Overexpression of the Multidrug Efflux Operon acrEF by Insertional Activation with IS 1 or IS 10 Elements in Salmonella enterica Serovar Typhimurium DT204 acrB Mutants Selected with Fluoroquinolones

Abstract

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ-resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8-to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1-or IS10-activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Notably, this mutation was present in clinical isolates from cystic fibrosis (CF) patients. Another interesting phenomenon was observed for Salmonella enterica serovar Typhimurium, in which the presence of the IS1 or IS10 insertion element in the promoter region upstream of the genes encoding the AcrEF pump enhanced the transcription of this operon, thereby conferring resistance to fluoroquinolones (191). Nonetheless, the vast majority of mutations affecting antibiotic export by efflux systems occur in genes encoding proteins with a regulatory function.…”

Regulation of Multidrug Efflux Systems Involved in Multidrug and Metal Resistance of Salmonella enterica Serovar Typhimurium

“…Eaves et al (14) reported that acrB, acrF, and acrD are coordinately regulated and that their expression influences the expression of the transcriptional activators marA and soxS. Furthermore, it has been reported that integration of an IS1 or an IS10 element in the upstream region of the acrEF operon causes increased expres-sion of acrEF (52). These examples illustrate the complexity and diversity of the mechanisms regulating bacterial multidrug efflux pumps.…”

Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

“…A spontaneous laboratory mutant of E. coli has also been selected with IS186 in acrR or IS2 upstream of acrEF (67). An IS element inserted in mexR has also been described for a clinical isolate of P. aeruginosa and for acrS (the putative repressor of acrEF) of S. enterica serovar Typhimurium DT204 (20,150). In the latter case, the mutant was selected in vitro on high concentrations of fluoroquinolone from a veterinary isolate.…”