Triclosan is a potent biocide that is included in a diverse range of products. This research was aimed to investigate the susceptibility of planktonic and biofilm-associated Salmonella enterica serovar Typhimurium to triclosan, and to identify potential mechanisms of adaptation. The effect of triclosan was studied on planktonic Salmonella (log and stationary phases), on biofilm-associated cells, on bacteria derived from disrupted biofilms and on a biofilm-deficient mutant. An eight-log reduction of exponentially growing cells was observed with 1000 micro g mL(-1) triclosan within 10 min, a 3.6-log reduction in stationary cells and a 6.3-log reduction in stationary cells of a biofilm-deficient mutant (P<0.05). Biofilm-associated cells were tolerant (1-log reduction). However, biofilm-derived cells showed sensitivity to triclosan similar to stationary-phase cells. Triclosan induced the transcription of fabI and micF. Within biofilms, triclosan also up-regulated the transcription of acrAB, encoding for an efflux pump, marA, and the cellulose-synthesis-coding genes bcsA and bcsE. Thus, Salmonella within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials.

Importance of the overexpression of AcrAB efflux pumps in the low-level resistance of pathogens to antimicrobials requires a better understanding of the AcrAB regulation. The goal of the present research was to study the transcription of acrAB, as well as the genes that play a role in its regulation in Salmonella enterica serovar Typhimurium. We monitored the transcription of these genes during growth at 30• C and 37• C, and thoroughly studied the effect of salicylate, paraquat and decanoate. The strengths of the promoters' activities were ordered from strong to weak as micF > rob > acrAB > soxS, marRAB. At both temperatures, marRAB was mainly upregulated by salicylate and decanoate, soxS by paraquat and acrAB and micF by all three compounds. rob was always downregulated. Transcription rates of all promoters were at the greatest level at the beginning of the stationary phase and, except for soxS, levels of transcription and induction were greater at 37• C. Results show that despite the promoters' similar activity and the sequence homology between Escherichia coli and S. typhimurium, regulation of the investigated genes of both strains differed in the response to temperature. This difference was found to be dependent on the promoters' sequence, as the marRAB and acrAB promoters maintained their original dependence on temperature when they were analyzed in the other strain. Hence, it is most likely that the nutrient-limited environment at 37• C in the human body will lead to increased transcription of marA, acrAB and micF in Salmonella, enhancing the resistance properties of the bacteria.

In intracellular bacteria, the expression of acrAB is already higher when compared with extracellular cells; therefore, salicylate does not result in significant acrAB induction intracellularly and subsequent resistance enhancement. Results show that conclusions raised from extracellular studies cannot be applied to intracellular bacteria, although the systems have similar functions.

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