Products from Photolysis Reactions of Tetracycline Mediated by Clay and Humic Substance Induce Contrasting Expressions of Target Resistance Genes

Phototransformation is a key process affecting the fate of many antibiotics in the environment, but little is known about whether their photoproducts exert selective pressure on bacteria by inducing antibiotic resistance genes (ARGs). Here, we examined the expression of tetracycline resistance gene tet(M) of a fluorescent Escherichia coli whole-cell bioreporter influenced by the phototransformation of tetracycline. The presence of suspended smectite clay (montmorillonite or hectorite, 1.75 g/L) or dissolved humic substance (Pahokee Peat humic acid or Pahokee peat fulvic acid, 10 mg C/L) in aqueous solutions markedly facilitated the transformation of tetracycline (initially at 400 μg/L) with half-life shortened by 1.4-2.6 times. Despite the similar phototransformation ratios (80-90%) of the total loaded tetracycline after 60 min irradiation, the decreased ratios of cell fluorescence intensity (which was proportional to the expression amount of ARG tet(M)) were much higher with the two clays (94 and 93%) than with the two humic substances (44 and 69%) when compared to the respective dark controls. As illustrated by mass spectroscopic and chemical analyses, tetracycline was proposed to be mainly transformed to amide (ineffective in inducing ARGs) with the presence of clays by reaction with self-photosensitized singlet oxygen (¹O₂), while the humic substances might catalyze the production of another two demethylated and/or deaminated compounds (still effective in inducing ARGs) in addition to the amide compound via reaction with triplet excited state dissolved organic matter (³DOM*). As clay minerals and humic substances are important soil constituents and ubiquitously present in surface environments, the observed clay and humic-dependent photooxidation pathways of tetracycline and the differing selective pressures of the associated products highlight the need for monitoring the transformation compounds of antibiotics and provide critical insight into the development of antibiotic treatment protocols.