OBJECTIVETo differentiate closely related pathogenic bacteria via phylogenetic method on the basis of gyrB gene sequences.

METHODSGyrB sequences of 19 strains of E.coli, 13 Shigella spp. 2 Aeromonas caviae, 2 Aeromonas hydrophilia,1 Aeromonas veronii were determined and combined with sequences retrieved from public databases to construct phylogenetic trees. For each sequence tested, the identification deduced from the clustering relation of sequences was compared with the phenetic identification.

RESULTSAll the tested sequences, except those of Shigella boydii and Shigella dysenteriae, were corresponded with the 5 closest sequences on the tree at the species level. While the BLAST queries returned some other bacteria organisms or undetermined entries.

CONCLUSIONPhylogenetics displays good discriminative power in bacterial sequences differentiation.

Aeromonas ; classification ; genetics ; Bacteria ; classification ; genetics ; Bacterial Typing Techniques ; DNA Gyrase ; genetics ; DNA, Bacterial ; genetics ; Escherichia coli ; classification ; genetics ; Phylogeny ; Sequence Analysis, DNA ; Shigella ; classification ; genetics

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.
Anti-Bacterial Agents/*pharmacology ; DNA Gyrase/*genetics ; DNA Topoisomerases, Type II/*genetics ; Haemophilus influenzae/*drug effects/pathogenicity ; Korea ; Microbial Sensitivity Tests ; Mutation ; Ofloxacin/*pharmacology

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.
Anti-Bacterial Agents/*pharmacology ; DNA Gyrase/*genetics ; DNA Topoisomerases, Type II/*genetics ; Haemophilus influenzae/*drug effects/pathogenicity ; Korea ; Microbial Sensitivity Tests ; Mutation ; Ofloxacin/*pharmacology

OBJECTIVESTo investigate the correlation between mutation of GyrA and ParC genes and quinolone resistance in Neisseria Gonorrhoeae.

METHODSThe gene fragments of quinolone resistance-determining region (QRDR) in GyrA and ParC genes in 20 Neisseria gonorrhoeae strains clinically isolated in Wuxi, China, were sequenced, and the susceptibility of the 20 strains to quinolone was examined by agar diffusion method.

RESULTSThe mutations at the Asp95 point in GyrA gene were found in 20 strains. Of the 19 stains examined, 16 had mutations at the 86, 87, 88, 91 points in ParC genes.

CONCLUSIONSThe mutations of Asp95 in GyrA gene and Asp86, Ser87, Ser88, Glu91 in ParC gene contribute to quinolone resistance in Neisseria Gonorrhoeae.

Base Sequence ; DNA Gyrase ; genetics ; DNA Topoisomerase IV ; genetics ; Drug Resistance, Bacterial ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Neisseria gonorrhoeae ; drug effects ; genetics ; isolation & purification ; Quinolones ; pharmacology

OBJECTIVETo study the phenotypic and genotypic resistance to Fluoroquinolones in Neisseria gonorrhoeae (NG) isolated in Jiangsu province of China.

METHODSIn-vitro, susceptibility testing of ciprofloxacin and levofloxacin against ninety-five clinical isolates were determined by agar dilution method. Detection of mutation in the gyrA and parC genes was performed by polymerase chain reaction (PCR) assay and sequence analysis.

RESULTSThe clinical isolates demonstrated 100% resistance to ciprofloxacin. Based on gyrA and parC mutations, 18 types could be categorized among the 54 isolates. Based on the same gyrA mutations,isolates with high MIC appeared to have had more mutations in parC gene.

CONCLUSIONThe status of resistance to ciprofloxacin in NG was quite serious, and ciprofloxacin treatment for the treatment of NG infections in Jiangsu province should not be recommended. The results from this study suggested that mutations in the parC gene had contributed to the development of high Fluoroquinolone resistance in NG.

China ; DNA Gyrase ; genetics ; DNA Topoisomerase IV ; genetics ; Drug Resistance, Bacterial ; Fluoroquinolones ; pharmacology ; Genotype ; Gonorrhea ; drug therapy ; Humans ; Neisseria gonorrhoeae ; drug effects ; genetics ; isolation & purification ; Phenotype

Escherichia coli (E. coli) is a clinically important causative organism that can lead to urinary tract infections. Quinolone antibiotics are among the first-line treatments for urinary tract infections. However, the frequency of resistance to quinolone in E. coli has been increasing. Therefore, new antimicrobial agents that can be used for treatment in lieu of quinolone antibiotics are needed. In this study, thirty-six compounds with higher scores in a virtual screening based on the three-dimensional structure of E. coli DNA gyrase were selected for in vitro antimicrobial activity testing. An in vitro test confirmed the antimicrobial activity of 4-[(1-methyl-6-nitroquinolin-1-ium-4-yl)amino]-N-[4-[(1-methylpyridin-1-ium-4-yl)amino]phenyl]benzamide (ZINC18057104) against E. coli among the 36 compounds. The minimum inhibitory concentration (MIC) of ZINC18057104 against E. coli ATCC® 25922™ was 2 μg/ml, and the MIC₅₀ and MIC₉₀ for the 72 quinolone-resistant E. coli clinical isolates were 4 and 64 μg/ml, respectively. ZINC18057104, which has a quinoline structure which is similar to the quinolone antibiotics, is predicted to exhibit antimicrobial activity in quinolone-resistant E. coli because it has different molecular interactions with the DNA gyrase than that of existing quinolone antibiotics.
Anti-Bacterial Agents ; Anti-Infective Agents ; DNA Gyrase* ; DNA* ; Drug Discovery ; Escherichia coli ; In Vitro Techniques ; Mass Screening ; Microbial Sensitivity Tests ; Urinary Tract Infections

