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Title The role of plasmid-located mucAB genes in emergence of quinolone resistant Escherichia coli (Mutators)
Author Andersen, Signe Tanja (Urban Water Engineering, Department of Environmental Engineering, Technical University of Denmark, DTU, DK-2800 Kgs. Lyngby, Denmark)
Supervisor Hasman, Henrik (Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark, DTU, DK-2800 Kgs. Lyngby, Denmark)
Christiansen, Lasse Engbo (Mathematical Statistics, Department of Informatics and Mathematical Modeling, Technical University of Denmark, DTU, DK-2800 Kgs. Lyngby, Denmark)
Institution Technical University of Denmark, DTU, DK-2800 Kgs. Lyngby, Denmark
Thesis level Master's thesis
Year 2009
Abstract Antibiotic resistance has in the recent years received an increasing attention, due to the decrease in effectiveness caused by development of bacterial resistance mechanisms. In the latest report from the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) in 2007, a strong correlation between the quantity of the antibiotics and the development of resistance has been found. The transfer of resistance genes have been found to be caused by mobile genetic elements such as plasmids, transposons and integrons. Escherichia coli are one common cause of infections in human and animals. In human diagnostic E. coli, which are resistant to beta-lactam antibiotics due to production of Extended-spectrum Beta-Lactamases (ESBL), has become more and more prominent. Fluoroquinolone is one alternative antibiotic used to treat infections caused by ESBL producers. But in the year 2000 until today the resistance towards fluoroquinolones has increased in correlation with the increased consumption. The main focus of this study was to investigate the role of plasmid-located mucAB genes in emergence of quinolone resistant E. coli. This was done by determining the distribution of the plasmidic mucAB genes in animal reservoirs and by analyzing in what degree mucAB is activated when fluoroquinolones are used. Isolates collected from pigs and cattle in Denmark were selected and screened for the presence of mucAB genes, which are carried on resistance plasmids. Isolates included 81 indicator and 58 diagnostic E. coli O149 isolated from pigs in 2008, 81 indicator and 45 diagnostic E. coli K99 isolated from cattle in 2008, 12 diagnostic E. coli ESBL producers from humans in Spain obtained in the period 2000-2003. Additionally, 33 diagnostic S. typhimurium isolated from pigs and 36 diagnostic S. ssp. isolated from cattle in 2008, were examined. It was found that approximately 26 % of the diagnostic E. coli from pigs and 8 % of the diagnostic Salmonella from cattle contained mucAB. None of the indicator strains contained mucAB, along with the diagnostic E. coli from cattle and human. When examining the impact of mucAB on resistance development, transformants of E. coli with a plasmid carrying mucAB, were constructed. Additionally two Salmonella strains from Ames test, TA1535 and TA100 were used. A fluctuation assay was constructed and the mutation frequency measured in the presence or absence of mucAB. The mutation frequency was expected to increase when ciprofloxacin was used as inducer by activation of the SOS response and thereby mucAB. The result did not show any significant increase of the mutation frequency in induced cells containing mucAB compared to the negative control strain. Neither was a significant increase in the mutation frequency found in cells induced by 4-nitroquinoline-N-oxide, a compound known to activate mucAB. The target modifications in gyrA and parC were found by PCR and sequencing. This was done to investigate if mucAB induce certain basepair substitution events. All the quinolone resistant strains had a mutation in gyrA, while none of them had a mutation in parC. None of them was fluoroquinolone resistant. Transversion events accounted for 50 % of the basepair substitutions where the bases adenine, guanine and thymine were substituted equally. Thymine was the most prevalent base inserted, accounting for 47 % of the inserted bases. In GyrA, Asp87 to Tyr87 and Gly87 as well as Ser83 to Leu83, were the most frequent amino acids substitutions found. The minimum inhibitory concentration (MIC) was examined to find out if the target specificity mediated by ciprofloxacin mediated strains with higher MIC values than strains produced when 4-nitroquinoline-N-oxide was used. No difference was found between the two compounds, both produced quinolone resistant strains with most predominant MIC values in the range of 0,125 to 0,5 μg/ml ciprofloxacin. Based on the data in this study the conclusions were that mucAB are distributed in animal reservoirs in Denmark, primarily in diagnostic E. coli from pigs. In the study mucAB were not activated by ciprofloxacin and 4-nitroquinoline-N-oxide and therefore a correlation between an increased mutation frequency and resistance development mediated by mucAB was not established. The target modifications could therefore not be correlated with the effects of mucAB, but was rather the result of spontaneous mutations. The MIC values in the quinolone resistant strains correlated with previous findings.
Imprint Technical University of Denmark : Kgs. Lyngby
Pages 92
Fulltext
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Admin Creation date: 2011-12-08    Update date: 2011-12-08    Source: dtu    ID: 313942    Original MXD