Degradation and deactivation of bacterial antibiotic resistance genes during exposure to (waste)water treatment and health-care disinfectants

Abstract

Conventional disinfectants applied in (waste)water treatment and healthcare practice can potentially provide important barriers to antibiotic resistance dissemination through inactivation of antibiotic resistant bacteria (ARB). However, limited information is available on whether and how these disinfection processes could effectively eliminate ARGs and their biological activities in disseminating resistance traits to non-resistant bacterial populations via horizontal gene transfer processes such as natural transformation. In this context, this work was undertaken to investigate the degradation (i.e., chemical modification as measured by qPCR) and/or deactivation (i.e., biological activity loss as measured by culture-based natural transformation assays) of various extra/intra-cellular ARGs, including blt, mecA, vanA, tet(A), ampC, blaNDM, and/or blaKPC (harbored by Bacillus subtilis, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, respectively) during treatment of commonly-used (waste)water disinfectants/oxidants, including free available chlorine (FAC), monochloramine (NH2Cl), ozone (O3), chlorine dioxide (ClO2), ultraviolet (UV) light, and hydroxyl radical (•OH), as well as healthcare disinfectants/antiseptics, including glutaraldehyde, chlorhexidine, ethanol, povidone-iodine, benzalkonium chloride, phenol, sodium hypochlorite, and hydrogen peroxide (H2O2). Kinetics results of (waste)water disinfections highlighted a wide range of ARG susceptibilities toward different chemical disinfectants, with reactivities declining in the order HO• >> O3 > FAC >> ClO2 > NH2Cl. For a given disinfectant, second-order or fluence-based first-order rate constants determined for various ARG or 16S rRNA gene amplicons (ranging from 143–1509 bp) increased linearly with contents of AT+GC base pairs (for FAC, NH2Cl, O3) or intrastrand doublets of 5’-TT-3’ (for UV) or 5’-GG-3’ (for ClO2) per amplicon. For FAC, NH2Cl, O3, ClO2, and UV, blt gene 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, due to its distinctive mechanism of DNA fragmentation rather than nucleobase alteration. These results enabled the development of a modeling approach for predicting degradation/deactivation rate constant and kinetics of a given DNA segment, based on its DNA sequence length or nucleotide content and elementary reactions toward each of the above disinfectants (except •OH). This kinetics-based modeling framework has been validated and utilized to predict degradation/deactivation profiles of a broad array of ARGs and 16S rRNA gene segments during bench-scale disinfections in clean buffer and/or wastewater matrixes, as well as their removal efficiencies in selected full-scale processes. At practical exposure ranges, extra- and intracellular ARGs/16S rRNA gene were 1-3 log10 degraded/deactivated by FAC, O3, and UV, but recalcitrant to NH2Cl and ClO2, which can be adequately predicted by the kinetics-based model under certain circumstances. Intracellular ARG degradation/deactivation always lagged behind host ARB inactivation. Bench-scale experimental and modeling results supported observations at local wastewater treatment plants (WWTPs) that UV provided ~1 log10 ARG elimination, while chlorination of high-ammonia (non-nitrified) wastewater yielded no significant ARG removal, due to rapid conversion of FAC into NH2Cl (non-reactive toward DNA) in excess ammonia. Last, this work was extended to healthcare disinfections by treating the model blt gene (primarily in its intracellular form) with each healthcare disinfectant on inanimate surface or in aqueous phase. Surface disinfection results demonstrated that typical healthcare conditions provided non-significant or minimal (< 1-log10) elimination of blt gene copy numbers on hard surfaces, although 2–5-log10 ARB inactivation was achieved by each disinfectant (except H2O2). Results of H2O2 treatment in aqueous phase elucidated that intracellular ARG deactivation in the presence of H2O2, also lagging behind ARB inactivation, is likely due to H2O2 decomposition to form •OH catalyzed by intracellular iron, leading to DNA strand fragmentation without base alteration. Findings of this work have provided systematic understanding of ARG fate during exposures of various disinfectants, and highlighted that ARGs could persist after effective ARB inactivation, potentially leading to resistance dissemination into downstream environments. Outcomes of this work provided significant implications on how to select, design, and improve disinfection practices in (waste)water treatment and clinical facilities.