longa ◽

Antibacterial Activities ◽

Blue Color ◽

Mean Size ◽

One Step ◽

Bactericidal Activities ◽

Bright Blue

Carbon dots (CDs) were obtained from medicinal turmeric leaves (Curcuma longa) by a facile one-step hydrothermal method and evaluated for their bactericidal activities against two gram-negative; Escherichia coli, Klebsiella pneumoniae, and two gram-positive counterparts; Staphylococcus aureus, S. epidermidis. The CDs exhibited spherical shapes with a mean size of 2.6 nm. The fluorescence spectra of CDs revealed intense fluorescence at λex/em = 362/429 nm with a bright blue color in an aqueous solution. The CDs showed strong photostability under various environmental conditions (pH, salt, and UV-radiation). The complete bactericidal potency of CDs was 0.25 mg/mL for E.coli and S. aureus after 8 h of exposure, while for K. pneumoniae, and S. epidermidis, the CDs at 0.5 mg/mL good antibacterial effect within 8 h and complete eradication after 24 h of exposure is observed. The release of reactive oxygen species played a crucial role in the death of the bacterial cell. The present study provides a strategy for the preparation of CDs from a medicinal plant and their potential antibacterial activities against four common contagious pathogens.

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High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-ResistantSalmonella enterica

BioMed Research International ◽

10.1155/2014/718084 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Soo Tein Ngoi ◽

Kwai Lin Thong

Keyword(s):

High Resolution ◽

Mutation Screening ◽

Rapid Screening ◽

Health Concern ◽

Wild Type ◽

High Resolution Melt ◽

Topoisomerase Iv ◽

Melting Analysis ◽

Type Strains ◽

Difference Plot

The increasedSalmonellaresistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62Salmonellastrains to identify mutations in the QRDR ofgyrA,gyrB,parC, andparEgenes. Mutations were detected in QRDR ofgyrA(n=52; S83F, S83Y, S83I, D87G, D87Y, and D87N) andparE(n=1; M438I).Salmonellastrains with mutations within QRDR ofgyrAare generally more resistant to nalidixic acid (MIC16>256 μg/mL). Mutations were uncommon within the QRDR ofgyrB,parC, andparEgenes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes inSalmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

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Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

International Journal of Molecular Sciences ◽

10.3390/ijms221910805 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10805

Author(s):

José L. Neira ◽

David Ortega-Alarcón ◽

Bruno Rizzuti ◽

Martina Palomino-Schätzlein ◽

Adrián Velázquez-Campoy ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Active Site ◽

Antibacterial Properties ◽

Helical Structure ◽

Titration Calorimetry ◽

Its Sequence ◽

Wild Type ◽

Phosphotransferase System ◽

Residual Structure ◽

Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

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A red herring in zebrafish genetics: allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants

10.1101/2021.08.06.455380 ◽

2021 ◽

Author(s):

Richard J White ◽

Eirinn Mackay ◽

Stephen W Wilson ◽

Elisabeth M Busch-Nentwich

Keyword(s):

Differentially Expressed Genes ◽

Differentially Expressed Gene ◽

Transcript Abundance ◽

Differentially Expressed ◽

Model Organisms ◽

Specific Gene ◽

Wild Type ◽

Specific Expression ◽

Genomic Location ◽

Allele Specific

In model organisms, RNA sequencing is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often overrepresented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that differentially expressed genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

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