Rapid method for detection of gyrA and grlA mutations in unrelated strains of Staphylococci susceptible and resistant to levofloxacin.

A panel of 150 clinical isolates of methicillin resistant and susceptible Staphylococci were investigated using a rapid and simple PCR-RFLPs technique to detect DNA nucleotide changes at the site of the most frequently reported mutations in grlA (codons 79, 80) and gyrA (codons 83, 84) genes which confer fluorquinolone resistance in Staphylococci. Convergent dual mutations in and gyrA and grlA were found in all strains exhibiting resistance to ciprofloxacin (MIC, 8 to > or =128 mg/l) and levofloxacin (MIC, 8 to > or =64 mg/l). Mutations in grlA and gyrA were also found in strains susceptible to levofloxacin and resistant to ciprofloxacin. In our sample no strains with only grlA mutations were found. Our data indicate that methicillin-resistant fluorquinolone-resistant strains are likely to have mutations in both grlA and gyrA. In contrast, methicillin-susceptible strains do not show any mutation. The genetic relatedness of a sample of representative epidemiologically unrelated MRSA strains, tested by PFGE and rep-PCR, are in agreement with the hypothesis of a clonal selection of these resistant strains.     

奶牛源MRSA肠毒素基因、耐药基因及分子分型分析

为了解宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌的肠毒素基因和耐药基因分布及其分子流行病学特征,本研究通过聚合酶链式反应(polymerase chain reaction, PCR)技术对前期分离于宁夏地区的9株奶牛源耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)进行了18种肠毒素基因和16种耐药基因的检测,同时采用脉冲场凝胶电泳(pulsed-field gel electrophoresis, PFGE)、正向重复序列(direct-repeat unit, dru)和辅助基因调节因子(accessory gene regulator, agr)分子分型技术对MRSA菌株进行分型研究。结果显示所有MRSA菌株均携带经典型肠毒素基因和新型肠毒素基因,共检出12种肠毒素基因,其中selk基因的检出率最高,达到了100%,未检出see、selj、selo、selp、ser和selu基因;11种耐药基因被检出,其中norA、gyrA、grlA和blaZ 4种基因的检出率均达到了100%,未检出tet (O)、optrA、Lin (A)、fexA和cfr基因。PFGE分型结果显示受试菌株间亲缘关系较近;dru分型检出dt11v和dt10a两种型,其中以dt11v(77.8%, 7/9)为主;agr分型主要为agr-Ⅰ型(88.9%, 8/9),agr-Ⅱ型仅有1株。研究表明宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌(MRSA)中的肠毒素基因和耐药基因分布广泛,菌株间亲缘关系较近,agr-Ⅰ-dt11v为MRSA菌株中的流行基因型。这为以后宁夏地区奶牛源MRSA的产毒性、耐药性和分子流行病学特征的进一步研究提供理论依据。     

Molecular epidemiology of quinolone resistance and comparative in vitro activities of new quinolones against European Staphylococcus aureus isolates

New fluoroquinolones (FQ) may possibly be used as alternative therapeutic options for Staphylococcus aureus infections. Our objectives were: (1) to define the in vitro activities of seven FQs in a collection of 434 methicillin-susceptible and 457 methicillin-resistant S. aureus from 23 European university hospitals; (2) to characterise the prevalence of mutations in the grlA and gyrA genes in all ciprofloxacin-resistant (n=433) isolates of S. aureus; (3) to determine the percentage of ciprofloxacin-resistant S. aureus strains with measurable quinolone efflux. Methods: (1) The in vitro activities of different FQs were determined by microdilution tests. (2) PCR-amplified DNA was sequenced. (3) Ciprofloxacin minimum inhibitory concentrations (MIC) were determined in the presence and absence of reserpine, which inhibits efflux pumps. Results: (1) Irrespective of the methicillin resistance of the isolates, sitafloxacin and clinafloxacin showed the best in vitro activities. (2) All ciprofloxacin-resistant isolates exhibited GrlA alterations, namely Ser-80-->Phe or Tyr or Glu-84-->Lys or Ala-116-->Glu or Pro or a combination of Ser-80-->Phe and Glu-84-->Val. These alterations in GrlA were combined with alterations in GyrA, namely Ser-84-->Leu or Lys or Glu-88-->Lys or Val. (3) Reserpine reduced ciprofloxacin MIC values in ca. 30% of the clinical isolates tested. Conclusions: (1) This current European overview of mutations involved in FQ resistance demonstrates that only a limited number of classical mutations in grlA and gyrA contributed to resistance in clinical isolates. (2) An efflux pump is involved in ca. 30% of ciprofloxacin-resistant S. aureus isolates. (3) Sitafloxacin and clinafloxacin are two very promising new FQs with good anti-staphylococcal activity. New FQs, perhaps in combination with efflux pump inhibitors, might play a role in the treatment of S. aureus infections.     

