Dataset Information

Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach.

ABSTRACT: As a result of the increasing resistance of Helicobacter pylori against first-line antibiotics, other drugs, such as quinolones, will be needed for eradication therapy in the future. We developed a real-time PCR to detect mutations in the gyrA gene associated with ciprofloxacin resistance of H. pylori, thereby contributing to the selection of patients who could be treated by ciprofloxacin-based therapy.

SUBMITTER: Glocker E

PROVIDER: S-EPMC404627 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach.

Glocker Erik E Kist Manfred M

Journal of clinical microbiology 20040501 5

As a result of the increasing resistance of Helicobacter pylori against first-line antibiotics, other drugs, such as quinolones, will be needed for eradication therapy in the future. We developed a real-time PCR to detect mutations in the gyrA gene associated with ciprofloxacin resistance of H. pylori, thereby contributing to the selection of patients who could be treated by ciprofloxacin-based therapy. ...[more]

PMID: 15131201

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Project description:AIM:To investigate the resistance of Helicobacter pylori (H. pylori) to ciprofloxacin (CIP), levofloxacin (LVX) and moxifloxacin (MOX) in the Beijing area and to elucidate the resistance mechanisms. METHODS:Seventy-nine H. pylori clinical strains, isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009, were tested for their susceptibility to CIP, LVX and MOX using the E-test method. H. pylori strain 26695 was included in the susceptibility testing as a control strain. According to the minimal inhibitory concentration (MIC) values, a strain was classified as resistant to CIP, LVX or MOX when the MIC was > 1 microg/mL. We amplified by polymerase chain reaction (PCR) and sequenced the quinolone resistance-determining regions of the gyrA and gyrB genes from 29 quinolone-resistant and 16 quinolone-susceptible H. pylori strains selected at random. RESULTS:In this study, the resistance rates of H. pylori to CIP, LVX or MOX were 55.7% (44/79), and the primary resistance rates were 26.6% (21/79). Patients with secondary resistance had received LVX in previous eradication treatments, but not MOX or CIP. Forty-five strains, including 29 CIP, LVX or MOX-resistant strains (MIC: 1.5-32 microg/mL) and 16 susceptible strains, were selected randomly from the 79 strains and used in PCR analysis. Among these 45 strains, 27 resistant strains had mutations in the gyrA gene, including 11 strains with mutations corresponding to Asp-91 (MIC: 2-32 microg/mL), one of which also had a mutation corresponding to Val-150, and 16 strains had mutations at Asn-87 (MIC: 4-32 microg/mL), three of which also had mutations corresponding to Arg-140 or Val-150. In addition, Arg-140, Val-150 or Ala-97 mutations were separately detected in three susceptible strains. Analysis of the gyrB gene showed that one strain of low resistance had a mutation corresponding to Ser-457 that coexisted with an Asp-91 mutation. There was a significant difference in the occurrence of mutations in the gyrA gene between CIP, LVX and MOX-resistant and -susceptible strains (P < 0.05), but 2 resistant strains were found to possess no quinolone resistance-determining region mutations. CONCLUSION:Resistance is primarily mediated through point mutations in gyrA. Whether other mechanisms are responsible for resistance in strains without mutations in the QRDR should be detected.

Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach.

Publications

Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach.

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