Mutations of DNA gyrase and topoisomerase IV in clinical isolates of fluoroquinolone-resistant Proteus mirabilis

The presence of fluoroquinolone resistance-associated mutations within the quinolone resistance-determining region of DNA gyrase and topoisomerase IV was investigated genetically in clinical isolates of Proteus mirabilis recovered from patients with urinay tract infections. Two isolates of fluoroquinolone-resistant P. mirabilis possessed the mutations in GyrA (Ser-83 --> Arg or Ile), GyrB (Ser-464 --> Tyr or Phe) and ParC (Ser-80 --> Ile). A novel mutation with Glu-87 --> Lys in GyrA, where suggested to be responsible for fluoroquinolone resistance, was identified. These results demonstrate that the presence of an additional mutation at Glu-87 in GyrA may contribute to high-level fluoroquinolone resistance, too.     

Alterations in the GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subunits of topoisomerase IV in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa

The presence of fluoroquinolone resistance-associated alterations in topoisomerase II and IV were investigated for 103 nfxC-like type Pseudomonas aeruginosa isolates. The most nfxC-like type isolates (98.1%) possessed the substitution of Ile for Thr-83 in GyrA. A single alteration in GyrA (Thr-83-->Ile) was the most frequently detected and the next common alteration was two alterations with Thr-83-->Ile in GyrA and Ser-87-->Leu in ParC. A novel alteration at position Glin-106 of GyrA, which was suggested to be responsible for fluoroquinolone resistance, was identified. Our study revealed that the alterations in GyrB (Glu-468-->Asp) and in ParE (Asp-419-->Asn or Glu-459-->Asp) play a complementary role in the acquisition of resistance to fluoroquinolone. There was a correlation between the ciprofloxacin MIC and the number of resistance-associated alterations in GyrA, GyrB, ParC and ParE of P. aeruginosa isolates.     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.     

人型支原体耐药性监测及耐药机制的研究

目的监测我院2008～2011年人型支原体(Mh)对喹诺酮类药物的耐药性,并从作用靶位氨基酸残基变异这一角度研究其耐药机制。方法收集我院经液体药敏一体法培养的单独Mh感染标本,分别对编码MhⅡ型拓扑异构酶GyrA、GyrB、ParC、ParE亚基的相应基因进行PCR-测序分析,将DNA序列与GenBank中Mh模式菌株ATCC23114翻译后氨基酸序列比对分析(blastn)。按照药敏表型与氨基酸残基变异间的规律对Mh进行分组,并计算各组热点氨基酸残基变异的变异率,用四格表确切概率法进行统计学分析。结果对司帕沙星(SPA)耐药组(R)28株Mh均检出GyrA丝氨酸残基(Ser)83→亮氨酸残基(Leu),变异率为100.0%,高于非耐药组(S+I),差异有统计学意义(P<0.05)。对氧氟沙星(OFX)和左氧氟沙星(LEV)非敏感组(R+I)的40株Mh有36株检测出ParC134位赖氨酸残基(Lys)→精氨酸残基(Arg),变异率为90.0%,高于敏感组(S),差异有统计学意义(P<0.05);而对OFX和LEV R+I组有16株检出ParE 426位天冬氨酸残基(Lys)→天冬酰胺残基(Arg),变异率为40.0%,高于敏感组(S),但差异无统计学意义(P>0.05)。结论 Mh GyrA亚基Ser83→Leu是其对SPA耐药的原因之一;ParC亚基Lys134→Arg是其对OFX和LEV耐药的原因之一。     

Structure-function studies of peptides inhibiting the ribonucleotide reductase activity of herpes simplex virus type I.

