Role of Tetracycline Speciation in the Bioavailability to <i>Escherichia coli</i> for Uptake and Expression of Antibiotic Resistance

Abstract

Tetracycline contains ionizable functional groups that manifest several species with charges at different locales and differing net charge; the fractional distribution of each species depends on pH-p<i>K</i>_a relationship in the aqueous phase. In nature, these species interact with naturally abundant cations (e.g., Ca²⁺ and Mg²⁺) to form metal-tetracycline complexes in water. In this study, we used <i>Escherichia coli</i> MC4100/pTGM whole-cell bioreporter to investigate tetracycline uptake from solution under varying conditions of pH, salt composition and concentration by quantifying the corresponding expression of antibiotic resistance gene. The expression of antibiotic resistance gene in the <i>E. coli</i> bioreporter responded linearly to intracellular tetracycline concentration. Less tetracycline entered <i>E. coli</i> cells at solution pH of 8.0 than at pH 6.0 or 7.0 indicating reduced bioavailability of the antibiotic at higher pH. Both Mg²⁺ and Ca²⁺ in solution formed metal-tetracycline complexes which reduced uptake of tetracycline by <i>E. coli</i> hence diminishing the bioresponse. Among the various tetracycline species present in solution, including both metal-complexed and free (noncomplexed) species, zwitterionic tetracycline was identified as the predominant species that most readily passed through the cell membrane eliciting activation of the antibiotic resistance gene in <i>E. coli</i>. The results indicate that the same total concentration of tetracycline in ambient solution can evoke very different expression of antibiotic resistance gene in the exposed bacteria due to differential antibiotic uptake. Accordingly, geochemical factors such as pH and metal cations can modulate the selective pressure exerted by tetracycline for development and enrichment of antibiotic resistant bacteria. We suggest that tetracycline speciation analysis should be incorporated into the risk assessment framework for evaluating environmental exposure and the corresponding development of antibiotic resistance