Degradation and Deactivation of Bacterial Antibiotic Resistance Genes during Exposure to Free Chlorine, Monochloramine, Chlorine Dioxide, Ozone, Ultraviolet Light, and Hydroxyl Radical

Abstract

This work investigated degradation (measured by qPCR) and biological deactivation (measured by culture-based natural transformation) of extra- and intracellular antibiotic resistance genes (eARGs and iARGs) by free available chlorine (FAC), NH₂Cl, O₃, ClO₂, and UV light (254 nm), and of eARGs by ^•OH, using a chromosomal ARG ( blt) of multidrug-resistant Bacillus subtilis 1A189. Rate constants for degradation of four 266-1017 bp amplicons adjacent to or encompassing the acfA mutation enabling blt overexpression increased in proportion to #AT+GC bps/amplicon, or in proportion to #5'-GG-3' or 5'-TT-3' doublets/amplicon, with respective values ranging from 0.59 to 2.3 (×10¹¹ M^-1 s^-1) for ^•OH, 1.8-6.9 (×10⁴ M^-1 s^-1) for O₃, 3.9-9.2 (×10³ M^-1 s^-1) for FAC, 0.35-1.2(×10¹ M^-1 s^-1) for ClO₂, and 2.0-8.8 (×10^-2 cm²/mJ) for UV at pH 7, and from 1.7-4.4 M^-1 s^-1 for NH₂Cl at pH 8. For FAC, NH₂Cl, O₃, ClO₂, and UV, ARG deactivation paralleled degradation of amplicons approximating a ∼800-1000 bp acfA-flanking sequence required for natural transformation in B. subtilis, whereas deactivation outpaced degradation for ^•OH. At practical disinfectant exposures, eARGs and iARGs were ≥90% degraded/deactivated by FAC, O₃, and UV, but recalcitrant to NH₂Cl and ClO₂. iARG degradation/ deactivation always lagged cell inactivation. These findings provide a quantitative framework for evaluating ARG fate during disinfection/oxidation, and support using qPCR as a proxy for tracking ARG deactivation under carefully selected circumstances.