Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.     

Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations

Abstract We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604 ), jointly with a gyrA mutation (denoted gyrA972 ) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.     

Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda

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 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→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.     

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.     

Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.     

Genetical conditioning of fluoroquinolone resistance mechanisms of clinical enterococcus faecalis strains. II. The mutations present in the qrdrs of gyrA, gyrB, parC and parE genes

The analyze selected fluoroquinolone resistance mechanisms of clinical E. faecalis strains was presented. In the second part of the study of genetic polymorphisms and mutations in the QRDRs of gyrA, gyrB, parC and parE genes were analyzed. The MSSCP technique and DNA sequencing were used. The activity (MICs) of ciprofloxacin, sparfloxacin and moxifloxacin were determined against 180 tested strains. The MSSCP method allows rapid screening of the genetic polymorphisms analyze of gyrA, gyrB, parC i parE genes. The amino acid substitutions of GyrA, GyrB and ParC were observed. The results indicate that mutations present among clinical E. faecalis strains associated with high level resistance to fluoroquinolons.     

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.     

The tip of the iceberg: quinolone-resistance conferred by mutations in gyrA gene in non-typhoidal Salmonella strains

Food-borne infections due to Salmonella spp. seldom require antimicrobial therapy, but this is compulsory in systemic salmonellosis. Salmonella resistance to a large panel of antibiotics has been described worldwide. Since the introduction of nalidixic acid in therapy, Salmonella spp. have steadily developed resistance, especially over the last three decades. The source of quinolone resistance is thought to be the selective pressure determined by the use of quinolones in both human and veterinary practices. Resistance acquisition of Salmonella strains is a stepwise process. Several mechanisms are described, which can lead to the development of quinolone resistance. The main mechanism is considered to be linked with mutations in the quinolone-resistance determining region (QRDR) of the target genes (gyrA and gyrB encoding DNA gyrase, and parC and parE encoding topoisomerase IV). This first step in mutational resistance usually determines a rise in the nalidixic acid minimal inhibitory concentration (MIC). The most common amino acid substitutions in the GyrA subunit, resulting in varied degrees of quinolone resistance, occur at codons Ser83 and Asp87. Higher levels of resistance may occur by further mutational steps, with amino acid changes in the same or a different target enzyme. Other mechanisms are as well involved, like increased efflux or plasmid-mediated resistance. Acknowledgement of the epidemiology and the onset mechanisms of quinolone resistance in Salmonella spp. is compulsory, and surveillance for resistant bacteria among human, animal and food sources remains critical.     

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.     

DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance.

Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously. DNA sequence analysis revealed close homology between the S. aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E. coli confers resistance to 4-quinolones. These results are discussed in regard to quinolone resistance mechanisms in S. aureus.     

Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target

The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.     

A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium

Abstract In order to study the role of gyrB in antibiotic resistance in post-ciprofloxacin therapy fluoroquinolone-resistant clinical isolates of Salmonella typhimurium , plasmid pBP548, which contains the Escherichia coli gyrB gene, was used in complementation studies. In a heterodiploid strain, the wild-type (quinolone sensitive) allele is dominant over the resistant allele therefore, eleven clinical isolates were complemented with gyrB encoded on pBP548. Only one transformant, L18pBP548, exhibited increased susceptibility to the quinolones nalidixic acid, ciprofloxacin and sparfloxacin. The amino acid sequence of the gyrase B protein from a wild-type and the pre-therapy S. typhimurium (deduced from the nucleotide sequence) was identical to that of E. coli from codons 436 to 470; however, a point mutation was identified in codon 463 of gyrB of the quinolone-resistant post-therapy isolate L18, giving rise to an amino acid substitution of serine to tyrosine.     

Implications of amino acid substitutions in GyrA at position 83 in terms of oxolinic acid resistance in field isolates of Burkholderia glumae, a causal agent of bacterial seedling rot and grain rot of rice

Oxolinic acid (OA), a quinolone, inhibits the activity of DNA gyrase composed of GyrA and GyrB and shows antibacterial activity against Burkholderia glumae. Since B. glumae causes bacterial seedling rot and grain rot of rice, both of which are devastating diseases, the emergence of OA-resistant bacteria has important implications on rice cultivation in Japan. Based on the MIC of OA, 35 B. glumae field isolates isolated from rice seedlings grown from OA-treated seeds in Japan were divided into sensitive isolates (OSs; 0.5 microg/ml), moderately resistant isolates (MRs; 50 microg/ml), and highly resistant isolates (HRs; > or =100 microg/ml). Recombination with gyrA of an OS, Pg-10, led MRs and HRs to become OA susceptible, suggesting that gyrA mutations are involved in the OA resistance of field isolates. The amino acid at position 83 in the GyrA of all OSs was Ser, but in all MRs and HRs it was Arg and Ile, respectively. Ser83Arg and Ser83Ile substitutions in the GyrA of an OS, Pg-10, resulted in moderate and high OA resistance, respectively. Moreover, Arg83Ser and Ile83Ser substitutions in the GyrA of MRs and HRs, respectively, resulted in susceptibility to OA. These results suggest that Ser83Arg and Ser83Ile substitutions in GyrA are commonly responsible for resistance to OA in B. glumae field isolates.     

Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.     

Characterization of the aac(6'')-Ik gene of Acinetobacter sp. 6

Abstract Chromosomal DNA of different species of mycobacteria, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium and Mycobacterium smegmatis , has been submitted to polymerase chain reaction using two oligonucleotide primers highly homologous to DNA sequences flanking the quinolone resistance-determining region in the gyrA gene of Escherichia coli and Staphylococcus aureus . For each of these mycobacterial species, a 150-bp DNA fragment hybridizing with an intragenic probe of the gyrA gene of E. coli K12 was obtained. The nucleotide sequences of the 108-bp fragments amplified from M. tuberculosis and M. avium were determined. The two sequences were 87% homologous. Except for one residue, their deduced amino acid sequences were identical and shared 67% homology with the quinolone resistance-determining region of the gyrase A subunits of E. coli and S. aureus . Sequencing of the 108-bp fragment amplified from an in vitro mutant of M. avium , highly resistant to fluoroquinolones, showed a point mutation leading to the substitution of Ala for Val at a position corresponding to residues involved in quinolone resistance in E. coli and S. aureus , i.e. Ser 83 for E. coli and Ser 84 for S. aureus .     

gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity

The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.     

Mutation at the "exit gate" of the salmonella gyrase a subunit suppresses a defect in the gyrase B subunit

In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.     

Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins

We have transferred the Escherichia coli gyrA and gyrB genes onto plasmids that allow the overproduction of the DNA gyrase A and B proteins and have designed relatively simple purification procedures for both proteins. The pure proteins are obtained in good yield; from 2 liters of culture (12 g of cells), one can recover 25 mg of GyrA or 3 mg of GyrB protein.