Abstract
The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10-10 - 9.9 x 10-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan.
Highlights
Biocides are broadly used to control microbial growth and, play an essential role in preventing the spread of infectious agents and disease [1]
Tolerance of bacteria to biocides is documented for most classes, and increasing percentages of tolerant strains might contribute to the survival of zoonotic pathogens in the food chain and might facilitate the emergence of bacterial persisters [3,5]
Among the Salmonella field isolates, MIC values of triclosan (MICTRC) varied between 0.125 - 0.5 μg/ml, these results being within the range recently reported for avian salmonellae in Germany [21]
Summary
Biocides are broadly used to control microbial growth and, play an essential role in preventing the spread of infectious agents and disease [1]. One mutant was selected from an agar plate supplemented with triclosan in a concentration of one dilution step below the MPC for further analysis.
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