Ac-Tyr298-Ala299-Gly300-Thr301-Val302-I le303-Asn304-Asp305-Leu306-OH (Ac-VZV R2-(298-306)) represents the acetylated form of the C-terminus of varicella-zoster virus (VZV) ribonucleotide reductase subunit 2 (R2). This peptide possesses a high degree of homology with the C-terminus nonapeptide of the herpes simplex virus (HSV) type I and II ribonucleotide reductase R2 protein and is 15 times more potent than the latter in its in vitro inhibition of HSV-1 reductase activity. Accordingly, a new series of analogues based on this structure was studied in vitro. The replacement of Asp305 by Asn, Glu, Gln, Ser, or Cys; of Asn304 by Gln or Ser; of Ile303 and Val302 by D-Val; and of Tyr298 by Cha induced an important loss of inhibitory potency. The substitution of Asn304 by Asp; of Thr301 by Cys, Ser, or Val; of Gly300 by Ala or Val; of Ala299 by Val; or of Tyr298 by homoPhe, 4'-fluoro-Phe, 4'-chloro-Phe, 3'-iodo-Tyr, Me-Tyr, or For-Trp led to a moderate decrease of the Ac-VZV R2-(298-306) potency. The replacement of Val302 by Ile; Ala299 by Cys, Ser, or Thr; or the insertion of a six- or eight-carbon chain between Tyr298 and the NH2 terminus either preserved or slightly increased the inhibitory potency of Ac-VZV R2-(298-306). Finally, the substitution of Tyr298 by Trp or the addition of 4'-nitro-Phe at the amino terminus resulted in a 3-fold increase of potency. Altogether, these results stress the importance of the structural integrity of the minimum active core 302-306 in preserving the inhibitory potency and suggest that further studies on monosubstitutions could be directed at the portion 298-301 of the peptide.     

Effectiveness of fluoroquinolones against gram-positive bacteria

Fluoroquinolones are broad-spectrum and therapeutically effective antibacterial agents that have retained high activity against methicillin-susceptible Staphylococcus aureus (MSSA) and streptococci. Primary targets of these agents in Gram-positive bacteria are DNA topoisomerase and DNA gyrase. High-level resistance is associated with at least two mutations in either target, or combinations of at least two mutations in multiple targets, particularly affecting the ParC subunit of topoisomerase IV and the GyrA subunit of DNA gyrase. This resistance may be enhanced when combined with mutations that increase fluoroquinolone efflux. Data from fluoroquinolone-resistant clinical isolates suggest that as yet unidentified mutations may also be involved in clinical resistance.     

Discovery and development of ATPase inhibitors of DNA gyrase as antibacterial agents

DNA gyrase is an attractive and well established target for the development of antibacterial agents. This bacterial enzyme, whose biological function is to control the topological state of DNA molecules, consists of two catalytic subunits; GyrA is responsible for DNA breakage and reunion, while the subunit GyrB contains the ATP-binding site. Coumarins and cyclothialidines are natural products that inhibit the ATPase activity of DNA gyrase by blocking the binding of ATP to subunit GyrB. The mechanism of action of these compounds was exhaustively characterized by biochemical methods and supported by protein crystallography. The abundance of crystallographic data on the N-terminal domain of GyrB in its complexes with various ligands has enabled the structure-based design of novel efficient chemotypes as inhibitors of the ATPase domain. This review summarizes the discovery of ATPase inhibitors of DNA gyrase B in the last decade and their development as potential antibacterial agents.     

Mutations in GyrA,ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa

The target enzymes GyrA and ParC and two efflux pump regulatory genes mexR and nfxB were analysed to determine changes associated with fluoroquinolone resistance in Pseudomonas aeruginosa. Both low- and high-level ciprofloxacin resistance was associated with a Thr-83Ile substitution in GyrA. A ParC Ser-80Leu substitution was found in highly resistant isolates in tandem with the Thr-83Ile substitution in GyrA. Mutations in the efflux regulatory genes were associated with resistance only when in tandem with a mutation in GyrA or ParC. These data show that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in GyrA, and that mutations in ParC and the efflux regulatory genes are secondary.     

Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population

Cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme of potential importance for the metabolism of estrogen and for metabolic activation of environmental carcinogens. We investigated an Ethiopian population for functional polymorphisms in the CYP1B1 gene using genomic DNA sequencing and detected three novel single nucleotide polymorphisms (SNPs). One of these (4360C-->G in exon 3) is present at a frequency of 7% and causes an Ala443Gly amino acid substitution. In addition, the four described previously missense mutations Arg48Gly, Ala119Ser, Leu432Val, and Asn453Ser were found with frequencies of 51, 50, 53, and 2%, respectively, yielding a total of 32 possible CYP1B1 haplotypes. Allele-specific PCR methods for haplotype analysis were developed and seven different CYP1B1 alleles were found: CYP1B1*1, *2, *3, *4, *5, *6, and *7 with frequencies of 8, 37, 39, 2, 0.7, 6, and 7%, respectively. The functional properties of different forms of CYP1B1, as well as of the Leu432Val + Asn453Ser and Leu432Val + Ala443Gly variants, were evaluated after heterologous expression of the corresponding cDNAs in Saccaromyces cerevisiae. The results revealed that CYP1B1.6 and CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, and Leu432Val in common, exhibited altered kinetics with significantly increased apparent K(m) and lowered V(max) values for both the 2- and 4-hydroxylation of 17 beta-estradiol, whereas the other constructs were indistinguishable from the CYP1B1.1 enzyme. The results emphasize the necessity of a complete haplotype analysis of enzyme variants for evaluation of functional consequences in vivo and for analyses of genetic polymorphisms in relation to, for example, cancer incidence.     

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

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance‐determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild‐type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni‐NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP‐dependent DNA supercoiling activity. A quinolone‐inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone‐resistant C. jejuni. An analysis of the structure‐activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R‐1 and a chloride substituent at R‐8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin‐resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP‐resistant C. jejuni. Copyright © 2016 John Wiley & Sons, Ltd.     

突变选择窗内筛选的大肠埃希菌耐药突变体靶位变异位点的研究

目的研究不同药物浓度、不同化学结构的氟喹诺酮类药物对突变选择窗（MSW）内筛选的大肠埃希菌耐药突变体的靶位基因的影响。方法应用5种氟喹诺酮类药物在突变选择窗内接种约1×10^10菌量的ATCC25922筛选耐药突变体；用琼脂平板二倍稀释法测定ATCC25922和耐药突变体的MIC；用PCR及DNA测序方法确定ATCC25922和耐药突变体耐药决定簇（QRDRs）的gyrA、parC的突变位点和相应的氨基酸变化。结果在ATCC25922的MSW中，筛选53株耐药突变体，所有突变体均为gyrA位点突变，无parC位点突变。其中有79％（42株）为Set-83→Leu位点突变，19％（10株）为Asp-87→Asn，2％（1株）为Gly81→cys位点突变；83位和87位为大肠埃希菌的常见突变位点。氟喹诺酮对Set-83→h突变体的MIC。较Gly81→Cys突变体的MIC90高2～8倍，较Asp-87→Asn突变体的MIC90高1～2倍；Ser-83→Leu是所有的突变位点中对耐药影响最重要的位点。结论氟喹诺酮对大肠埃希菌的主要靶位是GyrA；83位和87位突变为大肠埃希菌最常见突变位点。     

Isolation and characterization of insulin from the Brockmann body of Dissostichus mawsoni,an Antarctic teleost fish

Abstract: The Brockmann body of fish synthesizes and secretes insulin. The Brockmann body of Antarctic fish has been described anatomically and shown to contain insulin immunoreactive sites, however, the primary structure of an Antarctic fish insulin has yet to be reported. Insulin was isolated from the Brockmann bodies of the Antarctic perciform teleost, Dissostichus mawsoni. The peptide was purified to homogeneity by gel filtration and reversed‐phase HPLC. Insulin‐containing fractions were identified by radioimmunoassay using antisera raised against porcine insulin. Electrospray ionization‐mass spectrometry determined the mass of the isolated product to be 5725.27 a.m.u. The amino acid composition and primary structure were determined for the pyridylethylated A‐ and B‐ chains. The amino acid sequences of the A chain and B chain were H‐Gly‐Ile‐Val‐Glu‐Gln‐Cys‐Cys‐His‐Gln‐Pro¹⁰‐Cys‐Asn‐Ile‐Phe‐Asp‐Leu‐Gln‐Asn‐Tyr‐Cys²⁰‐Asn‐OH and H‐Ala‐Pro‐Gly‐Pro‐Gln‐His‐Leu‐Cys‐Gly‐Ser¹⁰‐His‐Leu‐Val‐Asp‐Ala‐Leu‐Tyr‐Leu‐Val‐Cys²⁰‐Gly‐Glu‐Arg‐Gly‐Phe‐Phe‐Tyr‐Asn‐Pro‐Lys³⁰‐OH, respectively. The primary structure of insulin from Antarctic fish is compared with known structures of insulin from other vertebrates.     

