Effect of the transcriptional activators SoxS, RobA and RamA

26 Control of membrane permeability is a key step in regulating the intracellular 27 concentration of antibiotics. Efflux pumps confer innate resistance to a wide 28 range of toxic compounds such as antibiotics, dyes, detergents and 29 disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux 30 pump is involved in multidrug resistance in E. cloacae. However, the underlying 31 mechanism that regulates the system in this microorganism remains unknown. 32 In Escherichia coli, transcription of acrAB is up-regulated under global stress 33 conditions by proteins such as MarA, SoxS and Rob. In the present study, two 34 different clinical isolates of E. cloacae, EcDC64 (MDR over-expressing the 35 AcrAB-TolC efflux pump) and Jc194 (with basal AcrAB-TolC expression level) 36 were used to determine whether similar global stress responses operate in E. 37 cloacae and also to establish the molecular mechanisms underlying this 38 response. A decrease in susceptibility to erythromycin, tetracycline, 39 telithromycin, ciprofloxacin and chloramphenicol was observed in clinical isolate 40 Jc194, and to a lesser extent in EcDC64, in the presence of salicylate, 41 decanoate, tetracycline and paraquat. Increased expression of the acrAB 42 promoter in the presence of the above described conditions was observed by 43 flow cytometry and RT-PCR, by using a reporter fusion protein (GFP). The 44 expression level of the AcrAB promoter decreased in E. cloacae EcDC64 45 derivates deficient in SoxS, RobA and RamA. Accordingly, the expression level 46 of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing 47 soxS, robA and ramA. Overall, the data showed that SoxS, RobA and RamA 48 regulators were associated with the up-regulation of acrAB, thus conferring 49 antimicrobial resistance as well as a stress response in E. cloacae. 50 on July 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom In summary, the regulatory proteins SoxS, RobA and RamA were cloned and 51 sequenced for the first time in this species. The involvement of these proteins in 52 conferring antimicrobial resistance through up-regulation of acrAB was 53 demonstrated in E. cloacae. 54 on July 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom