Effect of Transcriptional Activators SoxS, RobA, and RamA on Expression of Multidrug Efflux Pump AcrAB-TolC in Enterobacter cloacae

Abstract

Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.

Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

“…AcrABTolC is a well-characterized multidrug efflux pump of the RND family in E. cloacae (20,26,27). Based on the sequence of the protein AcrB (designated ECL_01233 in ECL13047), the inner membrane transporter protein of the system, we searched for the presence of other members of this family of transporters.…”

Genomics of KPC-Producing Klebsiella pneumoniae Sequence Type 512 Clone Highlights the Role of RamR and Ribosomal S10 Protein Mutations in Conferring Tigecycline Resistance

“…1A). Since RamR defects have been demonstrated to increase acrAB expression via RamA activation, the mutations observed in these two strains may account for their tigecycline resistance phenotype (19,22,(25)(26)(27)(28)(29).…”

“…The transcriptional activators of the acrAB genes are themselves controlled by other trans-acting factors. RamA expression is controlled by the negative regulator RamR; the depletion of RamR has been implicated in the activation of RamA, causing the overexpression of the AcrAB efflux pump (19,22,(25)(26)(27)(28)(29).…”

“…Recent studies demonstrated that tigecycline resistance can occur independently of AcrAB system upregulation (29,30), suggesting the presence of different mechanisms of resistance. The recent availability of whole-genome sequencing permits the discovery of genetic differences between pairs of genetically related susceptible and resistant bacterial strains.…”

Caracterização molecular dos mecanismos de resistência aos carbapenêmicos de isolados clínicos de Enterobacter aerogenes e Enterobacter cloacae

“…Esta bomba de efluxo foi identificada como mecanismo de resistência em isolados de E. aerogenes multiresistentes (125)(126)(127) . A atividade da bomba de efluxo em vários isolados clínicos resistentes foi caracterizada por meio de diferentes inibidores de bomba de efluxo que competem com o efluxo de antibióticos.…”