Isolates of 24 enterococci, 5 Enterococcus casseliflavus and 19 Enterococcus gallinarum, possessing vanC genes and showing low-level resistance to vancomycin were obtained from mice from commercial mouse breeding companies. Since some of these isolates showed resistance to other antibiotics, the purpose of this study was to clarify the resistant profiles of these isolates. One E. casseliflavus isolate showed resistance to erythromycin with a minimal inhibitory concentration (MIC) of 8 μg/mL and also showed apparent resistance to fluoroquinolones with an MIC of 32 μg/mL for ciprofloxacin. The MICs of 2 other fluoroquinolone-resistant E. casseliflavus and E. gallinarum isolates were 3 and 6 μg/mL, respectively. These 3 resistant isolates showed an absence of macrolide- and fluoroquinolone-resistant genes, including amino acid substitutions in the quinolone resistance determining regions of DNA gyrase and topoisomerase IV. Resistance to tetracycline was detected in 2 E. gallinarum isolates that were highly resistant, exhibiting MICs of 48 and 64 μg/mL and possessing tet(O) genes. The results indicate that antibiotic-resistant enterococci are being maintained in some laboratory mouse strains that have never been treated with an antibiotic.
Amino Acid Substitution ; Animals ; Anti-Bacterial Agents ; Breeding ; Ciprofloxacin ; DNA Gyrase ; DNA Topoisomerase IV ; Drug Resistance, Microbial ; Enterococcus ; Erythromycin ; Fluoroquinolones ; Mice ; Tetracycline ; Vancomycin

OBJECTIVETo induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.

METHODSMTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.

RESULTSMIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.

CONCLUSIONMTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.

Antitubercular Agents ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; genetics ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Ofloxacin ; pharmacology

Fluoroquinolone (FQ) antibiotics are an important class of synthetic antibacterial agents. These are the most extensively used drugs for treating bacterial infections in the field of both human and veterinary medicine. Herein, the antibacterial and pharmacological properties of four fluoroquinolones: lomefloxacin, norfloxacin, ciprofloxacin, and ofloxacin have been studied. The objective of this study was to analyze the antibacterial characteristics of the different fluoroquinolones. Also, the pharmacological properties of the compounds including the Lipinski rule of five, absorption, distribution, metabolism, and excretion, LD50, drug likeliness, and toxicity were evaluated. We found that among all four FQ molecules, ofloxacin showed the highest antibacterial activity through in silico assays with a strong interaction (−38.52 kJ/mol) with the antibacterial target protein (topoisomerase-II DNA gyrase enzyme). The pharmacological and pharmacokinetic analysis also showed that the compounds ciprofloxacin, ofloxacin, lomefloxacin and norfloxacin have good pharmacological properties. Notably, ofloxacin was found to possess an IGC50 (concentration needed to inhibit 50% growth) value of 0.286 μg/L against the Tetrahymena pyriformis protozoa. It also tested negative for the Ames toxicity test, showing its non-carcinogenic character.
Absorption ; Anti-Bacterial Agents ; Bacterial Infections ; Ciprofloxacin ; Computer Simulation ; DNA Gyrase ; Fluoroquinolones* ; Humans ; Lethal Dose 50 ; Metabolism ; Norfloxacin ; Ofloxacin ; Tetrahymena pyriformis ; Toxicity Tests ; Veterinary Medicine

BACKGROUNDThere is a growing interest in studying the relationship between intrinsic resistance and biofilms resistance to drugs. However, the relationship still remains unclear in the macroscopic bacterial growth. Our study is to illuminate the change of bacterial drug resistance of gyrA mutant and active efflux pump during the development of Pseudomonas aeruginosa (P. aeruginosa) biofilms.

METHODSThe strains of type II topoisomerase gene mutant (gyrA mutant) and multidrug resistance (MDR) efflux pump were clinical isolates and detected by polymerase chain reaction (PCR). The process of bacterial biofilms development was observed by scanning electron microscope. Triparental mating experiments were performed to transfer report gene of green fluorescent protein (GFP) into P. aeruginosa biofilms strains and followed by analysis of bacterial survival rate between intrinsic resistance and biofilms resistance.

RESULTSThe fluorescent strains with pGFPuv could develop mature biofilms on Teflon surface. Before a period of 72 hours, the survival rate of biofilms bacteria and intrinsic resistance strains in ciprofloxacin solution was significantly different (P < 0.05). The survival number of intrinsic resistance strains (gyrA mutation and active efflux pump) was illustriously higher than biofilm strain in the initial stage of biofilms development. After 72 hours incubation, there was no clearly difference between mutants and biofilms strains in the survival rate (P > 0.05). The carbonyl cyanide m-chlorophenylhydrazone and azithromycin could significantly reduce the drug resistance of biofilm strains and efflux pump strains.

CONCLUSIONSIn the development of P. aeruginosa biofilms, the strains of gyrA mutation and MDR efflux could be conferred with new level of drug resistance. When co-cultured mutated strains with biofilm strains, biofilms may play a major role in bacterial resistance. But after 72 hours incubation (a mature biofilms had been developed), there was no clearly difference between the number of mutant strains and biofilm strains.

Biofilms ; drug effects ; Carbonyl Cyanide m-Chlorophenyl Hydrazone ; pharmacology ; Ciprofloxacin ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; Mutation ; Pseudomonas aeruginosa ; drug effects ; genetics