• Biomedical Science Letters
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• v.10 no.4
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• pp.507-514
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• 2004

The Pseudomonas aeruginosa isolated from the clinical specimens has a mutation on the QRDR (quinolone resistance determining region). There were obvious mutations in both gyrA and parC gene which are major targets of quinolone. Simultaneous mutations were found two sites or more on these genes in all of ten strains. GyrB or parE gene had only silent mutation without converted amino acids. We confirmed that P. aeruginosa from clinical specimens exhibited decreased sensitivity to fluroquiolone due to changed Thr-83→lle and Asp-87→Asn types on gyrA and altered Ser-87→Leu type on parC. This is the first finding that a new Met-93→Thr type on parC as well as mutations on gyrB or parE genes differed from existing patterns. This study showed more mutations of gyrA rather than parC, suggesting that change of Type Ⅳ topoisomerase is more serious than that of type Ⅱ (DNA gyrase).

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1605-1612
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• 2016

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.3
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• pp.247-254
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• 2014

To investigate the acquisition of quinolone resistance, we examined mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase genes in ciprofloxacin (CIP)-resistant clinical isolates and in vitro mutants of Streptococcus parauberis. The CIP-resistant clinical isolates had one base change responsible for a Ser-79${\rightarrow}$Thr in the QRDR of parC. However, the CIP-resistant in vitro mutants had an altered QRDR of parC (Ser-79${\rightarrow}$Ile) that differed from that of the isolates. None of the CIP-resistant S. parauberis clinical isolates or in vitro mutants exhibited amino acid changes in gyrA or gyrB. However, even though involvement in the increased resistance was not clear, an Arg-449${\rightarrow}$Ser mutation outside of the QRDR of parE was detected in CIP-resistant mutant 2P1. These results suggest that the topoisomerase IV gene, parC (and possibly parE, as well), is the primary ciprofloxacin target in S. parauberis. Additionally we established a high-resolution melting (HRM) assay capable of detecting the dominant mutation in four type II topoisomerase genes conferring ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method of surveillance for genetic mutations conferring antibiotic resistance.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.185-190
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• 2014

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from raw bulk milk and to characterize the resistance determinants. In this study, the gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced from quinolone resistant E. coli isolates. Also, the presence of plasmid-mediated quinolone resistance (PMQR) and the expression of efflux pump genes based on quantitative real-time PCR (qRT-PCR) were investigated. Of the 487 coliform bacteria, 9 strains showed nalidixic acid resistance, and 6 of the 9 nalidixic acid resistant isolates were also ciprofloxacin resistant. These 9 strains had a single mutation at codon 83 (S83L) in gyrA, 2 of them had double mutations at codon 83 and 87 (S83L and D87N) in gyrA and 3 of the 9 isolates had single mutations at codon 80 (S80I) in parC. None of the 9 isolates harbored PMQR determinants. Compared with wild-type E. coli ATCC 25922, an over-expression of the acrB gene (2.15-5.74 fold), encoding the pump component of the AcrAB-TolC efflux pump was observed in 4 of 6 ciprofloxacin resistant isolates. This study identified the quinolone resistance mechanism of E. coli isolated from raw milk samples in Gyeonggi-do.

• Archives of Pharmacal Research
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• v.19 no.5
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• pp.353-358
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• 1996

E. coli 11 and E. coli 101, clinical isolates of Escherichia coli were resistant to various quinolones, especially MICs to norfloxacin of both strains were higher than 100 mg/ml. In the presence of carbonyl cyanide m-chlorophenylhydrazone, a proton gradient uncoupler, norfloxacin uptake in both strains was increased, suggesting that an efflux system play an important role in the norfloxacin resistance. Outer membrane proteins of the susceptible and resistant strains which could affect the route of norfloxacin entry into cells were different. When quinolone resistance determining region(QRDR) of gyrA was amplified using PCR and cut with Hinf I, QRDR in the susceptible strain yielded two fragments while QRDRs in E. coli 11 and E. coli 101 yielded only one uncut fragment. When DNA sequence of QRDR was analyzed, there were two mutations as Ser-83 and Asp-87 in both resistant strains. these residues were changed to Leu-83 and Asn-87, respectively. These results showed that the norfloxacin resistance of E. coli 11 and E. coli 101 was resulted from multiple changes-an altered DNA gyrase A subunit, a change in route of drug entry, and reduction in quinolone concentration inside cells due to an efflux system.

• Archives of Pharmacal Research
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• v.21 no.6
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• pp.671-676
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• 1998

Five hundred and seventy clinical strains of Pseudomonas aeruginosa were isolated from August 1993 to August 1994 in Korea and screened for their resistance to ciprofloxacin, norfloxacin, and ofloxacin. Among these, two P. aeruginosa strains (PA150 and PA300) were selected based on their strong resistance (MICs > 50mcg/ml) to all three quinolones. The susceptible strain as well as two resistant strains had proton gradient-dependent efflux system. Efflux system in PA300 showed different specificities to ofloxacin and ciprofloxacin while PA150 had less permeability for ofloxacin. Ofloxacin had a less inhibitory action on DNA synthesis in permeabilized cells of PA150 and PA300 than 1771M. When quinolone resistance determining region (QRDR) in gyrA was sequenced, PA300 had one missense mutation, Asn 116Tyr, which was newly reported in this work. The results showed that PA150 became ofloxacin resistant by reduced ofloxacin accumulation due to the existence of efflux system and low permeability, while resistance of PA300 was due to the efflux system and a mutation in QRDR of gyrA -the target site of quinolone.

