Overexpression of the MFS transporter pmrB in Streptococcus thermophilus improves the efficiency of ethidium bromide efflux and the growth-fitness under stressed condition

Genome analysis of S. thermophilus DSM20617 revealed the presence of several putative efflux pump systems belonging to the Major Facilitator Superfamily (MFS) potentially involved in the reduction of toxicity towards biocide and antibiotic molecules. Among them, pmrB orthologs have been detected in several S. thermophilus genomes analyzed to date, and S. macedonicus ACA-DC 198. RT-qPCR analysis revealed the induction of pmrB expression when cells were exposed to chlorhexidine or EB. Then pmrB gene was cloned in pNZ8048 under the control of the T5 promoter and the derived vector pNZ-T5-pmrB was used to transform strain DSM20617, thus obtaining the derivative MIM27 strain. The activity of pmrB was investigated by evaluating its ability to efflux EB. Compared to the strain harbouring the empty vector and named MIM20, the overall bacterial population of the pmrB-overexpressing MIM27 showed an improved efficiency of EB efflux as revealed by standard fluorometric approach and by flow cytometry. Unlike MIM27, the data obtained revealed that the MIM20 population is composed of two subpopulations probably due to a bistable expression of efflux pumps involved in EB detoxification. Interestingly, strain MIM27 showed an increased metabolic activity, compared to the MIM20, in presence of biocide chlorexidine, alexidine, and the antibiotics tetracycline and furaltadone as determined by a phenotype microarray analysis, thus indicating that the overexpression of pmrB in S. thermophilus could result in a cross resistance phenotype towards antibiotics and biocides. Finally, co-culturing MIM20 and MIM27 in a competition assay in presence of EB or tetracycline, MIM20 lost competition. It was therefore concluded that pmrB confers a competitive advantage when S. thermophilus is cultured in presence of antimicrobial compounds.