A Novel Approach to Combat Acquired Multiple Resistance in Escherichia coli by using EDTA as Efflux Pump Inhibitor

Abstract

In the present investigation, twenty three clinical isolates of Escherichia coli were analyzed for the presence of AcrAB-TolC efflux pump using the respective primer. In order to explore the possibility of EDTA as efflux inhibitor, the effect of EDTA on 4,6-diamidino-2-phenylindole (DAPI) efflux activity was measured. Thereafter, the minimum inhibitory concentration (MIC) of each antibacterial agent (drug) included in the study was determined with ethylene diamine tetraacetic acid (EDTA) and without EDTA according to Clinical and Laboratory Standards Institute, 2009. Next, the effect of EDTA and drugs with half of MIC on mRNA expressions of AcrAB-Tolc efflux pump was measured through semi quantitative reverse transcription-polymerase chain reaction (RT-PCR). Our results revealed that 10 mM EDTA when combined with ceftriaxone plus sulbactam (CSE1034; ceftriaxone plus sulbactam plus 10 mM EDTA) and meropenem, the MIC values reduced 32 and 8 fold, respectively, whereas other comparative drugs demonstrated only two fold reduction in MIC values. When, DAPI efflux activity was assessed with different concentration of EDTA, a concentration-dependent inhibition of DAPI efflux was observed with a significant inhibition at 10 mM. Further, increasing the concentration of EDTA, failed to produce any more inhibition. Similarly, 10 mM EDTA decreased the expression of AcrA, AcrB and TolC mRNA by 2.3, 2.2 and 2.4 folds, respectively. A significant (p<0.0001) reduction of mRNA levels of AcrAB-TolC in CSE1034 treated groups was observed compared to meropenem (p<0.01). However, ceftriaxone, amoxycillin plus clavulanate, piperacillin plus tazobactam, and cefoperazone plus sulbactam treated group exhibited a non-significant changes (p>0.05) in the expression of AcrAB-TolC mRNA compared with control. From the above results, it can be concluded that CSE1034 strongly decreased the expression of efflux pump. Hence, it can be one of the best choices to treat infections caused by the microorganisms over expressing AcrAB-TolC efflux pump. © 2012 Chaudhary M, et al.