Characterization of Campylobacter jejuni DNA gyrase as the target of quinolones

Abstract

Quinolones have long been used as the first-line treatment for Campylobacter infections. However, an increased resistance to quinolones has raised public health concerns. The development of new quinolone-based antibiotics with high activity is critical for effective, as DNA gyrase, the target of quinolones, is an essential enzyme for bacterial growth in several mechanisms. The evaluation of antibiotic activity against Campylobacter jejuni largely relies on drug susceptibility tests, which require at least 2 days to produce results. Thus, an in vitro method for studying the activity of quinolones against the C. jejuni DNA gyrase is preferred. To identify potent quinolones, we investigated the interaction of C. jejuni DNA gyrase with a number of quinolones using recombinant subunits. The combination of purified subunits exhibited DNA supercoiling activity in an ATP dependent manner. Drug concentrations that inhibit DNA supercoiling by 50% (IC50s) of 10 different quinolones were estimated to range from 0.4 (sitafloxacin) to >100 μg/mL (nalidixic acid). Sitafloxacin showed the highest inhibitory activity, and the analysis of the quinolone structure-activity relationship demonstrated that a fluorine atom at R-6 might play the important role in the inhibitory activity against C. jejuni gyrase. Measured quinolone IC50s correlated well with minimum inhibitory concentrations (R = 0.9943). These suggest that the in vitro supercoiling inhibition assay on purified recombinant C. jejuni DNA gyrase is a useful and predictive technique to monitor the antibacterial potency of quinolones. And furthermore, these data suggested that sitafloxacin might be a good candidate for clinical trials on campylobacteriosis.

Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni

“…Recombinant DNA gyrase subunits were expressed and purified as previously described . Briefly, expression vectors carrying the gyr A and gyr B genes of C. jejuni were transformed in E. coli BL 21(DE3)/pLysS.…”

“…Recombinant DNA gyrase subunits were expressed and purified as previously described. [16][17][18] Briefly, expression vectors carrying the gyrA and gyrB genes of C. jejuni were transformed in E. coli BL 21 (DE3)/pLysS. Transformants were grown to the log phase in Luria- Bertani (LB) medium with 100 μg/mL ampicillin, and the expression of GyrA and GyrB was induced by the addition of 1 mM of isopropyl β-D-thiogalactopyranoside (Wako Pure Chemicals Industries, Ltd.) and incubation at 18°C for 16 h. Harvested E. coli were sonicated on ice at 30% duty cycle, with 10 cycles of 40 s on and 40 s off (Sonifier 250; Branson, Danbury, CT, USA).…”

Fast quantification of fluoroquinolones in environmental water samples using molecularly imprinted polymers coupled with internal extractive electrospray ionization mass spectrometry

“…Fluoroquinolones (FQs) have been widely used in the treatment of bacterial infections. [1][2][3][4] However, some of the FQs cannot be absorbed completely, and are excreted through urine and excrement. 5 Thus, FQs have been found in soil, environmental water and even in drinking water.…”

Analysis of flumequine enantiomers in rat plasma by UFLC‐ESI‐MS/MS

“…Flumequine (Flu) is one of the quinolone broad‐spectrum antibacterial veterinary drugs. It inhibits topoisomerases needed for the transcription and replication of bacterial DNA . Quinolones and fluoroquinolones are the main antibiotic families used both in human and veterinary medicine.…”

“…It inhibits topoisomerases needed for the transcription and replication of bacterial DNA. 1,2 Quinolones and fluoroquinolones are the main antibiotic families used both in human and veterinary medicine. The presence of these drugs in food possibly results in bioaccumulation through the trophic chain.…”