Mutation of the androgen receptor at amino acid 708 (Gly-->Ala) abolishes partial agonist activity of steroidal antiandrogens

Mutation of a single amino acid in the ligand-binding domain (LBD) of the human androgen receptor (hAR) can induce functional abnormalities in androgen binding, stabilization of active conformation, or interaction with coactivators. The Gly708Ala and Gly708Val substitutions are associated with partial and complete androgen insensitivity syndromes, respectively. In this work, we introduced Ala, Val, and aromatic Phe mutations at position 708 on helix H3 of the hAR-LBD and tested the functional and structural consequences on hAR activity in the presence of steroidal or nonsteroidal agonists and antagonists. The residues involved in the specific recognition of these androgen ligands were identified and analyzed in the light of in vitro biological experiments and the 3D hAR-LBD structure. Our study demonstrated that the Gly708Ala mutation influenced the agonist versus antagonist activity of the ligands and confirmed the crucial role of this residue within the ligand-binding pocket (LBP) in the modulation of androgen agonists. The Gly708Ala mutation transformed the antiandrogen cyproterone acetate (CPA), a partial agonist, into a pure antiandrogen, and the pure nonsteroidal antiandrogen hydroxyflutamide in a partial agonist. From the docking studies, we suggest that CPA acts on AR through the novel mechanism called "passive antagonism".     

Specificity of 17beta-oestradiol and benzo[a]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms. 4. In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.     

Amino acid substitutions in GyrA affect quinolone susceptibility in Salmonella typhimurium

The prevalence of quinolone‐resistant Salmonella has become a public health concern. Amino acid substitutions have generally been found within the quinolone resistance‐determining region in subunit A of DNA gyrase (GyrA) of Salmonella Typhimurium. However, direct evidence of the contribution of these substitutions to quinolone resistance remains to be shown. To investigate the significance of amino acid substitutions in S. Typhimurium GyrA to quinolone resistance, we expressed recombinant wild‐type (WT) and five mutant DNA gyrases in Escherichia coli and characterized them in vitro. WT and mutant DNA gyrases were reconstituted in vitro by mixing recombinant subunits A and B of DNA gyrase. The correlation between the amino acid substitutions and resistance to quinolones ciprofloxacin, levofloxacin, nalidixic acid, and sitafloxacin was assessed by quinolone‐inhibited supercoiling assays. All mutant DNA gyrases showed reduced susceptibility to all quinolones when compared with WT DNA gyrases. DNA gyrase with a double amino acid substitution in GyrA, serine to phenylalanine at codon 83 and aspartic acid to asparagine at 87 (GyrA‐S83F‐D87N), exhibited the lowest quinolone susceptibility amongst all mutant DNA gyrases. The effectiveness of sitafloxacin was shown by the low inhibitory concentration required for mutant DNA gyrases, including the DNA gyrase with GyrA‐S83F‐D87N. We suggest sitafloxacin as a candidate drug for the treatment of salmonellosis caused by ciprofloxacin‐resistant S. Typhimurium. Copyright © 2015 John Wiley & Sons, Ltd.     

