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The Relation between the High-level Resistance to Fluoroquinolones and the Over-expression of the acrA among Quinolone-Resistant Escherichia coli -Quantification of acrA by Using Real time PCR and Northern hybridization
Byung Yeon Hwang, M.D.1, Sae Yoon Kee, M.D.1, Jeong Yeon Kim, M.D.1, Hye Won Jeong, M.D.1 Cheong Won Park, M.D.1, Yoon Hee Park, M.D.2, Meyoung Kon Kim, M.D.2 Hee Jin Cheong, M.D.1, and Woo Joo Kim, M.D.1
1Department of Internal Medicine, 2Department of Biochemistry, College of Medicine, Korea University, Seoul, Korea
Vol.37 Num.4 (p185~192)
Background:Target point mutation of DNA topoisomerase, which is the typical mode of quinolone resistance, cannot explain high level resistance to quinolones. Therefore, many authors looked into over expression of efflux pump as the possibility. After quantificating the arcA mRNA, which controls AcrAB- TolC, the authors tried to find out the difference in the expression of arcA mRNA according to MIC of ciprofloxacin. The authors also tried to determine the usefulness of real time PCR, which is more reproducible and takes less time than preexisting immunoblot assay, through quantification of acrA.
Material and Methods:Mutations in topoisomerase (GyrA, ParC) of 20 quinolone resistant E. coli isolates were identified by PCR and direct DNA sequencing. AcrA level was measured by real time PCR. GAPDH of E.coli was used as endogenous control. The expression of acrA was confirmed through northern hybridization method, the results obtained by real time PCR were compared.
Results:1) Topoisomerase mutations were found in all quinolone resistant E. coli strains. 2) AcrA expression in fluoroquinolone-resistant E. coli was quantified by using real time PCR. There was no relationship between the ratio of acrA expression to GAPDH and MIC of ciprofloxacin. 3) With Northern hybridization, we compared the band of acrA to that of GAPDH in compactness and area. No difference in the expression according to MIC could be found. 4) The results of AcrA/GAPDH were significantly correlated between the real-time PCR and northern blot (P<0.05, correlation coefficiency 0.98).
Conclusion:In this study, no relationship between overexpression of AcrA gene and high level fluoroquinolone resistance. Therefore, we assume that mechanism other than AcrAB efflux pump is involved in and contribute to high-level fluoroquinolone resistance. However, the degree of efflux pump expression could be confirmed with real time PCR using acrA mRNA. Therefore, real time PCR could be used in the molecular biologic study on the mechanism of resistance to antibiotics.
Keywords : AcrA, quinolone resistant E. coli, efflux pump, Real-time PCR, Northern hybridization