• Korean Journal of Veterinary Service
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• v.42 no.3
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• pp.153-159
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• 2019

The objective of this study was to identify mutations in the quinolone resistance determining region (QRDR) of the gyrA, gyrB, parC and parE genes, and the presence of plasmid-mediated quinolone resistance (PMQR) genes: qnrA, qnrB, qnrS, aac(6')-lb-cr and qepA in 40 nalidixic acid- resistant ($NA^R$) Salmonella isolates isolated from poultry slaughterhouse. The MIC of NA and ciprofloxacin for 40 $NA^R$ Salmonella isolates was $128{\sim}512{\mu}g/mL$ and < $0.125{\sim}0.25{\mu}g/mL$, respectively. The Salmonella isolates were resistant to NA (100%), gentamicin (5.0%) and ampicillin (2.5%). All $NA^R$ Salmonella isolates represented point mutation in codons Aspartic acid(Asp)-87 (90%) and Serine(Ser)-83 (10%) of QRDR of gyrA gene: $Asp87{\rightarrow}glycine$, $Ser83{\rightarrow}tyrosine$. No mutations were observed in QRDR of the gyrB, parC and parE gene. Moreover PMQR genes was not found in any of the tested isolates. Our findings showed that DNA gyrase is the primary target of quinolone resistance and a single mutation in codon Asp87 and Ser83 of the gyrA gene can confer resistance to NA and reduced susceptibility ciprofloxacin in Salmonella isolates.

• Biomedical Science Letters
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• v.13 no.2
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• pp.141-148
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• 2007

We determined the sequences of the quinolone resistance-determining region (QRDR) of gyrA and gyrB for 21 clinical strains of Enterobacteriaceae resistant to ciprofloxacin, norfloxacin and levofloxacin. The clinical strains were isolated from the specimens of three general hospitals in Busan. In the present study, we found mutations in type II topoisomerase (DNA gyrase) genes for all strains. We confirmed that some genera of Enterobacteriaceae of clinical specimen exhibited decreased sensitivity to fluroquinolone due to changes in Ser-83$\rightarrow$Leu and Asp-87$\rightarrow$Asn types on gyrA and alterations in Glu-465$\rightarrow$Arg and Ser-492$\rightarrow$Asn type on gyrB. All the twenty-one strains had a missense mutation in gyrA (codon 83 and 87). Three of them had an additional mutation in gyrB (codon 465 or 492), but one of them had an additional mutation in gyrB (codon 426, 427, 491, 495 and 496). The strains which had two mutations in type II topoisomerase genes (gyrA and gyrB) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA (mean MICs of ciprofloxacin: $\geq8\mu$g/ml, mean MICs of levofloxacin: $\geq16\mu$g/ml). Interestingly, the examination of silent nucleotide changes n the gyrA and gyrB genes revealed six different patterns of DNA polymorphism, respectively. Fifteen strains of the twenty-one strains bearing the gyrase A mutation shared the same polymorphism and eleven strains of the twenty-one strains bearing the gyrase B mutation shared the same polymorphism.

• Biomedical Science Letters
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• v.26 no.2
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• pp.120-126
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• 2020

Fluoroquinolone (FQ) resistant uropathogenic Escherichia coli (UPEC) have become a major problem in urinary tract infections (UTIs). The purpose of this study was to compare the quinolone resistance-determining region (QRDR) and plasmid mediated quinolone resistance (PMQR) determinants of FQ resistant UPEC between 1989 and 2010-2014. A total of 681 strains of UPEC clinical isolates was collected from Korean healthcare facility in 1989 (123 strains) and in 2010-2014 (558 strains). The minimum inhibitory concentrations (MICs) of FQs were determined by agar dilution method. QRDRs (gyrA, gyrB, parC and parE) and PMQR determinants (qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA) were analyzed polymerase chain reaction and sequencing method. Among 681 isolates, FQ resistant UPEC were 3 strains (2.4%) in 1989 isolates and 220 strains (39.4%) in 2010-2014 isolates. The rate of the FQ resistant UPEC strains in 2010-2014 isolates was increased than that of in 1989 isolates. UPEC isolates from 1989 and 2010-2014 were shown to carry mutations in gyrA (Ser83 and Asp87), gyrB (Ser464 and Thr469), parC (Ser80 and Glu84) and parE (Glu460, Ser458, Ile464 and Leu445). The most common mutations of QRDRs in 1989 isolates were Ser83Leu and Asp87Gly in gyrA and Ser80Ile in parC (2 strains: 66.7%) while those in 2010-2014 isolates were Ser83Leu and Asp87Asn in gyrA and Ser80Il2 and Glu84Val in parC (88 strains: 40.0%). PMQR determinants were detected only in 2010-2014 UPEC strains (47 strains: 21.4%).

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1735-1743
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• 2010

The full-length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity), and Salmonella Typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinolone-resistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83$\rightarrow$Arg). However, an alteration in the QRDR of ParC (Ser84$\rightarrow$Ile) following an amino acid substitution in GyrA (Asp87$\rightarrow$Gly) was detected in E. tarda mutants selected in vitro at $8{\mu}g/ml$ ciprofloxacin (CIP). A mutant with a GyrB (Ser464$\rightarrow$Leu) and GyrA (Asp87$\rightarrow$Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.