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.     

Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus.

We have isolated and cloned the gyrA and gyrB genes from Staphylococcus aureus. These adjacent genes encode the subunits of DNA gyrase. The nucleotide sequence of a 5.9-kb region which includes part of an upstream recF gene, the whole of gyrB and gyrA, and about 1 kb of unknown downstream sequence has been determined. The gyrB and gyrA gene sequences predict proteins of 886 and 644 amino acid residues, respectively, which have significant homologies with the gyrase subunits of Escherichia coli and Bacillus subtilis. Residues thought to be important to the structure and function of the subunits are conserved. These genes have been expressed separately by using a T7 promoter vector. N-terminal sequencing of the cloned gene products suggests that the mature GyrB subunit exists mainly with its initial five residues removed. Protein sequencing also supports the interpretation of our DNA sequencing data, which are inconsistent in several placed with the recently published sequence of the same genes (E. E. C. Margerrison, R. Hopewell, and L. M. Fisher, J. Bacteriol. 174:1596-1603, 1992).     

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.     

Primary levofloxacin resistance and gyrA/B mutations among Helicobacter pylori in Japan

BACKGROUND: Recent years have witnessed a decrease in the rate of Helicobacter pylori eradication due to antimicrobial resistance, clarithromycin or metronidazole resistance in particular. As one of the alternatives to the standard regimens, levofloxacin-containing therapy has been considered a promising regimen. Nevertheless, there is a little information concerning the prevalence of levofloxacin resistance and this resistance mechanism. MATERIALS AND METHODS: Levofloxacin susceptibility was examined using E-test in 507 H. pylori strains clinically isolated in Japan from 2001 to 2004. Mutation patterns in the quinolone resistance-determining regions of the gyrA and gyrB genes were evaluated, performing direct sequencing of 68 levofloxacin-resistant and 50 susceptible strains. RESULTS: Primary levofloxacin resistance was found in 76 (15.0%) strains. Fifty-seven (83.8%) of 68 levofloxacin-resistant strains analyzed had point mutations in gyrA at Asn-87 or Asp-91, while seven (14.0%) of 50 susceptible strains had gyrA mutations. There was a significant difference in the occurrence of gyrA mutations between levofloxacin-resistant and -susceptible strains (p < .001). In levofloxacin-resistant strains, the occurrence of gyrA mutations at Asn-87 was most common regardless of minimal inhibitory concentration levels, and that of gyrA mutations at Asp-91 tended to be associated with low-level resistance. A double gyrA mutation at Asn-87 and Asp-91 might have an additional impact. As for gyrB, three (4.4%) of 68 levofloxacin-resistant strains with no susceptible strains had mutations. CONCLUSIONS: Primary levofloxacin resistance was common in Japan and primarily related to gyrA mutations at Asn-87 and Asp-91.     

Rapid detection of gyrA and parC mutations in quinolone-resistant Salmonella enterica using Pyrosequencing technology

Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment of systemic Salmonella infection or for long-term salmonella carriage. Resistance to quinolones is commonly mediated by point mutations within the topoisomerase genes gyrA and parC. Pyrosequencing technology is a DNA sequencing method using 'sequencing by synthesis' and is suitable for the rapid detection of single nucleotide polymorphisms (SNPs). One hundred and ten Salmonella enterica isolates, representing 18 different serotypes, were used in this study. One hundred and four isolates had ciprofloxacin MICs of 0.25-32 microg/mL; the remaining six were ciprofloxacin-sensitive (ciprofloxacin MIC相似文献   

Characteristics of Klebsiella pneumoniae harboring QnrB32, Aac(6')-Ib-cr, GyrA and CTX-M-22 genes

