Genetic response of Salmonella enterica serotype Enteritidis to thioridazine rendering the organism resistant to the agent

Abstract

Thioridazine (TZ)-induced accumulation of the universal efflux pump substrate ethidium bromide and its subsequent efflux by Salmonella strains with various degrees of overexpressed efflux pumps takes place automatically at pH 7.4, is independent of a metabolic source, is not affected by a proton ionophore and is precluded by palmitic acid. Salmonella enterica serotype Enteritidis cultured in medium containing increasing concentrations of TZ does not grow during the first 6–8 h, after which time its growth is similar to unexposed controls. At the end of a 16-h exposure period, the organism is resistant to >250 mg/L TZ. Parallel assessment by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the activity of genes that regulate and code for the AcrB transporter of the main efflux pump (AcrAB) of the organism at periodic intervals suggests a sequence of activation beginning with the stress gene soxS, followed by the global regulator ramA, then by the local regulator marA and then by the transporter acrB. These activations take place during the period of no growth. By the end of a 16-h culture period, only the acrB transporter gene is still highly overexpressed. Assessment of the activity of genes of the two-component regulon PmrA/B indicates that TZ also activates this regulon. Because activation of pmrA/ B also activates acrB, development of high resistance to TZ during a 16-h culture period is in part due to activation of the two-component regulon.