Application of Nucleotide-Based Kinetic Modeling Approaches to Predict Antibiotic Resistance Gene Degradation during UV- and Chlorine-Based Wastewater Disinfection Processes: From Bench- to Full-Scale.

This study investigated antibiotic resistance gene (ARG) degradation kinetics in wastewaters during bench- and full-scale treatment with UV light and chlorine─with the latter maintained as free available chlorine (FAC) in low-ammonia wastewater and converted into monochloramine (NH2Cl) in high-ammonia wastewater. Twenty-three 142-1509 bp segments (i.e., amplicons) of seven ARGs (blt, mecA, vanA, tet(A), ampC, blaNDM, blaKPC) and the 16S rRNA gene from antibiotic resistant bacteria (ARB) strains Bacillus subtilis, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were monitored as disinfection targets by qPCR. Rate constants for ARG and 16S rRNA gene amplicon degradation by UV, FAC, and NH2Cl were measured in phosphate buffer and used to expand and validate several recently developed approaches to predict DNA segment degradation rate constants based solely on their nucleotide contents, which were then applied to model ARG degradation during bench-scale treatment in buffer and wastewater matrixes. Kinetics of extracellular and intracellular ARG degradation by UV and FAC were well predicted up to ∼1-2-log10 elimination, although with decreasing accuracy at higher levels for intracellular genes, while NH2Cl yielded minimal degradation under all conditions (agreeing with predictions). ARB inactivation kinetics varied substantially across strains, with intracellular gene degradation lagging cell inactivation in each case. ARG degradation levels observed during full-scale disinfection at two wastewater treatment facilities were consistent with bench-scale measurements and predictions, where UV provided ∼1-log10 ARG degradation, and chlorination of high-ammonia wastewater (dominated by NH2Cl) yielded minimal ARG degradation.

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

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.