Amino acid sequence of the tryptic SH-peptide of thermitase,a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins

The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val - Val - Gly - Gly - Trp - Asp - Phe - Val-Asp-Asn-Asp-Ser-Thr-Pro-Gln-Asn - Gly - Asn - Gly -His64- Gly - Thr - His -Cys68 - Ala-Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn - Asn - Ser - Thr - Gly - Ile - Ala - Gly - Thr - Ala - Pro - Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-¹⁴CH₂-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.     

Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments

Fluoroquinolones are broad-spectrum antimicrobials highly effective for treatment of a variety of clinical and veterinary infections. Their antibacterial activity is due to inhibition of DNA replication. Usually resistance arises spontaneously due to point mutations that result in amino acid substitutions within the topoisomerase subunits GyrA, GyrB, ParC or ParE, decreased expression of outer membrane porins, or overexpression of multidrug efflux pumps. In addition, the recent discovery of plasmid-mediated quinolone resistance could result in horizontal transfer of fluoroquinolone resistance between strains. Acquisition of high-level resistance appears to be a multifactorial process. Care needs to taken to avoid overuse of this important class of antimicrobial in both human and veterinary medicine to prevent an increase in the occurrence of resistant zoonotic and non-zoonotic bacterial pathogens that could subsequently cause human or animal infections.     

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.     

Selection of a gyrA mutation and treatment failure with gatifloxacin in a patient with Streptococcus pneumoniae with a preexisting parC mutation

An 81-year-old woman had pneumonia caused by Streptococcus pneumoniae (levofloxacin Etest minimum inhibitory concentration [MIC] 1.5 microg/ml) and was treated with intravenous gatifloxacin 200 mg/day. After 3 days of therapy, repeat sputum cultures were positive for S. pneumoniae, which was resistant to levofloxacin (Etest MIC > 32 microg/ml). The isolate obtained before therapy showed a preexisting parC mutation of aspartic acid-83 to asparagine (Asp83-->Asn), and the isolate obtained during therapy showed an acquired gyrA mutation from serine-81 to phenylalanine (Ser81-->Phe) and a second parC mutation from lysine-137 to Asn (Lys137-->Asn). Both isolates were the same strain, as determined with pulsed-field gel electrophoresis. This case demonstrates the potential for resistance to emerge during 8-methoxy fluoroquinolone therapy for fluoroquinolone-susceptible S. pneumoniae with a preexisting parC mutation. Additional clinical failures with a fluoroquinolone may occur unless these first-step parC mutants can be identified to assist clinicians in selecting appropriate antimicrobial therapy.     

Characterization of Salmonella Typhimurium DNA gyrase as a target of quinolones

Quinolones exhibit good antibacterial activity against Salmonella spp. isolates and are often the choice of treatment for life‐threatening salmonellosis due to multi‐drug resistant strains. To assess the properties of quinolones, we performed an in vitro assay to study the antibacterial activities of quinolones against recombinant DNA gyrase. We expressed the S. Typhimurium DNA gyrase A (GyrA) and B (GyrB) subunits in Escherichia coli. GyrA and GyrB were obtained at high purity (>95%) by nickel‐nitrilotriacetic acid agarose resin column chromatography as His‐tagged 97‐kDa and 89‐kDa proteins, respectively. Both subunits were shown to reconstitute an ATP‐dependent DNA supercoiling activity. Drug concentrations that suppressed DNA supercoiling by 50% (IC₅₀s) or generated DNA cleavage by 25% (CC₂₅s) demonstrated that quinolones highly active against S. Typhimurium DNA gyrase share a fluorine atom at C‐6. The relationships between the minimum inhibitory concentrations (MICs), IC₅₀s and CC₂₅s were assessed by estimating a linear regression between two components. MICs measured against S. Typhimurium NBRC 13245 correlated better with IC₅₀s (R = 0.9988) than CC₂₅s (R = 0.9685). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test to identify quinolones with promising activity against S. Typhimurium. The quinolone structure‐activity relationship demonstrated here shows that C‐8, the C‐7 ring, the C‐6 fluorine, and N‐1 cyclopropyl substituents are desirable structural features in targeting S. Typhimurium gyrase. Copyright © 2014 John Wiley & Sons, Ltd.