Quinolone resistance in members of the Enterobacteriaceae family is mostly due to mutations in the quinolone resistance-determining regions of topoisomerase genes. CTX-M-22 is a member of the CTX-M family which can reduce extended-spectrum β-lactamase (ESBL) production and modulate antibiotic resistance, resulting in low ceftazidime minimum inhibitory concentrations (MICs). There are four different genes in Klebsiella pneumoniae (KP4707) including qnrB32 (novel qnr allele gene, HQ704413), aac(6')-Ib-cr (novel aac(6')-Ib allele gene, HQ680690), gyrA (novel gyrA allele gene, HQ680691) and CTX-M-22 gene. Five point amino acid mutations Arn(N)27 → Leu(L), Val(V)129 → Ala(A), Iie(I)142 → Met(M), Gly(G)188 → Arg(R), Val(V)212 → Iie(I) were observed in the qnr32 gene when compared to qnrB1. Of all qnrB alleles, a novel variant of the qnrB32 gene, with qnrB31, had the highest amino acid homology. Three point amino acid mutations including Trp(W)105 → Arg(R), Asp(D)182 → Tyr(Y) and Val(V)201 → Asp(D) were observed in aac(6')-Ib-cr gene, when compared to GenBank number AF479774. New variants of qnr32, aac(6')-Ib-cr, gyrA and CTX-M-22 or other genotype determinants continuously appear in different genomic sites and also outside the Enterobacteriaceae family.     

Fluoroquinolone resistance and gyrA and parC mutations of Escherichia coli isolated from chicken

Escherichia coli is a common inhabitant of the intestinal tracts of animals and humans. The intestines of animals also represent an ideal environment for the selection and transfer of antimicrobial resistance genes. The aim of this study was to investigate the resistance of E. coli isolated from chicken fecal samples to fluoroquinolones and to analyze the characterization of mutations in its gyrA and parC gene related resistance. One hundred and twenty-eight E. coil isolates showed a high resistance to ciprofloxacin (CIP; 60.2%), enrofloxacin (ENO; 73.4%) and norfloxacin (NOR; 60.2%). Missense mutation in gyrA was only found in the amino acid codons of Ser-83 or Asp-87. A high percentage of isolates (60.2%) showed mutations at both amino acid codons. Missense mutation in parC was found in the amino acid codon of Ser-80 or Glu-84, and seven isolates showed mutations at both amino acid codons. Isolates with a single mutation in gyrA showed minimal inhibitory concentrations (MIC) for CIP (相似文献   

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins.

We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnaA-dnaN-recF-gyrB-gyrA, is similar to that found in the Bacillus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640- and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.     

Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review

Background

The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.

Methods

In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.

Results

Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.

Conclusions

Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Mutations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in clinical strains of fluoroquinolone-resistant Shigella in Anhui, China

In this research 26 Shigella isolates were examined by PCR and direct nucleotide sequencing for genetic alterations in the quinolone-resistance determining regions (QRDRs). We tested for the presence of qnr genes by PCR in 91 strains, but no qnr genes were found. The results did show, however, some novel mutations at codon 83 of gyrA (Ser-->Ile) and codon 64 of parC (Ala64-->Cys, Ala64-->Asp), which were related to fluroquinolone resistance.     

The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.     

Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition.

The letA (ccdA) and letD (ccdB) genes, located just outside the sequence essential for replication of the F plasmid, apparently contribute to stable maintenance of the plasmid. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. To identify the target of the letD gene product, temperature-sensitive growth-defective mutants were screened from bacterial mutants that had escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Of nine mutants analysed, three mutants were shown, by phage P1-mediated transduction and complementation analysis, to have mutations in the gyrA gene and the other six in the groE genes. The nucleotide sequence revealed that one of the gyrA mutants has a base change from G to A at position 641 (resulting in an amino acid change from Gly to Glu at position 214) of the gyrA gene. The mutant GyrA proteins produced by these gyrA(ts) mutants were trans-dominant over wild-type GyrA protein for letD tolerance. The wild-type GyrA protein, produced in excess amounts by means of a multicopy plasmid, overcame growth inhibition of the letD gene product. These observations strongly suggest that the A subunit of DNA gyrase is the target of the LetD protein.