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- Lindbäck, Emma
(author)
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Mechanisms of resistance to ciprofloxacin in Neisseria gonorrhoeae
- 2006
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Doctoral thesis (other academic/artistic)abstract
- A few years ago, most strains of Neisseria gonorrhoeae were susceptible to ciprofloxacin, but after introduction as a first line therapy, resistant strains emerged. Ciprofloxacin inhibit two enzymes necessary for DNA replication. Mutations in the quinolone resistance determining regions (QRDR) of genes encoding the subunit GyrA of the target enzyme DNA gyrase and ParC of topoisomerase IV are mechanisms of resistance in N. gonorrhoeae. These alterations do not explain why the minimum inhibitory concentrations (MICs) of ciprofloxacin in resistant strains vary so widely. The target enzymes also contain the subunits GyrB and ParE. Their role and other mechanisms of resistance in N. gonorrhoeae, such as increased efflux out of the cell, decreased uptake or competition of quinolone binding sites by protection, have not been fully investigated in N. gonorrhoeae. Several commercial kits for molecular diagnosis of N. gonorrhoeae are available and can be analyzed in a duplex PCR with Chlamydia trachomatis, but these methods are known to produce false positive results. Results obtained with AMPLICOR N. gonorrhoeae polymerase chain reaction (PCR) (Roche Diagnostics) were compared to cultivation results in 956 samples. In positive samples species verification of the 16S rRNA gene was compared to pyrosequencing of QRDR the gyrA gene, which was also evaluated as an indicator of ciprofloxacin susceptibility. Culture and the molecular method verified in gyrA produced two and one false negative result respectively and the molecular method verified in 16S rRNA produced four false positive results. QRDR of all eleven urine samples positive in AMPLICOR N. gonorrhoeae PCR, with corresponding isolates as well as 46 N. gonorrhoeae strains, were correctly diagnosed according to susceptibility to ciprofloxacin compared to MICs. Pyrosequencing of QRDR of gyrA of 40 isolates of nine other Neisseria spp. showed that QRDR in gyrA is not unique for N. gonorrhoeae. Sequencing of QRDR of gyrA, gyrB, parC, and parE in 25 highly ciprofloxacin resistant and five susceptible strains of N. gonorrhoeae, showed that all the resistant strains had two mutations in gyrA. Fourteen strains also had an additional mutation in parC, and 17 strains had an additional mutation in parE. No alterations were found in gyrB in any strain. In transformation experiments an alteration in GyrA was introduced in a ciprofloxacin susceptible N. gonorrhoeae strain (MIC 0.008 mg/L) and MIC increased to 0.064 mg/L. Two alterations, together, increased MIC to 0. 125 - 0.25 mg/L. Introduction of alterations in major outer membrane porin, PorB1b, and probably other alterations, in a moderately ciprofloxacin resistant strain (MIC 0.25 mg/L) gave transformants with MICs of ciprofloxacin 0.5-16 mg/L. In one transformant an alteration in ParE was also introduced. We conclude that verification of a molecular method by pyrosequencing in gyrA gene is superior to verification by PCR in 16S rRNA. The gene gyrA is not unique for N. gonorrhoeae. However, whether this region is possible to use also for verification also depends on the specificity of the primary method. QRDR of gyrA is a strong indicator of ciprofloxacin resistance in N. gonorrhoeae. Two alterations in gyrA only increases MIC of ciprofloxacin to 0. 125 - 0.25 mg/L. Additional alterations in QRDR of parC and parE as well as alterations in porB1b also contribute to ciprofloxacin resistance.
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| 2. |
- Lindbäck, Emma, et al.
(author)
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Pyrosequencing of the DNA gyrase gene in Neisseria species : effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae
- 2006
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In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 114:12, s. 837-841
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Journal article (peer-reviewed)abstract
- The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
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| 3. |
- Lindbäck, Emma, et al.
(author)
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Transformation of ciprofloxacin-resistant Neisseria gonorrhoeae gyrA, parE and porB1b genes
- 2006
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 28:3, s. 206-211
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Journal article (peer-reviewed)abstract
- In several transformation experiments, we have shown that introduction of an alteration in GyrA at position 95 of a ciprofloxacin-susceptible Neisseria gonorrhoeae strain (minimum inhibitory concentration (MIC) 0.008 mg/L) increases the MIC to 0.064 mg/L. Two alterations (positions 91 and 95) increase the MIC to 0.125-0.25 mg/L. Transformants with ciprofloxacin MICs of 0.5-16 mg/L were obtained from a moderately ciprofloxacin-resistant strain (MIC 0.25 mg/L). These transformants had alterations in the gene for PorB1b and probably other genes. In one transformant, an alteration in ParE was also introduced, which probably contributed to ciprofloxacin resistance. The ciprofloxacin-resistant transformants had the donor porB1b sequence, and most of them also had altered serovars. We conclude that alterations in N. gonorrhoeae PorB1b could be involved in ciprofloxacin resistance.
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| 4. |
- Tomasdottir, Maria, et al.
(author)
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Risk markers of incident atrial fibrillation in patients with coronary heart disease
- 2021
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In: American Heart Journal. - : Elsevier. - 0002-8703 .- 1097-6744. ; 233, s. 92-101
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Journal article (peer-reviewed)abstract
- BackgroundIn patients with coronary heart disease (CHD), atrial fibrillation (AF) is associated with increased morbidity and mortality. We investigated the associations between clinical risk factors and biomarkers with incident AF in patients with CHD.Methods and resultsAround 13,153 patients with optimally treated CHD included in the STabilization of Atherosclerotic plaque By Initiation of darapLadIb TherapY (STABILITY) trial with plasma samples obtained at randomization. Mean follow-up time was 3.5 years. The association between clinical risk factors and biomarkers with incident AF was estimated with Cox-regression models. Validation was performed in 1,894 patients with non-ST-elevation acute coronary syndrome included in the FRISC-II trial.The median (min-max) age was 64 years (range 26-92) and 2,514 (19.1%) were women. A total of 541 patients, annual incidence rate of 1.2%, developed AF during follow-up. In multivariable models, older age, higher levels of NT-proBNP, higher body mass index (BMI), male sex, geographic regions, low physical activity, and heart failure were independently associated with increased risk of incident AF with hazard ratios ranging from 1.04 to 1.79 (P ≤ .05). NT-proBNP improved the C-index from 0.70 to 0.71. In the validation cohort, age, BMI, and NT-proBNP were associated with increased risk of incident AF with similar hazard ratios.ConclusionsIn patients with optimally treated CHD, the incidence of new AF was 1.2% per year. Age, NT-proBNP as a marker of impaired cardiac function, and BMI were the strongest factors, independently and consistently associated with incident AF. Male sex and low physical activity may also contribute to the risk of AF in patients with CHD.
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| 5. |
- Unemo, Magnus, et al.
(author)
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Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain
- 2007
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In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 115:3, s. 231-240
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Journal article (peer-reviewed)abstract
- A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002-2003. In conclusion, a precise, i. e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.
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